Center for Biomolecular Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60607, USA.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA.
Int J Mol Sci. 2021 Sep 15;22(18):9984. doi: 10.3390/ijms22189984.
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 .
耐药性是对公众健康的一个迫在眉睫的威胁，因此利用未开发的细菌途径和酶靶标进行药物发现变得尤为重要。嘧啶生物合成是一种专门的、高度保守的途径，与几种临床相关病原体的存活和毒力都有关。I 类二氢乳清酸酶（DHOase）是一种存在于革兰氏阳性菌（即 ， ）中的独立而独特的酶，它将氨甲酰天冬氨酸（Ca-asp）转化为二氢乳清酸（DHO）——嘧啶生物合成途径中的一个重要步骤。本研究通过基于比色法的酶测定法对 3000 个片段化合物进行了高通量筛选（HTS），作为初步筛选，鉴定出 DHOase（DHOase）的小分子抑制剂，然后使用表面等离子体共振（SPR）进行直接结合分析对命中物进行验证。六种命中化合物和另外八种类似物与底物 Ca-asp 的竞争 SPR 研究确定了最佳化合物是具有 值为 11 µM 的竞争性抑制剂，比 Ca-asp 紧密 10 倍。初步的结构-活性关系（SAR）为基于结构的抗菌抑制剂设计提供了基础，以对抗 。
