Synthetic Drugs in TB
Synthetic Drugs in TB
Synthetic Drugs in TB
PII: S0010-4825(20)30178-5
DOI: https://doi.org/10.1016/j.compbiomed.2020.103811
Reference: CBM 103811
Please cite this article as: S.K. Kwofie, C. Adobor, E. Quansah, J. Bentil, M. Ampadu, W.A. Miller III.,
M.D. Wilson, Molecular docking and dynamics simulations studies of OmpATb identifies four potential
novel natural product-derived anti-Mycobacterium tuberculosis compounds, Computers in Biology and
Medicine (2020), doi: https://doi.org/10.1016/j.compbiomed.2020.103811.
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5 Samuel K. Kwofiea, b, c, d *
, Courage Adobora, e, Erasmus Quansaha, Joana Bentila, Michael
10 Cell and Molecular Biology, College of Basic and Applied Sciences, University of Ghana,
11 Accra, Ghana
c
12 Department of Medicine, Loyola University Medical Center, Maywood, IL, 60153, USA
d
13 Department of Physics and Engineering Science, Coastal Carolina University, Conway, SC
14 29528
e
15 Department of Parasitology, Noguchi Memorial Institute for Medical Research (NMIMR),
16 College of Health Sciences (CHS), University of Ghana, P.O. Box LG 581, Legon, Accra, Ghana
f
17 Department of Chemical and Biomolecular Engineering, School of Engineering and Applied
19
22
23
24
1
25 Abstract
27 that neutralizes the host pH to impede the uptake of hydrophilic antitubercular drugs. Identifying
28 natural compounds with the potential to inhibit OmpATb could allow circumvention of the
31 screening of 6394 diverse natural products. Characterization of the binding interactions of the
32 potential leads with OmpATb revealed nine critical residues comprising ARG86, LEU110,
33 LEU113, LEU114, ALA115, PHE142, SER145, VAL146, and PHE151. Molecular dynamics
34 simulations also revealed very stable protein-lead complexes. Most residues contributed lower
35 binding energies to the overall molecular mechanics Poisson–Boltzmann surface area (MM-
36 PBSA) binding free energies of the interactions between the molecules and OmpATb protein.
37 Induced Fit Docking (IFD) of the compounds regenerated poses of the molecular docking using
38 AutoDock Vina. These molecules could be starting templates for designing inhibitors to bypass
40 was suggested as a potential scaffold for designing efflux pump inhibitors of the gate mediating
41 activities of OmpATb and may enhance the uptake of hydrophilic drugs to reduce the duration
43 DrugBank database with a similarity threshold of 0.7 have been reported to exhibit antitubercular
45 experimentally to corroborate their antitubercular activity. Also, the skeletons of the molecules
47
2
48 Keywords: Mycobacterium tuberculosis, OmpATb; natural products; virtual screening;
50 docking
51 1 Introduction
52 Tuberculosis (TB) is one of the oldest known infectious disease dating back to 1000 BC and is
53 still one of the leading causes of deaths among infectious diseases [1,2]. An estimated one-third
54 of the world’s population are latently infected, of which nine million develop the infection
55 further to the active stage and about two million deaths arise each year [3,4]. Of those infected,
56 over one and half million cases occur annually in sub-Saharan Africa [5]. TB treatment protocols
57 are based on a combination of three or more of the first-line antitubercular drugs comprising
60 aerosol droplets from actively infected persons [3,8]. A person becomes infected through
61 inhalation of these aerosol droplets containing the bacteria, the inhaled bacteria enters the lungs
62 and is engulfed by the macrophages in the alveoli. These macrophages digest the bacteria by the
63 secretion of acid which results from the fusion of phagosome and lysosome inside the
64 macrophage and thereby making their inner environment acidic [1,2]. The host immune system
65 can attenuate the growth of M. tuberculosis at this stage, forming a granuloma around the
66 bacteria and causing it to enter into a state of dormancy. The host becomes latently infected for a
67 period or even a lifetime with the dormant bacteria awaiting reactivation. At this stage, the
68 bacteria cannot be transmitted [9,10]. For persons with efficient cell-mediated immunity, the
69 infection may be arrested permanently at this point [11]. However, if the host immunity cannot
70 control this initial stage of M. tuberculosis or if a latently infected person’s immune system is
3
71 compromised by some factors such as aging, HIV infection, or even by some drugs, the
72 granuloma formed around the bacteria becomes liquefied inside, giving the bacteria a suitable
73 condition to grow and multiply [9,10]. When the growth and multiplication of the bacteria
74 become uncontrollable, they burst out of the granuloma and infect other macrophages. At this
75 stage, the person becomes actively infected, showing signs and symptoms of tuberculosis and
77 In the macrophage, there is a lysosome and phagosome fusion which changes the pH of the inner
78 environment of the macrophage from about 6.2 to between 4.5 – 5.0 [12]. M. tuberculosis has
79 over the years developed some mechanisms to resist the pH change of the host macrophages.
80 These mechanisms which are not fully understood helps the bacteria to function and grow within
81 the host. Studies have shown that the outer membrane protein A (OmpATb), which is a pore-
82 forming protein is actively involved in these mechanisms [12-14]. OmpATb has two functions:
83 as a pore-forming protein with properties of a porin, and to help secrete ammonium into the
84 phagosomal environment to neutralize the acid [13,14]. Other studies further suggest that at low
85 pH, the OmpATb might be the only functioning porin and these pores close at this low pH
86 [13,15]. The closure of these hydrophilic pores causes the slow uptake of hydrophilic molecules
87 including current first-line drugs since the outer cell envelop is hydrophobic [13,15]. With this
88 slow drug intake of the bacterial, the time duration for the curing of TB infection is prolonged
89 than expected.
90 OmpATb has two terminal domains: the N-terminal and the C-terminal domains [3,15]. The
91 pore-forming activity and the resistance of the M. tuberculosis to acidic medium in macrophages
92 are mainly mediated by the N-terminal domain of OmpATb [3,15]. Due to the key role played by
94 plausible drug target [12,16,17]. Under normal conditions, the loss of OmpATb did not affect the
4
95 growth of M. tuberculosis, but its ability to grow was decreased at reduced pH [13]. The porin-
96 like activity of OmpATb plays a major role in mitigating the low pH environment of the host,
97 making it a worthy drug target. Therefore, any molecules which have the potential to disrupt the
98 normal activity of the porin at low pH could allow the host defense mechanisms to overcome the
100 inhibiting OmpATb, which could attenuate the growth of the bacteria due to host immune
101 mechanisms. Also, once the porin-like activity of OmpATb is impeded, the uptake of other
102 hydrophilic molecules under low pH could increase. The role of outer membrane protein A
103 (OmpA) as a therapeutic target is well corroborated in other bacterial organisms like
104 Acinetobacter baumannii and Pasteurella multocida [18-22]. Therefore, we hypothesized that
105 the binding of compounds (especially from natural extracts) to OmpATb would enable the
107 Natural products and their derivatives have historically been invaluable as sources of
108 therapeutic agents [23,24]. Also, natural product structures have high chemical diversity,
109 biochemical specificity, and other molecular properties that make them more favorable as lead
110 compounds for drug discovery, and which serve to differentiate them from libraries of synthetic
111 and combinatorial compounds [23]. To combat the dormant phenotype acquired by M.
