STRUCTURAL AND INTERACTION STUDIES OF RPIB FROM M. TUBERCULOSIS: SCREENING OF NEW COMPOUNDS TO A SPECIFIC TARGET

Tuberculosis annually approximately 10 million people worldwide and has been considered an epidemic in several countries. The use of outdated and long-term drugs has led to the emergence of resistant strains. Therefore, identifying new biological targets and inhibitors that are crucial to the microorganism is essential for developing specific drugs with minimal side effects. The Ribose-5- phosphate isomerase (Rpi) participates in the non-oxidative branch of the pentose phosphate pathway. This protein family has two types, and the RpiB is found in microorganisms but has no homologs in humans. Therefore, M. tuberculosis RpiB has been selected as a molecular target for structural and interaction studies using NMR. This study aims to investigate the interaction of MtRpiB through Fragment-Based Screening using a specific library of 696 fragments by Nuclear Magnetic Resonance (NMR) to discover and assess new compounds. MtRpiB was overexpressed in E. coli BL21(DE3) in LB and ¹⁵N isotopically labeled minimum medium, and purified by affinity chromatography. The fragment library was screened using Chemical Shift Perturbation (CSP), Waterlogsy, and T₂ Relaxation – CPMG experiments on a Bruker AVANCE 500 MHz spectrometer. Each experiment, which included pools of 12 compounds, was conducted both free and bound to MtRpiB, using a 10x excess of fragment to protein. Preliminary results identified 9 fragments with protein interaction. MtRpiB ¹⁵N-HSQC was performed in the presence of a fragment that showed significant interaction with the protein, indicating changes in the active site region. Repeating the experiments with the nine fragments and refining the analysis parameters revealed two additional fragments of high interest for further studies. Molecular docking studies are also in progress to visualize the interaction and measure potential changes in the presence of phosphate, which is essential for protein-substrate interaction. These results may provide new insights into the chemical space of the protein and could be crucial for developing new specific inhibitors against tuberculosis.