Alkyl-Branched Acyl-Lysine Peptides as Potent Cell-Permeable Slow-Substrate Inhibitors of SIRT2

Sirtuin 2 (SIRT2) is a nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylase implicated in cancer and neurodegeneration. Here, we report acyl-lysine peptide inhibitors bearing methyl-branched or cyclopropyl-modified acyl chains with low-nanomolar inhibitory potency. Their prolonged residence time and minimal catalytic turnover, confirmed by kinetic analysis, support a “slow substrate” inhibition mechanism. Ternary crystal structures of SIRT2−peptide−NAD+ complexes are consistent with a rate-limiting initial nucleophilic attack of the amide oxygen on NAD+. The inhibitors stabilize a catalytically competent yet unproductive enzyme state, distinct from thiocarbonyl inhibitors that form stalled intermediates with NAD+. Comparative structural and biochemical analyses with SIRT1 and SIRT3 reveal isoform-specific acyl chain trajectories underlying selectivity. Extension to potentially naturally occurring or drug-derived Nε-lysine modifications, i.e., phytanoyl- and ibuprofenyl-lysine, demonstrates that diverse branched acyl groups inhibit SIRT2. In cells, optimized inhibitors show low-nanomolar target engagement and induce robust α-tubulin hyperacetylation, establishing slow substrate inhibition as a strategy for selective sirtuin targeting.