Protein Biochemistry and

Proteomics
SCPAA3

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Week 6: Lesson 11
Introduction

In this lesson, you will learn about drug design.

 What will be covered
in today’s lesson?
Week 6
Drug Design
Lesson 11

Cry toxins
 Introduction

❑ Drug design, often known as rational drug design or simply rational design, is the innovative process of discovering
novel treatments based on a biological target's understanding.
❑ A therapeutic target is often a critical molecule engaged in a specific metabolic or signalling pathway related to a
disease state or pathology, or to the infectivity or survival of a microbial pathogen.
❑ Other techniques might include boosting certain molecules in the normal route that may have been altered in the
pathological stage.
 What is Drug Design?

❑ Drug design is a three-dimensional puzzle in which tiny drug molecules, known as ligands, are tailored to a
protein's binding site. Molecular docking and other quantitative structure-activity relationships (QSAR) approaches
can be used to characterise the parameters that influence the protein-ligand interaction.
 Molecular docking

❑ Molecular docking is a type of bioinformatic modelling in which two or more molecules combine to generate a
stable compound. It predicts the three-dimensional structure of any complex based on the binding characteristics
of the ligand and target.
 Quantitative structure-activity relationships
(QSAR)
❑ The quantitative structure activity relationship (QSAR) is a critical strategy for chemistry and pharmacology that is
founded on the premise that when we change the structure of a molecule, the activity or attribute of the
substance changes as well.
 Drug design approach
❑ Drug design denotes a methodical and focused approach. What exactly is a drug? A small molecule that interacts
with a target. Cellular proteins mediate numerous organism activities, including physiological systems and illness.

❑ Computer-aided drug design is one rational drug design approach that enables drug discovery based on
knowledge of target structures, functional qualities, and processes (CADD).
 Drug design approach
❑ CADD may be used to compare predicted and actual drug action in tests, with the results being utilised iteratively
to enhance compound attributes.
❑ The two primary CADD-based methodologies are structure-based drug design, which requires protein structures,
and ligand-based drug design, which uses ligands and ligand activities to create compounds that interact with
protein structures.
❑ Docking, de novo design, fragment-based drug discovery, and structure-based pharmacophore modelling are all
approaches in structure-based drug design.
❑ Quantitative structure-affinity connection and pharmacophore modelling based on ligand characteristics are two
approaches in ligand-based drug discovery.
 Drug design approach

❑ Different design methodologies may be adopted depending on whether the structure of the receptor and its
interaction with the ligand are known. Following the generation of lead compounds, the rule of five can be utilised
to determine whether they contain drug-like characteristics.
❑ To assess the metrics of alternative drug design methodologies, several quality validation approaches, including as
cost function analysis, Fisher's cross-validation analysis, and the goodness of hit test, may be utilised.
❑ To boost CADD performance even further, super-computers and graphics processing units may be used to save
money.
 Pharmacophore
❑ A pharmacophore is an abstract description of molecular characteristics required for ligand recognition by a
biological macromolecule. A pharmacophore is defined by the International Union of Pure and Applied Chemistry
(IUPAC) as "an ensemble of steric and electronic properties required to achieve optimum supramolecular
interactions with a given biological target and to trigger (or prevent) its biological response."
❑ A pharmacophore model describes how ligands with different structures can bind to the same receptor location.
Furthermore, pharmacophore models may be utilised to find new ligands that will bind to the same receptor via de
novo design or virtual screening.
 Ligand-based drug design
❑ Ligand-based drug design (also known as indirect drug design) is based on understanding of other compounds that
bind to the biological target of interest. These additional molecules can be utilised to create a pharmacophore
model, which outlines the minimal structural properties that a molecule must have in order to bind to the target.
❑ A quantitative structure-activity relationship (QSAR) may also be obtained, which is a connection between
estimated characteristics of molecules and their empirically determined biological activity. In turn, these QSAR
correlations may be utilised to forecast the activity of novel analogues.
 Structure-based drug design
❑ Structure-based drug design (or direct drug design) is based on knowledge of the biological target's three-
dimensional structure acquired through technologies such as x-ray crystallography or NMR spectroscopy.
❑ If an experimental structure of a target is not available, a homology model of the target based on the experimental
structure of a similar protein may be conceivable.
❑ Using interactive visuals and the intuition of a medicinal chemist, prospective medications that are projected to
bind with high affinity and selectivity to the biological target may be created.
❑ Alternatively, numerous automated computational approaches might be utilised to identify novel medication
candidates.
 Structure-based drug design
❑ Current methodologies for structure-based drug design may be broadly classified into three groups.
❑ The first strategy is to locate novel ligands for a specific receptor by exploring vast databases of 3D structures of
tiny molecules for those that fit the receptor's binding pocket using quick approximation docking tools. This is
referred to as virtual screening.
❑ A second category is the creation of novel ligands from scratch. By building tiny parts in a sequential fashion, ligand
molecules are formed up within the confines of the binding pocket. These fragments might be solitary atoms or
molecular fragments. The main advantage of such a strategy is that innovative structures that are not found in any
database may be proposed.
❑ The optimization of existing ligands by assessing putative analogues within the binding cavity is a third strategy.
 Cytochrome P450 family of enzymes

