Target Fishing Reveals a Novel Mechanism of <i>N</i>-Acylamino Saccharin Derivatives Targeting Glyceraldehyde-3-Phosphate Dehydrogenase toward Cyanobacterial Blooms Control

Abstract

Based on current challenges of poor targeting and limited choices in chemical control methods of cyanobacterial blooms (CBs), identifying new targets is an urgent and formidable task in the quest for target-based algaecides. This study discovered <i>N</i>-acylamino saccharin derivatives exhibiting potent algicidal activity. Thus, using <i>N</i>-acylamino saccharin as the probes, glyceraldehyde-3-phosphate dehydrogenase from cyanobacterial (<i>Cy</i>GAPDH) was identified as a new target of algaecides through the activity-based protein profiling (ABPP) strategy for the first time. Building upon the structure of <b>Probe2</b>, a series of derivatives were designed and synthesized, with compound <b>b6</b> demonstrating the most potent inhibitory activity against <i>Cy</i>GAPDH and <i>Synechocystis</i> sp. PCC6803 (IC₅₀ = 1.67 μM and EC₅₀ = 1.15 μM). Furthermore, the potential covalent binding model of <b>b6</b> to the cysteine residue C154 was explored through covalent possibility prediction, LC-MS experiments, substrate competitive inhibition experiments, and molecular docking. Especially, the results revealed C154 as a crucial covalent binding site, with residues T184 and R11 forming robust hydrophobic interactions and H181 establishing significant hydrogen-bonding interactions with <b>b6</b>, highlighting their potential as essential pharmacophores. In summary, this study not only identifies a novel target of algaecides for the control of CB but also lays the solid foundation for the development of targeted covalent algaecides.