Using molecular dynamic simulations and cryo-electron microscopy to understand antimicrobial resistance in Mycobacterium tuberculosis

• Grant number: 24/06177-4
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: November 04, 2024
• End date: November 03, 2025
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Nicolas de Oliveira Rossini
• Supervisor: Paulo Cesar Telles de Souza
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: École Normale Supérieure, Lyon (ENS), France
• Associated to the scholarship: 21/14205-0 - Determination of the structural bases for resistance to first-line drugs using missense mutations in Mycobacterium tuberculosis, BP.DD

Abstract

Among the main drugs used to treat tuberculosis, isoniazid (INH) is considered a first-line drug and fluoroquinolones (FQL), mainly Moxifloxacin (MFX), are gaining space in the most current treatment regimens. Particularly, for INH and MFX, their targets, which include the enzymes InhA encoded by inhA gene, and DNA gyrase, encoded by gyrA and GryB genes, respectively, have the characteristic of having missense mutations. However, little is known about the impact of these mutations on the molecular mechanism of M. tuberculosis resistance to INH and MFX. During the regular PhD project, we selected a series of missense mutations observed in inhA, gyrA and gyrB genes from clinical isolates from different countries, which have not yet been biophysically and biochemically characterized. To date, we have been able to make sixteen new mutant constructs for InhA and DNA gyrase (GyrBA) enzymes. Regarding InhA, we were able to obtain twelve new structures in complex with coenzyme NADH or the inhibitory adduct INH-NAD, carry out preliminary calorimetric studies using ITC and also investigate the thermal unfolding of these proteins using DSF, providing important information about novel mechanisms resistance associated with this enzyme. We were also able to optimize the expression of GyrBA, and we attempted to obtain the structure of this enzyme by cryo-electron microscopy in APO form. Therefore, we propose for this project to perform molecular dynamics simulations of the obtained InhA structures, using atomistic models. To study those mutations that may affect the oligomerization state of the enzyme, we will employ coarse-grained models to evaluate the tetrameric structure of the enzymes. On the other hand, for GyrBA and its mutants, we intend to obtain the structures of the mutant enzymes in complex with DNA and moxifloxacin using the cryo-electron microscopy technique and further perform molecular dynamic simulations. The application of these methods will contribute to a better understanding of the mutation effect on the structure of these targets.