Chemical Proteomics Reveals a Novel Long-Acting Maleimide Algaecide Targeting Glyceraldehyde-3-Phosphate Dehydrogenase for Cyanobacterial Bloom Control

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 EC 50 values of 0.85 and 0.66 μM, respectively, and sustained inhibitory activity lasting up to 60 days, significantly outperforming conventional algicides such as CuSO 4 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 ( Cy GAPDH) 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 Cy GAPDH, thereby inhibiting enzyme activity. These findings establish a promising scaffold and provide a framework for target-driven algaecide design.