Identification of small molecule inhibitors against staphylococcus aureus dihydroorotase via hts

Abstract

Drug‐resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts car-bamoyl‐aspartate (Ca‐asp) to dihydroorotate (DHO)—an integral step in the de novo pyrimidine bi-osynthesis pathway. This study sets forth a high‐throughput screening (HTS) of 3000 fragment compounds by a colorimetry‐based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca‐asp determined the best compound to be a competitive inhibitor with a KD value of 11 μM, which is 10‐fold tighter than Ca‐asp. Preliminary structure– activity relationship (SAR) provides the foundation for further structure‐based antimicrobial inhib-itor design against S. aureus.