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UNIT –– 1- MPC203T – Computer aided drug design

NIT – 1- MPC203T – Computer aided drug design

Drug targets
A biological target is anything within a living organism to which some other entity (like an
endogenous ligand or a drug) is directed and/or binds, resulting in a change in its behavior or
function. Examples of common classes of biological targets are proteins and nucleic acids.
The definition is context-dependent, and can refer to the biological target of
a pharmacologically active drug compound, the receptor target of a hormone (like insulin), or
some other target of an external stimulus. Biological targets are most commonly proteins such
as enzymes, ion channels, and receptors.
Mechanism
The external stimulus (i.e., the drug or ligand) physically binds to ("hits") the biological
target.[1][2] The interaction between the substance and the target may be:
 noncovalent – A relatively weak interaction between the stimulus and the target where no
chemical bond is formed between the two interacting partners and hence the interaction is
completely reversible.
 reversible covalent – A chemical reaction occurs between the stimulus and target in
which the stimulus becomes chemically bonded to the target, but the reverse reaction also
readily occurs in which the bond can be broken.
 irreversible covalent – The stimulus is permanently bound to the target through
irreversible chemical bond formation.
Depending on the nature of the stimulus, the following can occur:[3]
 There is no direct change in the biological target, but the binding of the substance
prevents other endogenous substances (such as activating hormones) from binding to the
target. Depending on the nature of the target, this effect is referred as receptor
antagonism, enzyme inhibition, or ion channel blockade.
 A conformational change in the target is induced by the stimulus which results in a
change in target function. This change in function can mimic the effect of the endogenous
substance in which case the effect is referred to as receptor agonism (or channel
or enzyme activation) or be the opposite of the endogenous substance which in the case
of receptors is referred to as inverse agonism.
The term "biological target" is frequently used in pharmaceutical research to describe the
native protein in the body whose activity is modified by a drug resulting in a specific effect,
which may be a desirable therapeutic effect or an unwanted adverse effect. In this context, the
biological target is often referred to as a drug target. The most common drug targets of
currently marketed drugs include:

Drug targets
 proteins
o G protein-coupled receptors (target of 50% of drugs)[7]
o enzymes (especially protein kinases, proteases, esterases, and phosphatases)
o ion channels
 ligand-gated ion channels
 voltage-gated ion channels
o nuclear hormone receptors
o structural proteins such as tubulin
o membrane transport proteins
 nucleic acids

Databases containing biological targets information:

 Therapeutic Targets Database (TTD)
 DrugBank
 Binding DB

Membrane proteins as drug targets

Membrane proteins are common proteins that are part of, or interact with, biological
membranes. Membrane proteins fall into several broad categories depending on their
location. Integral membrane proteins are a permanent part of a cell membrane and can either
penetrate the membrane (transmembrane) or associate with one or the other side of a
membrane (integral monotopic). Peripheral membrane proteins are transiently associated with
the cell membrane.
Membrane proteins are common, and medically important—about a third of all human
proteins are membrane proteins, and these are targets for more than half of all
drugs.[1] Nonetheless, compared to other classes of proteins, determining membrane protein
structures remains a challenge in large part due to the difficulty in establishing experimental
conditions that can preserve the correct conformation of the protein in isolation from its
native environment.
Function
Membrane proteins perform a variety of functions vital to the survival of organisms:[2]
 Membrane receptor proteins relay signals between the
cell's internal and external environments.
 Transport proteins move molecules and ions across the membrane. They can be
categorized according to the Transporter Classification database.
 Membrane enzymes may have many activities, such as oxidoreductase, transferase or
hydrolase.[3]
 Cell adhesion molecules allow cells to identify each other and interact. For example,
proteins involved in immune response
The localization of proteins in membranes can be predicted reliably using hydrophobicity
analyses of protein sequences, i.e. the localization of hydrophobic amino acid sequences.
Intergral membrane proteins
Integral membrane proteins are permanently attached to the membrane. Such proteins can be
separated from the biological membranes only using detergents, nonpolar solvents, or
sometimes denaturing agents. One such example of this type of protein which has not been
functionally characterized yet is SMIM23. They can be classified according to their
relationship with the bilayer:
 Integral polytopic proteins are transmembrane proteins that span across the membrane
more than once. These proteins may have different transmembrane topology.[4][5] These
proteins have one of two structural architectures:
o Helix bundle proteins, which are present in all types of biological membranes;
o Beta barrel proteins, which are found only in outer membranes of Gram-negative
bacteria, and outer membranes of mitochondria and chloroplasts.[6]
 Bitopic proteins are transmembrane proteins that span across the membrane only once.
Transmembrane helices from these proteins have significantly different amino acid
distributions to transmembrane helices from polytopic proteins.[7]
 Integral monotopic proteins are integral membrane proteins that are attached to only one
side of the membrane and do not span the whole way across
Peripheral membrane proteins
Peripheral membrane proteins are temporarily attached either to the lipid bilayer or to integral
proteins by a combination of hydrophobic, electrostatic, and other non-covalent interactions.
Peripheral proteins dissociate following treatment with a polar reagent, such as a solution
with an elevated pH or high salt concentrations.
Integral and peripheral proteins may be post-translationally modified, with added fatty
acid, diacylglycerol[8] or prenyl chains, or GPI (glycosylphosphatidylinositol), which may be
anchored in the lipid bilayer.
THE ROLE MEMBRANE PROTEINS PLAY AS DRUG TARGETS
The ligands can target membrane proteins to regulate their biological activity,
as drugs. The percentage of the es-tablished drugs acting on proteins to achieve
therapeutic ef-fects has reached 88%. For rational drug target discov-ery, we should
understand the role of MP target in biological pathways. In the review, we surveyed the
drug binding tar-gets in the DrugBank [21], if it could be traced from UniProt [28],
reviewed protein and annotated ‘membrane‘ (kw-0472), the drug binding protein
could be seen as membrane protein drug-target (statistics from April 20, 2018).
Conse-quently, for all drugs comprising approved and experi-mented, the proportion of
MPs is 42% (Fig. 1A). Whereas for approved drugs binding targets, 54% of them are
MPs (Fig. 1B). Protein targets are separated into different classes in dif-ferent kinds
of literature or databases according to their phylogenetics, functions and tissue
expression etc. Latest researches have shed light on the viewpoint that conven-tional
therapeutic drug targets are classified into about 130 protein families [29, 30]. Global
academics and experts gen-erally divide those protein targets mainly in four classes:
enzymes, transporters, ion channels, and receptors. Some articles hold one standpoint that
most drug targets are consti-tuted by GPCRs, ion channels, proteases, kinases or nuclear
hormone receptors [31].
Another classification method is DTO, the four sorts were GPCRs, kinases, ion
channels and nuclear receptors. In DrugBank, each drug can have one or more targets,
enzymes, transporters, and carriers asso-ciated with it. It divided a drug binding target as one
or more kind of the four types, target, enzyme, transporter, carrier, respectively. After
statistics, demonstrates the num-ber of nonredundant approved drug binding proteins (DBPs)
and MPs (only human proteins) in four types respectively. In (Fig. 3), approved drug
binding targets have been classified according to the DrugBank that are enzyme,
transporter, tar-get, carrier and their Venn diagram. (Fig. 3) clearly shows that a
protein belongs to one or more categories, which means that the data cannot be used
directly because of the redundancy. Overwhelming evidence now implicates that
GPCRs, ion channels and transporters are common MP tar-gets, all of which play crucial
roles in biochemical pathways and have an extraordinary tendency on diseases treatment
and to be drug targets [1]. The introduction of GPCRs, ion channels and transporters
are as follows. 2.1. G Protein-coupled Receptors (GPCRs) GPCRs also called 7TMs
has the similar architectural characteristic referring to a domain in which the protein
passes through the cell membrane seven times alternatively from the extracellular side
to intracellular and back [33]. GPCRs have been the mainstay biological target for
pharmacological therapy because they are the primary surface receptors to delivery signal and
little substance. Therefore, GPCRs are associated with many diseases, and there has been a
large number of researches focusing on the GPCR mechanisms and their biological
activities, such as interacting with drugs. Hauser et al. make statistics and manually curate
drugs in Clinical Trials database and other sources, resulting in that 475 drugs about
34% of the drugs that the US FDA approved act at GPCRs [34]. In 2008, twelve of the top-15
prescription drugs and top-15 generic drugs targeted GPCRs [35, 36]. The above
evidence indicates the importance of GPCRs.

Structure and function of receptors

• Globular proteins acting as a cell Globular proteins acting as a cell’s ‘letter boxes etter
boxes’
• Located mostly in the cell membrane Located mostly in the cell membrane
• Receive messages from chemical messengers coming from other Receive messages from
chemical messengers coming from other cells
• Transmit a message into the cell leading to a cellular effect Transmit a message into the cell
leading to a cellular effect
• Different receptors specific for different chemical messengers Different receptors specific
for different chemical messengers
• Each cell has a range of receptors in the cell membrane making it Each cell has a range of
receptors in the cell membrane making it responsive to different chemical messengers
responsive to different chemical messengers
 Receptors, which locate on both the cell surface and within the cell, are drug targets
where medicine produce their beneficial effects in various disease states.

Receptors are typically envisaged as cell surface recognition sites for endogenous hormones,
neurotransmitters, and neuromodulators. They are coupled to various signal transduction
systems located both within the membrane and intracellularly, and can therefore regulate
responses to the cellular/tissue microenvironment.

Receptors can be defined in terms of their selectivity, the saturability and reversibility of
ligand binding, and functionality. The definition of a receptor in both pharmacological and
physiological terms requires that it has specific interactions with ligands that belong to a
given pharmacological class.

Receptors are complex proteins with multiple potential ligand recognition sites, including
sites that may be distinct from the endogenous agonist recognition site and may actually
reside on distinct proteins that are part of the receptor complex.

Such receptor modulatory sites may represent novel drug targets, e.g., allosteric or
modulatory sites. The effect of benzodiazepines (BZs) on GABAA receptor function
illustrates the conceptualization of ancillary drug targets and the elusive nature of the
proposed endogenous modulator, presumed to be a "BZ-like" substance.

In septic shock, the induction of a toxic cytokine receptor-mediated cascade has significantly
complicated the search for new drugs to treat this condition. This emphasizes the need to
define key targets in critical pathways rather than attempt to treat their sequelae.

It is possible that many diseases are the result of multifactorial events that vary during the
pathophysiological course of the illness. For instance, >32 discrete gene loci have been
associated with schizophrenia. Therefore, drug targets that are downstream from key points in
the disease transduction pathway may not be the optimal targets for treating the disorder.
G protein-coupled receptors
G protein-coupled receptors (GPCRs), which include ~900 members, represent the most
leading family of validated drug targets in biomedicine. G protein-coupled receptors
(GPCRs) have an important role in multiple diseases, including the development of cancer
and cancer metastasis, and that's what makes GPCRs perfect drug targets for modern
medicinal drugs.

When some GPCRs ligands such as chemokine, thrombin, lysophosphatidic acid (LPA),
gastrin-releasing peptide and endothelin bind to their receptors, it causes a comformational
change in G protein-coupled receptors (GPCRs) , which are involved in two signal
transduction pathways: cAMP signaling pathway and phosphatidylinositol signaling pathway.

Recently, G protein-coupled receptors (GPCRs) have been an uprising star in drug therapy.
One example of such GPCRs is chemokine receptor type 4 (CXCR4) which has great
potential in drug target research. CXCR4 is thought to be involved in many disease states
including more than 23 types of cancer and several immunodeficiency disorders, including
head and neck cancer, breast cancer, small-cell lung cancer, non-small-cell lung cancer and
Acquired Immune Deficiency Syndrome (AIDS).

G-protein-coupled receptor/GPCR ligands are compounds that bind to a GPCRs. These

may be conceptualized as "keys" that fit into a specific "lock" on the cell surface, the latter
being the receptor, to modify cellular activity at the biophysical, biochemical, and/or genomic
level.

The complex signaling pathways modulated by G-protein-coupled receptors/GPCRs offer a

variety of potential drug targets. GPCRs are coupled to various members of the G protein
superfamily, so named because of their functional dependence on the hydrolysis of the purine
nucleotide, GTP, for activity.

GPCRs in mammals are classified into five main families, named Glutamate, Rhodopsin,
Adhesion, Frizzled, and Secretin according to the GRAFS classification.
GPCRs regulate physiological responses to a variety of stimuli that include endogenous
ligands such as biogenic amines, peptides, glycoproteins, lipids, nucleotides, Ca2+ ions, and
various exogenous ligands for sensory perception such as odorants, pheromones, and even
photons.

As a consequence, these receptors mediate multiple physiological processes such as

neurotransmission, cellular metabolism, secretion, cellular differentiation, growth,
inflammatory, and immune responses.

It is estimated that ~50% of clinically prescribed drugs and 25 of the 100 top selling drugs
target GPCRs. Yet, only a small fraction of all GPCRs are presently targeted by drugs.
Receptor Serine/Threonine Kinases (RSTKs
Receptor Serine/Threonine Kinases (RSTKs), respond to specific cytokines, including the
transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) families. With
the recent clinical success of drugs targeting protein kinase activity, drug discovery efforts are
focusing on the role of reversible protein phosphorylation in disease states.

Activins signal through a combination of type I and II transmembrane serine/threonine kinase

receptors. Activin receptors are shared by multiple transforming growth factor-β (TGF-β)
ligands such as myostatin, growth and differentiation factor-11 and nodal.
Enzymes as drug targets
 Medicine in twenty first century has become a science in which drug molecules are
directed against the macromolecules. Enzymes hold a prominent position among the
biological macromolecules that can be used as drug targets.
Some features of enzyme structures and reaction pathways which make them
attractive and primary target for drug action are as follows
 They play an essential role in biological life processes and pathophysiology
 The structures of active sites of enzymes and the ligand binding pockets are highly
amenable for high affinity interactions with small drug like molecules
 Presence of potential allosteric sites
 Conformational variations in the binding sites
 Can be directly used as a therapeutic agent
Enzymes are biological catalysts as well as can act as receptors by acting with the substrates.
These are the most efficient catalysts known in nature. They have the ability to enhance
reaction rates by lowering the activation energy of reactions and by stabilizing the reacting
molecules at their activated complex states. Stabilization theory of the activated complex by
enzymes was first proposed by Pauling. He concluded that the active site of enzyme is
complementary to the structure of the activated complex so that the binding of the enzyme to
the activated complex is extremely tight, which reduces the activation energy and enhance the
reaction rate.
Types of reversible inhibitors
Reversible inhibitors attach to enzymes with non-covalent interactions such as hydrogen
bonds, hydrophobic interactions and ionic bonds. Multiple weak bonds between the inhibitor
and the active site combine to produce strong and specific binding. In contrast
to substrates and irreversible inhibitors, reversible inhibitors generally do not undergo
chemical reactions when bound to the enzyme and can be easily removed by dilution
or dialysis.
There are four kinds of reversible enzyme inhibitors. They are classified according to the
effect of varying the concentration of the enzyme's substrate on the inhibitor.
Types of inhibition. This classification was introduced by W.W. Cleland.[5]
 In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the
same time, as shown in the figure on the right. This usually results from the inhibitor
having an affinity for the active site of an enzyme where the substrate also binds; the
substrate and inhibitor compete for access to the enzyme's active site. This type of
inhibition can be overcome by sufficiently high concentrations of substrate (Vmax remains
constant), i.e., by out-competing the inhibitor. However, the apparent Km will increase as
it takes a higher concentration of the substrate to reach the Km point, or half the Vmax.
Competitive inhibitors are often similar in structure to the real substrate (see examples
below).
 In uncompetitive inhibition, the inhibitor binds only to the substrate-enzyme complex.
This type of inhibition causes Vmax to decrease (maximum velocity decreases as a result of
removing activated complex) and Km to decrease (due to better binding efficiency as a
 result of Le Chatelier's principle and the effective elimination of the ES complex thus
decreasing the Km which indicates a higher binding affinity).
 In non-competitive inhibition, the binding of the inhibitor to the enzyme reduces
its activity but does not affect the binding of substrate. As a result, the extent of inhibition
depends only on the concentration of the inhibitor. Vmax will decrease due to the inability
for the reaction to proceed as efficiently, but Km will remain the same as the actual
binding of the substrate, by definition, will still function properly.
 In mixed inhibition, the inhibitor can bind to the enzyme at the same time as the
enzyme's substrate. However, the binding of the inhibitor affects the binding of the
substrate, and vice versa. This type of inhibition can be reduced, but not overcome by
increasing concentrations of substrate. Although it is possible for mixed-type inhibitors to
bind in the active site, this type of inhibition generally results from an allosteric effect
where the inhibitor binds to a different site on an enzyme. Inhibitor binding to
this allosteric site changes the conformation (i.e., tertiary structure or three-dimensional
shape) of the enzyme so that the affinity of the substrate for the active site is reduced.
These types can also be distinguished by the effect of increasing the substrate concentration
[S] on the degree of inhibition caused by a given amount of inhibitor. For competitive
inhibition the degree of inhibition is reduced by increasing [S], for noncompetitive inhibition
the degree of inhibition is unchanged, and for uncompetitive (also called anticompetitive)
inhibition the degree of inhibition increases with [S]
Irreversible inhibitors
Types of irreversible inhibition (covalent inactivation)
Reaction of the irreversible inhibitor diisopropylfluorophosphate (DFP) with a serine protease
Irreversible inhibitors usually covalently modify an enzyme, and inhibition can therefore not
be reversed. Irreversible inhibitors often contain reactive functional groups such as nitrogen
mustards, aldehydes, haloalkanes, alkenes, Michael acceptors, phenyl sulfonates,
or fluorophosphonates. These nucleophilic groups react with amino acid side chains to
form covalent adducts. The residues modified are those with side chains
containing nucleophiles such as hydroxyl or sulfhydryl groups; these include the amino
acids serine (as in DFP, right), cysteine, threonine, or tyrosine.
Irreversible inhibition is different from irreversible enzyme inactivation. Irreversible
inhibitors are generally specific for one class of enzyme and do not inactivate all proteins;
they do not function by destroying protein structure but by specifically altering the active site
of their target. For example, extremes of pH or temperature usually cause denaturation of
all protein structure, but this is a non-specific effect. Similarly, some non-specific chemical
treatments destroy protein structure: for example, heating in concentrated hydrochloric
acid will hydrolyse the peptide bonds holding proteins together, releasing free amino acids.[26]
Irreversible inhibitors display time-dependent inhibition and their potency therefore cannot be
characterised by an IC50 value.[1][27] This is because the amount of active enzyme at a given
concentration of irreversible inhibitor will be different depending on how long the inhibitor is
pre-incubated with the enzyme. Instead, kobs/[I] values are used,[28] where kobs is the observed
pseudo-first order rate of inactivation (obtained by plotting the log of % activity vs. time) and
[I] is the concentration of inhibitor. The kobs/[I] parameter is valid as long as the inhibitor does
not saturate binding with the enzyme (in which case kobs = kinact).

