The latest technology in direct binding assays helped identify a new class of Shc-dependent insulin sensitizers.
Jennifer Gray Tomilov, PhD
November 6, 2019 – Relevant to diabetes and insulin sensitization, the repurposed drug idebenone was shown from a library of 1680 compounds to directly bind to the p52Shc target and improve insulin signaling and cytoprotection.
Alexey Tomilov, PhD, with the University of California, Davis, in Davis, CA, and colleagues published their discovery in the November 2018 issue of Pharmacological Research.
According to researchers, “p52Shc is a signaling molecule that interacts directly with insulin receptor (IR) and competes for the same phosphotyrosine on the insulin receptor as IRS1.” Further, “a drug that blocks p52Shc binding to IR should allow greater access of IRS1 to the phosphotyrosine and a stronger insulin response per molecule of insulin bound.”
In an effort to identify a drug that disrupts the interaction between the inhibitory p52Shc and IR, the group first conducted a high-throughput screen (HTS) of 1680 small molecule drugs previously approved for human use. Drugs were applied to the mouse liver cell line FL83B, and in-cell western blotting with anti-phospho (Ser 473) Akt and anti-tubulin antibodies was utilized to detect phospho-Akt in the presence or absence of insulin. A total of 103 molecules passed this initial screening to become metabolic insulin sensitizer candidates, and 30 of these drugs were selected for downstream studies.
Notably, the team then utilized a real-time binding kinetics assay, Bio-Layer Interferometry (BLI), to determine the binding parameters of the 30 insulin sensitizer candidates for direct binding to the Phospho Tyrosine Binding domain (PTB) of p52Shc. Eleven compounds bound to the PTB domain of Shc with a response ratio of >1 when compared with binding to the mutated PTB Shc domain. Idebenone was selected as one of 11 compounds with binding to the PTB domain with an affinity greater than 5μM, having a binding affinity to the p52Shc PTB domain of ~100nM.
Real-time binding kinetics assays were additionally utilized in a follow-up target engagement assay. Idebenone was shown to block the interaction of the PTB region with the phosphopeptide NPEYp motif of IR in a dose-dependent manner. Inhibition of the interaction between p52Shc and NPEYp had an IC50 = 1.0E-7M. Co-immunoprecipitation studies validated the results seen with BLI, indicating that idebenone reduced the insulin-dependent association of IR with Shc.
Translating the direct binding study data to meaningful effects on physiology, researchers noted that the affinity by which idebenone bound to the Shc-PTB domain corresponded with its insulin sensitizing and cytoprotective effects in FL83B liver cell models.
Strikingly, idebenone also increased insulin sensitivity in vivo, except in Shc-deficient mice. Wild-type mice treated with insulin injection showed a decrease in blood glucose levels to 65% of initial levels, and idebenone-treated mice showed blood glucose levels decreased to 45% of initial levels. These results are in contrast with Shc knock-down (ShcKD) mice, which illustrated no insulin sensitivity increase upon idebenone administration.
“Type 2 diabetes affects ~30M in the US and more than 100M worldwide and is rapidly rising. New drug targets with new mechanisms of action are urgently needed”, stated Dr. Tomilov. He went on to explain, “we observed idebenone to increase insulin sensitivity in the context of two models of insulin resistance, and we show idebenone induces insulin sensitivity through blocking Shc’s interaction with insulin receptor.”
This study was funded by the National Institutes of Health.
Pharmacological Research. Published November 2018. Full text may be found here.