Preliminary structural characterization and screening of fragments for transpeptidase and transglycosylase domains of mycobacterial PonA1 protein

• Grant number: 20/07197-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2020
• End date: January 24, 2021
• Field of knowledge: Biological Sciences - Microbiology
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Paula da Silva Canôas
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Tuberculosis (TB), infectious disease caused by the microorganism Mycobacterium tuberculosis, is the major cause of death by a single infectious agent in the world. The dependence of an extensive administration scheme for the effectiveness of the chemotherapy currently used makes adhesion difficult, leading to the emergence of multidrug-resistant strains. This creates a need for research about new drug targets leading to the disease treatment. The importance of cell wall synthesis for the pathogenicity creates a new range of possibilities, among which the protein PonA1, responsible for cell wall proteoglycan synthesis, has shown to be a promising target since the depletion of ponA1 gene was capable of abolishing the pathogen dissemination in rats. Due to the difficulty of obtaining the soluble protein, as observed in a previous study in our laboratory, we are proposing to study the ortholog enzymes since they have conserved sequences and consequently, they should also have similar structures. This project aims to produce data about the structure of the protein PonA1 and its orthologs from M. thermoresistibile and M. smegmatis. For that, during this project, the coding regions of ponA1 gene from these mycobacteria will be amplified and cloned into expression vectors, which will be further transformed in competent cells for the enzyme production. The protein will be further purified using affinity and molecular exclusion chromatography and submitted to crystallization trials and screened against an in house library of small molecules. At the end of this project, we expect to contribute with relevant results for the understanding of PonA1 structure and also in identifying novel potential inhibitors for this enzyme.