Molecular docking and dynamics simulations studies of OmpATb identifies four potential novel natural product-derived anti-Mycobacterium tuberculosis compounds

https://doi.org/10.1016/j.compbiomed.2020.103811Get rights and content

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.

Abstract

The outer membrane protein A (OmpATb) of Mycobacterium tuberculosis is a virulence factor that neutralizes the host pH to impede the uptake of hydrophilic antitubercular drugs. Identifying natural compounds with the potential to inhibit OmpATb could allow circumvention of the porin-like activities of OmpATb. Four potential leads comprising ZINC000003958185, ZINC000000157405, ZINC000000001392 and ZINC000034268676 were obtained by virtual screening of 6394 diverse natural products. Characterization of the binding interactions of the potential leads with OmpATb revealed nine critical residues comprising ARG86, LEU110, LEU113, LEU114, ALA115, PHE142, SER145, VAL146, and PHE151. Molecular dynamics simulations also revealed very stable protein-lead complexes. Most residues contributed lower binding energies to the overall molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) binding free energies of the interactions between the molecules and OmpATb protein. Induced Fit Docking (IFD) of the compounds regenerated poses of the molecular docking using AutoDock Vina. These molecules could be starting templates for designing inhibitors to bypass the pore mediating activities of OmpATb. Based on structural similarity, ZINC000034268676 was suggested as a potential scaffold for designing efflux pump inhibitors of the gate mediating activities of OmpATb and may enhance the uptake of hydrophilic drugs to reduce the duration time of tuberculosis treatment. Furthermore, structurally similar compounds available in the DrugBank database with a similarity threshold of 0.7 have been reported to exhibit antitubercular and anti-mycobacterial activities. These biomolecules can be further characterized experimentally to corroborate their antitubercular activity. Also, the skeletons of the molecules can be adopted as sub-structures for the design of future anti-mycobacterial drugs.

Introduction

Tuberculosis (TB) is one of the oldest known infectious disease dating back to 1000 BCE and is still one of the leading causes of deaths among infectious diseases [1,2]. An estimated one-third of the world's population are latently infected, of which nine million develop the infection further to the active stage and about two million deaths arise each year [3,4]. Of those infected, over one and half million cases occur annually in sub-Saharan Africa [5]. TB treatment protocols are based on a combination of three or more of the first-line antitubercular drugs comprising streptomycin, isoniazid, rifampicin, pyrazinamide and ethambutol [6,7].

The etiological agent of tuberculosis is Mycobacterium tuberculosis which is transmitted by aerosol droplets from actively infected persons [3,8]. A person becomes infected through inhalation of these aerosol droplets containing the bacteria, the inhaled bacteria enters the lungs and is engulfed by the macrophages in the alveoli. These macrophages digest the bacteria by the secretion of acid which results from the fusion of phagosome and lysosome inside the macrophage and thereby making their inner environment acidic [1,2]. The host immune system can attenuate the growth of M. tuberculosis at this stage, forming a granuloma around the bacteria and causing it to enter into a state of dormancy. The host becomes latently infected for a period or even a lifetime with the dormant bacteria awaiting reactivation. At this stage, the bacteria cannot be transmitted [9,10]. For persons with efficient cell-mediated immunity, the infection may be arrested permanently at this point [11]. However, if the host immunity cannot control this initial stage of M. tuberculosis or if a latently infected person's immune system is compromised by some factors such as aging, HIV infection, or even by some drugs, the granuloma formed around the bacteria becomes liquefied inside, giving the bacteria a suitable condition to grow and multiply [9,10]. When the growth and multiplication of the bacteria become uncontrollable, they burst out of the granuloma and infect other macrophages. At this stage, the person becomes actively infected, showing signs and symptoms of tuberculosis and can also transmit the infection [10].

In the macrophage, there is a lysosome and phagosome fusion which changes the pH of the inner environment of the macrophage from about 6.2 to between 4.5 and 5.0 [12]. M. tuberculosis has over the years developed some mechanisms to resist the pH change of the host macrophages. These mechanisms which are not fully understood helps the bacteria to function and grow within the host. Studies have shown that the outer membrane protein A (OmpATb), which is a pore-forming protein is actively involved in these mechanisms [[12], [13], [14]]. OmpATb has two functions: as a pore-forming protein with properties of a porin, and to help secrete ammonium into the phagosomal environment to neutralize the acid [13,14]. Other studies further suggest that at low pH, the OmpATb might be the only functioning porin and these pores close at this low pH [13,15]. The closure of these hydrophilic pores causes the slow uptake of hydrophilic molecules including current first-line drugs since the outer cell envelop is hydrophobic [13,15]. With this slow drug intake of the bacterial, the time duration for the curing of TB infection is prolonged than expected.