Gene silencing

As the name implies, gene silencing is a technique that aims to reduce or eliminate the
production of a protein from its corresponding gene. Genes are sections of DNA that contain the
instructions for making proteins. Proteins are essential molecules that perform an array of
functions including signaling between cells, speeding up biochemical reactions, and providing
structural support for the cell. Each gene is responsible for producing a corresponding protein in
a two-step process. First, a copy of the information encoded in a gene is made in the form
of messenger RNA (mRNA), a process known as transcription. This occurs in the nucleus of
the cell, the cellular structure where all of the cell’s genetic material is
contained. The mRNA subsequently travels out of the nucleus, and the genetic information it
carries is used to produce a specific protein, a process known as translation. (For more
information about proteins and how they are made, click here.)
Instead of directly editing DNA or inhibiting the transcription process, the key idea
behind gene silencing is intervening in gene expression prior to translation. By designing
a molecule that can specifically identify and breakdown the mRNA carrying instructions for
making a certain protein, scientists have been able to effectively decrease levels of
that protein. Imagine the gene silencing molecule as a censor and mRNA as messages from genes
that are broadcast into proteins: the molecule will censor out a specified mRNA message,
preventing the corresponding protein from being broadcast into the cell, and thus silencing
the gene that is providing these instructions. The ability to significantly lower the levels of a
specific protein opens up many possibilities in scientific research and drug development, since
proteins are critically involved in the proper function and structure of cells.
Types of Gene Silencing Techniques^
There are various gene silencing methods currently employed in research and being
developed as potential disease therapeutics. Nearly all of them involve disabling the function
of mRNA by preventing it from being translated into a protein. However, they differ in the
design of the molecule used to disrupt mRNA and the manner of mRNA breakdown. As a result,
different silencing methods have specific advantages and drawbacks. Two of the leading and
most understood methods of gene silencing are RNA interference (RNAi) and antisense
oligonucleotides (ASOs).
RNA Interference
In RNAi, the molecules that identify the target mRNA are called small-interfering RNAs
(siRNAs). Unlike normal single-stranded RNA found in cells – such as mRNA – siRNAs are
short, synthetically made double-stranded RNA molecules designed to pair with a
specific mRNA strand. This association of the siRNAs with a particular target mRNA causes the
breakdown of the target mRNA by recruiting other proteins
that degrade the mRNA target. Because siRNAs are double-stranded, they are more stable and
less susceptible to degradation than ASOs, allowing them to continue to perform their silencing
function for a longer period of time in the cell. For a more detailed description of how RNAi
works, click here.
Antisense Oligonucleotides
 Similar to siRNAs, ASOs are engineered by scientists to associate with a
target mRNA strand. The binding of the ASO to mRNA directs a protein to breakdown
the mRNA. However, unlike siRNAs, ASOs are smaller, single-stranded RNA molecules. As
mentioned above, single-stranded RNAs are not as stable as double-stranded ones; thus, ASOs are
often chemically modified to increase their durability in a biological environment. However,
their smaller size and chemical structure allow ASOs to be transported in cells and living tissues
much more effectively than siRNAs. For a more detailed description of how ASOs work,
click here.
Is one gene silencing method better than the other?
In terms of developing a drug therapy based on gene silencing, how do RNAi and ASOs
compare to each other in effectiveness? In cell culture experiments, gene silencing is often used
to intentionally decrease levels of a certain protein for research purposes. In such applications,
siRNAs have sometimes been shown to produce stronger and longer lasting gene silencing than
ASOs. However, when developing silencing therapeutics, the strength and duration of gene
silencing needed for treatment may vary; sometimes a shorter-acting or less complete gene
silencing may be required. Furthermore, when considering the efficacy of each method in
live animal models, the results are not as clear-cut. For example, as mentioned earlier, ASOs can
often be distributed more easily than siRNAs throughout the target tissue because of their size and
structure. This observation would be expected to simplify delivery and lower costs of a
therapeutic application. The fact that there is no definitive answer to which gene
silencing method is more effective has resulted in continued active research and development of
both areas.

Antisense technology:
 Antisense technology is a recent approach to specific modification or inhibition of gene
expression in vitro or in vivo.
 It is a tool to study gene function and utilize it to manipulate the gene expression within
cells to treat an endless number of diseases.
 The antisense approach utilizes antisense agents to alter the expression of viral
genome inside the host cell or regulate the expression of specific genes that causes that
particular disease.
  Sense strand or sequence: it is the coding strand within double-stranded DNA that
carries the translatable code in the 5′ to 3′ direction. It is complementary to the template
strand. The sense strand have sequences similar to that of mRNA.
 Antisense strand: it is the template strand of ds DNA, from which mRNA is
transcribed. Thus, the antisense strand is complementary to mRNA.
 In simple term ‘sense’ refers to original sequence of DNA or RNA molecule and
‘antisense’ refers to Complementary copy.
 The antisense strand base pairs with its complementary mRNA strands (sense RNA)
and thus prevents it from being translated into a protein.
 Therefore in antisense technology, the complementary nucleic acid sequence (antisense
agents) is utilized to silence gene expression. The binding, or hybridization, of
antisense nucleic acid sequences to a specific mRNA target will inhibit normal gene
expression (ie. either interrupt transcription or translation) resulting in flow of message
from DNA to protein.
 There are several mechanisms to interrupt the gene expression by antisense technology.
Sometime the gene expression is completely inhibited known as knock-out and other
time it is partially interrupted known as knock-down.
 Some of the antisense agents are:
 Antisense oligonucleotide: like oligodeoxyribonucleotides (ODN) having less
than 30 mucleotides or longer antisense RNA (a RNA) sequences
 First generation
 Second generation
 Third generation
 Ribozymes
 RNA interference (RNAi)
1. Antisense oligonucleotide:
 Zamecnik and Stephenson first demonstrated the antisense effect of synthetic
oligonucleotide
 Zamecnik and Stephenson identified a repeated sequence of 21 nucleotides that was
crucial to viral integration with the help of nucleotide sequences from the 5’ and 3’
ends of the 35S RNA of Rous sarcoma virus (RSV).
 They synthesized a 13-mer oligonucleotide, d(AATGGTAAAATGG), complement to
the portion of this viral sequence.
  Viral production got inhibited when synthetic oligonucleotide was introduced into
cultured fibroblast cells. Thus, they concluded that oligonucleotide was inhibiting viral
integration by hybridizing to the crucial sequences and blocking them. They introduced
the term ‘hybridon’ to describe such oligonucleotides.
 At the same time, Tennant et al and Miller et al reported similar effects for synthetic
oligonucleotides in other systems.
 Criteria for successful oligonucleotide
 Specific target recognition by Watson-Crick pairing
 Good structural Mimicry
 Activation of RNaseH
 Enhanced cellular uptake
 Enhanced resistance to various nucleases: Synthetic oligonucleotides are foreign
to the cells into which they are introduced and thus becomes prey for endogenous
nucleases.
 Synthetic oligonucleotides were protected from endogenous nuclease when they
attained the persistence level in cell.
 There are three possible sites on a nucleotide where protective modifications could be
introduced.
Antisense Oligonucleotide modification: first, second and third generation
 The three possible sites for oligonucleotide modification are- at the position of
Nitrogenous Base, Ribose sugar (2’ OH group) and the Phosphate backbone.
 The main purpose of modification is to protect the antisense nucleotide from nuclease
degradation when introduced inside cells. And at the same time it should be considered
that the modification do not alter the inhibit hybridization ability of the antisense
nucleotide.
First generation modification:
 The first generation antisense-motivated nucleotide modification is made by Eckstein
and colleagues in the late 1960s by replacing one of the oxygen atom (non-bridging
oxygen) of the phosphate backbone with a sulfur atom.
 This modified antisense agent is known as phosphorothioate.
 Phosphorothioate oligonucleotide is more nuclease resistant than the original
oligonucleotide. The nuclease resistance was measured by an increased half-life for a
phosphorothioated oligonucleotide upto ten hours in human serum as compared to that
of one hour of an unmodified oligonucleotide having the same sequence.
  However, the phosphorothionated nucleotide displayed slight reduced hybridization
ability and also a tendency to bind un-specifically to certain proteins in cell. High
concentration of phosphrothionated nucleotide thus result in cytotoxicity.
 Matsukura and colleagues demonstrated that phosphorothioated oligonucleotides were
effective hybridons against the HIV replication in the cultured cells.
 The first FDA-approved antisense drug is Vitravene from ISIS (Carlsbad, CA, USA.)
Second generation modification:
 The second generation modification focused on non-specific bind with certain proteins
and cytotoxic effect of phosphorothioated nucleotides.
 In this modification, the antisense oligonucleotide undergoes alkyl modifications at the
carbon no 2 (C2 position) of the ribose sugar.
 The two most important of these modifications are 2’-O-methyl and 2’-O-methoxy-
ethyl at the C2 position.
 After alkylation at the C2 position of ribose sugar, the antisense oligonucleotides
become resistant to nuclease degradation and shows low cytotoxicity effect.
 However, the antisense oligonucleotide with 2’-O-alkyl modification are unavailable
for RNase H cleavage after hybridization with sense RNA.
 Since RNase H cleavage is the most desirable mechanism for antisense effect, A hybrid
oligonucleotide is constructed containing both the desirable characteristics of nuclease
resistance and RNase cleavage and it is known as gapmer antisense oligonucleotide.
 Gapmer antisense oligonucleotide:
 This hybrid antisense oligonucleotide contains a central block of
deoxynucleotides sufficient to induce RNase H cleavage flanked by blocks of 2’-
O-methyl modified nuclease resistance ribonucleotide.
Third generation modification:
 In this modification a.
 Antisense oligonucleotide forms either DNA: DNA homo-duplex or DNA: RNA
hetero-duplex depending upon the nature of oligonucleotide.
 The unmodified oligo-deoxynucleotides form such desired DNA: DNA or DNA:RNA
duplexes.
 However variety of nucleic acid analogs have been developed having high affinity with
target DNA or RNA and as a modification these analogs are utilized for antisense
oligonucleotide construction.
  Some of these nucleic acid analogs are peptide nucleic acids (PNAs), 2’-fluoro N3-P5’-
phosphoramidites, 1’, 5’- anhydrohexitol nucleic acids (HNAs) and locked nucleic
acids.
 Among these analogs locked nucleic acid (LNA) is very desirable as shows promising
effect. The LNA is composed of nucleotides that is locked into a single conformation
through a 2’-0’, 4’-C methylene linkage in 1,2:5,6-di-O-isopropylene-α-allofuranose.
LNA has increased the thermodynamic stability and enhanced nucleic acid recognition.
2. Ribozymes:
 Ribozymes are known as catalytic RNA, first described by Tom Cech (1982) from
ribosomal RNA precursor from Tetrahymena thermophilia.
 Ribozymes acts as an enzymes to processes RNA precursors. And catalyze the
modification or alteration of RNA or even DNA.
3. RNA Interference (RNAi):
 RNA interference (RNAi) was first described by Fire and colleagues in Caenorhabditis
elegans.
 Several types of very short RNAs repress or silence the expression of genes and such
silencing is known as RNA interference.
 RNA interference manifest in different ways; some time by inhibiting translation of
mRNA and in other case by destruction of mRNA or silencing of promoter.
Antisense mediated gene silencing
 The basic concept in gene silencing utilizing antisense technology is that an antisense
oligonucleotide (a short DNA or RNA) is synthesized and introduced into a cell. Since
the antisense oligonucleotide is complementary to the targeted mRNA, it will bind
forming RNA dimers in the cytoplasm and halts protein synthesis. This occurs because
the mRNA no longer has access to the ribosome and cytoplasm. In the same time, it is
degraded by nucleases.
 Therefore, the introduction of antisense oligonucleotide results in silencing the
expression of gene.
 The events of gene expression is the flow of information from DNA into proteins
through transcription (post transcriptional modification) and translation (post-
translational modification).
 In the first step DNA is transcribed into pre-mRNA
 Pre-mRNA undergoes post transcriptional modification including- 5’ capping, removal
of intron (intron excision) and poly-adenylation to form mature functional mRNA.
 Then mRNA is transported to ribosome for translation.
 For silencing gene expression, the antisense oligonucleotide acts on each step of gene
expression to achieve antisense knock-down or knock-out of the targeted gene.
 Gene silencing by antisense mechanism includes-Blocking RNA splicing, accelerating
degradation of the RNAmolecule, and preventing introns from being spliced out of the
mRNA, impeding the exportation of mRNA into the cytoplasm, hindering translation,
and the triplex formation in DNA.
Silencing transcription:
 In this step, antisense oligonucleotide turn off the gene expression by binding either of
the three region- minor groove binding, Strand displaying by nucleic acid analogs, and
major groove binding , triplex forming oligonucleotides.
 Pyrrole-imidazole are minor groove binding antisense polypeptides that binds with
specific sequence in minor groove.
 Nucleic acid analogs containing antisense oligonucleotide binds with complementary
strand of DNA helix, displacing the other strand. This is because the affinity and
stability of nucleic acid analog with DNA is more than DNA: DNA duplex.
 Some antisense oligonucleotide are triplex forming and they create stable triplex DNA
helix. Triplex forming oligonucleotides binds to duplex through Hoogsteen hydrogen
binding: T-A:T and C-G:C triplets.
 Silencing post-transcription:
 In this step, antisense oligonucleotides inhibit post-transcriptional modification or RNA
splicing.
 Once the mRNA is transcribed from DNA, it undergoes several modification including
5’ capping, polyA tail and intron removal. After post transcriptional modification,
 mRNA is transported out from nucleus to cytoplasm. Once the mRNA is hybridized
with antisense oligonucleotide, the double stranded RNA duplex cannot be transported
out to cytoplasm.
 Removal of intron is essential for RNA splicing because intron are non-coding
sequences. In this process, the oligonucleotide-based antisense agent is used which
binds with specific sequence of pre-mRNA preventing intron-excision.
 Silencing translation:
 In this step, antisense oligonucleotide inhibit translation by complement pairing with
targeted mRNA.
 Binding of antisense oligonucleotide with mRNA results in duplex formation which
interferes with the transcription apparatus and prevents formation of the ribosomal
complex. Thus translation is halted.
 When an antisense strand binds to a mRNA strand, it form a double helix structure
which is recognized as faulty and degraded by RNase H preventing the production of
undesired protein.
 RNase H degradation mechanism is most used mechanism in gene silencing. RNase H
is an endogenous enzyme which cleaves the RNA moiety of an RNA: DNA duplex.
 RNase is present in both nucleus and cytoplasm. It cannot degrade single stranded
mRNA but can degrade dsRNA duplex.