❑ Cytochrome P450 is an enzyme family present in both prokaryotic and eukaryotic species. They are located in both
the ER and the mitochondria of mammalian cells and participate in a variety of metabolic processes, including
cholesterol and steroid hormone production.
❑ Several hundred CYP450 (CYP) isoforms are involved in drug metabolism; they constitute the most significant
enzyme group in phase I metabolism. These isoforms are primarily present in the ER.
❑ The CYP enzymes catalyse processes that begin with the abstraction of hydrogen from NADPH by cytochrome P450
reductase, an auxiliary enzyme.
❑ CYP uses hydrogen to convert one of the two atoms of molecular oxygen to water. The other oxygen atom is kept in
a highly reactive state and is then employed to drive one of two types of reactions on a substrate.
 Pregnane X receptor (PXR; NR1I2)

❑ The nuclear hormone receptor – vitamin D like receptor family – pregnancy X receptor (PXR; NR1I2) is a key
component of the body's adaptive defence mechanism against harmful compounds such as foreign toxins
(xenobiotics).
❑ A wide range of endogenous and exogenous substances, including steroids, antibiotics, antimycotics, bile acids,
and the herbal antidepressant St. John's wort, activate PXR.
❑ The PXR ligand binding domain's three-dimensional structure revealed that it contains a wide, spherical ligand
binding cavity that allows it to interact with a diverse spectrum of hydrophobic substances.
❑ Thus, unlike other nuclear receptors that engage with their physiological ligands selectively, PXR operates as a
generalist sensor of hydrophobic poisons. PXR interacts to DNA response elements in the regulatory areas of
cytochrome P450 3A monooxygenase genes and a number of other genes involved in the metabolism and removal
of xenobiotics as a heterodimer with the 9-cis retinoic acid receptor (NR2B).
 Pregnane X receptor (PXR; NR1I2)

❑ Although PXR developed to defend the body, its activation by a range of prescription medications is the molecular
foundation for a significant class of potentially hazardous drug-drug interactions. Assays that detect PXR activity
will thus be important in the development of safer prescription medications.
 CYP - Pregnane X receptor Importance

