Functional and structural preliminary characterization of the interaction from the transpeptidase domain of PonA1 from mycobacteria with beta-lactam antibiotics

• Grant number: 22/03967-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2022
• End date: August 31, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Luís Victor Santana Rosa
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Tuberculosis (TB) is an infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB), one of the major causes of death in the world. The treatment of the disease involves the association of several drugs for long periods, which results in the interruption of the treatment and contributes to the emergence of multidrug-resistant strains. Consequently, the discovery of new drugs is urgently required. Part of the success of MTB as a pathogen is due to the structure of its complex cell wall, which includes the peptidoglycan, a layer of arabinomannan and mycolic acids. During peptidoglycan biosynthesis, it was observed that the enzyme PonA1, a penicillin-binding protein (PBP), influences the growth and proliferation process of MTB. Since PBPs are enzymes inhibited by ²-lactam antibiotics, a class not used for the treatment of tuberculosis, it is interesting to study and attempt to reposition these drugs, as they are safe molecules. Due to the difficulty in obtaining MTB PonA1 in its soluble form, which was verified by previous studies in the laboratory, the functional and structural study with orthologous proteins becomes an interesting strategy, since they have conserved sequences and can be produced in the soluble form. For this, constructions already available in our laboratory will be introduced into competent cells to produce the enzymes and they will be purified by affinity, ion exchange, and molecular exclusion purification methods. The pure form of the transpeptidase domains of PonA1 will be used in crystallization tests, as well as be analyzed by differential scanning fluorimetry against an in-house library of ²-lactams. Finally, the enzyme activity and inhibition will also be assayed using a hydrolyzable ²-lactam, nitrocefin. Thus, this project aims to produce knowledge about the interaction of PonA1 of mycobacteria, particularly M. tuberculosis, M. thermoresistibile and M. smegmatis with different classes of ²-lactam antibiotics, contributing to the repositioning of antimicrobials for the treatment of tuberculosis.(AU)