Molecular mechanism of SIRT1 activation by polymethoxyflavonoids derived from citrus peels

Sirtuin 1 (SIRT1) activation by small molecules is linked to aging-related health benefits. We investigated how citrus peel–derived polymethoxyflavonoids (PMFs), 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF) and 5,6,7,8,3′,4′-hexamethoxyflavone (nobiletin), activate SIRT1. Both compounds modestly but reproducibly increased deacetylase activity toward acetylated p53 and PGC-1α peptides. HMF and nobiletin significantly increased catalytic efficiency ( k cat / K m ) toward both substrates, whereas K m decreased significantly only for the PGC-1α peptide. Residue-resolved NMR analyses localized the PMF-responsive surface to the α2–loop–α3 region of the SIRT1 N-terminal domain (NTD), with overlapping perturbations at T209, E214, L220, I223, and V224. Tryptophan fluorescence-quenching measurements provided complementary evidence. Docking and molecular-dynamics simulations showed that both PMFs remained on this NTD surface while adopting multiple binding modes. Integrative models combining AlphaFold 3–derived SIRT1–substrate complexes with MD-derived PMF poses suggest that NTD-bound PMFs may stabilize a catalytically favorable SIRT1–substrate state, providing molecular insight into SIRT1 activation by citrus-derived PMFs.