RNA interference
 RNA interference is a natural phenomenon by which an mRNA is silenced thereby
inhibiting the protein coded from that particular mRNA.
 Scientists have been working on this field from 1990 and finally, two scientists named
Andrew Fire and Creg C. Mello shared the Nobel Prize in Physiology or Medicine for
their work on RNAi on the nematode worm, Caenorhabditis elegans, which they had
published on 1998.
 It has been serving as a very effective tool for the suppression of the desired protein by
silencing the required mRNA.
 The basic macro-molecules involved in the RNAi mechanism are two types of RNA
molecules: Micro RNA (miRNA) and small interfering RNA (siRNA).
 These two types of RNA can bind to specific mRNA through complementary bonding
and inhibit the translation by ribosomes.
 The miRNA are short non-coding RNA nucleotides produced from the chromosome
inside the nucleus and have hairpin sequences thereby folding it into double strands.
 The pre-miRNA is bound by a complex of protein called microprocessing unit which
contains an RNase III enzyme and a dsRNA binding subunit; this microprocessing
unit takes the pre-miRNA from the nucleus to the cytoplasm through the Nuclear pore
complex (NPC) where it is subjected to a protein called Dicer, which contains a dsRNA-
specific endonuclease subunit, which cleaves some portions of the pre-miRNA and a
mature miRNA is produced.
 On the other hand, siRNAs molecules which are exogenous dsRNA nucleotides are
similar in sequences to that of miRNA but are not produced inside the nucleus of the cell.
 It comes from external sources. After the processing of miRNA, both miRNA and siRNA
contains a passenger strand and a guide strand.
 A complex of protein and RNA called RNA induced silencing complex (RISC) is
bound to the guide RNA strand and the passenger RNA strand is degraded.
 This guide strand contains sequences that are complementary to a specific mRNA. It
binds to the mRNA and the RISC makes cleavages on the mRNA separating the guide-
mRNA double strand.
 The remaining mRNA is degraded by cytosolic exonucleases.
 Hence, the protein coded by the mRNA does not come into existence as the mRNA is
silenced.

miRNA
A microRNA (abbreviated miRNA) is a small single-stranded non-coding RNA molecule
(containing about 22 nucleotides) found in plants, animals and some viruses, that functions
in RNA silencing and post-transcriptional regulation of gene expression.[1] miRNAs function
via base-pairing with complementary sequences within mRNA molecules.[2] As a result, these
mRNA molecules are silenced, by one or more of the following processes: (1) Cleavage of
the mRNA strand into two pieces, (2) Destabilization of the mRNA through shortening of
its poly(A) tail, and (3) Less efficient translation of the mRNA into proteins
by ribosomes.[2][3]
miRNAs resemble the small interfering RNAs (siRNAs) of the RNA interference
(RNAi) pathway, except miRNAs derive from regions of RNA transcripts that fold back on
themselves to form short hairpins, whereas siRNAs derive from longer regions of double-
stranded RNA.[4] The human genome may encode over 1900 miRNAs,[5] although more
recent analysis indicates that the number is closer to 600.[6]
miRNAs are abundant in many mammalian cell types[7][8] and as extracellular circulating
miRNAs.[9] Circulating miRNAs are released into body fluids including blood and
cerebrospinal fluid and have the potential to be available as biomarkers in a number of
diseases.[9][10] MiRNAs appear to target about 60% of the genes of humans and other
mammals.[11][12] Many miRNAs are evolutionarily conserved, which implies that they have
important biological functions.[6][1] For example, 90 families of miRNAs have been conserved
since at least the common ancestor of mammals and fish, and most of these conserved
miRNAs have important functions, as shown by studies in which genes for one or more
members of a family have been knocked out in mice
UNIT – 3- – Computer aided drug design
Drug Designing is
1)challenging
2)Expensive
3)Time consuming
So, Multidisciplinary approach:
Computational tools, methodologies for structure guided approach + Global gene expression
data analysis by softwares.
Hence,
1) Efficiency increased
2) Cost effectiveness
3) Time saved
4) Strategies to overcome toxic side effects
• Rational drug design or simply rational design, is the inventive process of finding
new medications based on the knowledge of a biological target
• The drug is most commonly an organic small molecule that activates or inhibits the
function of a biomolecule such as a protein, which in turn results in a therapeutic
benefit to the patient.
• Drug design involves the design of small molecules that are complementary in shape
and charge to the biomolecular target with which they interact and therefore will bind
to it.
• Drug design frequently but not necessarily relies on computer modeling techniques.
• This type of modeling is often referred to as computer-aided drug design.
• Finally, drug design that relies on the knowledge of the three-dimensional structure of
the biomolecular target is known as structure-based drug design.
• A more accurate term is ligand design (i.e., design of a small molecule that will bind
tightly to its target)
 • Although modeling techniques for prediction of binding affinity are reasonably
successful, there are many other properties, such as bioavailability, metabolic half-
life, side effects, etc., that first must be optimized before a ligand can become a safe
and efficacious drug.
• Typically a drug target is a key molecule involved in a particular metabolic or
signaling pathway that is specific to a disease condition or pathology or to the
infectivity or survival of a microbial pathogen.
• Some approaches attempt to inhibit the functioning of the pathway in the diseased
state by causing target molecule to stop functioning.
• Drugs may be designed that bind to the active region and inhibit this target molecule.
• Another approach may be to enhance the normal pathway by promoting specific
molecules in the normal pathways that may have been affected in the diseased state.
• All drugs should also be designed so as not to affect any other important "off-target"
molecules or antitargets, since drug interactions with off-target molecules may lead to
undesirable side effects. Sequence homology is often used to identify such risks.
• Most commonly, drugs are organic small molecules produced through chemical
synthesis, but biopolymer-based drugs (also known as biologics) produced through
biological processes are becoming increasingly more common. In addition, mRNA-
based gene silencing technologies may have therapeutic applications
Types
There are two major types of drug design. The first is referred to as ligand-based drug
design and the second, structure-based drug design

Ligand-based
• Ligand-based drug design (or indirect drug design) relies on knowledge of other
molecules that bind to the biological target of interest.
• These other molecules may be used to derive a pharmacophore model that defines the
minimum necessary structural characteristics a molecule must possess in order to bind
to the target.
• In other words, a model of the biological target may be built based on the knowledge
of what binds to it, and this model in turn may be used to design new molecular
entities that interact with the target.
• Alternatively, a quantitative structure-activity relationship (QSAR), in which a
correlation between calculated properties of molecules and their experimentally
 determined biological activity, may be derived. These QSAR relationships in turn
may be used to predict the activity of new analogs.
Structure-based
• Structure-based drug design (or direct drug design) relies on knowledge of the three
dimensional structure of the biological target obtained through methods such as x-ray
crystallography or NMR spectroscopy.
• If an experimental structure of a target is not available, it may be possible to create a
homology model of the target based on the experimental structure of a related protein.
• Using the structure of the biological target, candidate drugs that are predicted to bind
with high affinity and selectivity to the target may be designed using interactive
graphics and the intuition of a medicinal chemist. Alternatively various automated
computational procedures may be used to suggest new drug candidates.
• Current methods for structure-based drug design can be divided roughly into two
categories.
• The first category is about “finding” ligands for a given receptor, which is usually
referred as database searching. In this case, a large number of potential ligand
molecules are screened to find those fitting the binding pocket of the receptor. This
method is usually referred as ligand-based drug design. The key advantage of
database searching is that it saves synthetic effort to obtain new lead compounds.
• Another category of structure-based drug design methods is about “building” ligands,
which is usually referred as receptor-based drug design. In this case, ligand molecules
are built up within the constraints of the binding pocket by assembling small pieces in
a stepwise manner. These pieces can be either individual atoms or molecular
fragments. The key advantage of such a method is that novel structures, not contained
in any database, can be suggested
Active site identification
• Active site identification is the first step in this program.
• It analyzes the protein to find the binding pocket, derives key interaction sites within
the binding pocket, and then prepares the necessary data for Ligand fragment link.
• The basic inputs for this step are the 3D structure of the protein and a pre-docked
ligand in PDB format, as well as their atomic properties.
• Both ligand and protein atoms need to be classified and their atomic properties should
be defined, basically, into four atomic types:
• hydrophobic atom: All carbons in hydrocarbon chains or in aromatic groups.
 • H-bond donor: Oxygen and nitrogen atoms bonded to hydrogen atom(s).
• H-bond acceptor: Oxygen and sp2 or sp hybridized nitrogen atoms with lone electron
pair(s).
• Polar atom: Oxygen and nitrogen atoms that are neither H-bond donor nor H-bond
acceptor, sulfur, phosphorus, halogen, metal, and carbon atoms bonded to hetero-
atom(s).
• The space inside the ligand binding region would be studied with virtual probe atoms
of the four types above so the chemical environment of all spots in the ligand binding
region can be known
Ligand fragment link
• A fragments database can enable drug design. The term “fragment” refers to
functional groups or portions of molecules which might have bioactivity.
• Organic molecules can be decomposed into basic chemical fragments. The number of
kinds of fragment structures is limited.
• There are a large number of possible fragment combinations. A small perturbation of
the previous fragment conformation would cause great difference in activity. In order
to find the lowest binding energy on the Potential energy surface (PES) between
fragments and a receptor pocket, the scoring function calculation would be performed
for every step of conformation change of the fragments derived from every type of
possible fragments combination. Since this requires a large amount of computation,
using different tricks may use less computing power and let the program work more
efficiently.
• When a ligand is inserted into the pocket site of a receptor, groups on the ligand that
bind tightly with the receptor should have the highest priority in finding their lowest-
energy conformation. This allows us to put several seeds into the program at the same
time and optimize the conformation of those seeds that form significant interactions
with the receptor, and then connect those seeds into a continuous ligand in a manner
that make the rest of the ligand have the lowest energy.
• The pre-placed seeds ensure high binding affinity and their optimal conformation
determines the manner in which the ligand will be built, thus determining the overall
structure of the final ligand. This strategy efficiently reduces the calculation burden
for fragment construction.
 • The two strategies above are widely used in most structure-based drug design
programs. They are described as “Grow” and “Link”. The two strategies are always
combined in order to make the construction result more reliable
Scoring method
• Structure-based drug design attempts to use the structure of proteins as a basis for
designing new ligands by applying accepted principles of molecular recognition.
• The basic assumption underlying structure-based drug design is that a good ligand
molecule should bind tightly to its target. Thus, one of the most important principles
for designing or obtaining potential new ligands is to predict the binding affinity of a
certain ligand to its target and use it as a criterion for selection.
• One early method was developed by Böhm to develop a general-purposed empirical
scoring function in order to describe the binding energy. The following “Master
Equation” was derived:
• where:
• desolvation – enthalpic penalty for removing the ligand from solvent
• motion – entropic penalty for reducing the degrees of freedom when a ligand binds to
its receptor
• configuration – conformational strain energy required to put the ligand in its "active"
conformation
• interaction – enthalpic gain for "resolvating" the ligand with its receptor
• The basic idea is that the overall binding free energy can be decomposed into
independent components that are known to be important for the binding process. Each
component reflects a certain kind of free energy alteration during the binding process
between a ligand and its target receptor. The Master Equation is the linear
combination of these components. According to Gibbs free energy equation, the
relation between dissociation equilibrium constant, Kd, and the components of free
energy was built.

Lead Molecule
• Target validation (TV) → assay development → high-throughput screening → hit to
lead (H2L) → lead optimization (LO) → preclinical drug development → clinical
drug development
• Hit to lead (H2L) also known as lead generation is a stage in early drug
discovery where small molecule hits from a high throughput screen (HTS) are
 evaluated and undergo limited optimization to identify promising lead compounds.
These lead compounds undergo more extensive optimization in a subsequent step of
drug discovery called lead optimization(LO)
• The hit to lead stage starts with confirmation and evaluation of the initial screening
hits and is followed by synthesis of analogs (hit expansion).
• Typically the initial screening hits display binding affinities for their biological
target in the micromolar (10-6 molar concentration) range.
• Through limited H2L optimization, the affinities of the hits are often improved by
several orders of magnitude to the nanomolar (10-9 M) range.
• The hits also undergo limited optimization to improve metabolic half life so that the
compounds can be tested in animal models of disease and also to
improve selectivity against other biological targets binding that may result in
undesirable side effects.
Hit confirmation
After hits are identified from a high throughput screen, the hits are confirmed and evaluated
using the following methods:
• Re-testing: compounds that were found active against the selected target are re-tested
using the same assay conditions used during the HTS.
• Dose response curve generation: several compound concentrations are tested using the
same assay, an IC50 or EC50value is then generated. Methods are being developed that
may allow the reuse of the compound that generated the hit in the initial HTS step.
These molecules are removed from beads and transferred to a microarray for
quantitative assessment of binding affinities in a "seamless" approach that could allow
for the investigation of more hits and larger libraries.[4]
• Orthogonal testing: Confirmed hits are assayed using a different assay which is
usually closer to the target physiological condition or using a different technology.
• Secondary screening: Confirmed hits are tested in a functional assay or in a cellular
environment. Membrane permeability is usually a critical parameter.
• Chemical amenability: Medicinal chemists evaluate compounds according to their
synthesis feasibility and other parameters such as up-scaling or costs
• Biophysical testing: Nuclear magnetic resonance (NMR), Isothermal Titration
Calorimetry, dynamic light scattering,surface plasmon resonance, dual polarisation
interferometry, microscale thermophoresis (MST) are commonly used to assess
 whether the compound binds effectively to the target, the stoïchiometry of binding,
any associated conformational change and to identify promiscuous inhibitors.
• Hit ranking and clustering: Confirmed hit compounds are then ranked according to the
various hit confirmation experiments.
• Freedom to operate evaluation: Hit compound structures are quickly checked in
specialized databases to determine if they are patentable
Hit expansion
Following hit confirmation, several compound clusters will be chosen according to
their characteristics in the previously defined tests. An Ideal compound cluster will:
• have compound members that exhibit a high affinity towards the target (less than 1
µM)
• Moderate molecular weight and lipophilicity (usually measured as cLogP). Affinity,
molecular weight and lipophilicity can be linked in single parameter such as ligand
efficiency and lipophilic efficiency to assess druglikeness
• show chemical tractability
• be free of Intellectual property
• not interfere with the P450 enzymes nor with the P-glycoproteins
• not bind to human serum albumin
• be soluble in water (above 100 µM)
• be stable
• have a good druglikeness
• exhibit cell membrane permeability
• show significant biological activity in a cellular assay
• not exhibit cytotoxicity
• not be metabolized rapidly
• show selectivity versus other related targets

LEAD OPTMIZATION
• A lead compound (i.e. a "leading" compound, not lead metal) in drug discovery is
a chemical compound that has pharmacological or biological activity likely to be
therapeutically useful, but may still have suboptimal structure that requires
modification to fit better to the target.
 • Its chemical structure is used as a starting point for chemical modifications in order to
improve potency, selectivity, or pharmacokinetic parameters.
• Furthermore, newly invented pharmacologically active moieties may have
poor druglikeness and may require chemical modification to become drug-like enough
to be tested biologically or clinically.
Discovering lead compounds
• A lead compound may arise from a variety of different sources.
• Lead compounds are found by characterizing natural products,
employing combinatorial chemistry, or by molecular modeling as in rational drug
design.
• Lead compounds are often tested by high-throughput screenings ("hits") which can
screen compounds for their ability to inhibit (antagonist) or stimulate (agonist) a
receptor of interest as well as determine their selectivity for them
• Traditional library screening, in house library, out sourced library, fragment based
screening
• Virtual screening or in silico screening –computerised models of both the target and
the molecule – no library required; followed by docking – estimate the binding energy
using a scoring function
What makes a good Lead
 Ideally multiple discrete series from High Throughput Screening
 Confirmed activity and structure using fresh, pure sample
 < 3uM in vitro, appropriate functional activity
 Reversibility
 Key assays available for selectivity
 Singletons need to be confirmed by small library synthesis
 Need to be 5-10 compounds to confirm options for diveristy
 No undesirable functional groups or chemical reactivity
 Toxicologically suspect groups
 Scope for expansion (IP position and suitable chemistry)
 Tractable physical properties
 Molecular Weight, Solubility, Polarity
 ADME profiles available for representative analogues
 Particularly oral absorption, solubility for i.v. and CNS penetration as
appropriate.
  Evidence that any ADME issues are tractable

Lead optimization
• Lead identification/optimization is the one of the most important steps in drug
development.
• The chemical structure of the lead compound is used as a starting point for chemical
modifications in order to improve potency, selectivity, or pharmacokinetic parameters.
• Once a molecule is identified, the next step is to check its ADMET (Adsorption,
Distribution, Metabolism, Excretion and Toxicity) properties.
• If the molecule has no toxicity and no mutagenicity either, it has potential for use as
lead molecule. Further optimization gives better quality of lead molecules. These may
subsequently be developed as drug(s).
• The objective of this drug discovery phase is to synthesize lead compounds, new
analogs with improved potency, reduced off-target activities, and
physiochemical/metabolic properties suggestive of reasonable in
vivo pharmacokinetics. This optimization is accomplished through chemical
modification of the hit structure, with modifications chosen by employing knowledge
of the structure–activity relationship (SAR) as well as structure-based design if
structural information about the target is available.
• Lead optimization is concerned with experimental testing and confirmation of the
compound based on animal efficacy models and ADMET (in vitro and in situ) tools
that may be followed by target identification and target validation.
•
Pharmacophore

