Structural and Biochemical Studies of the ThiL Enzyme on Vitamin B1 Biosynthesis in Gram-negative Pathogens and Mycobacterium tuberculosis.

• Grant number: 22/04298-3
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: December 01, 2022
• End date: November 30, 2026
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Alessandro Silva Nascimento
• Grantee: Erick Giancarlo Suclupe Farro
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

Antibiotic resistance is a global and urgent problem. The World Health Organization (WHO) estimates that by 2050, we will reach 10 million deaths annually as a direct result of infections caused by antibiotic-resistant organisms. The problem is compounded by the limited number of pathways/targets on which currently available antibiotics act. According to Walsh and Wencevicz, currently existing antibiotics are restricted to only five targets/pathways of action. This adds up to (i) the spread of resistant strains, (ii) the decreasing number of new antibiotics or innovative molecules, and (iii) the reduced number of targets on which antibiotics act. Recently, the enzyme thiamine phosphate kinase (ThiL) was validated as a new target for the development of antibiotics against Pseudomonas aeruginosa. This enzyme is part of the thiamine (vitamin B1) synthesis pathway present in many microorganisms and in plants, but absent in mammals. Deletion (or inhibition) of the ThiL enzyme has recently been shown to cause growth defects in P. aeruginosa and reduce the virulence of the bacterium. However, the results are still limited to studies in this organism. As the research groups of profs. Alessandro S. Nascimento (IFSC/USP) and Carsten Wrenger (ICB/USP) have been dedicating themselves to the structural and biochemical studies of the enzymes of the thiamine synthesis pathway, in this project we propose a study of the structure-function relationship on the enzyme ThiL in other clinically important organisms, including Klebsiella pneumoniae, Escherichia coli and Mycobacterium tuberculosis. In the scope of this project, we propose the determination of the enzyme structure of these organisms and the screening of potential inhibitors that allow a more complete evaluation of the enzyme's role from a biochemical and functional point of view, i.e., in cell culture. For this purpose, genes for the ThiL enzymes from K. pneumoniae, E. coli and M. tuberculosis were synthesized with codon optimization for bacterial expression. Preliminary expression tests indicate that the first two proteins are well expressed in E. coli and are purified in the soluble fraction. In this project, we intend to proceed with the determination of the structure and, taking advantage of the group's previous experience and structural information, to screen potential ligands of this enzyme that could be inhibitors for later biochemical tests, as well as in cell cultures. The final objective of the project is the validation of the chemical inhibition of this enzyme in vitro from experimental structural models.