Identification of Small Molecule Inhibitors against Dihydroorotase via HTS.

Drug-resistant is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. 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., , ) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)-an integral step in the pyrimidine biosynthesis 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 DHOase (DHOase), 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 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 inhibitor design against .