• A pharmacophore is an abstract description of molecular features which are necessary

for molecular recognition of a ligand by a biological macromolecule.
• The IUPAC defines a pharmacophore to be "an ensemble of steric and electronic
features that is necessary to ensure the optimal supramolecular interactions with a
specific biological target and to trigger (or block) its biological response”
• Group on a molecule that interacts with receptor and responsible for biological
activity
• A pharmacophore model explains how structurally diverse ligands can bind to a
common receptor site. Furthermore pharmacophore models can be used to identify
through denovo design or virtual screening novel ligands that will bind to the same
receptor.
FEATURES
• Typical pharmacophore features include hydrophobic centroids, aromatic rings,
hydrogen bond acceptors or donor, cations, and anions.
 • These pharmacophoric points may be located on the ligand itself or may be projected
points presumed to be located in the receptor.
• The features need to match different chemical groups with similar properties, in order
to identify novel ligands.
• Ligand-receptor interactions are typically “polar positive”, “polar negative” or
“hydrophobic”. A well-defined pharmacophore model includes both hydrophobic
volumes and hydrogen bond vectors.
Process of pharmacophore development

The process for developing a pharmacophore model generally involves the following steps:
• Select a set of molecules
Choose a set of molecules that will be used for developing the pharmacophore model. As a
pharmacophore model should be able to discriminate between molecules with and without
bioactivity, the set of molecules should include both active and inactive compounds.
• Conformational analysis
Generate a set of low energy conformations that is likely to contain the bioactive
conformation for each of the selected molecules.
• Molecular superimposition
Superimpose ("fit") all combinations of the low-energy conformations of the molecules.
Similar functional groups common to all molecules in the set might be fitted (e.g., phenyl
rings or carboxylic acid groups). The conformation that results in the best fit is presumed to
be the active conformation.
• Abstract description
Transform the superimposed molecules into an abstract representation. For example,
superimposed phenyl rings might be referred to more conceptually as an 'aromatic ring'
pharmacophore element. Likewise, hydroxy groups could be designated as a 'hydrogen-bond
donor/acceptor' pharmacophore element.
• Validation
A pharmacophore model is a hypothesis accounting for the observed biological activities of a
set of molecules that bind to a common biological target. The model is only valid insofar as it
is able to account for differences in biological activity of a range of molecules.
• As the biological activities of new molecules become available, the pharmacophore
model can be updated to further refine it.
Molecular Docking

Molecular docking is a well established computational technique which predicts the

interaction energy between two molecules. Molecular docking studies are used to determine
the interaction of two molecules and to find the best orientation of ligand which would form a
complex with overall minimum energy. The small molecule, known as ligand usually fits
within protein’s cavity which is predicted by the search algorithm. These protein cavities
become active when come in contact with any external compounds and are thus called as
active sites.
 The results are analyzed by a statistical scoring function which converts interacting
energy into numerical values called as the docking score; and also the interacting energy is
calculated. The 3D pose of the bound ligand can be visualized using different visualizing
tools like Pymol, Rasmol etc which could help in inference of the best fit of ligand. Predicting
the mode of protein-ligand interaction can assume the active site of the protein molecule and
further help in protein annotation. Moreover molecular docking has major application in drug
discovery and designing.
This technique mainly incorporates algorithms like molecular dynamics, Monte
Carlo stimulation, fragment based search methods.

Different types of Interactions

Interactions between particles can be defined as a consequence of forces
between the molecules contained by the particles. These forces are divided into four
categories:
• Electrostatic forces - Forces with electrostatic origin due to the charges residing in
the matter. The most common interactions are charge-charge, charge-dipole and
dipole-dipole.
• Electrodynamics forces-The most widely known is the Van der Waals interactions.
• Steric forces - Steric forces are generated when atoms in different molecules come
into very close contact with one another and start affecting the reactivity of each
other. The resulting forces can affect chemical reactions and the free energy of a
system.
• Solvent-related forces - These are forces generated due to chemical reactions
between the solvent and the protein or ligand. Examples are Hydrogen bonds
(hydrophilic interactions) and hydrophobic interactions.
• A common characteristic of all these forces is their electromagnetic nature.
• Other physical factors - Conformational changes in the protein and the ligand are
often necessary for successful docking.

Molecular docking
Molecular docking can be divided into two separate sections.
1) Search algorithm – These algorithms determine all possible optimal conformations
 for a given complex (protein-protein, protein-ligand) in a environment i.e. the position and
orientation of both molecules relative to each other. They can also calculate the energy of
the resulting complex and of each individual interaction.
The different types of algorithms that can be used for docking analysis are given below.
• Molecular dynamics
• Monte Carlo methods
• Genetic algorithms
• Fragment-based methods
• Point complementary methods
• Distance geometry methods
• Systematic searches

2) Scoring function – These are mathematical methods used to predict the strength of
the non-covalent interaction called as binding affinity, between two molecules after they
have been docked. Scoring functions have also been developed to predict the strength of
other types of intermolecular interactions, for example between two proteins or between
protein and DNA or protein and drug. These configurations are evaluated using scoring
functions to distinguish the experimental binding modes from all other modes explored
through the searching algorithm.
For example:
• Binding Energy
∆G bind = ∆Gvdw + ∆Ghbond + ∆Gelect + ∆Gconform + ∆G tor + ∆G sol

General concept of the algorithm:

1) A 'negative' image of the binding site is made - a collection of spheres of varying
radii, each touching the molecular surface at just 2 points.
 2) Ligand atoms are then matched to sphere centers where at least four distances
between ligand atoms are matched to sphere center distances.

3) Proper orientation is achieved by a least squares fit of ligand atoms to the sphere centers.
4) Orientation is checked for any steric clashes between ligand and receptor.
5) If acceptable, then interaction energy is computed as a 'score' for that binding mode
6) New orientations are obtained by matching different sets of atoms and sphere centers
7) Top-scoring orientations are retained for subsequent analysis

Types of Docking -
The following are majorly used type of docking are-

• Lock and Key or Rigid Docking – In rigid docking, both the internal geometry of the
 receptor and ligand is kept fixed during docking.
• Induced fit or Flexible Docking - In this model, the ligand is kept flexible and the energy
for different conformations of the ligand fitting into the protein is calculated. Though more
time consuming, this method can evaluate many different possible conformations which
make it more reliable.
Major steps in molecular docking:
Step I – Building the Receptor
In this step the 3D structure of the receptor should be downloaded from PDB; and modified.
This should include removal of the water molecules from the cavity, stabilizing charges,
filling in the missing residues, generation the side chains etc according to the parameters
available. After modification the receptor should be biological active and stable.
Step II – Identification of the Active Site
After the receptor is built, the active site within the receptor should be identified. The
receptor may have many active sites but the one of the interest should be selected. Most of
the water molecules and heteroatoms if present should be removed.
Step III – Ligand Preparation
Ligands can be obtained from various databases like ZINC, PubChem or can be sketched
using tools like Chemsketch. While selecting the ligand, the LIPINSKY’S RULE OF 5
should be applied. The rule is important for drug development where a pharmacologically
active lead structure is optimized stepwise for increased activity and selectivity, as well as
drug-like properties, as described.
For the selection of a ligand using LIPINSKY’S RULE:
• Not more than 5 –H bond donors.
• Molecular Weight NOT more than 500 Da.
• Log P not over 5 for octanol water partition coefficient.
• NOT more than 10 H bond acceptors.
Step IV- Docking
This is the last step, where the ligand is docked onto the receptor and the interactions are
checked. The scoring function generates scores depending on which the ligand with the
best fit is selected.
Software available for Molecular Docking:
SCHRODINGER, DOCK, AUTOLOCK TOOLS, DISCOVERY STUDIO, iGemDock

QSAR - Quantitative structure–activity relationship

Quantitative structure–activity relationship models (QSAR models) are regression or
classification models used in the chemical and biological sciences and engineering. Like
other regression models, QSAR regression models relate a set of "predictor" variables
(X) to the potency of the response variable (Y), while classification QSAR models relate
the predictor variables to a categorical value of the response variable.

In QSAR modeling, the predictors consist of physico-chemical properties or theoretical

molecular descriptors of chemicals; the QSAR response-variable could be abiological
activity of the chemicals. QSAR models first summarize a supposed relationship between
chemical structures and biological activity in a data-set of chemicals. Second, QSAR
models predict the activities of new chemicals.

Related terms include quantitative structure–property relationships (QSPR) when a

chemical property is modeled as the response variable.

As an example, biological activity can be expressed quantitatively as the concentration of a

substance required to give a certain biological response. Additionally, when
physicochemical properties or structures are expressed by numbers, one can find a
mathematical relationship, or quantitative structure-activity relationship, between the two.
The mathematical expression, if carefully validated can then be used to predict the
modeled response of other chemical structures.

A QSAR has the form of a mathematical model:

Activity = f(physiochemical properties and/or structural properties) + error

The error includes model error (bias) and observational variability, that is, the
variability in observations even on a correct model.
Examples of biological activity that can be used for QSAR studies include:
 Enzyme activity
 Minimum effective dose
 Toxicity

Possible molecular descriptors that can be used for building QSAR models may include:

 Dipole moment
 Atomic volume
 Number of carbons
 Number of aromatic moieties
 Molar volume
 Wang octanol-water partition coefficient
 Molecular weight
 Quantum chemical descriptors such as molecular orbital energies (HOMO &
LUMO) and atomic net charge
Advantages and Disadvantages of QSAR
Advantages of predicting biological activity with quantitative structure-activity
relationships modelling include:

 Able to predict activities of a large number of compounds with little to

no prior experimental data on activity.
 Can reveal which molecular properties may be worth investigating further.
 Regarded as a “green chemistry” approach since chemical waste is not generated
when performing in silico predictions.
 In vivo and in vitro experimentation can be very expensive and time-consuming.
QSAR modelling reduces the need for testing on animals and/or on cell cultures
and saves time.

Disadvantages of predicting biological activity with QSAR modelling include:

 Does not provide an in-depth insight on the mechanism of biological action.

 Some risk of highly inaccurate predictions of pharmacological or biological activity.
Applications of QSAR in Pharmacology and Medicinal Chemistry

 Quantitative structure-activity relationships (QSAR) can be used during the drug

design and drug discovery process. QSAR models can be used as a screening tool
to test a large set of compounds or for eliminating test compounds which do not
show promise in terms of predicted biological activity.
 Toxicity endpoints of compounds towards organisms can be predicted using
QSAR-based methodologies. [1] For instance, the oral rat 50% lethal dose (LD50)
Molecular Descriptors used in QSAR

Molecular descriptors can be defined as a numerical representation of chemical

information encoded within a molecular structure via mathematical procedure.267
This mathematical representation has to be invariant to the molecule’s size and
number of atoms to allow model building with statistical methods. The information
content of structure descriptors depends on two major factors:
(1) The molecular representation of compounds.
(2) The algorithm which is used for the calculation of the descriptor.

The three major types of parameters initially suggested are, (1) Hydrophobic (2)
Electronic (3) Steric
PARAMETERS
• The parameters used in QSAR is a measure of the potential contribution of its group
to a particular property of the parent drug.
• Activity is expressed as log(1/C). C is the minimum concentration required to cause a
defined biological response.
• Physicochemical property as log p.
Various parameters used in QSAR studies are
• 1.Lipophilic parameters: partition coefficient, π- substitution constant.
• 2.Electronic parameters: Hammet constant, dipole moment.
• 3.Steric parameters: Taft’s constant, molar refractivity, Verloop steric parameter
LIPOPHILIC PARAMETERS
• Lipophilicity is partitioning of the compound between an aqueous and non-aqueous
phase.
• Partition coefficient: P=[drug] in octanol/[drug] in water
• High P High hydrophobicity
 •  value depends on inductive and resonance effects
•  value depends on whether the substituent is meta or para
• ortho values are invalid due to steric factors
STERIC SUBSTITUTION CONSTANT
• It is a measure of the bulkiness of the group it represents and it effects on the
closeness of contact between the drug and receptor site. It is much harder to
quantitate.
• v Taft’s steric factor (Es') •Measured by comparing the rates of hydrolysis of
substituted aliphatic esters against a standard ester under acidic conditions
• Es = log kx - log ko
• kx represents the rate of hydrolysis of a substituted ester
• ko represents the rate of hydrolysis of the parent ester
• Molar refractivity (MR)--measure of the volume occupied by an atom or group--
equation includes the MW, density(d), and the index of refraction(n)– MR=(n²-
1)MW/(n²+2)d
• Verloop steric parameter--computer program uses bond angles, van der Waals radii,
bond lengths
Advantages
• No need for physicochemical constants or tables
• Useful for structures with unusual substituents
• Useful for quantifying the biological effects of molecular features that cannot be
quantified or tabulated by the Hansch method
Disadvantages
• A large number of analogues need to be synthesised to represent each different
substituent and each different position of a substituent
• It is difficult to rationalise why specific substituents are good or bad for activity
3D QSAR
Evaluation of the quality of QSAR models
QSAR modeling produces predictive models derived from application of statistical tools
correlating biological activity (including desirable therapeutic effect and undesirable side
effects) or physico-chemical properties in QSPR models of chemicals
(drugs/toxicants/environmental pollutants) with descriptors representative of molecular
structure or properties. QSARs are being applied in many disciplines, for example: risk
assessment, toxicity prediction, and regulatory decisions in addition to drug
discovery and lead optimization.[30] Obtaining a good quality QSAR model depends on many
factors, such as the quality of input data, the choice of descriptors and statistical methods for
modeling and for validation. Any QSAR modeling should ultimately lead to statistically
robust and predictive models capable of making accurate and reliable predictions of the
modeled response of new compounds.
For validation of QSAR models, usually various strategies are adopted:[31]
1. internal validation or cross-validation (actually, while extracting data, cross validation
is a measure of model robustness, the more a model is robust (higher q2) the less data
extraction perturb the original model);
2. external validation by splitting the available data set into training set for model
development and prediction set for model predictivity check;
3. blind external validation by application of model on new external data and
4. data randomization or Y-scrambling for verifying the absence of chance correlation
between the response and the modeling descriptors.
The success of any QSAR model depends on accuracy of the input data, selection of
appropriate descriptors and statistical tools, and most importantly validation of the developed
model. Validation is the process by which the reliability and relevance of a procedure are
established for a specific purpose; for QSAR models validation must be mainly for
robustness, prediction performances and applicability domain (AD) of the models.
Some validation methodologies can be problematic. For example, leave one-out cross-
validation generally leads to an overestimation of predictive capacity. Even with external
validation, it is difficult to determine whether the selection of training and test sets was
manipulated to maximize the predictive capacity of the model being published.
Different aspects of validation of QSAR models that need attention include methods of
selection of training set compounds,[34] setting training set size[35] and impact of variable
selection[36] for training set models for determining the quality of prediction. Development of
novel validation parameters for judging quality of QSAR models is also important.

Drug binding kinetics

Binding affinity
Three important notes about binding affinity

Binding affinity is the concentration of a drug required to occupy 50% of the target
molecules at equilibrium. This value quantifies the extent of target occupancy, which
is used to predict in-vivo efficacy.1
 Residence time is a quantitative value representing the time a drug-receptor
interaction takes to reach target occupancy. It is usually calculated as dissociation half
time: t1/2 = 0.693/koff, where koff is the dissociation rate of the interaction (Hoare et
al., 2019). An interaction is widely considered to be at equilibrium when five
dissociation half times have passed.1
 In simplified terms, very slow dissociation (off) rates frequently result in erroneous
affinity measurements due to not actually reaching equilibrium. This is just as critical
when designing SPR experiments to measure steady-state affinity, but can be
circumvented by measuring kinetics directly.
Enzyme inhibitors
Various compounds can reduce the activity of enzymes. They may act in a variety of
different ways, and indeed may be reversible or irreversible inhibitors of the enzyme.
On this page there are notes about:
 Competitive inhibition
 Non-competitive inhibition
 Uncompetitive inhibition
 The choice of a competitive or non-competitive inhibitor as a drug
 Ki, the inhibitor constant
An irreversible inhibitor causes covalent modification of the enzyme, so that its activity is
permanently reduced. Compounds that act as irreversible inhibitors are often useful as drugs
that need be taken only every few days, although adjusting the dose to suit the patient’s
response is a lengthy process with such compounds. By contrast, the effect of a reversible
inhibitor can be reversed by removing the inhibitor, e.g. by dialysis or gel filtration.
The normal sequence of an enzyme reaction can be represented as:

where:
E = enzyme
S = substrate
E-S = enzyme-substrate complex
E-P = enzyme-product complex
P = product
There are three main types of reversible inhibitor:
 competitive inhibitor
 non-competitive inhibitor
 uncompetitive inhibitor
They interact with the enzyme or enzyme-substrate complex at different stages in the
sequence
Competitive inhibition
A competitive inhibitor competes with the substrate for the active site of the enzyme:
This means that increasing the concentration of substrate will decrease the chance of inhibitor
binding to the enzyme. Hence, if the substrate concentration is high enough the enzyme will
reach the same Vmax as without the inhibitor. However, it will require a higher concentration
of substrate to achieve this and so the Km of the enzyme will also be higher. Reacting the
enzyme with a range of concentrations of substrate at different concentrations of a
competitive inhibitor will give a family of curves as shown below:

The Lineweaver-Burk double reciprocal plot for this set of data shows a series of lines
crossing the y (1/v) axis at the same point - i.e. Vmax is unchanged, but with a decreasing
value of 1/Km (and hence a higher Km) in the presence of the inhibitor:
Non-competitive inhibition
A non-competitive inhibitor reacts with the enzyme-substrate complex, and slows the rate of
reaction to form the enzyme-product complex.

