Structural bases of the control of cellular elongation and cell division in Mycobacterium tuberculosis and identification of drug hits based on fragment screening

Author(s)

Catharina dos Santos Silva

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2023-05-26

Examining board members

Marcio Vinícius Bertacine Dias; Frederico José Gueiros Filho; Ana Marcia de Sá Guimarães; Vinicius Gonçalves Maltarollo

• Advisor: Marcio Vinícius Bertacine Dias

Abstract

Tuberculosis is the world\'s deadliest infection caused by a single infectious agent, the bacillus Mycobacterium tuberculosis. The increasing in resistance and the current global burden of tuberculosis highlight the need for drug discovery and the search for novel treatment regimens. The components of macromolecular complexes in the control of cellular growth and division in mycobacteria have emerged as interesting targets for novel anti-tuberculosis agents. In this study, we propose to study the role of protein-protein interactions in the control of peptidoglycan biosynthesis during mycobacteria cell cycle. The hypothesis is that the penicillin-binding protein PonA1 and the peptidoglycan hydrolase RpfB compete for the same site of interaction with the endopeptidase RipA. These interactions suggest a post-translational mechanism regulating peptidoglycan synthesis and remodelling in mycobacteria. Furthermore, RipA and PonA1 are attractive targets for the development of new anti-tuberculosis drugs because they are essential for growth and cell separation, respectively. Thus, the study aims to elucidate the three-dimensional structure of the active conformation of RipA and also to obtain RipA:RpfB and RipA:PonA1 complexes. Moreover, in this work we also propose to apply the fragment-based drug discovery strategy against the catalytic domain of RipA and to screen &#946-lactam compounds against the transpeptidase domain of PonA1. Through the production of recombinant proteins and the use of biophysical techniques, the study of the target enzymes were performed, as well as the research on protein:protein and protein:ligand interactions. The work noted structural alterations in the active conformation of RipA and identified 11 ligands against the endopeptidase. We also presented preliminary data on RipA:RpfB interaction. This study concludes that proteolytic activation of RipA results in conformational alterations of the enzyme and may lead to the formation of a quaternary structure, suggesting a new hypothesis for the control of RipA enzymatic activity. This is also the first work that identified active molecules with potential for the development of the first leading-compounds inhibiting Rip enzymes from mycobacteria. (AU)

• FAPESP's process: 16/18721-4 - Structural basis of the elongation and cell division control in Mycobacterium tuberculosis and identification of new drug hits based in fragment trials
• Grantee: Catharina dos Santos Silva
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)