Novel molecular hybrids of EGCG and quinoxaline: Potent multi-targeting antidiabetic agents that inhibit α-glucosidase, α-amylase, and oxidative stress.

Diabetes mellitus is a multifactorial disease and its effective therapy often demands several drugs with different modes of action. Herein, we report a rational design and synthesis of multi-targeting novel molecular hybrids comprised of EGCG and quinoxaline derivatives that can effectively inhibit α-glucosidase, α-amylase as well as control oxidative stress by scavenging ROS. The hybrids showed superior inhibition of α-glucosidase along with similar α-amylase inhibition as compared to standard drug, acarbose. Most potent compound, 15c showed an IC50 of 0.50 µM (IC50 of acarbose 190 µM) against α-glucosidase. Kinetics studies with 15c revealed a competitive inhibition against α-glucosidase. Binding affinity of 15c (-9.5 kcal/mol) towards α-glucosidase was significantly higher than acarbose (-7.7 kcal/mol). 15c exhibited remarkably high antioxidant activity (IC50 = 18.84 µM), much better than vitamin C (IC50 = 33.04 µM). Of note, acarbose shows no antioxidant activity. Furthermore, α-amylase activity was effectively inhibited by 15c with an IC50 value of 16.35 µM. No cytotoxicity was observed for 15c (up to 40 µM) in MCF-7 cells. Taken together, we report a series of multi-targeting molecular hybrids capable of inhibiting carbohydrate hydrolysing enzymes as well as reducing oxidative stress, thus representing an advancement towards effective and novel therapeutic approaches for diabetes.

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.