Target Fishing\nReveals a Novel Mechanism of <i>N</i>‑Acylamino Saccharin\nDerivatives Targeting Glyceraldehyde-3-Phosphate\nDehydrogenase toward Cyanobacterial Blooms Control

Based on current challenges of poor targeting and limited\nchoices\nin chemical control methods of cyanobacterial blooms (CBs), identifying\nnew targets is an urgent and formidable task in the quest for target-based\nalgaecides. This study discovered <i>N</i>-acylamino saccharin\nderivatives exhibiting potent algicidal activity. Thus, using <i>N</i>-acylamino saccharin as the probes, glyceraldehyde-3-phosphate\ndehydrogenase from cyanobacterial (<i>Cy</i>GAPDH) was identified\nas a new target of algaecides through the activity-based protein profiling\n(ABPP) strategy for the first time. Building upon the structure of <b>Probe2</b>, a series of derivatives were designed and synthesized,\nwith compound <b>b6</b> demonstrating the most potent inhibitory\nactivity against <i>Cy</i>GAPDH and Synechocystis sp. PCC6803 (IC<sub>50</sub> = 1.67 μM and EC<sub>50</sub> = 1.15 μM). Furthermore, the potential covalent binding model\nof <b>b6</b> to the cysteine residue C154 was explored through\ncovalent possibility prediction, LC–MS experiments, substrate\ncompetitive inhibition experiments, and molecular docking. Especially,\nthe results revealed C154 as a crucial covalent binding site, with\nresidues T184 and R11 forming robust hydrophobic interactions and\nH181 establishing significant hydrogen-bonding interactions with <b>b6</b>, highlighting their potential as essential pharmacophores.\nIn summary, this study not only identifies a novel target of algaecides\nfor the control of CB but also lays the solid foundation for the development\nof targeted covalent algaecides.