OmpATb has two terminal domains: the N-terminal and the C-terminal domains [3,15]. The pore-forming activity and the resistance of the M. tuberculosis to acidic medium in macrophages are mainly mediated by the N-terminal domain of OmpATb [3,15]. Due to the key role played by OmpATb in the survival of the mycobacterium in macrophages, it has been suggested as a plausible drug target [12,16,17]. Under normal conditions, the loss of OmpATb did not affect the growth of M. tuberculosis, but its ability to grow was decreased at reduced pH [13]. The porin-like activity of OmpATb plays a major role in mitigating the low pH environment of the host, making it a worthy drug target. Therefore, any molecules which have the potential to disrupt the normal activity of the porin at low pH could allow the host defense mechanisms to overcome the virulence of M. tuberculosis [13]. The pH mediating mechanisms could be exploited by inhibiting OmpATb, which could attenuate the growth of the bacteria due to host immune mechanisms. Also, once the porin-like activity of OmpATb is impeded, the uptake of other hydrophilic molecules under low pH could increase. The role of outer membrane protein A (OmpA) as a therapeutic target is well corroborated in other bacterial organisms like Acinetobacter baumannii and Pasteurella multocida [[18], [19], [20], [21], [22]]. Therefore, we hypothesized that the binding of compounds (especially from natural extracts) to OmpATb would enable the circumvention of its pH mediated pore-forming activity.

Natural products and their derivatives have historically been invaluable as sources of therapeutic agents [23,24]. Also, natural product structures have high chemical diversity, biochemical specificity, and other molecular properties that make them more favorable as lead compounds for drug discovery, and which serve to differentiate them from libraries of synthetic and combinatorial compounds [23]. To combat the dormant phenotype acquired by M. tuberculosis during infection and the reduced bacilli tolerance to front-line drugs, bioassay directed methods have been adopted to screen natural products from higher plant extracts against tuberculosis [25]. Marine natural products have also been screened against M. tuberculosis using luciferase reporter assay and enumerated colony-forming unit (CFU) bioassays [26]. Computationally, natural products from the Philippines and those in Ambinter database have been screened against S-adenosyl-l-homocysteine hydrolase (SAHH) protein of M. tuberculosis [27]. Moreover, the structural-based virtual approach has also been used to screen commercial libraries from Asinex database against the l-alanine dehydrogenase protein of M. tuberculosis to identify novel compounds [28].

This study aimed to identify novel small molecules of natural origin with the potential to inhibit the pore-forming activities of OmpATb by using computational structure-based drug design. This involved computational protocols including virtual screening, molecular docking simulations, pharmacological profiling of hits, and molecular dynamics simulation of potential lead compounds.

Section snippets

Protein structure retrieval

The three-dimensional (3D) molecular structure file of the N-terminal domain of OmpATb was retrieved from Protein Data Bank (PDB; http://www.rcsb.org/pdb/) [29] with PDB ID 2KGS [30].

Small molecules retrieval

Ligand molecular structural files were retrieved from eleven catalogues in ZINC15 database [31] comprising AfroDB [32], AnalytiCon Discovery NP [33], Herbal Ingredients In-Vivo Metabolism [34], Herbal Ingredients Targets [35], Interbioscreen Natural Compounds [36], INDOFINE Natural Products [37], NPACT Database [38

Protein structure and characterization of the active site

The 3D structure of the N-terminal domain contained three α-helices and six β-sheets connected by loops (Fig. 1a) [30]. The characterization of the active site using KVFinder [44] and MetaPocket 2.0 [45] revealed various binding cavities (Supplementary Table S6). One of the cavities was considered as a plausible binding pocket because of the large volume of 53.57 Å3 and surface area of 83.52 Å2 (Fig. 1a), which makes it possible for small compounds to dock firmly into it [65,66]. The active

Conclusion

OmpATb is a crucial target since it enables M. tuberculosis to survive in the harsh acidic environment of the macrophages by impeding the uptake of hydrophilic compounds, including some antitubercular molecules. This study predicted four novel natural products ZINC000003958185, ZINC000000157405, ZINC000000001392 and ZINC000034268676, which could be utilized as templates for the design of potential OmpATb inhibitors. ZINC000034268676 was specifically suggested as a potential scaffold for

Author contributions

S.K.K. and M.D.W. conceptualized the research project. Data analysis was predominantly 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 molecular dynamics simulations. S.K.K., M.D.W., C.A., E.Q., J.B., and M.A. co-wrote the first draft. All authors contributed to the revision of the drafts and agreed on the final version of the manuscript before submission.

Funding

The study was not funded.

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgments

The authors express their gratitude to the faculty members of the Department of Biomedical Engineering, University of Ghana, for all advice on the project. The West African Centre for Cell Biology of Infectious Pathogens (WACCBIP) at University of Ghana made available Zuputo, a Dell EMC high performance computing cluster for the molecular dynamics simulations.

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