112 tuberculosis during infection and the reduced bacilli tolerance to front-line drugs, bioassay
113 directed methods have been adopted to screen natural products from higher plant extracts against
114 tuberculosis[25]. Marine natural products have also been screened against M. tuberculosis using
115 luciferase reporter assay and enumerated colony-forming unit (CFU) bioassays[26].
116 Computationally, natural products from the Philippines and those in Ambinter database have
118 [27]. Moreover, the structural-based virtual approach has also been used to screen commercial
5
119 libraries from Asinex database against the l-alanine dehydrogenase protein of M. tuberculosis to
121 This study aimed to identify novel small molecules of natural origin with the potential to inhibit
122 the pore-forming activities of OmpATb by using computational structure-based drug design.
123 This involved computational protocols including virtual screening, molecular docking
124 simulations, pharmacological profiling of hits, and molecular dynamics simulation of potential
128 The three-dimensional (3D) molecular structure file of the N-terminal domain of OmpATb was
129 retrieved from Protein Data Bank (PDB; http://www.rcsb.org/pdb/)[29] with PDB ID 2KGS [30].
131 Ligand molecular structural files were retrieved from eleven catalogues in ZINC15 database [31]
135 Princeton BioMolecular Research Natural Products[40], Specs Natural Products[41] and UEFS
136 Natural Products[42]. The screening libraries were categorized into two A and B. Library A
137 contained 831 African natural products from the AfroDB database, whilst library B contained
138 5,493 small molecules from ten ZINC15 catalogues (Table 1). The compounds in library B were
139 obtained by pre-filtering using Lipinski’s rule of five (log P ≤ 5, hydrogen bond donors ≤ 5,
140 hydrogen bond acceptors ≤ 10 and molecular weight ≤ 500 Daltons) [43]. Compounds that
6
141 violated any of these rules were eliminated from downstream analysis. A total of 6,324 small
144 The putative ligand binding sites of OmpATb were computed using KVFinder [44] and
145 confirmed with MetaPocket 2.0 [45]. KVFinder applies a geometry-based method for the
146 identification of protein cavities. MetaPocket combines LIGSITEcs, PASS, Q-SiteFinder and
149 The 3D crystal structure of the N-terminal domain of the OmpATb protein was prepared using
150 the AutoDockTools version 1.5.6 [46] utility script “prepare_receptor4.py”, which assigned
151 partial Gasteiger charges and added polar hydrogen atoms to the protein structure. Also,
152 AutoDockTools were used to generate a grid box of size 26Å x 30Å x 30Å around the active site
153 to provide a search space for the small molecules during the virtual screening process.
154 The 3D structures of the 6,324 small molecules were converted to “.pdbqt” file format using
155 AutoDockTools utility script “prepare_ligand4.py”. This utility script allows the addition of
156 polar hydrogens, assignment of Gasteiger charges, and set free rotatable and torsional bonds for
159 A custom python script was used to automate the screening of the libraries by docking them into
160 the active site of the receptor using AutoDock Vina 1.1.2 [47]. The virtually screened small
161 molecules were ranked based on their binding affinity to the protein in each of the two libraries.
7
162 The results of the binding poses of all ligands were visualized using PyMOL 2.0.6 [48]. Taking
163 into consideration the binding poses and the binding affinity ranking, hits were selected from
165 2.6 Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) prediction
166 Derek Nexus version 2.1[49] was used to predict the toxicity of the virtual screening hits for
167 three animal species: mouse, monkey and human [50]. A total toxicity weight (TTW) was
168 computed from the Derek Nexus toxicity results, which was used to assess the toxicological
169 profiles of the compounds. Also, the mutagenicity of the hits was predicted using Sarah Nexus
170 version 2.1 [51]. After combining the results from Derek Nexus and Sarah Nexus, the best
171 scoring compounds in terms of their toxicological profiles were shortlisted for the Absorption,
172 Distribution, Metabolism and Excretion (ADME) testing. The ADME profiles of the selected
173 small molecules were evaluated using ADMET Predictor 8.1[52] and SwissADME [53]. Also,
174 the Lipinski’s rule of five was used for physicochemical profiling of library A via SwissADME.
175 The ligands with reasonably good ADMET profiles were selected for downstream analysis.
177 The molecular interactions between the hits and OmpATb protein were elucidated for each
180 Molecular dynamics (MD) simulations were performed using GROMACS version 2018 [55]
181 separately on the unbound OmpATb receptor and the OmpATb receptor complexed with the
182 potential leads. PRODRG[56] (settings: Chirality=Yes, Charges=Full and EM=No) was used to
8
183 generate the GROMACS topology files for each compound. The simulations were run using the
184 Gromos 43a1 force field in a 1.0 nm cube water solvent box and five sodium ions were added to
185 the systems to neutralize them. The systems were equilibrated at a temperature of 300 K and a
186 pressure of 1 bar. The production MD was run for 100 ns for each simulation. The root mean
187 square deviation (RMSD) and root mean square fluctuation (RMSF) graphs were generated and
188 plotted using XMGRACE[57] version 5.1.25 after the production run for each simulation.
189 2.9 Free binding energy calculation using the MM-PBSA method
190 The binding free energies of the complexes over the 100 ns MD simulations with a time step of 1
191 ns were calculated using the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA)
192 method implemented in g_mmpbsa [58,59] (Supplementary Table S5). Also, the binding free
193 energy contributions of different OmpATb residues involved in the binding of each compound
194 was estimated. The results were plotted using the R programming package [60].
196 Schrödinger Suite 2018-2 was used to generate the Induced Fit-Docking (IFD) [61]. The
197 potential lead compounds were re-docked with the OmpATb structure using the IFD technique.
198 The poses of the ligands from the IFD were superimposed and fitted onto those of AutoDock
201 Structural similarity searches were done at a threshold of 0.7 via DrugBank 5.0 database [63] to
202 evaluate the potential anti-mycobacterial activity and possible mechanisms of action from similar
9
203 compounds. Also, Prediction of Activity Spectra for Substances (PASS) [64] was used to further
207 The 3D structure of the N-terminal domain contained three α-helices and six ß-sheets connected
208 by loops (Figure 1a) [30]. The characterization of the active site using KVFinder [44] and
209 MetaPocket 2.0 [45] revealed various binding cavities (Supplementary Table S6). One of the
210 cavities was considered as a plausible binding pocket because of the large volume of 53.57 Å3
211 and surface area of 83.52 Å2 (Figure 1a), which makes it possible for small compounds to dock
212 firmly into it [65,66]. The active site residues comprised LE84, ARG86, ASN89, THR90,
213 VAL91, LEU93, ALA109, LEU110, ASN111, LEU113, LEU114, ALA115, VAL118, ASN119,
214 VAL120, VAL141, PHE142, THR143, SER145, VAL146, PRO147, ILE148 and PHE151
216
217
10
218 Figure 1. The (a) Cartoon and (b) Surface representations of the N-terminal domain of OmpATb (PDB
219 ID: 2KGS) retrieved from the Protein Data Bank. The active site residues are in multi-colors and the
220 volume of sphere colored in magenta denotes the buried active site.
