5 We selected two closely related START domain proteins.21 StARD10 is overexpressed in some primary human breast cancers,22 and like PC-TP, it binds and transfers

phosphatidylcholines in vitro.23 StARD10 activity was inhibited by compound A1, but less effectively. StARD7 is a highly expressed protein in a choriocarcinoma cell line24 and exhibits phosphatidylcholine transfer activity.25 It was only modestly inhibited by compounds A1 and B1, although precise IC50 values for these weakly active compounds could not be quantified under conditions of the assay. These findings suggest that small molecule inhibition was at least relatively selective for the structural characteristics of PC-TP. An important unresolved question from the initial high-throughput screen20 was the mechanism of inhibition. HM781-36B in vivo The in vitro activity of PC-TP in the fluorescence quench assay reflects a multistep process20: The protein must first associate with a donor small unilamellar vesicle, exchange a phosphatidylcholine molecule, dissociate from the vesicle, associate with an acceptor small unilamellar vesicle, again exchange a phosphatidylcholine, and then dissociate. Therefore, it was possible that the small molecule inhibitors might not bind directly to PC-TP, but may have instead inserted into the membrane bilayer and

disrupted membrane association of the protein. Because such a mechanism would likely reduce the therapeutic potential of an inhibitor in vivo, we explored whether the compounds

bound directly to PC-TP. Surface plasmon resonance, Epigenetics Compound Library mw which is a sensitive biophysical technique for the measurement of ligand-protein interactions,26 revealed that inhibitors of PC-TP bound the protein with KD values that were in good agreement with respective IC50 values. In further support of an inhibitory mechanism that involved direct binding to the 上海皓元医药股份有限公司 protein, compound A1 displaced a fluorescent phosphatidylcholine analog from the PC-TP lipid binding pocket, displaying similar relative affinity for PC-TP as natural phosphatidylcholines. Compound A1 also increased the thermal stability of PC-TP, providing additional independent evidence for inhibitor-protein binding. Because of its favorable metabolic and pharmacokinetic characteristics, we selected compound A1 for in vivo testing. Consistent with a PC-TP-dependent mechanism of glucose regulation, the administration of this compound to high-fat-fed mice led to significant reductions in fasting plasma glucose concentrations and improved glucose tolerance tests for wildtype, but not Pctp−/− mice. Due to prohibitive logistical issues, we were unable to perform hyperinsulinemic euglycemic clamp studies to determine whether inhibitor treatment reduced hepatic glucose production. We therefore used pyruvate tolerance tests as a more facile surrogate measure of hepatic glucose production.