Functional Identification of 1-Deoxy-<scp>d</scp>-Xylulose-5-Phosphate Synthase (DXPS) as an Effective Herbicide Target for the Discovery of Novel Potent Inhibitors through Virtual Screening

1-Deoxy-d-xylulose-5-phosphate synthase (DXPS) is a key-limiting enzyme in the methylerythritol phosphate (MEP) pathway. With only two commercially available herbicides targeting DXPS, there is still a great amount of chemical space to explore new inhibitors targeting DXPS with different skeletons. In this work, we first used CRISPR/Cas9 and virus-induced gene silencing (VIGS) methods to identify the essential function of DXPS. Based on structure-based virtual screening and herbicidal activity assay, we screened two active inhibitors (<b>19</b> and <b>24)</b> with novel chemotypes. The analysis of chlorophyll content, surface plasmon resonance (SPR), and molecular docking revealed the mechanism of action of <b>19</b> and <b>24</b> targeting DXPS. In greenhouse tests, the postemergence and pre-emergence herbicidal activities of <b>24</b> (100 and 65%) and Bixlozone (100 and 61%) on BRACL (<i>Brassica chinensis</i> L.) at 750 g a.i./ha were comparable. This work provides a novel chemical framework and optimization basis for the development of herbicides based on DXPS.