221 3.2 Virtual screening and filtering of libraries using physicochemical and ADMET profiles
222 The virtual screening results were compiled and ranked based on binding energy scores.
223 Compounds with the lowest binding energies were ranked as having the highest binding affinity
224 based on the scoring function of AutoDock Vina. The highest-ranked compounds docked firmly
225 in the binding pocket of the OmpATb protein upon visualization using PyMOL (Figure 2). Out
226 of a total of 831 small molecules in library A, 14 docked firmly in the active site pocket of the
227 protein and also did not violate any of the Lipinski’s Rule of Five. For the 5493 small molecules
228 in library B, 870 compounds docked firmly in the active site pocket. The binding affinity and the
229 results of the physicochemical profiling of predicted leads using Lipinski’s Rule of Five are
231 For a good ADME profile, a compound should be absorbed easily through the gastrointestinal
232 tract so that it can be available in the systemic circulation. It should be metabolized by more than
233 one metabolism enzymes so that it can be excreted easily from the body without causing
234 undesirable adverse effects. Also, it should not inhibit an enzyme’s functions in the body so as
235 not to impede the normal biological processes [67]. Thus, compounds with logP less than 5 were
236 considered more water-soluble and have high gastrointestinal (GI) absorption. Also, compounds
237 that inhibited not more than one Cytochrome P450 (CYP) metabolism enzymes and were
238 substrate to more than one CYP metabolism enzymes were selected as compounds that were in
239 the range of drug-likeness. Applying these selection criteria, one compound from library A and
240 three compounds from library B passed these pharmacodynamics selection criteria (Table 2).
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241 A merged hit list composed of the 14 compounds from library A and the first 35 from library B
242 were profiled using ADMET. Therefore, a total of 49 hits were obtained from the virtual
244
245 Figure 2. PyMOL visualization of the top hit compounds superimposed on each other (multi-colored in
246 the middle) from the virtual screening protocol showing the hits as being docked firmly within the active
249 The toxicity profiles of the virtual screening hits were evaluated using Derek Nexus and Sarah
250 Nexus [49,51]. Derek Nexus is a structure-activity relationship (SAR) expert knowledge-based
251 system that searches its database to determine whether a compound possesses toxicophores
252 [50,67,68]. Derek Nexus predicts toxicity endpoints into categories (Table 3) for multiple animal
12
253 species [50]. Sarah Nexus predicts the mutagenicity of chemical compounds using the self-
254 organizing hypothesis network (SOHN) based on the presence of structural fragments of these
255 compounds, which have been associated with activity or inactivity in its training dataset [70].
256 The predictions of Sarah Nexus are categorized as either positive (mutagenic), negative (non-
259 The toxicity endpoints of the forty-nine virtual screening hits were predicted into seven
260 categories by Derek Nexus for three animal species (human, monkey and mouse) and were
261 assigned category weights (Table 3). The category weights of the seven categories were
262 interpolated linearly between 0 and 1 (inclusive) [Table 3]. Also, the different animal species
263 were assigned weighted values summing up to ten as follows: human = 5, monkey = 3 and
264 mouse = 2, based on the closeness of the species to human in terms of their coding DNA
265 similarity (~97% with mouse [70,71] and ~98% with monkey [72,73]). The highest weight was
266 assigned to human species, followed by monkey and mouse. For easy interpretation, the assigned
267 category weights and the weighted values of the different animal species (species weight) were
268 used to transform the qualitative toxicity results (Table 4) from Derek Nexus to quantitative
269 results (Table 5). The following conversion formula was used to calculate the total toxicity
= 1× 1 + 2× 2 + 3× 3 1
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274
275 For example, the compound ZINC000003958185 had a toxicity endpoint prediction of HERG
276 channel inhibition in vitro with category Doubted (= 0.3334) for all the three animal species. By
277 substituting the values into the formula (1), TTW was calculated as follows:
278 Therefore, ZINC000003958185 had a TTW of 3.334 for the toxicity endpoint prediction (Table
279 5). These TTW values which are out of ten were used to assess how toxic the compounds were,
280 with compounds having TTW weights closest to 10 considered to be more toxic and compounds
281 closest to zero considered to be less toxic. Thus, compounds with TTW above 6.0 were
283
285 The total toxicity weight (TTW) evaluation and the mutagenicity of the virtual screening hits
286 were considered in the selection of compounds for downstream analysis (Table 6). Compounds
287 with prediction confidence above 50% for positive mutagenicity by Sarah Nexus were
288 eliminated. The filtering of compounds using toxicity further reduced the hits in library A to four
289 compounds and those in library B to twelve compounds, making a total of 16 small molecules
290 that complied with the toxicity test (Supplementary Tables S2 and S3).
291
293 The binding interactions of the four potential lead compounds were elucidated to evaluate the
294 important intermolecular bonds involved in each of the complexes. ZINC000003958185 formed
14
295 hydrophobic bonds with six OmpATb residues comprising ARG86, LEU110, ASN111, LEU113,
296 PHE142 and VAL146, and one hydrogen bond with SER145 of bond length 2.45Å (Figures 3 and
297 4a). ZINC000000157405 formed hydrophobic bonds with eight residues ILE84, LEU114,
298 ALA115, PHE142, SER145, VAL146, ILE148 and PHE151 (Supplementary Figure S1a).
299 ZINC000000001392 formed hydrophobic bonds with six residues ARG86, LEU110, LEU113,
300 VAL118, PHE142 and PHE151 (Supplementary Figure S1b). Additionally, ZINC000034268676
301 formed hydrophobic bonds with six residues ASN89, LEU113, LEU114, THR143, VAL146 and
302 PRO147, and two hydrogen bonds with ARG86 and ALA115 with bond lengths of 3.07Å and
303 3.27Å, respectively (Figure 4b). Of all the binding interactions, nine residues ARG86, LEU110,
304 LEU113, LEU114, ALA115, PHE142, SER145, VAL146 and PHE151 interacted with at least
306
307
308
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309 Figure 3. PyMOL visualization of the predicted top lead compound, ZINC000003958185 (colored in
310 brown sticks) docked into the predicted active site (surrounded by sticks) of OmpATb protein (cartoon
311 representation).
