Protein S-palmitoylation is markedly inhibited by 4″-alkyl ether lipophilic derivatives of EGCG, the major green tea polyphenol: In vitro and in silico studies

S-palmitoylation is a dynamic lipid-based protein post-translational modification facilitated by a family of protein acyltransferases (PATs) commonly known as DHHC-PATs or DHHCs. It is the only lipid modification that is reversible, and this very fact uniquely qualifies it for therapeutic interventions through the development of DHHC inhibitors. Herein, we report that 4″-alkyl ether lipophilic derivatives of EGCG can effectively inhibit protein S-palmitoylation in vitro. With the help of metabolic labeling followed by copper(I)-catalyzed azide-alkyne cycloaddition Click reaction, we demonstrate that 4″-C₁₄ EGCG and 4″-C₁₆ EGCG markedly inhibited S-palmitoylation in various mammalian cells including HEK 293T, HeLa, and MCF-7 using both in gel fluorescence as well as confocal microscopy. Further, these EGCG derivatives were able to attenuate the S-palmitoylation to the basal level in DHHC3-overexpressed cells, suggesting that they are plausibly targeting DHHCs. Confocal microscopy data qualitatively reflected spatial and temporal distribution of S-palmitoylated proteins in different sub-cellular compartments and the inhibitory effects of 4″-C₁₄ EGCG and 4″-C₁₆ EGCG were clearly observed in the native cellular environment. Our findings were further substantiated by in silico analysis which revealed promising binding affinity and interactions of 4″-C₁₄ EGCG and 4″-C₁₆ EGCG with key amino acid residues present in the hydrophobic cleft of the DHHC20 enzyme. We also demonstrated the successful inhibition of S-palmitoylation of GAPDH by 4″-C₁₆ EGCG. Taken together, our in vitro and in silico data strongly suggest that 4″-C₁₄ EGCG and 4″-C₁₆ EGCG can act as potent inhibitors for S-palmitoylation and can be employed as a complementary tool to investigate S-palmitoylation.