Screening for Small Molecules to Treat Type 2 Diabetes, Based on Disruption of the Interaction between Protein Kinase C epsilon and RACK2 — ASN Events
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Screening for Small Molecules to Treat Type 2 Diabetes, Based on Disruption of the Interaction between Protein Kinase C epsilon and RACK2 (#70)

Dale Hancock 1 , Jonathan Baell 2 , Carsten Schmitz-Peiffer 3 4
  1. School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia
  2. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Melbourne, NSW, Australia
  3. Diabetes and Metabolism Division, Garvan Institute of Medical Research, Sydney, NSW, Australia
  4. St Vincent's Clinical School, University of New South Wales, Sydney, NSW, Australia

Introduction: We and others have demonstrated causative roles for the lipid-activated kinase PKC epsilon in the generation of insulin resistance and beta cell dysfunction, making it a potential therapeutic target. However, catalytic site inhibitors of PKC isoforms show poor specificity due to the conservation of this domain between PKCs and other kinases.

Objectives: (1) To develop a screen for PKCε inhibitors based on the specific interaction of the kinase regulatory domain with its anchoring protein RACK2, which is required to target PKCε to its substrates. (2) To perform a compound library screen and further investigate selected hits.

Methods: 6xHis-tagged PKCε and biotinylated RACK2 were expressed in Sf9 insect cells and affinity-purified. An AlphaScreen bead-based proximity assay was developed to measure protein-protein interaction by chemiluminescence. Library screening was performed at the WEHI Screening Laboratory using a Known Drug boutique library (3707 compounds) and a Pilot Compound library (5120 compounds). Selected hits were reconfirmed, tested for assay interference and titrated to examine potency.

Results: Two replicate screens were performed independently at 20 µM. Compounds were defined as hits when inhibition was greater than 24.5% (3 standard deviations from the mean). 108 compounds were identified, including Known Drugs and novel compounds. Among compounds selected for further testing, a Known Drug was confirmed by re-assay, shown not to be assay-interfering and found to exhibit dose-dependent inhibition. Interestingly, though not used to treat T2D, this drug has previously been reported to increase insulin secretion and also improve insulin action.

Conclusions: This pilot screen demonstrates that the PKCε-RACK2 protein interaction can be successfully targeted to identify compounds for further development into therapeutic agents to treat T2D. Because many compound families were omitted from the pilot library, a full screen (>100,000 compounds) is required to obtain the best starting points for medicinal chemistry optimization and drug development.

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