16
312
313 Figure 4. Binding interactions between OmpATb protein residues and two of the potential lead
314 compounds (joined with purple bonds): (a) ZINC000003958185 and (b) ZINC000034268676. The red
17
315 dotted lines indicate hydrophobic contacts and the green dotted lines indicate hydrogen bonds with bond
317
319 The conformational stability of the native OmpATb protein and its complexes with the predicted
320 leads were evaluated over 100 ns simulation time. The root mean square deviation (RMSD)
321 values obtained for all the simulations rose sharply to within a range of 0.4 – 0.07 nm over the
322 first 5 ns and stayed within this range for the rest of the simulation time (Figure 5). This narrow
323 RMSD range indicated the conformational stability of the protein and its complexes over the
324 simulation time. Although, ZINC000000157405 complex experienced fewer fluctuations after 50
325 ns. Generally, there were not many fluctuations in the residues as shown in the root mean square
326 fluctuation (RMSF) plot (Figure 6), except for the two terminal regions and predicted active site
327 cavity. The loop at the C-terminus region of the protein experienced more fluctuations, whereas
328 the loop at the N-terminus region and the central residues (between 115 and 120) in the active
329 site cavity experienced fewer level of fluctuations (Figures 1a and 6). These show that the
330 residues in the C-terminal loop region were more flexible than other residues. Also, the minimal
331 fluctuations of residues in the active cavity reveal fewer residue flexibility indicative of ligand-
18
333
334 Figure 5. RMSD graphs for the backbone atoms of the protein after 100 ns MD simulations for the
335 OmpATb protein (black) and the OmpATb protein complexed with ZINC000003958185 (blue),
337
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338
339 Figure 6. RMSF graphs for the protein residues after 100 ns MD simulations for the OmpATb protein
340 (black) and OmpATb protein complexed with ZINC000003958185 (blue), ZINC000000157405 (green),
342 3.6 Free binding energy calculation using the MM-PBSA method
344 approach used in estimating the free binding energies of ligands to macromolecules. This method
345 has been applied to several protein-ligand systems with varying degrees of success [79,80]. The
346 binding free energy components for each complex of the OmpATb with the ligands were
347 calculated over 100 ns molecular dynamics simulation at a time step of 1 ns (Table 7). The
348 predicted lead from AfroDB, ZINC000034268676 had the lowest binding free energy (-126.238
349 kJ/mol), which was largely contributed by van der Waal forces. The other compounds,
20
351 -31.688 kJ/mol, -118.821 kJ/mol and -102.356 kJ/mol, respectively, which were also largely
352 contributed by van der Waal forces. Therefore, per the MM-PBSA energy calculations,
353 ZINC000034268676 established stronger binding interactions with the OmpATb protein.
354 The MM-PBSA binding free energies of the interactions between the predicted leads and
355 OmpATb protein were decomposed into the energy contribution of each protein residue, to
356 evaluate the critical binding residues, which are residues with higher energy contributions to the
357 overall binding energy. The critical residues were LEU82, ILE84, SER85, ARG86, VAL91,
358 LEU93, PHE97, LEU106, MET107, ALA109, LEU110, ASN111, LEU113, LEU114, ALA115,
359 PRO116, VAL118, ASP122, ILE124, LEU133, ASP134, ALA138, GLU139, PRO140,
360 VAL141, PHE142, THR143, ALA144, SER145, VAL146, ILE148, PRO149, ASP150, PHE151,
361 GLY152, LEU153, LEU162, VAL175, ALA178, ALA179, TRP183, PRO199 and PRO200
362 (Figure 7, Supplementary Table S4 and Supplementary Figure S2). The predicted active site
363 residues contributed the highest energies to the MM-PBSA binding free energies between the
364 OmpATb protein and the four predicted lead compounds. Also, the nine residues comprising
365 ARG86, LEU110, LEU113, LEU114, ALA115, PHE142, SER145, VAL146 and PHE151,
366 which formed binding interactions with at least two of the potential lead compounds from the
367 molecular dockings, were among the highest contributors to the MM-PBSA binding free
368 energies. All the residues contributed highly negative binding energies except residue ARG85,
369 which contributed positive binding energies in all cases. MM-PBSA results were used to validate
370 the molecular docking since it is an efficient metric for distinguishing between good binders and
372
373
21
374
375 Figure 7. A plot of MM-PBSA binding free energy decomposition of each OmpATb residue contribution
376 to the binding interaction between OmpATb and ZINC000034268676. The predicted active site residues
378 3.7 Comparison of the binding of the predicted leads to those of known TB drugs
379 The binding affinity between the predicted leads and OmpATb were compared to those of known
380 TB drugs and targets. The binding energies of known TB drugs docked against receptors
381 inhibited by these drugs range from -4.2 to -7.3 kcal/mol (Supplementary Figure S4 and Table
382 S7), compared to those of the potential leads that range from -5.3 to -7.3 (Table 2). Rifampicin
383 had the highest binding energy of -7.3 kcal/mol amongst the known drugs, which was the same
385 obtained using RMSD computations over 100 ns. There were fluctuations in RMSD from 0 ns
22
386 until after 30 ns when the complex appeared to be fairly stable (Supplementary Figure S5A). The
387 binding free energy obtained from the MM-PBSA calculations for the complex of rifampicin and
388 DNA-directed RNA polymerase subunit beta was -1345.984 +/- 63.247 kJ/mol (Supplementary
389 Table S8 and Figures S5B and C), which was lower than those of the predicted leads and
390 OmpATb (Table 7). This implies that rifampicin showed stronger binding interactions to its
391 receptor than the predicted leads and OmpATb. Nevertheless, the predicted leads demonstrated a
393
395 Molecular docking protocol is a plausible means to predict the binding poses and affinities of
396 docked ligands. However, this technique is constrained by efficient modeling of protein
397 flexibility, causing some inaccuracies in predicting the most plausible active site geometries and
398 poses of the docked ligands [82]. To account for these molecular docking constraints, Induced-
399 Fit Docking (IFD) techniques account for the movement of the side chains and consider both the
400 ligands and protein as flexible. IFD was used to re-dock the predicted lead compounds against
401 the active site of OmpATb. The IFD protocol reproduces the most plausible binding pose of a
402 ligand in the active site with very small deviations compared to experimental binding poses
403 [83,84]. The IFD output structures were superimposed onto those of AutoDock Vina to compare
404 the RMSDs of both the ligand poses and the protein atoms. The IFD generated protein structures
405 had minimal atomic deviations compared to the original protein structure (Table 8 and
406 Supplementary Figure S3). Minimal structural deviations were observed between the IFD and
407 the pre-IFD proteins, which suggest that the predicted leads docked firmly into the active pocket.
