Abstract

Computational Saturation Mutagenesis is an in-silico approach that employs systematic mutagenesis of each amino acid residue in the protein to all other amino acid types, and predicts changes in thermodynamic stability and affinity to the other subunits/protein counterparts, ligands and nucleic acid molecules. The data thus generated are useful in understanding the functional consequences of mutations in antimicrobial resistance phenotypes. In this study, we applied computational saturation mutagenesis to three important drug-targets in Mycobacterium leprae (M. leprae) for the drugs dapsone, rifampin and ofloxacin namely Dihydropteroate Synthase (DHPS), RNA Polymerase (RNAP) and DNA Gyrase (GYR), respectively. M. leprae causes leprosy and is an obligate intracellular bacillus with limited protein structural information associating mutations with phenotypic resistance outcomes in leprosy. Experimentally solved structures of DHPS, RNAP and GYR of M. leprae are not available in the Protein Data Bank, therefore, we modelled the structures of these proteins using template-based comparative modelling and introduced systematic mutations in each model generating 80,902 mutations and mutant structures for all the three proteins. Impacts of mutations on stability and protein-subunit, protein-ligand and protein-nucleic acid affinities were computed using various in-house developed and other published protein stability and affinity prediction software. A consensus impact was estimated for each mutation using qualitative scoring metrics for physicochemical properties and by a categorical grouping of stability and affinity predictions. We developed a web database named HARP (a database of Hansen's Disease Antimicrobial Resistance Profiles), which is accessible at the URL - https://harp-leprosy.org and provides the details to each of these predictions.

Highlights

  • Mycobacterium leprae (M. leprae) is a pathogenic species of mycobacterium that causes leprosy in tropical countries

  • Computational saturation mutagenesis [18] is an approach that aids in systematically analysing the impacts of all possible substitution mutations at a given residue position in the protein. We have applied this approach earlier to the beta subunit of RNA Polymerase (RNAP) [19]. We have extended this approach and applied it to the three known drug-targets in M. leprae, the Dihydropteroate Synthase (DHPS), RNA Polymerase (RNAP) and DNA Gyrase (GYR) that are the targets of dapsone, rifampin and ofloxacin respectively

  • HARP embodies systematic computational saturation mutagenesis of all the three known drug-target proteins in M. leprae namely DHPS, RNAP and GYR with predicted impacts resulting from mutations on thermodynamic stability and affinity to other proteins, ligands and nucleic acids

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Summary

Introduction

Mycobacterium leprae (M. leprae) is a pathogenic species of mycobacterium that causes leprosy ( known as Hansen’s disease) in tropical countries. Earlier monotherapies with dapsone and rifampin have led to the emergence of resistant strains of M. leprae for dapsone in the year 1964 and for rifampin in 1976 [3]. This has led to the introduction of multidrug therapy (MDT) by the World Health Organisation (WHO) in 1983.

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