Target-based chemotherapeutics development against Mycobacterium tuberculosis ABSTRACT Tuberculosis (TB) has been a global burden for thousands of years that is currently costing 1.6 million lives every year. The current therapies for TB are inadequate to control this epidemic and there is an urgent demand for the development of new effective anti-TB drugs, now more than ever, to overcome the problems of antimicrobial resistance. We have identified MraY as an attractive drug target, but one that is rather unexploited. MraY is essential for the biosynthesis of the peptidoglycan of bacterial cell walls. The limitations of current nucleoside MraY inhibitors are several: (i) poor in vivo efficacy; (ii) promiscuity (off-target activities); and (iii) accessibility only by semi-synthesis or total synthesis (>20 steps). We hypothesize that using structural insights on mechanism of MraY catalysis and inhibition, we could address those limitations and more importantly convert the existing broad-spectrum and promiscuous antibacterials to design new MraY inhibitors with narrow-spectrum activity against Mycobacterium tuberculosis (Mtb). The following aims will aid our discovery of new generations of anti-TB drugs that act through inhibition of MraY enzyme. Aim1. Develop MraY inhibitors as narrow-spectrum antibiotics against Mtb using structure-based design. We will utilize molecular modeling and docking studies on the recently published X-ray crystal structures of MraY from Aquifex aeolicus and Clostridium bolteae and design structurally simple analogues using a pharmacophore hybridization approach that will retain the key elements required for binding to the active site of MraY protein and critical for Mtb inhibitory activity. We will perform systematic structure-activity relationship studies to improve the inhibitory activity of the analogues against MraY and against replicating and multi-drug resistant Mtb strains. Aim 2. Identify new inhibitors of MraYMtb using protein-target-based virtual screening protocols. We will construct and validate homology models of MraYMtb using molecular dynamics simulations, which will be used to screen small molecule databases to identify and develop novel hits as MraY inhibitors. The outcome of these aims will establish a new strategy for TB therapy, where antibiotic resistance is impeding our ability to effectively control this epidemic. Notably, MraY is a validated target that is conserved in all bacteria and is not present in humans. Through this work, we will gain insight into how structure-based rational drug design strategies can be used effectively to convert broad-spectrum antibacterials to species-specific narrow-spectrum drug candidates and will provide a new therapeutic avenue for treatment of drug-sensitive and drug-resistant TB cases. This proposal will provide a critical step in treating other pathogenic and notoriously drug-resistant bacteria including methicillin-resistant Staphylococcus aureus, Vancomycon-resistant Enterococci, and Pseudomonas aeruginosa. The proposed research will advance the NIAID emphasis areas of basic and applied research to better understand, treat, and ultimately prevent infectious diseases.