Study of the mechanism of action and resistance to pyrazinamide in Mycobacterium tuberculosis

• Grant number: 24/19817-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: January 01, 2025
• End date: December 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Rafaela Kaskanlian Pires do Rio
• 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 a high-impact disease, diagnosed in 10.6 million patients and with a fatality rate of 1.3 million people in 2022 alone. The main drugs used to combat the disease are: isoniazid, rifampicin, ethambutol and pyrazinamide. Various studies have been carried out to understand the mechanism of action of these drugs, but there is no consensus on pyrazinamide (PZA). Two hypotheses are proposed for its mechanism of action: firstly, there is evidence that pyrazinoic acid (POA), which is the active form of PZA, ensures its efficacy by binding directly to the active site of PanD, acting as a competitive inhibitor; on the other hand, it is also proposed that POA binds to PanD and directs it to the ClpP1P2C1 enzyme complex so that it undergoes degradation mediated by recognition of the C-terminal portion of PanD. Since the mechanism of action of PZA is not fully elucidated, its resistance mechanism also has several gaps. Furthermore, there are few studies using clinical isolates of PanD that show a mechanism of resistance to pyrazinamide. Most of the mutations found in PZA-resistant clinical isolates have mutations in the panD and clpC1 gene, corroborating the involvement of the PanD and ClpC1 enzymes. The aim of this project is to advance our understanding of the mechanism of action and resistance to PZA through biochemical and biophysical studies of wild-type PanD and the missense mutants V138A, H21R, I49V, E130G and P134S. Thus, in this project, we intend to: analyze the secondary structure content of mutant PanD enzymes compared to wild-type protein, in the presence and absence of POA, the thermal stability of mutants when exposed to pyrazinamide compared to wild-type protein; obtain POA dissociation constants for mutant and wild-type proteins and standardize an enzymatic degradation assay for PanD in the presence of POA by the ClpP1P2C1 complex.