408 Also, the RMSDs obtained from the overlap of the ligands from both the IFD and AutoDock
23
409 Vina docking protocols showed minimal deviations, as two compounds had RMSD values below
410 2.0 Å (a generally accepted threshold) and the other two compounds had slightly higher RMSD
411 values than the 2.0 Å (Table 8, Figure 8) [85]. This showed that the poses from the molecular
412 docking protocol can easily be regenerated using IFD. However, the superimposition of the
413 ligands from the IFD and virtual screening (Figure 8) showed better fitting of these compounds
414 onto each other despite the slightly higher RMSDs of the other two compounds. These similar
415 binding poses generated by the two different docking protocols provide further confidence to the
416 predicted lead compounds as plausible lead-like molecules worthy of further exploration using
418
419
24
420
421 Figure 8. Superimposition and fitting between the ligand poses obtained with Induced Fit Docking (red)
422 and AutoDock Vina docking (green) for (a) ZINC000003958185, (b) ZINC000000157405, (c)
424
25
425 3.9 Exploring the anti-mycobacterial potential of the predicted leads
426 To support the computationally identified molecules in this study, chemical structure similarity
427 searches were carried out via DrugBank 5.0 database [63]. Analogs or similar compounds with
429 Spectra for Substances (PASS) [64] was used to evaluate the antitubercular and anti-
433 are plausible scaffolds for antitubercular drug development [86,87]. Therefore,
435 A search with ZINC000000157405 yielded five compounds belonging to the benzenoids class,
437 with the highest structural similarity score of 0.793 and was known to have activity against
438 Lanosterol 14-alpha demethylase protein of M. tuberculosis [88]. With the high structural
441 OmpATb and Lanosterol 14-alpha demethylase receptors, making it a more probable potent
443 A search with ZINC000000001392 yielded six compounds belonging to the benzenoids class.
444 None of these six similar compounds had been associated with tuberculosis in the DrugBank
446 combined with other antitubercular drugs had higher potency against Mycobacterium smegmatis,
26
448 potentially novel hydrobenzoate analog that may also show high potency when combined with
449 other antitubercular drugs to reduce the duration of tuberculosis treatment. Furthermore,
451 activity (Pa) and probability of inactivity (Pi) of 0.436 and 0.022, respectively, Also,
452 ZINC000000001392 was predicted as an anti-mycobacterial with Pa and Pi of 0.400 and 0.039,
453 respectively. Since Pa > Pi in both cases, ZINC000000001392 is a potential scaffold for the
455 A search with ZINC000034268676 retrieved two similar structures which are terpenoids
456 comprising Farnesol and Squalene with similarity scores of 0.8 and 0.75, respectively. Farnesol
458 potential efflux pump inhibitor in M. smegmatis [91], a surrogate model of M. tuberculosis.
460 pump inhibitors of the gating activities of the OmpATb receptor of M. tuberculosis. Also,
461 Farnesol and squalene have been shown to inhibit the growth of M. tuberculosis up to 99% at
462 100 µg/mL concentration in vitro [92]. Squalene has also been shown to possess M. tuberculosis
463 inhibition at 100 µg/mL concentration in vitro [93]. Generally, terpenoid derivatives have anti-
464 mycobacterial activity against M. tuberculosis [94]. With the high structural similarity scores
467 Interestingly, the predicted leads and structurally similar compounds have been associated with
468 anti-mycobacterial activity which makes them plausible compounds or scaffolds for
469 antitubercular drug design. Also, this study supports the need to exploit the pore-forming
27
471 3.10 Drug-likeness evaluations of the predicted leads
472 As previously stated, all compounds which violated Lipinski’s rule of five were eliminated from
473 the virtual screening. We further evaluated the drug-likeness of the predicted leads using other
474 metrics such as those of the Comprehensive medicinal chemistry database (CMC), Modern drug
475 data report (MDDR), World drug index (WDI), Ghose, Veber, and Egan violations
476 (Supplementary Table S9). These drug-likeness metrics have been comprehensively described
478 none of the CMC, Veber, Ghost, and Egan rules. ZINC000000157405 violated one of the CMC
479 and Ghose rules, whilst ZINC000034268676 violated two of WDI like rules. All the four
480 compounds violated two of the MDDR like rules. Since the compounds complied with most of
481 the drug-like indices, the predicted leads have the propensity to be drug-like. Nevertheless,
482 compliance of any of these metrics does not necessarily render a compound drug-like [96].
484 Historically, natural products and their derivatives have been invaluable sources of
485 therapeutic agents because of their high chemical diversity, biochemical specificity, and other
486 essential properties [23,97,98]. Our study has prioritized four novel natural compounds out of
487 over 6,000 different molecules with the potential to inhibit the pore-forming mechanism of
488 OmpATb. The methodology adopted involved carefully chosen pharmacoinformatics techniques
489 that allow the validation of the predicted leads using molecular dynamics protocols including
490 MM-PBSA and IFD. Furthermore, rigorous pharmacological profiling was used to evaluate the
491 physicochemical and pharmacokinetic properties. These molecules have been predicted as
492 anti-mycobacterial as well as antitubercular and are worthy of experimental validation
493 [83,84]. Even though other M. tuberculosis receptors have been exploited as drug targets, this
494 work primarily focused on the screening of natural compounds computationally against
495 OmpATb. OmpATb is a virulence factor that mediates the pH-dependent pore activities of the
496 bacteria. Inhibiting the OmpATb prevents the bacteria from neutralizing the acidic environment
497 of the host cells, thereby impeding the survivability of the bacteria. Blocking the OmpATb could
28
498 allow the passage of inhibitors since they can bypass the difficult impermeable outer membrane
499 [16]. Also, one of the predicted leads ZINC000034268676 shares significant structural similarity
500 with Farnesol which is a potential efflux pump inhibitor in M. smegmatis [91], a surrogate model
501 for M. tuberculosis. Therefore, the potential exists for ZINC000034268676 to be explored as an
502 efflux pump inhibitor of the gating activities of OmpATb receptor of M. tuberculosis.
503 Experimental assays include the screening of these compounds against cell lines of M.
504 tuberculosis to ascertain their inhibition constants and against human cell lines to evaluate
505 their toxicity [99]. The potential lead compounds can be purchased or synthesized for
506 experimental validation. The stringent thresholds applied in the in silico analysis and the
507 promising results obtained so far shows a higher likelihood of the templates being used as
508 antitubercular fragments. Also, these fragments could form scaffolds for the de novo design of
509 new inhibitors against OmpATb. Chemically similar structures, derivatives, or analogs can be
510 explored in experimental assays in cases where the actual compounds are not readily available
511 [100–103]. The reported work consolidates existing efforts towards discovery of
512 antitubercular agents and has the potential to greatly reduce the high cost and time consuming
513 low-throughput experimental assays.
514 4 Conclusion
515 OmpATb is a crucial target since it enables M. tuberculosis to survive in the harsh acidic
516 environment of the macrophages by impeding the uptake of hydrophilic compounds, including
517 some antitubercular molecules. This study predicted four novel natural products
519 which could be utilized as templates for the design of potential OmpATb inhibitors.
520 ZINC000034268676 was specifically suggested as a potential scaffold for designing efflux pump
521 inhibitors of OmpATb gating. These potential leads have appreciably high binding affinity to
522 OmpATb and exhibited similar active site binding pose when re-docked using the IFD protocol.
523 Also, the compounds had reasonably good pharmacological profiles with negligible toxicity.
524 Molecular dynamics simulations showed minimal deviations and fluctuations in the backbone
29
525 atoms between the unbound OmpATb receptor and its complexes. Most residues contributed to
526 the overall low binding free energies of the interactions of the predicted lead compounds with the
527 OmpATb protein via MM-PBSA. This suggests that the complexes were stable experimentally.
528 Furthermore, structurally similar compounds have been reported to have antitubercular and anti-
529 microbial activities. The scaffolds of the molecules merit further studies for antitubercular drug
530 development.
