The Covalent Binding of Splicing Inhibitors occurs via a Zinc- Assisted Enzymatic-like mechanism

Drugs modulating pre-mRNA splicing hold anticancer therapeutic potential by compensating splicing defects and rebalancing gene expression. Spliceostatins are potent antitumor agents that inhibit splicing by covalently binding to the SF3b complex via an elusive mechanism. Here, multi-scale all-atom simulations resolved the molecular mechanism underlying the covalent linkage of spliceostatin A (SSA). We uncover that SSA binds to SF3b through a metal-assisted enzymatic-like reaction, with specific binding site residues and a zinc finger ion actively promoting the chemical process. Namely, while the zinc ion activates the Cys-S nucleophile, a flanking Lys-Asp residues pair cooperatively activates the SSA epoxy warhead, increasing its susceptibility to be attacked by Cys-S nucleophile. A bioinformatic analysis of the Protein Data Bank revealed that zinc finger motifs flanked by nearby acidic residues are present in other spliceosome proteins, suggesting that SSA-like inhibitors could be designed for other zinc finger-containing targets. Our findings carry important implications for future drug design campaigns.