Artificial Genetically Encoded Peptides and Proteins as Next-Generation Therapeutics: Selection ofligands for Mycobacterium tuberculosis UDP-Galactopyranose Mutase as Potential Inhibitors.

Peptides and small proteins provide a dynamic platform for drug discovery and therapeutics. They have a wide range of applications including inhibition of protein-protein interaction, inhibition of transporter and enzyme activity, imaging, and as co-crystallisation ligand for structural studies. In this study, we employed in vitro selection of ligands, using mRNA display, from an artificial genetically encoded library of peptides/proteins to identify candidates that bind the enzyme Mycobacterium tuberculosis UDPGalactopyranose mutase (MtUGM). The enzyme catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf), which is then assembled as the galactofuran layer in Mycobacterium tuberculosis (Mtb) cell wall. Cell wall biosynthesis is essential for Mtb survival and pathogenicity, deletion in genes involved in this process have proven lethal. We successfully identified macrocyclic peptides (MCPs) and affibodies specific for MtUGM from a library with a diversity >1012 clones through random non-standard peptide integrated discovery (RaPID) system and mRNA display, respectively. Enrichment of positive binders was observed for both selection processes suggesting binding specificity. Previous studies reveal that natural product-like MCPs can inhibit enzyme activity; their small size, conformational stability and high affinity for the target makes them attractive ligands. Thus, we hypothesize that identified MCPs may show inhibition of MtUGM thereby halting cell wall biosynthesis. Discovered affibodies will be used for structural analysis of MtUGM. The World Health Organisation (WHO) reported tuberculosis (TB) caused by Mtb to be one of the leading causes of death worldwide, with increasing incidences of drug resistant strains necessitating discovery of novel therapeutics. Through our study, we present a novel approach for discovering peptide/protein-based ligands for MtUGM and hope to develop an assay for screening MCPs for potential inhibitors.