Dynamics of fluoroquinolones induced resistance in DNA gyrase of Mycobacterium tuberculosis

DNA gyrase is a validated target of fluoroquinolones which are key components of multidrug resistance tuberculosis (TB) treatment. Most frequent occurring mutations associated with high level of resistance to fluoroquinolone in clinical isolates of TB patients are A90V, D94G, and A90V–D94G (double mutant [DM]), present in the larger subunit of DNA Gyrase. In order to explicate the molecular mechanism of drug resistance corresponding to these mutations, molecular dynamics (MD) and mechanics approach was applied. Structure-based molecular docking of complex comprised of DNA bound with Gyrase A (large subunit) and Gyrase C (small subunit) with moxifloxacin (MFX) revealed high binding affinity to wild type with considerably high Glide XP docking score of ?7.88 kcal/mol. MFX affinity decreases toward single mutants and was minimum toward the DM with a docking score of ?3.82 kcal/mol. Docking studies were also performed against 8-Methyl-moxifloxacin which exhibited higher binding affinity against wild and mutants DNA gyrase when compared to MFX. Molecular Mechanics/Generalized Born Surface Area method predicted the binding free energy of the wild, A90V, D94G, and DM complexes to be ?55.81, ?25.87, ?20.45, and ?12.29 kcal/mol, respectively. These complexes were further subjected to 30 ns long MD simulations to examine significant interactions and conformational flexibilities in terms of root mean square deviation, root mean square fluctuation, and strength of hydrogen bond formed. This comparative drug interaction analysis provides systematic insights into the mechanism behind drug resistance and also paves way toward identifying potent lead compounds that could combat drug resistance of DNA gyrase due to mutations.     

Refined homology model of cytochrome bcc complex B subunit for virtual screening of potential anti-tuberculosis agents

Abstract

Cytochrome bcc complex is important for ATP synthesis and cellular activity, as a crucial step in the terminal reduction of oxygen in aerobic electron transport chains. The b subunit of cytochrome bcc complex (QcrB) has been reported as a promising anti-tuberculosis target, with many novel anti-tuberculosis scaffolds reported. However, the 3D structure of mycobacterium tuberculosis (M. tuberculosis) QcrB has not been released, making it hard to understand the interactions between QcrB and its inhibitors as well as to develop novel anti-tuberculosis scaffolds. Herein we built the optimal homology model of M. tuberculosis QcrB using the M. smegmatis QcrB structure as template, which was refined through all-atom molecular dynamics simulation. Then, the binding modes of known inhibitors were predicted through molecular docking method, along with molecular dynamics simulation and binding free energy calculation to verify the accuracy of docking results and stability of the protein-inhibitor complexes. The informative key residues within QcrB site enabled us to perform structure-based virtual library screening to obtain potential M. tuberculosis QcrB inhibitors, which were validated through molecular dynamics simulation and MM-GBSA calculation and analyzed through pharmacokinetic properties prediction. Our research would provide a deeper insight into the interactions between M. tuberculosis QcrB and its inhibitors, which boosts to develop novel therapy against tuberculosis.

Communicated by Ramaswamy H. Sarma     

肺外结核病微生物学诊断方法的研究和应用进展

肺外结核病指由结核分枝杆菌（Mycobacterium tuberculosis, MTB）感染所引起的发生在肺部以外器官和部位的结核病。近年来肺外结核的发病率逐渐升高,未能得到早期有效治疗的肺外结核病患者可能并发畸形、截瘫甚至死亡等严重后果。微生物学检测方法对从病原学角度诊断肺外结核病至关重要。基于此,总结了近年来肺外结核病细菌学检查方法、结核分枝杆菌的抗原检测与分子生物学检测等微生物学诊断方法的概况及应用进展,并对这些检测方法的优缺点及适用范围进行了分析、比较,以期为今后肺外结核病病原学诊断的研究提供相关信息。     

Halting ionic shuttle to disrupt the synthetic machinery—Structural and molecular insights into the inhibitory roles of Bedaquiline towards Mycobacterium tuberculosis ATP synthase in the treatment of tuberculosis