This means that increasing the concentration of substrate will not relieve the inhibition, since
the inhibitor reacts with the enzyme-substrate complex. Reacting the enzyme with a range of
concentrations of substrate at different concentrations of a non-competitive inhibitor will give
a family of curves as shown below:
The Lineweaver-Burk double reciprocal plot for this set of data shows a series of lines
converging on the same point on the X (1/S) axis - i,.e. Km is unchanged, but Vmax is
reduced:
Uncompetitive inhibition
This is a very rare class of inhibition. An uncompetitive inhibitor binds to the enzyme and
enhances the binding of substrate (so reducing Km), but the resultant enzyme-inhibitor-
substrate complex only undergoes reaction to form the product slowly, so that Vmax is also
reduced:

Reacting the enzyme with a range of concentrations of substrate at different concentrations of

an uncompetitive inhibitor will give a family of curves as shown below:

The Lineweaver-Burk double reciprocal plot for this set of data shows a series of parallel
lines - both Km and Vmax are reduced:
The choice of a competitive or non-competitive inhibitor as a drug
If the requirement is to increase the intracellular concentration of the substrate, then either a
competitive or non-competitive inhibitor will serve, since both will inhibit the utilisation of
substrate, so that it accumulates.
However, if the requirement is to decrease the intracellular concentration of the product, then
the inhibitor must be non-competitive. As unused substrate accumulates, so it will compete
with a competitive inhibitor, and the final result will be a more or less normal rate of
formation of product, but with a larger pool of substrate. Increasing the concentration of
substrate does not affect a non-competitive inhibitor.
Ki, the inhibitor constant
The inhibitor constant, Ki, is an indication of how potent an inhibitor is; it is the
concentration required to produce half maximum inhibition.
Plotting 1/v against concentration of inhibitor at each concentration of substrate (the Dixon
plot) gives a family of intersecting lines.
For a competitive inhibitor, the lines converge above the x axis, and the value of [I] where
they intersect is -Ki
For a non-competitive inhibitor, the lines converge on x axis, and the value of [I] where they
intersect is -Ki
UNIT – 4- SBIA5302 – Computer aided drug design
 Molecular modelling encompasses all methods, theoretical and computational, used
to model or mimic the behaviour of molecules. The methods are used in the fields
of computational chemistry, drug design, computational biology and materials science to
study molecular systems ranging from small chemical systems to large biological molecules
and material assemblies. The simplest calculations can be performed by hand, but inevitably
computers are required to perform molecular modelling of any reasonably sized system. The
common feature of molecular modelling methods is the atomistic level description of the
molecular systems. This may include treating atoms as the smallest individual unit
(a molecular mechanics approach), or explicitly modelling protons and neutrons with its
quarks, anti-quarks and gluons and electrons with its photons (a quantum
chemistry approach).

Molecules can be modelled either in vacuum, or in the presence of a solvent such as

water. Simulations of systems in vacuum are referred to as gas-phase simulations, while those
that include the presence of solvent molecules are referred to as explicit solvent simulations.
In another type of simulation, the effect of solvent is estimated using an empirical
mathematical expression; these are termed implicit solvation simulations.
Most force fields are distance-dependent, making the most convenient expression for
these Cartesian coordinates. Yet the comparatively rigid nature of bonds which occur
between specific atoms, and in essence, defines what is meant by the designation molecule,
make an internal coordinate system the most logical representation. In some fields the IC
representation (bond length, angle between bonds, and twist angle of the bond as shown in
the figure) is termed the Z-matrix or torsion angle representation. Unfortunately, continuous
motions in Cartesian space often require discontinuous angular branches in internal
coordinates, making it relatively hard to work with force fields in the internal coordinate
representation, and conversely a simple displacement of an atom in Cartesian space may not
be a straight line trajectory due to the prohibitions of the interconnected bonds. Thus, it is
very common for computational optimizing programs to flip back and forth between
representations during their iterations. This can dominate the calculation time of the potential
itself and in long chain molecules introduce cumulative numerical inaccuracy. While all
conversion algorithms produce mathematically identical results, they differ in speed and
numerical accuracy.[3] Currently, the fastest and most accurate torsion to Cartesian
conversion is the Natural Extension Reference Frame (NERF) method.
Molecular mechanics

Molecular Mechanics Force Field

The "mechanical" molecular model was developed out of a need to describe molecular
structures and properties in as practical a manner as possible. The range of applicability of
molecular mechanics includes:

Molecules containing thousands of atoms.

Organics, oligonucleotides, peptides, and saccharides (metallo-
organics and inorganics in some cases).
Vacuum, implicit, or explicit solvent environments.
Ground state only.
Thermodynamic and kinetic (via molecular dynamics) properties.

The great computational speed of molecular mechanics allows for its use in
procedures such as molecular dynamics, conformational energy searching, and docking. All
the procedures require large numbers of energy evaluations.

Molecular mechanics methods are based on the following principles:

Nuclei and electrons are lumped into atom-like particles.

Atom-like particles are spherical (radii obtained from measurements or theory) and
have a net charge (obtained from theory).
Interactions are based on springs and classical potentials.
Interactions must be preassigned to specific sets of atoms.

Interactions determine the spatial distribution of atom-like particles and their energies
To define a force field one must specify not only the functional form but also the
parameters (i.e.the various constants). Two force fields may use an identical functional form
yet have very different parameters. A force field should be considered as a single entity; it is
not strictly correct to divide the energy into its individual components, let alone to take some
of the parameters from one forcefield and mix them with parameters from another force field.
The forcefields used in molecular modelling are primarily designed to reproduce structural
properties but they can also be used to predict other properties, such as molecular spectra.
However, molecular mechanics force fields can rarely predict spectra with great accuracy
(although the more recent molecular. mechanics force fields are much better in this
regard). A force field is generally designed to predict certain properties and will be
parametrised accordingly. While it is useful to try to predict other quantities which have not
been included in the parametrisation process it is not necessarily a failing if a force field is
unable to do so. Transferability of the functional form and parameters is an important feature
of a forcefield. Transferability means that the same set of parameters can be used to model a
series of related molecules, rather than having to define a new set of parameters for each
individual molecule. A concept that is common to most force fields is that of an atom type.
When preparing the input for a quantum mechanics calculation it is usually necessary to
specify the atomic numbers of the nuclei present, together with the geometry of the system
and the overall charge and spin multiplicity. For a force field the overall charge and spin
multiplicity are not explicitly required, but it is usually necessary to assign an atom type to
each atom in the system. The atom type is more than just the atomic number of an atom; it
usually con• tains information about its hybridisation state and sometimes the local
environment. For example, it is necessary in most force fields to distinguish between sp3 -
hybridised carbon atoms (which adopt a tetrahedral geometry), sp2-hybridised carbons
(which are trigonal) and sp-hybridised carbons (which are linear).

The mechanical molecular model considers atoms as spheres and bonds as springs.
The mathematics of spring deformation can be used to describe the ability of bonds to stretch,
bend, and twist:
 Non-bonded atoms (greater than two bonds apart) interact through van der Waals
attraction, steric repulsion, and electrostatic attraction/repulsion. These properties are easiest
to describe mathematically when atoms are considered as spheres of characteristic radii.

The object of molecular mechanics is to predict the energy associated with a given
conformation of a molecule. However, molecular mechanics energies have no meaning as
absolute quantities. Only differences in energy between two or more conformations have
meaning. A simple molecular mechanics energy equation is given by:

Energy = Stretching Energy + Bending Energy + Torsion Energy + Non-Bonded

Interaction Energy
 These equations together with the data (parameters) required to describe the behavior
of different kinds of atoms and bonds, is called a force-field. Many different kinds of force-
fields have been developed over the years. Some include additional energy terms that
describe other kinds of deformations. Some force-fields account for coupling between
bending and stretching in adjacent bonds in order to improve the accuracy of the mechanical
model.
 The mathematical form of the energy terms varies from force-field to force-field. The
more common forms will be described.

Stretching Energy

The stretching energy equation is based on Hooke's law. The "kb" parameter
controls
the stiffness of the bond spring, while "ro" defines its equilibrium length. Unique "kb"
and "ro" parameters are assigned to each pair of bonded atoms based on their types (e.g. C-C,
C-H, O-C, etc.). This equation estimates the energy associated with vibration about the
equilibrium bond length. This is the equation of a parabola, as can be seen in the following
plot
 Notice that the model tends to break down as a bond is stretched toward the point of
dissociation.
Bending Energy
 The bending energy equation is also based on Hooke's law. The "ktheta" parameter
controls the stiffness of the angle spring, while "thetao" defines its equilibrium angle. This
equation estimates the energy associated with vibration about the equilibrium bond angle:
 Unique parameters for angle bending are assigned to each bonded triplet of atoms
based on their types (e.g. C-C-C, C-O-C, C-C-H, etc.). The effect of the "kb" and "ktheta"
parameters is to broaden or steepen the slope of the parabola. The larger the value of "k", the
more energy is required to deform an angle (or bond) from its equilibrium value. Shallow
potentials are achieved for "k" values between 0.0 and 1.0. The Hookeian potential is shown
in the following plot for three values of "k":
 Torsion Energy

The torsion energy is modeled by a simple periodic function, as can be seen in the
following plot:
 The torsion energy in molecular mechanics is primarily used to correct the remaining
energy terms rather than to represent a physical process. The torsional energy represents the
amount of energy that must be added to or subtracted from the Stretching Energy + Bending
Energy
+ Non-Bonded Interaction Energy terms to make the total energy agree with
experiment or rigorous quantum mechanical calculation for a model dihedral angle (ethane,
for example
might be used a a model for any H-C-C-H bond).

The "A" parameter controls the amplitude of the curve, the n parameter controls its
periodicity, and "phi" shifts the entire curve along the rotation angle axis (tau). The
parameters are determined from curve fitting. Unique parameters for torsional rotation are
assigned to each bonded quartet of atoms based on their types (e.g. C-C-C-C, C-O-C-N, H-C-
C-H, etc.). Torsion potentials with three combinations of "A", "n", and "phi" are shown in the
following plot:

Notice that "n" reflects the type symmetry in the dihedral angle. A CH3-CH3 bond,
for example, ought to repeat its energy every 120 degrees. The cis conformation of a dihedral
angle is assumed to be the zero torsional angle by convention. The parameter phi can be used
to synchronize the torsional potential to the initial rotameric state of the molecule whose
energy is being computed.
Cross terms

The presence of cross terms in a forcefield reflects coupling between the internal
coordinates. For example, as a bond angle is decreased it is found that the adjacent bonds
stretch to reduce the interaction between the 1,3 atoms, as illustrated in Figure.

One should in principle include cross terms between all contributions to a force
field. However, only a few cross terms are generally found to be necessary in order to
reproduce structural properties accurately; more may be needed to reproduce other properties
such as vibrational frequencies, which are more sensitive to the presence of such terms. In
general, any interactions involving motions that are far apart in a molecule can usually be set
to zero. Most cross terms are functions of two internal coordinates, such as stretch-stretch,
stretch-bend and stretch-torsion terms, but cross terms involving more than two internal
coordinates such as the bend- bend- torsion have also been used.
 Various functional forms are possible for the cross terms. For
example,the stretch- stretch cross term between two bonds 1 and 2 can be modelled as:

Non-Bonded Energy

Independent molecules and atoms interact through non-bonded forces, which also play an important
role in determining the structure of individual molecular species. The non-bonded interactions do not
depend upon a specific bonding relationship between atoms. They are 'through-space' interactions and are
usually modelled as a function of some inversepower of the distance. The non-bonded terms in a forcefield
are usually considered in two groups, one comprising electrostatic interactions and the other van der Waals
interactions.
The non-bonded energy represents the pair-wise sum of the energies of all possible interacting non-
bonded atoms i and j:

The non-bonded energy accounts for repulsion, van der Waals attraction, and electrostatic
interactions.

Van der Waals attraction occurs at short range, and rapidly dies off as the interacting atoms move
apart by a few Angstroms. Repulsion occurs when the distance between interacting atoms becomes even
slightly less than the sum of their contact radii. Repulsion is modeled by an equation that is designed to
rapidly blow up at close distances. The energy term that describes attraction/repulsion provides for a
smooth transition between these two regimes. These effects are often modeled using a 6-12 equation, as
shown in the following plot:
The "A" and "B" parameters control the depth and position (interatomic distance) of the potential
energy well for a given pair of non-bonded interacting atoms (e.g. C:C, O:C, O:H, etc.). In effect, "A"
determines the degree of "stickiness" of the van der Waals attraction and "B" determines the degree of
"hardness" of the atoms (e.g marshmallow-like, billiard ball-like, etc.).
14
 Electrostatic interactions

Electrostatic interactions also arise from changes in the charge distribution of a molecule
or atom caused by an external field, a process called polarisation. The primary effect of the
external electric field (which in our case will be caused by neighbouring molecules) is to induce
a dipole in the molecule. The magnitude of the induced dipole moment µind is proportional to
the electric field E, with the constant of proportionality being the polarisability a:

The electrostatic contribution is modeled using a Coulombic potential. The electrostatic

energy is a function of the charge on the non-bonded atoms, their interatomic distance, and a
molecular dielectric expression that accounts for the attenuation of electrostatic interaction by the
environment (e.g. solvent or the molecule itself). Often, the molecular dielectric is set to a
constant value between 1.0 and 5.0. A linearly varying distance-dependent dielectric (i.e. 1/r) is
sometimes used to account for the increase in environmental bulk as the separation distance
between interacting atoms increases.

Conformational analysis
The most important concerns in Medicinal chemistry and pharmaceutical research are structure
elucidation, conformational analysis, physicochemical characterization and biological activity
determination. The determination of molecular structure is essential as the structure of the
molecule predicts the physical, chemical, and biological properties of the molecule.

Conformational search methods find applications in the design of targeted chemical hosts and
drug discovery 2. Conformations are different 3D spatial arrangements of the atoms in a
molecule are interconvertible by free rotation of single bonds 3.

The major objective of conformational analysis is to gain insight on conformational

characteristic of flexible biomolecules and drugs but to also identify the relation between the role
of conformational flexibility and their activity. Therefore, it plays a significant role in computer
aided design as well. The significance of conformational analysis not just extends to
computational docking and screening but also for lead optimization
 Energy minimization

In the field of computational chemistry, energy minimization (also called energy

optimization, geometry minimization, or geometry optimization) is the process of finding an
arrangement in space of a collection of atoms where, according to some computational model of
chemical bonding, the net inter-atomic force on each atom is acceptably close to zero and the
position on the potential energy surface (PES) is a stationary point. The collection of atoms
might be a single molecule, an ion, a condensed phase, a transition state or even a collection of
any of these. The computational model of chemical bonding might, for example, be quantum
mechanics.

The motivation for performing a geometry optimization is the physical significance of the
obtained structure: optimized structures often correspond to a substance as it is found in nature
and the geometry of such a structure can be used in a variety of experimental and theoretical
investigations in the fields of chemical structure, thermodynamics, chemical
kinetics, spectroscopy and others.

Typically, but not always, the process seeks to find the geometry of a particular arrangement of
the atoms that represents a local or global energy minimum. Instead of searching for global
energy minimum, it might be desirable to optimize to a transition state, that is, a saddle point on
the potential energy surface. Additionally, certain coordinates (such as a chemical bond length)
might be fixed during the optimization.

• Energy minimization methods can precisely locate minimum energy conformations by

mathematically "homing in" on the energy function minima (one at a time).
• The goal of energy minimization is to find a route (consisting of variation of the
intramolecular degrees of freedom) from an initial conformation to the nearest minimum
energy conformation using the smallest number of calculations possible.
• The way in which the energy varies with the coordinates is usually referred to as PES or
hyper surface
• Energy of any conformation is a function of its internal or cartesian coordinates
• N atoms – energy is a function of 3N-6 internal coordinates or 3N cartesian coordinates

• Changes in the energy are a function of its nuclear coordinates.

