Integration of Experimental and in silico Methods in the Study of Antimicrobials Mechanisms Resistance in Mycobacterium tuberculosis

• Grant number: 24/09645-9
• Support Opportunities: Regular Research Grants
• Start date: October 01, 2024
• End date: September 30, 2026
• Field of knowledge: Biological Sciences - Microbiology
• Mobility Program: SPRINT - Projetos de pesquisa - Mobilidade
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Marcio Vinicius Bertacine Dias
• Principal researcher abroad: Paulo Cesar Telles de Souza
• Institution abroad: Centre National de la Recherche Scientifique, France
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

Tuberculosis is an infectious disease caused predominantly by Mycobacterium tuberculosis and leads to the death of more than a million people every year worldwide. The treatment of tuberculosis consists of the use of four different antimicrobials for at least six months. Although antimicrobial resistance mechanisms in tuberculosis do not involve horizontal transfer, mutations in responsible genes for activating pro-drugs, promoter regions, and drug targets are constantly reported in antimicrobial resistance clinical isolates. Particularly to isoniazid (INH) and pyrazinamide (PZA), which are among the first-line antimicrobials to treat tuberculosis, missense mutations in the gene coding regions to the enzymatic target represent an essential fraction of those found in resistant strains. INH and PZA are both pro-drug and need activation to inhibit mycolic acid and CoA biosynthesis, respectively. INH is activated by KatG, a peroxidase, and forms the adduct NAD-INH, which inhibits an ACP enoyl reductase, InhA. On the other hand, the active form of PZA is pyrazinoic acid (POA), which inhibits the enzyme aspartate decarboxylase, PanD. Although there are biochemical and biophysical studies to understand the effect of missense mutations on the structure and activity of InhA and PanD, the sampling of analyzed substitutions is minimal, considering the large number of those that have been reported. Consequently, the molecular mechanisms of resistance based on missense mutation to these two antimicrobials could not be understood entirely. Thus, in this project, we aim to join expertise, including experimental biochemical and biophysical studies from the Brazilian group with in silico methods of coarse grain and atomistic molecular dynamic simulations from the French group to understand the effect of a large number of missense mutations that have been described in the literature and that were not studied so far. Thus, we aim to employ crystallography, isothermal titration calorimetry, differential scanning fluorimetry, and biochemical assays combined with trajectories of molecular dynamic simulations to dissect the impact of the mutation on antituberculosis drugs on their targets. With this information in hand, novel molecular mechanisms of resistance might be identified, and we will also be able to provide data to propose, in the future, a more personalized tuberculosis treatment. (AU)