Therapeutic targeting of the adenosine triphosphate (ATP) machinery of Mycobacterium tuberculosis (Mtb) has recently presented a potent and alternative measure to halt the pathogenesis of tuberculosis. This has been potentiated by the development of bedaquiline (BDQ), a novel small molecule inhibitor that selectively inhibits mycobacterial F₁F_o-ATP synthase by targeting its rotor c-ring, resulting in the disruption of ATP synthesis and consequential cell death. Although the structural resolution of the mycobacterial C₉ ring in co`mplex with BDQ provided the first-hand detail of BDQ interaction at the c-ring region of the ATP synthase, there still remains a need to obtain essential and dynamic insights into the mechanistic activity of this drug molecule towards crucial survival machinery of Mtb. As such, for the first time, we report an atomistic model to describe the structural dynamics that explicate the experimentally reported antagonistic features of BDQ in halting ion shuttling by the mycobacterial c-ring, using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Surface Area methods. Results showed that BDQ exhibited a considerably high ΔG while it specifically maintained high-affinity interactions with Glu65_B and Asp32_B, blocking their crucial roles in proton binding and shuttling, which is required for ATP synthesis. Moreover, the bulky nature of BDQ induced a rigid and compact conformation of the rotor c-ring, which impedes the essential rotatory motion that drives ion exchange and shuttling. In addition, the binding affinity of a BDQ molecule was considerably increased by the complementary binding of another BDQ molecule, which indicates that an increase in BDQ molecule enhances inhibitory potency against Mtb ATP synthase. Taken together, findings provide atomistic perspectives into the inhibitory mechanisms of BDQ coupled with insights that could enhance the structure-based design of novel ATP synthase inhibitors towards the treatment of tuberculosis.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

Tuberculosis vaccine design: influence of the completed genome sequence

Tuberculosis continues to be a major health problem, with more adults dying from Mycobacterium tuberculosis than any other pathogen world-wide.With the onset of the HIV epidemic and an increase in drug-resistant M. tuberculosis strains, the need for an improved vaccine has become an international priority.The recent completion of the genome sequences for two M. tuberculosis strains provides a wealth of information that can be used to design new strategies for vaccine development. The challenge comes in making rational choices from among the 4,000 genes of the most probable candidate immunogens or virulence genes.Thus, a well-designed screen is needed to reduce the number of candidates that must be tested. Presently, the most valuable role that bioinformatics can play is to provide such a screen.     

Molecular detection, identification and drug resistance detection in Mycobacterium tuberculosis

This minireview presents recent developments in molecular methods for the diagnosis of tuberculosis, including detection, identification and determination of drug resistance of Mycobacterium tuberculosis . Tuberculosis remains one of the major causes of global death from a single infectious agent. This situation is worsened by the HIV/AIDS pandemic because one-third of HIV/AIDS patients are coinfected with M. tuberculosis . Also of great concern is the emergence of drug-resistant tuberculosis because there are almost no treatment options available for patients affected by highly resistant strains of M. tuberculosis . Advances in molecular biology techniques and a better knowledge of the molecular mechanisms of drug resistance have provided new tools for the rapid diagnosis of tuberculosis. Several nucleic acid amplification technologies have been developed and evaluated. New molecular approaches are being introduced continuously. This minireview will also comment on the future perspectives for the molecular diagnosis of tuberculosis and the feasibility for the implementation of these newer techniques in the clinical diagnostic laboratory.     

结核分枝杆菌基因组学与基因组进化

在后基因组时代,特别是在新的测序理论和设备大发展的背景下,一些重大传染性致病微生物基因组序列正在被逐一测定,并且随后的基因功能注释,蛋白质三维结构重建等工作也正在开展,以期对致病微生物的生物学特性、诊断策略和治疗方法等有突破性的认识.作为对人类健康一直存在严重威胁的结核分枝杆菌,其基因组在进化中所发生的各种遗传事件对其生物学性质、致病能力和抗药性等各方面有重要作用.本文旨在阐述结核分枝杆菌的起源及其基因组特征,论述其基因组进化的研究进展.     

