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Targeted protein degradation (TPD) through a proteasome-dependent pathway induced by heterofunctional small molecules is initiated by the formation of a ternary complex with recruited E3 ligases. This complex formation affects the degradation ability of TPD molecules, and thus we tested for visualization of the intracellular dynamics of ternary complex formation. In this study, we applied the fluorescent-based technology detecting protein-protein interaction (Fluoppi) system, in which detectable fluorescent foci are formed when ternary complex formation induced by TPD molecules occurs in cells. We show here that cells coexpressing BRD4 and cereblon (CRBN) tagged with the Fluoppi system formed detectable foci in both live and fixed cells only when treated with BRD4-targeting degraders utilizing CRBN as an E3 ligase in dose- and time-dependent manners. Notably, the maintenance and efficacy of TPD molecule-induced foci formation correlated with the ability to degrade target proteins. Furthermore, we demonstrated that BRD4-targeting and FKBP12F36V-targeting degraders formed ternary complexes mainly in the nucleus and cytoplasm, respectively, suggesting that TPD molecules utilize the proteasome to degrade target proteins in their corresponding localized region. Our results also suggest that the Fluoppi system is a powerful tool for characterizing TPD molecules by visualizing the spatiotemporal formation of ternary complex.

To obtain compounds that promote glucose uptake in muscle cells, the novel cell lines A31-IS derived from Balb/c 3T3 A31 and C2C12-IS from mouse myoblast C2C12 were established. In both cell lines, glucose consumption was induced by insulin and suppressed by the addition of Akt-activating kinase inhibitor. The A31-IS cells highly express the insulin receptor β chains, Glut4, and uncoupling protein-3, as compared to the parent Balb /c 3T3 A31 cells, and C2C12-IS cells highly express the insulin receptor β chain as compared to its parent cell line. Using A31-IS cells, we screened our library compounds and obtained three compounds, DF-4394, DF-4451, and DG-5451. These compounds dose-dependently promoted glucose consumption in A31-IS cells and facilitated [3H]-2-deoxyglucose uptake in differentiated C2C12-IS cells. The compounds that we obtained from the library screening will be good candidates for improving insulin resistance in muscle cells. Keywords:: skeletal muscle cell, insulin, glucose uptake

To obtain compounds that inhibit the interaction of von Willebrand factor (vWF) and glycoprotein (GP) Ib, a novel binding assay was established. The binding of fixed platelets to vWF-R497 mutant was quantified by a solid phase assay. In this assay, fixed platelets bound to the vWF-R497 mutant, carrying the deletion of Glu497-Tyr508 and the missense mutation of Arg545 to Ala, without binding modulators such as ristocetin. The Kd value of the binding was 2.8 nM, which was consistent with the result from liquid binding assay. The binding was inhibited by aurin tricarboxylic acid (ATA) and an anti GPIb antibody, AK2. Using this binding assay, we screened our library compounds and obtained D74-3736. This compound also inhibited ristocetin-induced platelet aggregation in the human platelet-rich plasma. Keywords:: biotinylation of fixed platelets, binding assay of von Willebrand factor (vWF) to glycoprotein Ib (GPIb)