Potential energy Surface

A potential energy surface (PES) describes the energy of a system, especially a collection of
atoms, in terms of certain parameters, normally the positions of the atoms. The surface might
define the energy as a function of one or more coordinates; if there is only one coordinate, the
surface is called a potential energy curve or energy profile. An example is the Morse/Long-range
potential.
It is helpful to use the analogy of a landscape: for a system with two degrees of freedom (e.g.
two bond lengths), the value of the energy (analogy: the height of the land) is a function of two
bond lengths (analogy: the coordinates of the position on the ground).[1]

PES for water molecule: Shows the energy minimum corresponding to optimized molecular
structure for water- O-H bond length of 0.0958nm and H-O-H bond angle of 104.5°
The PES concept finds application in fields such as chemistry and physics, especially in the
theoretical sub-branches of these subjects. It can be used to theoretically explore properties of
structures composed of atoms, for example, finding the minimum energy shape of a molecule or
computing the rates of a chemical reaction.
The geometry of a set of atoms can be described by a vector, r, whose elements represent the
atom positions. The vector r could be the set of the Cartesian coordinates of the atoms, or could
also be a set of inter-atomic distances and angles.
Given r, the energy as a function of the positions, E(r), is the value of E(r) for all r of interest.
Using the landscape analogy from the introduction, E gives the height on the "energy landscape"
so that the concept of a potential energy surface arises.
To study a chemical reaction using the PES as a function of atomic positions, it is necessary to
calculate the energy for every atomic arrangement of interest. Methods of calculating the energy
of a particular atomic arrangement of atoms are well described in the computational
chemistry article, and the emphasis here will be on finding approximations of E(r) to yield fine-
grained energy-position information.
For very simple chemical systems or when simplifying approximations are made about inter-
atomic interactions, it is sometimes possible to use an analytically derived expression for the
energy as a function of the atomic positions.
• Changes in the energy of a system can be considered as movements on
a multidimensional surface called energy surface.
• coordinates.
• Movement of the nuclei influences change in energy
• Mathematical function that gives the energy of a molecule as a function of
its geometry
• Energy is plotted on the vertical axis, geometric coordinates (e.g bond
lengths, valence angles, etc.) are plotted on the horizontal axes
• A PES can be thought of it as a hilly landscape, with valleys, mountain passes and
peaks
• Real PES have many dimensions, but key feature can be represented by a 3
dimensional PES
 • Equilibrium molecular structures correspond to the positions of the minima in the
valleys on a PES
• Energetics of reactions can be calculated from the energies or altitudes of the minima
for reactants and products
• A reaction path connects reactants and products through a mountain pass
• A transition structure is the highest point on the lowest energy path
• Reaction rates can be obtained from the height and profile of the potential energy
surface around the transition structure
• The shape of the valley around a minimum determines the vibrational spectrum
• Each electronic state of a molecule has a separate potential energy surface, and the
separation between these surfaces yields the electronic spectrum
• Properties of molecules such as dipole moment, polarizability, NMR shielding, etc.
depend on the response of the energy to applied electric and magnetic fields

• Minima, lowest – global energy minima

• Minimization algorithms
• Highest point in the pathway between 2 minima is saddle point represents the
transition state
• Minima and saddle points are stationary states on PES where the first derivative of
energy function is 0
• E = f(x)
• E is a function of coordinates either cartesian or internal
• At minimum the first derivatives are zero and the second derivatives are all positive
Minimization Methods

Several methods exist for finding a minimum of an arbitrary continuous function. One way
to classify a minimization method is based on what kind of derivatives are used to guide
the minimization. In this classification, we can distinguish between:

ds that use no derivatives (function values, such as the energy)

Computer simulation

Computer simulation is the process of mathematical modelling, performed on a computer, which

is designed to predict the behaviour of or the outcome of a real-world or physical system. Since
they allow to check the reliability of chosen mathematical models, computer simulations have
become a useful tool for the mathematical modeling of many natural systems
in physics (computationalphysics), astrophysics, climatology, chemistry, biology and manufactur
ing, as well as human systems in economics, psychology, social science, health
care and engineering. Simulation of a system is represented as the running of the system's model.
It can be used to explore and gain new insights into new technology and to estimate the
performance of systems too complex for analytical solutions.

A computer model is the algorithms and equations used to capture the behavior of the system
being modeled. By contrast, computer simulation is the actual running of the program that
contains these equations or algorithms. Simulation, therefore, is the process of running a model.
Thus one would not "build a simulation"; instead, one would "build a model", and then either
"run the model" or equivalently "run a simulation"

Benefits

 Gain greater understanding of a process

 Identify problem areas or bottlenecks in processes
 Evaluate effect of systems or process changes such as demand, resources, supply, and
constraints
 Identify actions needed upstream or downstream relative to a given operation,
organization, or activity to either improve or mitigate processes or events
 Evaluate impact of changes in policy prior to implementation
Types

 Discrete Models – Changes to the system occur at specific times

 Continuous Models – The state of the system changes continuously over time
 Mixed Models – Contains both discrete and continuous elements

Types of Data/Information Needed to Develop a Simulation Model:

 The overall process flow and its associated resources

 What is being produced, served, or acted upon by the process (entities)
 Frequency at which the entities arrive in the process
 How long do individual steps in the process take
 Probability distributions that characterize real life uncertainties and variations in the
process
 Computer simulation is the use of a computer to represent the dynamic responses of one
system by the behavior of another system modeled after it.
 A simulation uses a mathematical description, or model, of a real system in the form of a
computer program.
 This model is composed of equations that duplicate the functional relationships within the
real system.
 When the program is run, the resulting mathematical dynamics form an analog of the
behavior of the real system, with the results presented in the form of data.
 A simulation can also take the form of a computer-graphics image that represents dynamic
processes in an animated sequence.
 Computer simulations have become a useful part of mathematical modeling of many natural
systems in physics, astrophysics, chemistry, biology, climatology, psychology, social
science, etc

USES

 Computer simulations are used to study the dynamic behavior of objects or systems in
response to conditions that cannot be easily or safely applied in real life.
 Simulations are especially useful in enabling observers to measure and predict how the
functioning of an entire system may be affected by altering individual components within
that system.
 Simulations have great military applications also. Many uses for a computer simulation can
be found within various scientific fields of study such as meteorology, physical sciences, etc

Process of building a computer model, and the interplay between experiment, simulation, and
theory.

Basic Simulation Techniques

To explore the energy landscape described by the molecular mechanics force field, i.e. to sample
molecular conformations, a simulation is required. This is also the route to relate the microscopic
movements and positions of the atoms to the macroscopic or thermodynamic quantities that can
be measured experimentally. There are two major simulation methods to sample biomolecular
systems: molecular dynamics (MD) and Monte Carlo (MC)

Molecular dynamics (MD) is a computer simulation method for analyzing the physical
movements of atoms and molecules. The atoms and molecules are allowed to interact for a fixed
period of time, giving a view of the dynamic "evolution" of the system.
 Pharmacokinetics
Pharmacokinetics (from Ancient Greek pharmakon "drug" and kinetikos "moving,
putting in motion"; see chemical kinetics), sometimes abbreviated as PK, is a branch
of pharmacology dedicated to determine the fate of substances administered to a living organism.
The substances of interest include any chemical xenobiotic such as: pharmaceutical
drugs, pesticides, food additives, cosmetics, etc. It attempts to analyze chemical metabolism and
to discover the fate of a chemical from the moment that it is administered up to the point at
which it is completely eliminated from the body. Pharmacokinetics is the study of how an
organism affects a drug, whereas pharmacodynamics (PD) is the study of how the drug affects
the organism. Both together influence dosing, benefit, and adverse effects, as seen in PK/PD
models.
Pharmacokinetics describes how the body affects a specific xenobiotic/chemical after
administration through the mechanisms of absorption and distribution, as well as the metabolic
changes of the substance in the body (e.g. by metabolic enzymes such as cytochrome
P450 or glucuronosyltransferase enzymes), and the effects and routes of excretion of
the metabolites of the drug.[2] Pharmacokinetic properties of chemicals are affected by the route
of administration and the dose of administered drug. These may affect the absorption rate.[3]

Topics of Pharmacokinetics
Models have been developed to simplify conceptualization of the many processes that take
place in the interaction between an organism and a chemical substance. One of these, the multi-
compartmental model, is the most commonly used approximations to reality; however, the
complexity involved in adding parameters with that modelling approach means
that monocompartmental models and above all two compartmental models are the most-
frequently used. The various compartments that the model is divided into are commonly referred
to as the ADME scheme (also referred to as LADME if liberation is included as a separate step
from absorption):
 Liberation – the process of release of a drug from the pharmaceutical formulation.[4][5] See
also IVIVC.
 Absorption – the process of a substance entering the blood circulation.
 Distribution – the dispersion or dissemination of substances throughout the fluids and tissues
of the body.
 Metabolism (or biotransformation, or inactivation) – the recognition by the organism that a
foreign substance is present and the irreversible transformation of parent compounds into
daughter metabolites.
 Excretion – the removal of the substances from the body. In rare cases,
some drugs irreversibly accumulate in body tissue.[citation needed]
The two phases of metabolism and excretion can also be grouped together under the title
elimination. The study of these distinct phases involves the use and manipulation of basic
concepts in order to understand the process dynamics. For this reason, in order to fully
comprehend the kinetics of a drug it is necessary to have detailed knowledge of a number of
factors such as: the properties of the substances that act as excipients, the characteristics of the
appropriate biological membranes and the way that substances can cross them, or the
characteristics of the enzyme reactions that inactivate the drug.
All these concepts can be represented through mathematical formulas that have a
corresponding graphical representation. The use of these models allows an understanding of the
characteristics of a molecule, as well as how a particular drug will behave given information
regarding some of its basic characteristics such as its acid dissociation
constant (pKa), bioavailability and solubility, absorption capacity and distribution in the
organism.
The model outputs for a drug can be used in industry (for example, in
calculating bioequivalence when designing generic drugs) or in the clinical application of
pharmacokinetic concepts. Clinical pharmacokinetics provides many performance guidelines for
effective and efficient use of drugs for human-health professionals and in veterinary medicine

Metrics
The following are the most commonly measured pharmacokinetic metrics: [6] The units of
the dose in the table are expressed in moles (mol) and molar (M). To express the metrics of the
table in units of mass, instead of Amount of substance, simply replace 'mol' with 'g' and 'M' with
'g/dm3'. Similarly, other units in the table may be expressed in units of an
equivalent dimension by scaling.

In pharmacokinetics, steady state refers to the situation where the overall intake of a drug is
fairly in dynamic equilibrium with its elimination. In practice, it is generally considered that
steady state is reached when a time of 3 to 5 times the half-life for a drug after regular dosing is
started.
Pharmacokinetic models

Pharmacokinetic modelling is performed by noncompartmental

or compartmental methods. Noncompartmental methods estimate the exposure to a drug by
estimating the area under the curve of a concentration-time graph. Compartmental methods
estimate the concentration-time graph using kinetic models. Noncompartmental methods are
often more versatile in that they do not assume any specific compartmental model and produce
accurate results also acceptable for bioequivalence studies. The final outcome of the
transformations that a drug undergoes in an organism and the rules that determine this fate
depend on a number of interrelated factors. A number of functional models have been developed
in order to simplify the study of pharmacokinetics. These models are based on a consideration of
an organism as a number of related compartments. The simplest idea is to think of an organism
as only one homogenous compartment. This monocompartmental model presupposes that blood
plasma concentrations of the drug are a true reflection of the drug's concentration in other fluids
or tissues and that the elimination of the drug is directly proportional to the drug's concentration
in the organism (first order kinetics).
However, these models do not always truly reflect the real situation within an organism.
For example, not all body tissues have the same blood supply, so the distribution of the drug will
be slower in these tissues than in others with a better blood supply. In addition, there are some
tissues (such as the brain tissue) that present a real barrier to the distribution of drugs, that can be
breached with greater or lesser ease depending on the drug's characteristics. If these relative
conditions for the different tissue types are considered along with the rate of elimination, the
organism can be considered to be acting like two compartments: one that we can call the central
compartment that has a more rapid distribution, comprising organs and systems with a well-
developed blood supply; and a peripheral compartment made up of organs with a lower blood
flow. Other tissues, such as the brain, can occupy a variable position depending on a drug's
ability to cross the barrier that separates the organ from the blood supply.
This two compartment model will vary depending on which compartment elimination
occurs in. The most common situation is that elimination occurs in the central compartment as
the liver and kidneys are organs with a good blood supply. However, in some situations it may
be that elimination occurs in the peripheral compartment or even in both. This can mean that
there are three possible variations in the two compartment model, which still do not cover all
possibilities.
This model may not be applicable in situations where some of the enzymes responsible
for metabolizing the drug become saturated, or where an active elimination mechanism is present
that is independent of the drug's plasma concentration. In the real world each tissue will have its
own distribution characteristics and none of them will be strictly linear. If we label the
drug's volume of distribution within the organism VdF and its volume of distribution in a
tissue VdT the former will be described by an equation that takes into account all the tissues that
act in different ways, that is:

This represents the multi-compartment model with a number of curves that express
complicated equations in order to obtain an overall curve. A number of computer programs have
been developed to plot these equations.[8] However complicated and precise this model may be, it
still does not truly represent reality despite the effort involved in obtaining various distribution
values for a drug. This is because the concept of distribution volume is a relative concept that is
not a true reflection of reality. The choice of model therefore comes down to deciding which one
offers the lowest margin of error for the drug involved.

Non compartmental model

Noncompartmental PK analysis is highly dependent on estimation of total drug exposure.
Total drug exposure is most often estimated by area under the curve (AUC) methods, with
the trapezoidal rule (numerical integration) the most common method. Due to the dependence on
the length of x in the trapezoidal rule, the area estimation is highly dependent on the
blood/plasma sampling schedule. That is, the closer time points are, the closer the trapezoids
reflect the actual shape of the concentration-time curve. The number of time points available in
order to perform a successful NCA analysis should be enough to cover the absorption,
distribution and elimination phase to accurately characterize the drug. Beyond AUC exposure
measures, parameters such as Cmax (maximum concentration), Tmax(time at maximum
concentration), CL and Vd can also be reported using NCA methods.
Compartmental analysis
Compartmental PK analysis uses kinetic models to describe and predict the
concentration-time curve. PK compartmental models are often similar to kinetic models used in
other scientific disciplines such as chemical kinetics and thermodynamics. The advantage of
compartmental over some noncompartmental analyses is the ability to predict the concentration
at any time. The disadvantage is the difficulty in developing and validating the proper model.
Compartment-free modelling based on curve stripping does not suffer this limitation. The
simplest PK compartmental model is the one-compartmental PK model with IV bolus
administration and first-order elimination. The most complex PK models (called PBPK models)
rely on the use of physiological information to ease development and validation.

Single-compartment model
Linear pharmacokinetics is so-called because the graph of the relationship between the
various factors involved (dose, blood plasma concentrations, elimination, etc.) gives a straight
line or an approximation to one. For drugs to be effective they need to be able to move rapidly
from blood plasma to other body fluids and tissues.
The change in concentration over time can be expressed as
Multi-compartmental models[edit]

Graphs for absorption and elimination for a non-linear pharmacokinetic model.

The graph for the non-linear relationship between the various factors is represented by
a curve; the relationships between the factors can then be found by calculating the dimensions of
different areas under the curve. The models used in non-linear pharmacokinetics are largely
based on Michaelis–Menten kinetics. A reaction's factors of non-linearity include the following:
 Multiphasic absorption: Drugs injected intravenously are removed from the plasma through
two primary mechanisms: (1) Distribution to body tissues and (2) metabolism + excretion of
the drugs. The resulting decrease of the drug's plasma concentration follows a biphasic
pattern (see figure).
 Plasma drug concentration vs time after an IV dose
o Alpha phase: An initial phase of rapid decrease in plasma concentration. The decrease is
primarily attributed to drug distribution from the central compartment (circulation) into
the peripheral compartments (body tissues). This phase ends when a pseudo-equilibrium
of drug concentration is established between the central and peripheral compartments.
o Beta phase: A phase of gradual decrease in plasma concentration after the alpha phase.
The decrease is primarily attributed to drug elimination, that is, metabolism and
excretion.[9]
o Additional phases (gamma, delta, etc.) are sometimes seen.[10]
 A drug's characteristics make a clear distinction between tissues with high and low blood
flow.
 Enzymatic saturation: When the dose of a drug whose elimination depends on
biotransformation is increased above a certain threshold the enzymes responsible for its
metabolism become saturated. The drug's plasma concentration will then increase
disproportionately and its elimination will no longer be constant.
 Induction or enzymatic inhibition: Some drugs have the capacity to inhibit or stimulate their
own metabolism, in negative or positive feedback reactions. As occurs
with fluvoxamine, fluoxetine and phenytoin. As larger doses of these pharmaceuticals are
administered the plasma concentrations of the unmetabolized drug increases and
the elimination half-life increases. It is therefore necessary to adjust the dose or other
treatment parameters when a high dosage is required.
 The kidneys can also establish active elimination mechanisms for some drugs, independent
of plasma concentrations.
It can therefore be seen that non-linearity can occur because of reasons that affect the entire
pharmacokinetic sequence: absorption, distribution, metabolism and elimination.