Inhibitor binding studies of Mycobacterium tuberculosis MraY (Rv2156c): Insights from molecular modeling,docking, and simulation studies

Abstract

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis (M.tb) or tubercule bacillus, and H37Rv is the most studied clinical strain. The recent development of resistance to existing drugs is a global health-care challenge to control and cure TB. Hence, there is a critical need to discover new drug targets in M.tb. The members of peptidoglycan biosynthesis pathway are attractive target proteins for antibacterial drug development. We have performed in silico analysis of M.tb MraY (Rv2156c) integral membrane protein and constructed the three-dimensional (3D) structure model of M.tb MraY based on homology modeling method. The validated model was complexed with antibiotic muraymycin D2 (MD2) and was used to generate structure-based pharmacophore model (e-pharmacophore). High-throughput virtual screening (HTVS) of Asinex database and molecular docking of hits was performed to identify the potential inhibitors based on their mode of interactions with the key residues involved in M.tb MraY–MD2 binding. The validation of these molecules was performed using molecular dynamics (MD) simulations for two best identified hit molecules complexed with M.tb MraY in the lipid bilayer, dipalmitoylphosphatidyl-choline (DPPC) membrane. The results indicated the stability of the complexes formed and retained non-bonding interactions similar to MD2. These findings may help in the design of new inhibitors to M.tb MraY involved in peptidoglycan biosynthesis.     

Targeting the cell wall of Mycobacterium tuberculosis: a molecular modeling investigation of the interaction of imipenem and meropenem with L,D-transpeptidase 2

The single crystal X-ray structure of the extracellular portion of the L,D-transpeptidase (ex-Ldt_Mt2 – residues 120–408) enzyme was recently reported. It was observed that imipenem and meropenem inhibit activity of this enzyme, responsible for generating L,D-transpeptide linkages in the peptidoglycan layer of Mycobacterium tuberculosis. Imipenem is more active and isothermal titration calorimetry experiments revealed that meropenem is subjected to an entropy penalty upon binding to the enzyme. Herein, we report a molecular modeling approach to obtain a molecular view of the inhibitor/enzyme interactions. The average binding free energies for nine commercially available inhibitors were calculated using MM/GBSA and Solvation Interaction Energy (SIE) approaches and the calculated energies corresponded well with the available experimentally observed results. The method reproduces the same order of binding energies as experimentally observed for imipenem and meropenem. We have also demonstrated that SIE is a reasonably accurate and cost-effective free energy method, which can be used to predict carbapenem affinities for this enzyme. A theoretical explanation was offered for the experimental entropy penalty observed for meropenem, creating optimism that this computational model can serve as a potential computational model for other researchers in the field.     

Identification of peptidomimetic compounds as potential inhibitors against MurA enzyme of Mycobacterium tuberculosis

Abstract

Increasing prevalence of resistance to anti-tubercular drugs has become the foremost challenge now. According to WHO, over half a million of multidrug resistance cases (rifampicin, isoniazid, etc.) were reported in 2017, mostly emerging from countries such as China, India, and Russia. Therefore, developing new drugs or repurposing existing ones is need of the hour. The Mycobacterium cell wall biogenesis pathway offers many attractive targets for drug discovery against Tuberculosis (TB). MurA, a transferase enzyme that catalyzes the initial step of peptidoglycan (PG) biosynthesis, is one among them. A peptidoglycan layer resides over the plasma membrane and is an integral component of the bacterial cell wall. Therefore, disruption of their formation through inhibition of MurA enzyme should lead to deficiency in Mycobacterium cell synthesis. Based on this strategy, we have designed this study where two libraries of peptidomimetic compounds (Asinex & ChemDiv) were first screened against our modeled MurA structure and then validated through molecular dynamic simulations. From our virtual screening, top four compounds (ChemDiv: D675-0102, D675-0217; Asinex: BDE25373574, BDE 26717803) were selected based on their docking scores, binding energies, and interactions with catalytic site residues, for further evaluation. Results revealed stable ligand-MurA interactions throughout 50?ns of MD simulation and also druggability acceptable pharmacokinetic profile for all four compounds. Thus, based on our findings, these compounds could be considered as potential inhibitors of Mycobacterium MurA enzyme and hence be further tested for in vitro experimental validation as TB therapeutic drug candidate.

Communicated by Ramaswamy H. Sarma