541 PDBQT: Protein Data Bank, Partial Charge (Q), & Atom Type (T)
30
545 PDB: Protein Data Bank
556
561 undertaken by S.K.K., M.D.W., C.A., E.Q., J.B., and M.A. with inputs from W.A.M.III on
562 molecular dynamics simulations. S.K.K., M.D.W., C.A., E.Q., J.B., and M.A. co-wrote the first
563 draft. All authors contributed to the revision of the drafts and agreed on the final version of the
31
565 8 Funding
566 The study was not funded.
567 9 Acknowledgments
568 The authors express their gratitude to the faculty members of the Department of Biomedical
569 Engineering, University of Ghana, for all advice on the project. The West African Centre for Cell
570 Biology of Infectious Pathogens (WACCBIP) at University of Ghana made available Zuputo, a
571 Dell EMC high performance computing cluster for the molecular dynamics simulations.
574 11 References
575 [1] S. Brighenti and M. Lerm, “How Mycobacterium tuberculosis Manipulates Innate and Adaptive
576 Immunity : New Views of an Old Topic Manipulates Innate and Adaptive Immunity – New Views
578 [2] E. A. Talbot and B. J. Raffa, “Mycobacterium tuberculosis,” in Molecular Medical Microbiology:
581 Milián-Suazo, “What Can Proteomics Tell Us about Tuberculosis?,” vol. 25, no. 8, pp. 1181–
583 [4] K. K. Addo, P. Caulley, I. Mensah, M. Minamikawa, C. Lienhardt, and F. A. Bonsu, “SPECIAL
586 [5] A. Zumla et al., “Tuberculosis treatment and management--an update on treatment regimens,
587 trials, new drugs, and adjunct therapies.,” Lancet. Respir. Med., vol. 3, no. 3, pp. 220–34, Mar.
32
588 2015.
589 [6] S. Caño-Muñiz, R. Anthony, S. Niemann, and J. W. C. Alffenaar, “New approaches and
590 therapeutic options for mycobacterium tuberculosis in a dormant state,” Clinical Microbiology
591 Reviews, vol. 31, no. 1. American Society for Microbiology, 01-Jan-2018.
592 [7] C. C. Leung, H. L. Rieder, C. Lange, and W. W. Yew, “Treatment of latent infection with
593 Mycobacterium tuberculosis: Update 2010,” European Respiratory Journal, vol. 37, no. 3.
595 [8] R. Cloete, E. Oppon, E. Murungi, W. Schubert, and A. Christoffels, “Resistance related metabolic
596 pathways for drug target identification in Mycobacterium tuberculosis,” BMC Bioinformatics, pp.
598 [9] G. Ravi Kr., B. Kumar, B. Deepa, K. Katoch, M. Kalyan, and R. Srivastava, “Differentially
601 [10] P. K. Drain et al., “Incipient and subclinical tuberculosis: A clinical review of early stages and
602 progression of infection,” Clinical Microbiology Reviews, vol. 31, no. 4. American Society for
604 [11] P. P. Salvatore and Y. Zhang, “Tuberculosis: Molecular Basis of Pathogenesis,” in Reference
606 [12] O. H. Vandal, C. F. Nathan, and S. Ehrt, “Acid Resistance in Mycobacterium tuberculosis ᰔ ,” vol.
608 [13] C. Raynaud et al., “The functions of OmpATb , a pore-forming protein of Mycobacterium
610 [14] H. Song et al., “Expression of the ompATb operon accelerates ammonia secretion and adaptation
611 of Mycobacterium tuberculosis to acidic environments,” vol. 80, no. 4, pp. 900–918, 2012.
33
612 [15] A. Alahari, N. Saint, S. Campagna, and V. Molle, “The N-Terminal Domain of OmpATb Is
613 Required for Membrane Translocation and Pore-Forming Activity in Mycobacteria ᰔ ,” vol. 189,
615 [16] M. Niederweis, O. Danilchanka, J. Huff, C. Hoffmann, and H. Engelhardt, “Mycobacterial outer
616 membranes: in search of proteins.,” Trends Microbiol., vol. 18, no. 3, pp. 109–16, Mar. 2010.
617 [17] V. Molle et al., “pH-dependent pore-forming activity of OmpATb from Mycobacterium
618 tuberculosis and characterization of the channel by peptidic dissection,” Mol. Microbiol., vol. 61,
620 [18] X. Vila-Farrés et al., “Combating virulence of Gram-negative bacilli by OmpA inhibition,” Sci.
622 [19] A. M. Viale and B. A. Evans, “Microevolution in the major outer membrane protein OmpA of
624 [20] A. Kubera, A. Thamchaipenet, and M. Shoham, “Biofilm inhibitors targeting the outer membrane
625 protein A of Pasteurella multocida in swine,” Biofouling, vol. 33, no. 1, pp. 14–23, Jan. 2017.
626 [21] D. Nie et al., “Outer membrane protein A (OmpA) as a potential therapeutic target for
627 Acinetobacter baumannii infection,” Journal of Biomedical Science, vol. 27, no. 1. BioMed
629 [22] R. Parra-Millán et al., “Synergistic activity of an OmpA inhibitor and colistin against colistin-
630 resistant Acinetobacter baumannii: mechanistic analysis and in vivo efficacy.,” J. Antimicrob.
632 [23] A. A. Siddiqui, F. Iram, S. Siddiqui, and K. Sahu, “Role of natural products in drug discovery
633 process,” Int. J. Drug Dev. Res., vol. 6, no. 2, pp. 172–204, 2014.
634 [24] S. Mushtaq, B. H. Abbasi, B. Uzair, and R. Abbasi, “Natural products as reservoirs of novel
635 therapeutic agents,” EXCLI Journal, vol. 17. Leibniz Research Centre for Working Environment
34
636 and Human Factors, pp. 420–451, 04-May-2018.
637 [25] C. E. Salomon and L. E. Schmidt, “Natural Products as Leads for Tuberculosis Drug
638 Development,” Curr. Top. Med. Chem., vol. 12, no. 7, pp. 735–765, Mar. 2012.
639 [26] C. Rodrigues Felix et al., “Selective killing of dormant Mycobacterium tuberculosis by marine
640 natural products,” Antimicrob. Agents Chemother., vol. 61, no. 8, pp. 1–14, 2017.
641 [27] A. R. B. Sampaco and J. B. Billones, “Virtual screening of natural products, molecular docking
644 [28] S. Saxena, P. B. Devi, V. Soni, P. Yogeeswari, and D. Sriram, “Identification of novel inhibitors
646 based virtual screening,” J. Mol. Graph. Model., vol. 47, pp. 37–43, 2014.
647 [29] S. K. Burley et al., “RCSB Protein Data Bank: biological macromolecular structures enabling
648 research and education in fundamental biology, biomedicine, biotechnology and energy.,” Nucleic
649 Acids Res., vol. 47, no. D1, pp. D464–D474, Jan. 2019.
650 [30] V. Molle et al., “Structure of the Mycobacterium tuberculosis OmpATb protein: A model of an
651 oligomeric channel in the mycobacterial cell wall ´,” Proteins Struct. Funct. Bioinforma., vol. 79,
653 [31] T. Sterling and J. J. Irwin, “ZINC 15--Ligand Discovery for Everyone.,” J. Chem. Inf. Model., vol.