Bioavailabilty

In pharmacology, bioavailability is a subcategory of absorption and is the fraction (%) of

an administered drug that reaches the systemic circulation.[1]
By definition, when a medication is administered intravenously, its bioavailability is
100%.[2][3] However, when a medication is administered via routes other than intravenous, its
bioavailability is generally[TH] lower than that of intravenous due to intestinal endothelium
absorption and first-pass metabolism. Thereby, mathematically, bioavailability equals the ratio of
comparing the area under the plasma drug concentration curve versus time (AUC) for the
extravascular formulation to the AUC for the intravascular formulation.[4] AUC is used because
AUC is proportional to the dose that has entered the systemic circulation.[5]
Bioavailability of a drug is an average value; to take population variability into
account, deviation range is shown as ±.[4] To ensure that the drug taker who has poor absorption
is dosed appropriately, the bottom value of the deviation range is employed to represent real
bioavailability and to calculate the drug dose needed for the drug taker to achieve systemic
concentrations similar to the intravenous formulation.[4] To dose without knowing the drug
taker's absorption rate, the bottom value of the deviation range is used in order to ensure the
intended efficacy, unless the drug is associated with a narrow therapeutic window.[4]
For dietary supplements, herbs and other nutrients in which the route of administration is
nearly always oral, bioavailability generally designates simply the quantity or fraction of the
ingested dose that is absorbed.
Absolute bioavailability
Absolute bioavailability compares the bioavailability of the active drug in systemic
circulation following non-intravenous administration (i.e., after oral, buccal, ocular, nasal,
rectal, transdermal, subcutaneous, or sublingual administration), with the bioavailability of the
same drug following intravenous administration. It is the fraction of the drug absorbed through
non-intravenous administration compared with the corresponding intravenous administration of
the same drug. The comparison must be dose normalized (e.g., account for different doses or
varying weights of the subjects); consequently, the amount absorbed is corrected by dividing the
corresponding dose administered.
In pharmacology, in order to determine absolute bioavailability of a drug,
a pharmacokinetic study must be done to obtain a plasma drug concentration vs time plot for the
drug after both intravenous (iv) and extravascular (non-intravenous, i.e., oral) administration.
The absolute bioavailability is the dose-corrected area under curve (AUC) non-intravenous
divided by AUC intravenous. The formula for calculating the absolute bioavailability, F, of a
drug administered orally (po) is given below (where D is dose administered).

Therefore, a drug given by the intravenous route will have an absolute bioavailability of 100%
(f = 1), whereas drugs given by other routes usually have an absolute bioavailability of less than
one. If we compare the two different dosage forms having same active ingredients and compare
the two drug bioavailability is called comparative bioavailability.[citation needed]
Although knowing the true extent of systemic absorption (referred to as absolute
bioavailability) is clearly useful, in practice it is not determined as frequently as one may think.
The reason for this is that its assessment requires an intravenous reference; that is, a route of
administration that guarantees all of the administered drug reaches systemic circulation. Such
studies come at considerable cost, not least of which is the necessity to conduct preclinical
toxicity tests to ensure adequate safety, as well as potential problems due to solubility
limitations. These limitations may be overcome, however, by administering a very low dose
(typically a few micrograms) of an isotopically labelled drug concomitantly with a therapeutic
non-isotopically labelled oral dose (the isotopically-labelled intravenous dose is sufficiently low
so as not to perturb the systemic drug concentrations achieved from the non-labelled oral dose).
The intravenous and oral concentrations can then be deconvoluted by virtue of their
different isotopic constitution, and can thus be used to determine the oral and intravenous
pharmacokinetics from the same dose administration. This technique eliminates pharmacokinetic
issues with non-equivalent clearance as well as enabling the intravenous dose to be administered
with a minimum of toxicology and formulation. The technique was first applied using stable-
isotopes such as 13C and mass-spectrometry to distinguish the isotopes by mass difference. More
 14
recently, C labelled drugs are administered intravenously and accelerator mass spectrometry
(AMS) used to measure the isotopically labelled drug along with mass spectrometry for the
unlabelled drug.

There is no regulatory requirement to define the intravenous pharmacokinetics or

absolute bioavailability however regulatory authorities do sometimes ask for absolute
bioavailability information of the extravascular route in cases in which the bioavailability is
apparently low or variable and there is a proven relationship between the pharmacodynamics and
the pharmacokinetics at therapeutic doses. In all such cases, to conduct an absolute
bioavailability study requires that the drug be given intravenously.[18]
Intravenous administration of a developmental drug can provide valuable information on
the fundamental pharmacokinetic parameters of volume of distribution (V) and clearance (CL)

Relative bioavailability and equivalence

In pharmacology, relative bioavailability measures the bioavailability (estimated as
the AUC) of a formulation (A) of a certain drug when compared with another formulation (B) of
the same drug, usually an established standard, or through administration via a different route.
When the standard consists of intravenously administered drug, this is known as absolute
bioavailability (see above).

Relative bioavailability is one of the measures used to assess bioequivalence (BE)

between two drug products. For FDA approval, a generic manufacturer must demonstrate that
the 90% confidence interval for the ratio of the mean responses (usually of AUC and the
maximum concentration, Cmax) of its product to that of the "brand name drug"[OB] is within the
limits of 80% to 125%. Where AUC refers to the concentration of the drug in the blood over
time t = 0 to t = ∞, Cmax refers to the maximum concentration of the drug in the blood.
When Tmax is given, it refers to the time it takes for a drug to reach Cmax.
 While the mechanisms by which a formulation affects bioavailability and bioequivalence
have been extensively studied in drugs, formulation factors that influence bioavailability and
bioequivalence in nutritional supplements are largely unknown.[19] As a result, in nutritional
sciences, relative bioavailability or bioequivalence is the most common measure of
bioavailability, comparing the bioavailability of one formulation of the same dietary ingredient
to another.

Factors affecting bioavailability

The absolute bioavailability of a drug, when administered by an extravascular route, is
usually less than one (i.e., F< 100%). Various physiological factors reduce the availability of
drugs prior to their entry into the systemic circulation. Whether a drug is taken with or without
food will also affect absorption, other drugs taken concurrently may alter absorption and first-
pass metabolism, intestinal motility alters the dissolution of the drug and may affect the degree
of chemical degradation of the drug by intestinal microflora. Disease states affecting liver
metabolism or gastrointestinal function will also have an effect.

Other factors may include, but are not limited to:

 Physical properties of the drug (hydrophobicity, pKa, solubility)
 The drug formulation (immediate release, excipients used, manufacturing methods, modified
release – delayed release, extended release, sustained release, etc.)
 Whether the formulation is administered in a fed or fasted state
 Gastric emptying rate
 Circadian differences
 Interactions with other drugs/foods:
o Interactions with other drugs (e.g., antacids, alcohol, nicotine)
o Interactions with other foods (e.g., grapefruit juice, pomello, cranberry
juice, brassica vegetables
 Transporters: Substrate of efflux transporters (e.g. P-glycoprotein)
 Health of the gastrointestinal tract
 Enzyme induction/inhibition by other drugs/foods:
 o Enzyme induction (increased rate of metabolism),
e.g., Phenytoin induces CYP1A2, CYP2C9, CYP2C19, and CYP3A4
o Enzyme inhibition (decreased rate of metabolism), e.g., grapefruit juice inhibits CYP3A
→ higher nifedipine concentrations
 Individual variation in metabolic differences
o Age: In general, drugs are metabolized more slowly in fetal, neonatal, and geriatric
populations
o Phenotypic differences, enterohepatic circulation, diet, gender
 Disease state
o E.g., hepatic insufficiency, poor renal function

Each of these factors may vary from patient to patient (inter-individual variation), and indeed in
the same patient over time (intra-individual variation). In clinical trials, inter-individual variation
is a critical measurement used to assess the bioavailability differences from patient to patient in
order to ensure predictable dosing.

Bioavailability of drugs vs dietary supplements

In comparison to drugs, there are significant differences in dietary supplements that
impact the evaluation of their bioavailability. These differences include the following: the fact
that nutritional supplements provide benefits that are variable and often qualitative in nature; the
measurement of nutrient absorption lacks the precision; nutritional supplements are consumed
for prevention and well-being; nutritional supplements do not exhibit characteristic dose-
response curves; and dosing intervals of nutritional supplements, therefore, are not critical in
contrast to drug therapy.[11]
In addition, the lack of defined methodology and regulations surrounding the
consumption of dietary supplements hinders the application of bioavailability measures in
comparison to drugs. In clinical trials with dietary supplements, bioavailability primarily focuses
on statistical descriptions of mean or average AUC differences between treatment groups, while
often failing to compare or discuss their standard deviations or inter-individual variation. This
failure leaves open the question of whether or not an individual in a group is likely to experience
the benefits described by the mean-difference comparisons. Further, even if this issue were
discussed, it would be difficult to communicate meaning of these inter-subject variances to
consumers and/or their physicians.

LADME
A number of phases occur once the drug enters into contact with the organism, these are
described using the acronym LADME:
 Liberation of the active substance from the delivery system,
 Absorption of the active substance by the organism,
 Distribution through the blood plasma and different body tissues,
 Metabolism that is inactivation of the xenobiotic substance, and finally
 Excretion or elimination of the substance or the products of its metabolism.
Some textbooks combine the first two phases as the drug is often administered in an active
form, which means that there is no liberation phase. Others include a phase that combines
distribution, metabolism and excretion into a disposition phase. Other authors include the drug's
toxicological aspect in what is known as ADME-Tox or ADMET.

Each of the phases is subject to physico-chemical interactions between a drug and an

organism, which can be expressed mathematically. Pharmacokinetics is therefore based on
mathematical equations that allow the prediction of a drug's behavior and which place great
emphasis on the relationships between drug plasma concentrations and the time elapsed since the
drug's administration.
 Knowledge of these processes and the ways that they can vary between individuals is an
important part of understanding how and why a drug is selected for a patient. To investigate the
pharmacokinetic characteristics of a study drug (drug X), researchers will give a group of healthy
adults a standard dose of drug X intravenously (IV) or orally at the start of the study. Blood is
drawn from the study subjects repeatedly, at predetermined times, and analyzed for the amount
of drug per volume of blood at each point in time. The value obtained is the serum or plasma
concentration of the drug at the time the blood was drawn. When serum drug concentrations are
graphed versus time, the result is the serum concentration versus time curve illustrated in Figure
3.2.
Urine is also collected from the study subjects to monitor for the appearance of drug x or
related metabolites. Immediately after intravenous administration and at a later time after oral or
other routes of administration, the amount of drug in the blood will reach a peak (peak serum
concentration) and then begin to fall off, eventually disappearing from the blood completely. The
time it takes for the serum concentration to fall by one half is called the t1 /2, or half-life, of
elimination. At some point after the drug is no longer detectable in the blood, the last of the drug
and its metabolites, which have been filtered out by the kidneys, will also disappear from the
urine. Plasma concentration data collected from this type of study is plotted against time and
analyzed in order to understand the behavior of a specific drug in the body. This type of
pharmacokinetic data, collected from average adults, is the basis for determining dose, dosing
intervals, and limitations on the safe use of a drug. However, it is important for the technician
student to remember that individuals do not always behave the way the average adult does. It is
individual differences in ADME processes that create the need to modify doses or select different
drugs in order to prevent poor treatment outcomes and adverse effects.

tion. When a drug is administered intravenously, absorption is not required because the drug is
transferred from the administration device directly into the bloodstream. In the case of
intravenous administration, the entire dose of the drug is available to move to the sites of drug
action. Administration by other routes may result in less availability due to incomplete
absorption. When this occurs, less of the drug is delivered by the bloodstream to the site of
action. When a tablet or capsule is swallowed it must dissolve before it can be absorbed. The
dissolving of a tablet or capsule is referred to as dissolution. Manufacturing processes and the
water solubility of the drug affect dissolution rates. Highly water-soluble medications dissolve
more readily in the gastrointestinal (GI) tract, while fat-soluble drugs dissolve more slowly.
Drugs with smaller particle sizes go into solution more readily. The inert ingredients added to
formulations can also affect their dissolution. Manufacturers must avoid producing tablets so
compacted that they pass through the GI tract without ever dissolving. Tablets that dissolve too
early are also problematic, because they taste bad and are difficult to swallow. Special
formulations or coatings can be used to delay dissolution, thereby protecting the drug from
stomach acid or allowing the gradual release of the drug to intentionally lengthen the absorption
process. These are referred to as delayed or sustained release formulations. Liquid preparations
do not require the step of dissolution. This explains the more rapid onset of action seen with
liquid formulations as compared with the same drug given in tablet or capsule form.
Once dissolution has occurred, the drug molecules must pass through the selectively
permeable membranes of the cells lining the gastrointestinal tract to reach the bloodstream.
Depending on their chemical and physical properties, drugs will be absorbed either by passive
diffusion or carrier-mediated transport across these membranes. Passive diffusion occurs when
there is a high concentration of the drug on one side of the membrane and a low concentration on
the other side. This difference from one side of the membrane to the other is called a
concentration gradient. It is the natural tendency of substances to move from a region of higher
concentration to a region of lower concentration; in other words, substances move down the
concentration gradient. Drug molecules move across membranes or move through pores between
the epithelial cells (Fig. 3.3). Diffusion is most efficient with drugs that are small molecules.
This movement is solely driven by the kinetic energy within molecules, and continues until
concentrations reach equilibrium. When equilibrium exists, the concentration of the substance is
approximately equal on both sides of the membrane.
Passive diffusion does not involve a carrier molecule and the process is not a saturable
process. Saturable processes are limited to a certain rate of activity by some aspect of the
process. The vast majority of drugs gain access to the blood stream by diffusion. Drugs, which
are usually somewhat lipid-soluble (fat-soluble), readily move across most biological
membranes. Those drugs that are highly water-soluble penetrate the cell membrane through
aqueous channels. A few drugs that closely resemble naturally occurring compounds are
absorbed via carrier-mediated transport. This process requires carrier proteins that attach to and
actively carry the drug molecules across the membrane, utilizing a natural “pump” mechanism.
This method of absorption is limited by the availability of the carrier protein and is therefore,
saturable. Carrier-mediated transport requires energy and can move molecules against the
concentration gradient. For an illustration of passive diffusion and carrier-mediated transport see
Figure 3.4.

Bioavailability
The relationship between the drug dose and the amount ultimately delivered to the
bloodstream is defined as bioavailability and is generally expressed as a percentage. If a 1 gram
dose of a drug is administered by mouth, and half of that reaches the systemic circulation, the
drug is 50% bioavailable. Bioavailability is calculated, not measured directly. Previously, the
half-life of elimination and how it is determined was discussed. The same graph of serum
concentrations against time also provides the data necessary to derive bioavailability. The area
under the plasma concentration versus time curve represents the total amount of the drug
reaching the circulatory system (see Fig. 3.2). This curve will have a different shape depending
on the route of drug administration. The curve obtained from plotting values after intravenous
administration of a drug serves as a reference for complete bioavailability. To determine
bioavailability for nonintravenous formulations, the area under the curve obtained after drug
administration is compared with the area achieved when the same dose is given intravenously.
The ratio of the two is the bioavailability of the formulation tested. The acid environment or
presence of food in the stomach, the solubility and other chemical properties of the drug, and the
effect of the initial exposure to metabolic processes in the liver may all reduce the amount of
drug that reaches the systemic circulation after oral administration, thereby reducing the
bioavailability of the drug. When a drug is absorbed through the GI tract, it must travel through
the liver before entering the systemic circulation (see Fig. 3.5). If the drug is subject to
metabolism by the liver, the amount of drug that reaches the systemic circulation is decreased.
Some drugs, such as propranolol or enalapril, undergo significant metabolism during a single
passage through the liver. This is called the first-pass effect. When drugs are highly susceptible
to the first-pass effect, the oral dose needed to cause a response will be significantly higher than
the intravenous dose used to cause the same response.
Bioavailability becomes important in drug product selection. While two generic products
may contain the same active ingredients, they may not have the same dissolution or absorption
characteristics and therefore cannot be considered bioequivalent. In the case of extended-release
products, the change in dissolution characteristics is intentional. In some cases, however,
products are simply poorly manufactured and should be avoided. Generic equivalent products
approved by the Food and Drug Administration (FDA) must meet a standard of less than 20%
variation from the comparison product, an amount that does not significantly affect therapeutic
efficacy or safety in most cases. In practice, however, most generic products typically vary from
the original by less than 5%. Bioequivalency data is published by the FDA’s Center for Drug
Evaluation and Research in the “Approved Drug Products with Therapeutic Equivalence
Evaluations” publication, universally referred to as the “Orange Book” and available on the web.

Factors Affecting Absorption

A number of patient-specific factors can affect absorption. Absorption from any site of
administration requires blood flow. A patient who is in shock or cardiopulmonary arrest will
need to have medications administered intravenously to achieve the desired response because of
the reduced blood circulation in these situations. Most absorption after oral administration occurs
in the small intestine because of its larger surface area and therefore greater blood flow.
 Significant impairment of absorption can result when sections of the small intestine are
removed for medical reasons. Contact time with the epithelial lining of the GI tract is also an
important factor in drug absorption. In people with a very rapid transit time through the GI tract,
due to severe diarrhea for example, medication cannot be effectively absorbed. Conversely,
anything that delays stomach emptying (for instance, a large meal) will also delay and potentially
reduce absorption.

