Identification of Small Molecule Inhibitors against <i>Staphylococcus aureus</i> Dihydroorotase via HTS

• Main Authors: Amy J. Rice, Russell P. Pesavento, Jinhong Ren, Isoo Youn, Youngjin Kwon, Kassapa Ellepola, Chun-Tao Che, Michael E. Johnson, Hyun Lee
• Format: Article
• Language: English
• Published: MDPI AG 2021-09-01
• Series: International Journal of Molecular Sciences
• Subjects: <i>S. aureus</i>; antimicrobial; dihydroorotase; Class I DHOase; inhibitors
• Online Access: https://www.mdpi.com/1422-0067/22/18/9984

Drug-resistant <i>Staphylococcus aureus</i> is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. <i>De novo</i> 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., <i>S. aureus</i>, <i>B. anthracis</i>) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)—an integral step in the <i>de novo</i> 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 <i>S. aureus</i> DHOase (<i>Sa</i>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 <i>K</i>_D 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 <i>S. aureus</i>.