655 [32] F. Ntie-Kang et al., “AfroDb: A Select Highly Potent and Diverse Natural Product Library from
656 African Medicinal Plants,” PLoS One, vol. 8, no. 10, p. e78085, 2013.
657 [33] Y.-W. Chin, M. J. Balunas, H. B. Chai, and A. D. Kinghorn, “Drug discovery from natural
35
659 [34] H. Kang et al., “HIM-herbal ingredients in-vivo metabolism database,” J. Cheminform., vol. 5, no.
660 5, 2013.
661 [35] H. Ye et al., “HIT: linking herbal active ingredients to targets.,” Nucleic Acids Res., vol. 39, no.
667 [38] M. Mangal, P. Sagar, H. Singh, G. P. S. Raghava, and S. M. Agarwal, “NPACT: Naturally
668 Occurring Plant-based Anti-cancer Compound-Activity-Target database.,” Nucleic Acids Res., vol.
670 [39] A. C. Pilon et al., “NuBBEDB: an updated database to uncover chemical and biological
671 information from Brazilian biodiversity,” Sci. Rep., vol. 7, no. 1, p. 7215, Dec. 2017.
677 [43] C. A. Lipinski, F. Lombardo, B. W. Dominy, and P. J. Feeney, “Experimental and computational
678 approaches to estimate solubility and permeability in drug discovery and development settings,”
679 Adv. Drug Deliv. Rev., vol. 46, no. 1–3, pp. 3–26, Mar. 2001.
680 [44] R. V. Honorato et al., “KVFinder : Steered identification of protein cavities as a PyMOL plugin
681 KVFinder : steered identification of protein cavities as a PyMOL plugin,” no. July, 2014.
36
682 [45] Z. Zhang, Y. Li, B. Lin, M. Schroeder, and B. Huang, “Identification of cavities on protein surface
683 using multiple computational approaches for drug binding site prediction.,” Bioinformatics, vol.
685 [46] G. M. Morris et al., “AutoDock4 and AutoDockTools4: Automated docking with selective
686 receptor flexibility.,” J. Comput. Chem., vol. 30, no. 16, pp. 2785–91, Dec. 2009.
687 [47] O. Trott and A. J. Olson, “AutoDock Vina: Improving the speed and accuracy of docking with a
688 new scoring function, efficient optimization, and multithreading,” J. Comput. Chem., vol. 31, no.
690 [48] PyMOL, “The PyMOL Molecular Graphics System, Version 2.0.6 Schrödinger, LLC.”
694 [50] P. M. Sancheti and S. P. Pawar, “IN SILICO TOXICITY PREDICTION OF TROGLITAZONE,
695 ROSIGLITAZONE AND PIOGLITAZONE USING DEREK NEXUS,” vol. 3, no. 2, pp. 129–
699 [52] J. Ghosh, M. S. Lawless, M. Waldman, V. Gombar, and R. Fraczkiewicz, “Modeling ADMET,” in
701 [53] A. Daina, O. Michielin, and V. Zoete, “SwissADME: a free web tool to evaluate
702 pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules,” Sci.
704 [54] R. A. Laskowski and M. B. Swindells, “LigPlot+: Multiple ligand-protein interaction diagrams for
705 drug discovery,” J. Chem. Inf. Model., vol. 51, no. 10, pp. 2778–2786, 2011.
37
706 [55] M. J. Abraham et al., “Gromacs: High performance molecular simulations through multi-level
707 parallelism from laptops to supercomputers,” SoftwareX, vol. 1–2, pp. 19–25, Sep. 2015.
708 [56] A. W. Schüttelkopf and D. M. F. van Aalten, “PRODRG: a tool for high-throughput
709 crystallography of protein–ligand complexes,” Acta Crystallogr. Sect. D Biol. Crystallogr., vol.
712 27-Feb-2020].
714 nanosystems: application to microtubules and the ribosome.,” Proc. Natl. Acad. Sci. U. S. A., vol.
716 [59] R. Kumari, R. Kumar, A. Lynn, and A. Lynn, “g_mmpbsa —A GROMACS Tool for High-
717 Throughput MM-PBSA Calculations,” J. Chem. Inf. Model., vol. 54, no. 7, pp. 1951–1962, Jul.
718 2014.
719 [60] R. C. Team, “R: A Language and Environment for Statistical Computing.” R Foundation for
721 [61] Schrödinger, “Schrödinger Suite 2018-2 Induced Fit Docking protocol; Glide, Schrödinger, LLC,
722 New York, NY, 2018; Prime, Schrödinger, LLC, New York, NY, 2018.” New York, NY, 2018.
723 [62] A. Heifets and R. H. Lilien, “LigAlign: Flexible ligand-based active site alignment and analysis,”
724 J. Mol. Graph. Model., vol. 29, no. 1, pp. 93–101, Aug. 2010.
725 [63] D. S. Wishart et al., “DrugBank 5.0: A major update to the DrugBank database for 2018,” Nucleic
726 Acids Res., vol. 46, no. D1, pp. D1074–D1082, 2018.
727 [64] D. A. Filimonov et al., “Prediction of the Biological Activity Spectra of Organic Compounds
728 Using the Pass Online Web Resource,” Chem. Heterocycl. Compd., vol. 50, no. 3, pp. 444–457,
38
730 [65] A. Stank, D. B. Kokh, J. C. Fuller, and R. C. Wade, “Protein Binding Pocket Dynamics,” 2016.
731 [66] M. Cammisa, A. Correra, G. Andreotti, and M. V. Cubellis, “Identification and analysis of
732 conserved pockets on protein surfaces,” BMC Bioinformatics, vol. 14, no. SUPPL7, p. S9, Apr.
733 2013.
734 [67] H. Wan, “What ADME tests should be conducted for preclinical studies?,” Admet Dmpk, vol. 1,
736 [68] N. Greene, P. N. Judson, J. J. Langowski, and C. A. Marchant, “Knowledge-Based Expert Systems
737 for Toxicity and Metabolism Prediction: DEREK, StAR and METEOR,” SAR QSAR Environ.
738 Res., vol. 10, no. 2–3, pp. 299–314, Jul. 1999.
739 [69] J. E. Ridings et al., “Computer prediction of possible toxic action from chemical structure: an
740 update on the DEREK system.,” Toxicology, vol. 106, no. 1–3, pp. 267–79, Jan. 1996.
741 [70] C. Barber et al., “Evaluation of a statistics-based Ames mutagenicity QSAR model and
742 interpretation of the results obtained,” Regul. Toxicol. Pharmacol., vol. 76, pp. 7–20, Apr. 2016.
744 and the Human Genome,” Science (80-. )., vol. 296, no. 5573, pp. 1661–1671, May 2002.
