Structural and interaction studies of RpiB from M. tuberculosis: screening and development of new compounds to a specific target

Tuberculosis annually affects about 10 million people worldwide and has been considered an epidemic in several countries. The treatment with old and long-term drugs has generated resistant strains.Thus, searching for new biological targets and inhibitors with high essentiality to the microorganism is paramount to finding specific drugs with low side effects. Ribose-5-phosphate isomerase (Rpi) participates in the non-oxidative branch of the pentose.This protein family has two types, and the RpiB is found in microorganisms with no homologs in humans. Therefore, M. tuberculosis RpiB was chosen as a molecular target for structural and interaction studies using NMR. Recently, our group assigned the dimeric MtRpiB, described the structural and biochemical features of this enzyme, and suggested a mechanism for the uptake of the substrate. This work aims to study the interaction of MtRpiB using Fragment-based Screening using a specific library of 768 fragments by Nuclear Magnetic Resonance (NMR) to understand and search for new compounds. MtRpiB was overexpressed in E.coli BL21(DE3) at 37oC and induced with 0.5mM of IPTG in LB and 15N isotopically labeled minimum medium and purified by affinity and molecular exclusion chromatography. The fragments library was screened using Chemical Shift Perturbation (CSP), Waterlogsy and T2 Relaxation – CPMG experiments using a Bruker AVANCE 500 MHz spectrometer . Each experiment containing pools of 12 compounds was performed free and bounded to MtRpiB using a 10x excess fragment:protein (232mM:20 mM). Positive interaction (Hit) was considered when at least two experiments showed differences between free and bound states. Preliminary results showed at least ten fragments with protein interaction. MtRpiB 15N-HSQC was performed in the presence of two fragments that showed interaction. Molecular docking studies are ongoing. These results may provide new information about the chemical space of the protein and could be essential to the development of new specific inhibitors against tuberculosis.