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Rational design of drug-like compounds targeting Mycobacterium marinum MelF protein.

ABSTRACT: The mycobacterial mel2 locus (mycobacterial enhanced infection locus, Rv1936-1941) is Mycobacterium marinum and M. tuberculosis specific, which can withstand reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced stress. A library of over a million compounds was screened using in silico virtual ligand screening (VLS) to identify inhibitors against the modeled structure of MelF protein expressed by melF of mel2 locus so that M. marinum's ability to withstand ROS/RNS stress could be reduced. The top ranked 1000 compounds were further screened to identify 178 compounds to maximize the scaffold diversity by manually evaluating the interaction of each compound with the target site. M. marinum melF was cloned, expressed and purified as maltose binding protein (MBP)-tagged recombinant protein in Escherichia coli. After establishing the flavin dependent oxidoreductase activity of MelF (~ 84 kDa), the inhibitors were screened for the inhibition of enzyme activity of whole cell lysate (WCL) and the purified MelF. Amongst these, 16 compounds could significantly inhibit the enzyme activity of purified MelF. For the six best inhibitory compounds, the minimal inhibitory concentration (MIC) was determined to be 3.4-19.4 ?M and 13.5-38.8 ?M for M. marinum and M. tuberculosis, respectively. Similarly, the minimal bactericidal concentration (MBC) was determined to be 6.8-38.8 ?M and 27-38.8 ?M against M. marinum and M. tuberculosis, respectively. One compound each in combination with isoniazid (INH) also showed synergistic inhibitory effect against M. marinum and M. tuberculosis with no cytotoxicity in HeLa cells. Interestingly, these inhibitors did not display any non-specific protein-structure destabilizing effect. Such inhibitors targeting the anti-ROS/RNS machinery may facilitate the efficient killing of replicating and nonreplicating mycobacteria inside the host cells.

SUBMITTER: Dharra R

PROVIDER: S-EPMC5584760 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Rational design of drug-like compounds targeting Mycobacterium marinum MelF protein.

Dharra Renu R Talwar Sakshi S Singh Yogesh Y Gupta Rani R Cirillo Jeffrey D JD Pandey Amit K AK Kulharia Mahesh M Mehta Promod K PK

PloS one 20170905 9

The mycobacterial mel2 locus (mycobacterial enhanced infection locus, Rv1936-1941) is Mycobacterium marinum and M. tuberculosis specific, which can withstand reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced stress. A library of over a million compounds was screened using in silico virtual ligand screening (VLS) to identify inhibitors against the modeled structure of MelF protein expressed by melF of mel2 locus so that M. marinum's ability to withstand ROS/RNS stress coul ...[more]

PMID: 28873466

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Project description:SARS-CoV-2 caused the current COVID-19 pandemic and there is an urgent need to explore effective therapeutics that can inhibit enzymes that are imperative in virus reproduction. To this end, we computationally investigated the MPD3 phytochemical database along with the pool of reported natural antiviral compounds with potential to be used as anti-SARS-CoV-2. The docking results demonstrated glycyrrhizin followed by azadirachtanin, mycophenolic acid, kushenol-w and 6-azauridine, as potential candidates. Glycyrrhizin depicted very stable binding mode to the active pocket of the Mpro (binding energy, -8.7 kcal/mol), PLpro (binding energy, -7.9 kcal/mol), and Nucleocapsid (binding energy, -7.9 kcal/mol) enzymes. This compound showed binding with several key residues that are critical to natural substrate binding and functionality to all the receptors. To test docking prediction, the compound with each receptor was subjected to molecular dynamics simulation to characterize the molecule stability and decipher its possible mechanism of binding. Each complex concludes that the receptor dynamics are stable (Mpro (mean RMSD, 0.93 Å), PLpro (mean RMSD, 0.96 Å), and Nucleocapsid (mean RMSD, 3.48 Å)). Moreover, binding free energy analyses such as MMGB/PBSA and WaterSwap were run over selected trajectory snapshots to affirm intermolecular affinity in the complexes. Glycyrrhizin was rescored to form strong affinity complexes with the virus enzymes: Mpro (MMGBSA, -24.42 kcal/mol and MMPBSA, -10.80 kcal/mol), PLpro (MMGBSA, -48.69 kcal/mol and MMPBSA, -38.17 kcal/mol) and Nucleocapsid (MMGBSA, -30.05 kcal/mol and MMPBSA, -25.95 kcal/mol), were dominated mainly by vigorous van der Waals energy. Further affirmation was achieved by WaterSwap absolute binding free energy that concluded all the complexes in good equilibrium and stability (Mpro (mean, -22.44 kcal/mol), PLpro (mean, -25.46 kcal/mol), and Nucleocapsid (mean, -23.30 kcal/mol)). These promising findings substantially advance our understanding of how natural compounds could be shaped to counter SARS-CoV-2 infection.

Project description:Multi-drug therapy is the standard-of-care treatment for tuberculosis. Despite this, virtually all studies of the pharmacodynamics (PD) of mycobacterial drugs employed for the design of treatment protocols are restricted to single agents. In this report, mathematical models and in vitro experiments with Mycobacterium marinum and five antimycobacterial drugs are used to quantitatively evaluate the pharmaco-, population and evolutionary dynamics of two-drug antimicrobial chemotherapy regimes. Time kill experiments with single and pairs of antibiotics are used to estimate the parameters and evaluate the fit of Hill-function-based PD models. While Hill functions provide excellent fits for the PD of each single antibiotic studied, rifampin, amikacin, clarithromycin, streptomycin and moxifloxacin, two-drug Hill functions with a unique interaction parameter cannot account for the PD of any of the 10 pairs of these drugs. If we assume two antibiotic-concentration dependent functions for the interaction parameter, one for sub-MIC and one for supra-MIC drug concentrations, the modified biphasic Hill function provides a reasonably good fit for the PD of all 10 pairs of antibiotics studied. Monte Carlo simulations of antibiotic treatment based on the experimentally-determined PD functions are used to evaluate the potential microbiological efficacy (rate of clearance) and evolutionary consequences (likelihood of generating multi-drug resistance) of these different drug combinations as well as their sensitivity to different forms of non-adherence to therapy. These two-drug treatment simulations predict varying outcomes for the different pairs of antibiotics with respect to the aforementioned measures of efficacy. In summary, Hill functions with biphasic drug-drug interaction terms provide accurate analogs for the PD of pairs of antibiotics and M. marinum. The models, experimental protocols and computer simulations used in this study can be applied to evaluate the potential microbiological and evolutionary efficacy of two-drug therapy for any bactericidal antibiotics and bacteria that can be cultured in vitro.

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Rational design of drug-like compounds targeting Mycobacterium marinum MelF protein.

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Rational design of drug-like compounds targeting Mycobacterium marinum MelF protein.

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