745 [72] S. Batzoglou, L. Pachter, J. P. Mesirov, B. Berger, and E. S. Lander, “Human and mouse gene
746 structure: comparative analysis and application to exon prediction.,” Genome Res., vol. 10, no. 7,
748 [73] C. Gunter and D. Ritu, “CHIMPANZEE,” Nature, vol. 437, no. 7055, p. 7055, 2005.
749 [74] K. Prüfer et al., “The bonobo genome compared with the chimpanzee and human genomes.,”
750 Nature, vol. 486, no. 7404, pp. 527–31, Jun. 2012.
752 the Connection between Dynamics Properties of Ribosomal Proteins and Ribosome Assembly,”
39
753 PLoS Comput. Biol., vol. 8, no. 5, p. e1002530, May 2012.
754 [76] H. Shukla, R. Shukla, A. Sonkar, T. Pandey, and T. Tripathi, “Distant Phe345 mutation
755 compromises the stability and activity of Mycobacterium tuberculosis isocitrate lyase by
756 modulating its structural flexibility /631/45/56 /631/92/606 /631/114/2411 /9 /82/83 /82 /82/16
757 /101 article,” Sci. Rep., vol. 7, no. 1, pp. 1–11, Dec. 2017.
758 [77] S. Shukla, K. Bafna, D. Sundar, and S. S. Thorat, “The bitter barricading of prostaglandin
760 inhibition by amarogentin, a secoiridoid glycoside from Swertia chirayita,” PLoS One, vol. 9, no.
762 [78] C. V. Kumar, R. G. Swetha, A. Anbarasu, and S. Ramaiah, “Computational Analysis Reveals the
763 Association of Threonine 118 Methionine Mutation in PMP22 Resulting in CMT-1A,” Adv.
765 [79] S. Genheden and U. Ryde, “The MM/PBSA and MM/GBSA methods to estimate ligand-binding
766 affinities,” Expert Opin. Drug Discov., vol. 10, no. 5, p. 449, 2015.
767 [80] S. Borkotoky, C. K. Meena, and A. Murali, “Interaction Analysis of T7 RNA Polymerase with
768 Heparin and Its Low Molecular Weight Derivatives - An In Silico Approach.,” Bioinform. Biol.
770 [81] B. Kuhn, P. Gerber, T. Schulz-Gasch, and M. Stahl, “Validation and use of the MM-PBSA
771 approach for drug discovery,” J. Med. Chem., vol. 48, no. 12, pp. 4040–4048, Jun. 2005.
772 [82] M. Xu and M. A. Lill, “Induced fit docking, and the use of QM/MM methods in docking,” 2013.
773 [83] W. Sherman, T. Day, M. P. Jacobson, R. A. Friesner, and R. Farid, “Novel Procedure for
774 Moldeing Ligand/Receptor Induced Fit Effects,” J Med Chem, vol. 49, no. 2, pp. 534–553, 2006.
775 [84] A. J. Clark et al., “Prediction of Protein-Ligand Binding Poses via a Combination of Induced Fit
776 Docking and Metadynamics Simulations,” J. Chem. Theory Comput., vol. 12, no. 6, pp. 2990–
40
777 2998, 2016.
778 [85] K. Liu and H. Kokubo, “Exploring the Stability of Ligand Binding Modes to Proteins by
779 Molecular Dynamics Simulations: A Cross-docking Study,” J. Chem. Inf. Model., vol. 57, no. 10,
781 [86] S. Singh, G. Kaur, V. Mangla, and M. K. Gupta, “Quinoline and quinolones: Promising scaffolds
782 for future antimycobacterial agents,” J. Enzyme Inhib. Med. Chem., vol. 30, no. 3, pp. 492–504,
783 2015.
784 [87] R. S. Keri and S. A. Patil, “Quinoline: A promising antitubercular target,” Biomed.
789 “Antimycobacterial activity of ferutinin alone and in combination with antitubercular drugs
790 against a rapidly growing surrogate of Mycobacterium tuberculosis,” vol. 6419, 2011.
791 [90] E. Tatar et al., “Design, Synthesis, and Molecular Docking Studies of a Conjugated Thiadiazole–
792 Thiourea Scaffold as Antituberculosis Agents,” Biol. Pharm. Bull., vol. 39, no. 4, pp. 502–515,
793 2016.
794 [91] J. Jin et al., “Farnesol, a Potential Efflux Pump Inhibitor in Mycobacterium smegmatis,”
795 Molecules, vol. 15, no. 11, pp. 7750–7762, Oct. 2010.
796 [92] A. Jiménez, M. Meckes, V. Alvarez, J. Torres, and R. Parra, “Secondary metabolites from
797 Chamaedora tepejilote (Palmae) are active against Mycobacterium tuberculosis,” Phyther. Res.,
800 “Antitubercular triterpenes and phytosterols from Pandanus tectorius Soland. var. laevis,” J. Nat.
41
801 Med., vol. 62, no. 2, pp. 232–235, Apr. 2008.
802 [94] D. B. dos Reis et al., “Synthesis and biological evaluation against Mycobacterium tuberculosis and
803 Leishmania amazonensis of a series of diaminated terpenoids,” Biomed. Pharmacother., vol. 84,
805 [95] N. Roy and R. Kadam, “Recent trends in drug-likeness prediction: A comprehensive review of In
806 silico methods,” Indian J. Pharm. Sci., vol. 69, no. 5, p. 609, 2007.
807 [96] “Prediction of Drug-Like Properties - Madame Curie Bioscience Database - NCBI Bookshelf.”
809 [97] P. Wangchuk, “Therapeutic Applications of Natural Products in Herbal Medicines, Biodiscovery
810 Programs, and Biomedicine,” Journal of Biologically Active Products from Nature, vol. 8, no. 1.
812 [98] D. A. Dias, S. Urban, and U. Roessner, “A Historical overview of natural products in drug
813 discovery,” Metabolites, vol. 2, no. 2. MDPI AG, pp. 303–336, 16-Apr-2012.
814 [99] G. Khare, P. Kumar, and A. K. Tyagi, “Whole-cell screening-based identification of inhibitors
816 Chemother., vol. 57, no. 12, pp. 6372–6377, Dec. 2013.
817 [100] J. P. Renaud, T. Neumann, and L. Van Hijfte, “Fragment-Based Drug Discovery,” in Small
818 Molecule Medicinal Chemistry: Strategies and Technologies, Hoboken, NJ: Wiley Blackwell,
821 discovery and design,” Journal of Chemical Information and Modeling, vol. 54, no. 3. American
823 [102] B. J. Davis and S. D. Roughley, “Fragment-Based Lead Discovery,” in Annual Reports in
824 Medicinal Chemistry, vol. 50, Academic Press Inc., 2017, pp. 371–439.
42
825 [103] Y.-C. Lo and J. Z. Torres, “Chemical Similarity Networks for Drug Discovery,” in Special Topics
827
43
Highlights
• New insights into the binding mechanisms of OmpATb
• Four scaffolds for antitubercular drug design
• A scaffold for designing efflux pump inhibitors against OmpATb
• Four potential novel anti-tubercular molecules for in vitro activity testing
Authors declare no conflict of interest