Some medications exhibit drug-food or drug-drug interactions with other compounds

present in the GI tract. Specific foods or other drugs may bind a drug and prevent absorption.
The interaction between tetracycline and dairy products or antacids is a good example of this
effect. Although excess exposure to stomach acid may negatively impact some drugs, patients
with very low levels of stomach acid (achlorhydria) may experience inadequate tablet dissolution
and therefore poor drug absorption. Achlorhydria is most common in the elderly population.
Distribution
Once a drug is absorbed into the bloodstream it can be carried throughout the body. This
process is called distribution, and is a reversible process; while some molecules may be
interacting with receptors on cell membranes or inside of cells, other molecules may move back
into the bloodstream. The delivery of a drug from the bloodstream to the site of drug action
primarily depends on blood flow, capillary permeability, the degree of binding (attachment) of
the drug to blood and tissue proteins, and the relative lipid-solubility of the drug molecule. Blood
flow to different organs of the body is not equal. The most vitally important organs of the body
receive the greatest supply of blood. These organs include the brain, liver, and kidneys. Skeletal
muscle and bone receive less blood, and adipose tissue (fat) receives the least. If blood flow were
the only factor affecting distribution, it would be reasonable to expect that high concentrations of
administered medications would always appear in the brain and liver. In reality, few drugs
exhibit good penetration of the central nervous system.
The anatomical structure of the capillary network in the brain creates a significant barrier
to the passage of many drugs and is commonly referred to as the blood-brain barrier. This barrier
is an adaptation that for the most part protects brain tissue from invasion by foreign substances.
To readily penetrate into the brain, drugs must be fairly small and lipidsoluble or must be picked
up by the carrier-mediated transport mechanism in the central nervous system. This explains why
the small and highly fat-soluble anesthetic gases quickly and easily penetrate the brain to cause
anesthesia, while other larger and water soluble molecules like penicillin antibiotics penetrate the
central nervous system to a much lesser degree. Compare the fairly impermeable capillaries of
the brain to the highly permeable capillary walls in the liver and spleen (Fig. 3.6). These
capillaries have gaps between their cells that allow large proteins to pass through to the capillary
basement membrane. Capillary structure here is well adapted to the function of the liver, the key
protein producer in the body and a center for chemical change of other compounds. In order to
function, the liver must have access to amino acids, sugars, and other large molecules from the
bloodstream. These molecules undergo chemical processing in the liver, and then must be moved
out of the hepatic cells and back to the bloodstream.
Factors Affecting Distribution
The blood is composed of a number of elements, including plasma, red and white blood
cells, and plasma proteins. Most drugs reversibly bind to plasma proteins in varying degrees.
Albumin is the plasma protein with the greatest capacity for binding drugs. Binding to plasma
proteins affects drug distribution into tissues, because only drug that is not bound is available to
penetrate tissues, bind to receptors, and exert activity. As free drug leaves the bloodstream, more
bound drug is released from binding sites. In this way, drugs maintain a balance between free
and bound drug that is unique to each compound, based on its affinity for plasma proteins.
Albumin, then, acts as a reservoir of an administered drug.

Some drugs have a high affinity for binding to serum proteins and may be 95% to 98% protein
bound. With highly protein bound drugs, low albumin levels (as in protein-calorie malnutrition,
or chronic illness) may lead to toxicity because there are fewer than the normal sites for the drug
to bind. The amount of free drug is significantly increased in that case. The physician or
pharmacist must consider the patient’s serum albumin level when the dose of a highly protein
bound medication is selected. Competition for binding sites is one important way that drugs
might interact. If a patient is using two highly protein bound drugs at the same time, there will be
competition for binding sites on the albumin. The drug with the greatest affinity for the albumin
will bind, and is thought to disrupt the normal ratio of free to bound drug for the second
medication. As a result, the second medication will be more available to distribute to the site of
action and potentially cause side effects.
Other patient variables that can affect distribution include body composition, cardiac
decompensation (heart failure), and age of the patient. These factors all affect the apparent
volume of distribution (Vd) of a drug and ultimately play a role in determining the appropriate
dose of a drug. Volume of distribution is the hypothetical volume needed to account for all of the
drug in the body based on the serum concentration in a blood sample. For example, assume that a
1-gram dose of drug X is given by IV injection to a patient. Thirty minutes later the serum
concentration (Cp) is 25mcg/ml. If the drug were evenly distributed through the body, the
apparent volume of distribution would have to be 40 liters (L) to account for the entire dose of
the drug.
Cp = Dose (amount in the body)/Vd
The adult human body is about 60% water. Therefore, the body of an average adult who weighs
70 kg (about 150 lbs) contains 42 L, or 10 gallons, of water. Total body water can be
conceptually divided into three spaces or compartments. The fluid contained in the bloodstream
makes up about 5 L, or about 9% of the volume of an average sized adult. The total water outside
of the cells (extracellular fluid) includes the plasma volume plus the fluid in the interstitial space
and is about 14 L. Intracellular fluid makes up the remaining 28 L. Drugs with high molecular
weights or drugs that are extremely hydrophilic (with a strong affinity for water) tend to stay
within the circulatory system and organs with a rich blood supply, and have a smaller apparent
volume of distribution. Small, highly lipophilic drugs have a large apparent volume of
distribution.
Volume of distribution often becomes important when dosing calculations are made based on
weight. A short, obese woman who weighs 250 lbs cannot handle the same dose of an
aminoglycoside antibiotic that a tall, muscular man of the same weight would require. This is
because a greater portion of the woman’s body is made up of fat. Aminoglycoside drugs are
water-soluble and stay mainly in extracellular fluid, therefore dosing must be based on adjusted
body weight, which will more correctly reflect the true volume of extracellular fluid in the body.
Dosing of medications in infants and children requires special consideration. It is the often-
repeated wisdom of pediatric health care specialists that “children are not simply small adults.”
This means that dosing cannot simply be adjusted based on the lower weight of children. The
body composition of children is very different from adults. Their bodies contain a much higher
percentage of water and a lower percentage of muscle and fat. Albumin levels may also be lower,
especially in neonates. These variations result in different values for volume of distribution and
significantly affect drug dosing.

Metabolism
Drugs are eliminated from the body either unchanged through the kidneys and bile, or
they may undergo chemical changes that allow them to be more easily excreted. The process of
undergoing chemical changes is called biotransformation, or metabolism. As previously noted,
anything absorbed through the GI tract goes directly into the portal circulation that feeds into the
liver. The liver is adapted to clear toxins from the body and is the major site for drug
metabolism, but specific drugs may undergo biotransformation in other tissues. The kidneys
cannot efficiently excrete highly fat-soluble drugs that readily cross cell membranes because they
are reabsorbed in the last stages of filtration. These compounds must first be metabolized in the
liver to more water-soluble compounds and then removed. There are two types of metabolic
processes drugs undergo in the liver. Most undergo one or both types of reactions. In the first
type of reaction drugs are made more polar through oxidation-reduction reactions or hydrolysis.
These reactions use metabolic enzymes, most often those of the cytochrome P450
enzyme system, to catalyze the biotransformation. In enzyme-catalyzed reactions, the rate of the
reaction is accelerated by the presence of enzymes. A limited amount of enzyme is present at any
given time in the liver. Since the rate of enzyme-catalyzed drug metabolism is limited by the
quantity of available enzyme, metabolism in these cases is considered a saturable process. This
means that the rate of conversion will only continue at the normal pace until the available supply
of enzyme is used. At that point, metabolism is slowed until enzyme becomes available again.
For the usual doses of most drugs, these reactions never reach saturation. There are a few drugs
where doses may reach the saturation point of the enzymes. Once enzymes become saturated,
blood levels increase exponentially toward toxicity. Examples include metabolism of alcohol and
phenytoin.
The second type of metabolism involves conjugation reactions. In this type of reaction
the drug undergoing change is joined with another substance, such as glucuronic acid, sulfuric
acid, acetic acid, or an amino acid. Glucuronidation is the most common conjugation reaction.
The result of conjugation is a more water-soluble compound that is easier for the kidneys to
excrete. These metabolites are most often therapeutically inactive. Some agents are initially
administered as an inactive compound (prodrug) in order to improve availability or reduce side
effects. Metabolism converts the prodrug to the active form. Fosphenytoin, for example, is a
prodrug of phenytoin, a drug used for seizure disorders. Fosphenytoin is more completely and
quickly absorbed when given by IM injection than phenytoin and can be used in critical
situations with greater ease because it dose not require insertion of an intravenous catheter.

Factors Affecting Metabolism

Metabolism of drugs can vary widely between population groups. Deficiency of some
drug metabolizing enzymes is genetic and will result in poor tolerance of certain drugs. For
example, many Asians and Native Americans have difficulty metabolizing drugs that require
acetylation, such as ethanol. These individuals will exhibit a low tolerance of such drugs, and can
suffer adverse drug reactions at a much higher rate than the average population. Age is another
important variable that has a bearing on metabolism. Organ function gradually declines with age
and the elderly may poorly tolerate drugs that require metabolism. The very young require
special consideration of drug dosing because of immaturity of their organ systems. This subject
matter is worthy of further study for those technicians who will be serving these special
populations.
Drug interactions may occur between two drugs that are metabolized by the same enzyme
systems in the liver. Because there is a limit on available enzymes for metabolism, excess drug
will remain active and free to exert an effect elsewhere in the body. Usually, of two drugs that
are metabolized by the same enzyme system, one has a higher affinity for the enzyme, and levels
of the second drug build up. In some cases, the drug being metabolized will induce the
production of more of the enzymes. Enzyme induction sets the stage for another type of drug
interaction, because the increased production of metabolizing enzymes may result in higher rates
of removal and the need for an increased dose of the second drug. A good example of a drug that
stimulates production of metabolizing enzymes is phenobarbital, a drug used to treat some forms
of epilepsy.

Excretion
When a drug is taken into and distributed throughout the body, it must be subsequently
removed, or concentrations of the drug would continue to rise with each successive dose. The
complete removal of the drug from the body is referred to as elimination. Elimination of the drug
encompasses both the metabolism of the drug, and excretion of the drug through the kidneys, and
to a much lesser degree into the bile. Excretion into the urine is one of the most important
mechanisms of drug removal.
The kidneys act as a filter for the blood and create urine as a vehicle for removal of
waste. Blood enters the kidney through renal arteries and then is filtered by the glomerulus. The
glomerular filtrate becomes concentrated and substances are removed as it passes through the
renal tubule and eventually becomes urine. Drug molecules in the bloodstream that are not bound
to albumin are also filtered out into the glomerular filtrate. When drugs have not been converted
to water soluble compounds in the liver, they are likely to be reabsorbed back into the
bloodstream at the end of the filtration process, and will cycle through the body again. If they are
water soluble, they will end up in the urine and be excreted.
When a medication is given repeatedly, as most are in real patients, the total amount of
drug in the body will increase up to a point and then stabilize. At this point, the amount being
taken in by the patient is equal to the amount being removed by the liver and kidneys (Fig. 3.7).
This state of equilibrium is called steady state, and drug levels will remain fairly constant unless
there is a dose change, an interruption in treatment, or failure of the organs of elimination. The
therapeutic effects of many drugs are closely correlated to a specific range of steady state serum
drug levels, and physicians or clinical pharmacists will monitor these levels and adjust doses
when necessary so that patients obtain the appropriate drug response

Factors Affecting Excretion

 The complete elimination of a drug from the body is dependent on normal liver and
kidney function. The kidney is the major organ of excretion; however, the liver also contributes
to elimination through metabolism and excretion into the feces via the bile. When a patient has
reduced kidney function or another problem that lengthens the half-life of a drug, dosage
adjustment is required. If the dosage is not adjusted, the drug will accumulate in the body.
Kidney or liver failure, or conditions where blood flow to these organs is reduced, complicate
drug and dose selection. Drugs that are dependent upon excretion through the kidneys are not the
best choice for patients with renal failure. Patients with liver disease will better tolerate drugs
that can be cleared exclusively through the kidneys. Age must be considered in a discussion of
drug excretion. The very young and very old will have lower rates of excretion; the old because
of deterioration in organ function and the very young, because the kidneys have not reached full
maturity. Doses often require reduction in these patients. Drug interactions, such as when
multiple drugs compete for metabolic processes, can also reduce drug removal. Clearly, the
interactions between a drug and the human body are incredibly complex. This makes choosing
the most appropriate medication and dose complicated, and that choice becomes more obscure
when a patient is taking many other medications. This complexity makes it all the more essential
that prescriptions be filled carefully and accurately at all times.

Pharmacodynamics
Pharmacodynamics (PD) is the study of the biochemical and physiologic effects
of drugs (especially pharmaceutical drugs). The effects can include those manifested
within animals (including humans), microorganisms, or combinations of organisms (for
example, infection).
Pharmacodynamics and pharmacokinetics are the main branches of pharmacology, being itself a
topic of biology interested in the study of the interactions between both endogenous and
exogenous chemical substances with living organisms.
In particular, pharmacodynamics is the study of how a drug affects an organism, whereas
pharmacokinetics is the study of how the organism affects the drug. Both together
influence dosing, benefit, and adverse effects. Pharmacodynamics is sometimes abbreviated as
PD and pharmacokinetics as PK, especially in combined reference (for example, when speaking
of PK/PD models).
Pharmacodynamics places particular emphasis on dose–response relationships, that is, the
relationships between drug concentration and effect.[1] One dominant example is drug-receptor
interactions as modeled by

where L, R, and LR represent ligand (drug), receptor, and ligand-receptor complex

concentrations, respectively. This equation represents a simplified model of reaction
dynamics that can be studied mathematically through tools such as free energy maps.

Effects on the body

Desired activity
The desired activity of a drug is mainly due to successful targeting of one of the following:
 Cellular membrane disruption
 Chemical reaction with downstream effects
 Interaction with enzyme proteins
 Interaction with structural proteins
 Interaction with carrier proteins
 Interaction with ion channels
 Ligand binding to receptors:
o Hormone receptors
o Neuromodulator receptors
o Neurotransmitter receptors
General anesthetics were once thought to work by disordering the neural membranes, thereby
altering the Na+ influx. Antacids and chelating agents combine chemically in the body. Enzyme-
substrate binding is a way to alter the production or metabolism of key endogenous chemicals,
for example aspirin irreversibly inhibits the enzyme prostaglandin synthetase
(cyclooxygenase) thereby preventing inflammatory response. Colchicine, a drug for gout,
interferes with the function of the structural protein tubulin, while Digitalis, a drug still used in
heart failure, inhibits the activity of the carrier molecule, Na-K-ATPase pump. The widest class
of drugs act as ligands that bind to receptors that determine cellular effects. Upon drug binding,
receptors can elicit their normal action (agonist), blocked action (antagonist), or even action
opposite to normal (inverse agonist).
In principle, a pharmacologist would aim for a target plasma concentration of the drug for a
desired level of response. In reality, there are many factors affecting this goal. Pharmacokinetic
factors determine peak concentrations, and concentrations cannot be maintained with absolute
consistency because of metabolic breakdown and excretory clearance. Genetic factors may exist
which would alter metabolism or drug action itself, and a patient's immediate status may also
affect indicated dosage.

Undesirable effects
Undesirable effects of a drug include:
 Increased probability of cell mutation (carcinogenic activity)
 A multitude of simultaneous assorted actions which may be deleterious
 Interaction (additive, multiplicative, or metabolic)
 Induced physiological damage, or abnormal chronic conditions

Therapeutic Window
The therapeutic window is the amount of a medication between the amount that gives an effect
(effective dose) and the amount that gives more adverse effects than desired effects. For instance,
medication with a small pharmaceutical window must be administered with care and control, e.g.
by frequently measuring blood concentration of the drug, since it easily loses effects or gives
adverse effects.

Duration of action
The duration of action of a drug is the length of time that particular drug is effective.[4] Duration
of action is a function of several parameters including plasma half-life, the time to equilibrate
between plasma and target compartments, and the off rate of the drug from its biological target

Therapeutic Index
The therapeutic index (TI; also referred to as therapeutic ratio) is a quantitative
measurement of the relative safety of a drug. It is a comparison of the amount of a therapeutic
agent that causes the therapeutic effect to the amount that causes toxicity.[1] The related
terms therapeutic window or safety window refer to a range of doses which optimize between
efficacy and toxicity, achieving the greatest therapeutic benefit without resulting in unacceptable
side-effects or toxicity.
Classically, in an established clinical indication setting of an approved drug, TI refers to
the ratio of the dose of drug that causes adverse effects at an incidence/severity not compatible
with the targeted indication (e.g. toxic dose in 50% of subjects, TD50) to the dose that leads to
the desired pharmacological effect (e.g. efficacious dose in 50% of subjects, ED50). In contrast,
in a drug development setting TI is calculated based on plasma exposure levels.
In the early days of pharmaceutical toxicology, TI was frequently determined in animals
as lethal dose of a drug for 50% of the population (LD50) divided by the minimum effective
dose for 50% of the population (ED50). Today, more sophisticated toxicity endpoints are used.

For many drugs, there are severe toxicities that occur at sublethal doses in humans, and
these toxicities often limit the maximum dose of a drug. A higher therapeutic index is preferable
to a lower one: a patient would have to take a much higher dose of such a drug to reach the toxic
threshold than the dose taken to elicit the therapeutic effect.
Generally, a drug or other therapeutic agent with a narrow therapeutic range (i.e. having little
difference between toxic and therapeutic doses) may have its dosage adjusted according to
measurements of the actual blood levels achieved in the person taking it. This may be achieved
through therapeutic drug monitoring (TDM) protocols. TDM is recommended for use in the
treatment of psychiatric disorders with lithium due to its narrow therapeutic range.

Term Meaning

ED Effective Dose

TD Toxic Dose

LD Lethal Dose

TI Therapeutic Index
TR Therapeutic Ratio