❑ Given that human cells contain several dozen distinct CYP enzymes, it is impressive that a single isoform, CYP3A4, is
involved in the metabolism of about 50% of all clinically prescribed medicines.
❑ CYP3A4, like several other isoforms, is inducible, which means that some medicines enhance the rate of gene
transcription.The nuclear hormone receptor that causes induction in CYP3A4 is the pregnane X receptor (PXR).
❑ When an appropriate medication interacts to this receptor, it translocates from the cytosol to the nucleus and
binds to its cognate regulatory DNA sequences, known as xenobiotic response elements (XRE), increasing
transcription of genes in the region.
❑ The PXR reacts to a wide range of medications, which helps to explain why CYP3A4 plays such an important role in
drug metabolism.
Cry toxins
BT maize in SA
 Activity
1.Define Cry toxin –Article 1 p. 33
2.Explain how the toxin works –
•Article 2 p. 24
•Two proposed mechanism of action
‒article 1 p. 41, article 2 p. 23
‒article 1 p. 41, article 2 p. 23
3.Explain how this toxin is used in Biotechnology
•article 1 abstract and p. 31
4.Define Cadherin
•article 1 p. 38
 Cry toxins

•Cry toxin (monomer) capable of oligomerization

•BT crystals made out of protoxinsubunits

•Encoded by cry genes found on plasmids

•Can be integrated into genome

•Cyttoxins also present

Cry toxin structure
 Cry toxin structure
• Domain I –7 α-helices
• Domain II –3 antiparallel β-sheets
• Domain III –2 antiparallel β-sheets that
adopts β-sandwich fold with jelly roll
topology
 Functions of domains?
•Domain I –Forms ion channels in the cell membrane – Most
conserved

•Domain II –Most divergent –toxin-receptor interaction –major

determinant of toxicity

•Domain III –Receptor binding, channel formation and linked to

toxicity
End of domain II and start of domain III
Two proposed mode of actions

1. Induces cell death by forming ionic pores

following insertion into the membrane –osmotic
lysis of midgut epithelial cells

2. Activation of Mg2+ dependent signal cascade

triggered by the interactions of Cry toxin with
primary receptor Cadherin
What is in common?

•Bt crystals must be solubilised in vivo or in vitro

•Protoxins must be activated by proteases before

and/or after binding to receptors such as cadherin

 How is crystals solubilised?

• Alkaline midgut of insects enhances solubility of Cry

toxins
• In vitro solubilisation needed for insects with
neutral/acidic midguts…
 Mechanism 1

Fig. 2.3 Model of the mode of action of Cry1A toxins. 1 Crystal toxin solubilisation, 2 Initial cleavage by gut
proteases, 3 Toxin monomer binding to receptors and second cleavage by membrane
bound protease, 4 Membrane insertion-competent oligomer formation, 5 Binding of oligomerictoxin to
receptors, 6 Lytic pore formation. (Bravo et al. 2004)
Mechanism 2

Lets first look at Cadherin…..

Calcium dependent transmembrane glycoproteins
•Cell-cell adhesion
•Cell migration
•Regulation of tissue organization
•Morphogenesis
 Structure of Cadherin
4 different domains
1.Ectodomain(EC)
•Repeating calcium-binding cadherin repeats (110 aa
in length) -5 to 34 repeats
2.Membrane proximal extracellular domain (MPED)
3.Transmembrane domain (TM)
4.Cytoplasmic domain (CYTO)
Figure 5. Domain structure and putative Cry
toxin-binding regions in cadherin-like
proteins from mosquito (red), moth (green)
and beetle (golden). EC, ectodomain;
MPED,membrane proximal extracellular
domain; TM, transmembrane domain;
CYTO, cytoplasmic domain, TBR, toxin
binding region.
Where does Cry toxin bind onto Cadherin?

• EC modules adjacent to the MPED (Toxin binding

regions –TBR)

• Two adjacent β-strands and loop within the β-barrel fold

of either EC11/EC12
Similarity to human Cadherin?

Bioinformatics exercise

Is Bt protein safe for human consumption?

Cry docking onto Cadherin
Back to Mechanism 2..
Cell death…
What Happens Next?

•Next week we will learn about receptors and signal

transduction.
•Ensure that you engage with your week 7 myLMS content
before your week 7 lecturer led session.