identification of potential inhibitors of Mycobacterium tuberculosis MmpS5L5 from the ReFRAME database: a structure-based virtual screening, molecular docking and molecular dynamics approach.

INTRODUCTION: Tuberculosis (TB), caused by(), is the leading infectious cause of death globally, disproportionately impacting low- and medium-income countries (LMICs). The emergence and transmission of drug resistantstrains has rendered a majority of the current anti-TB agents ineffective and significantly complicated TB treatment. Thus, the development of new anti-TB remedies with novel modes of action is a pressing priority. An attractive, viable strategy is the development of potentiators of anti-TB drugs that reverse drug efflux, a key intrinsicdrug resistance mechanism. TargetingMmpS5L5, a critical efflux pump (EP) implicated in the mycobacterial expulsion of various anti-TB drugs including bedaquiline, tetracyclines, azoles and clofazimine would likely enhance the efficacy of current anti-TB drugs by preventing the development of drug resistance.

METHODS: The recent determination of a high-resolution crystal structure ofMmpS5L5 (PDBID: 8ZKP) enables the utilisation of structure-anchored approaches for the uncovering of probable efflux inhibitors. In this study, pharmacophore models developed using theMmpS5L5 three-dimensional (3-D) structure and its known inhibitors, verapamil and norverapamil, were thereafter utilised for the screening of the REFRAME database, a comprehensive drug repurposing library, to identify novel ligand scaffolds with putative activity against the EP. Predicted target binding affinity for the top candidates was ascertained and validated using molecular docking and 100 ns molecular dynamics (MD) simulations, respectively. Further, post-MD analysis including Molecular Mechanics/Generalized Born Surface Area calculations (MMGBSA), Principal Component Analysis, and Free Energy Landscapes were done to study thermodynamic and conformational dynamics of the complexes.

RESULTS: Six compounds (406, 3920, 4031, 4787, 7104, 10367) had stronger predicted binding affinities for MmpS5L5 than the known inhibitors, with docking scores ranging from -8.70 to -5.01 kcal/mol and had predicted protein contacts similar to those of the validated inhibitors. Molecular dynamic simulations and MMGBSA analyses demonstrated stable and energetically favourable protein-ligand interaction. Among the six compounds, 3920 and 4031 emerged as the most promising hits as their average total ΔG bind (-111.81 ± 8.98 kcal/mol and -109.56 ± 8.40 kcal/mol respectively) and ligand efficiency (-16.46 ± 4.06 kcal/mol and -17.63 ± 1.27 kcal/mol) were lower than those of the reference inhibitors.

DISCUSSION: This study identified compounds from the ReFRAME database that may provide putative scaffolds for the development ofefflux inhibitors that can potentiate the treatment efficacy of current anti-TB drugs. Furtherandstudies are needed to validate their inhibition potential.