Chemical Proteomics Reveals a Novel Long-Acting Maleimide Algaecide Targeting Glyceraldehyde-3-Phosphate...

Cyanobacterial blooms pose severe threats to aquatic ecosystems and public health, driving an urgent need for targeted and environmentally benign control agents. Herein, we report a series of maleamide-based algaecides developed through phenotype-guided structural optimization. Among them, compound A4 exhibited exceptional potency against Synechocystis sp. PCC 6803 and Microcystis aeruginosa FACHB 905, with EC₅₀ values of 0.85 and 0.66 μM, respectively, and sustained inhibitory activity lasting up to 60 days, significantly outperforming conventional algicides such as CuSO₄ and prometryn. Zebrafish assays indicated low acute toxicity, supporting a favorable biosafety profile. Employing activity-based protein profiling, LC-MS/MS, and overexpression assays, cyanobacterial glyceraldehyde-3-phosphate dehydrogenase (CyGAPDH) was identified as a major target. Mechanistic investigations, integrating enzymatic assays, site-directed mutagenesis, and theoretical calculation, revealed that A4 covalently modifies the catalytic cysteine residue (C154) of CyGAPDH, thereby inhibiting enzyme activity. These findings establish a promising scaffold and provide a framework for target-driven algaecide design.