on of GCK translocation to GKRP-positive cells. The basal nuclear localization of GCK from lot FOS was approximately three-fold higher than lot TRZ. Both lots showed modest translocation in response to glucose, with the 10 GCK/GKRP Assays number of translocation-positive cells approximately 10% greater at 25 mM glucose compared to 2.5 mM glucose. However, even at 25 mM glucose, the percentage of translocation-positive cells from lot TRZ was lower than the percentage from lot FOS under any condition tested. Lot FOS showed some evidence of translocation in response to GKA, but no translocation was observed in the presence of sorbitol. Lot TRZ demonstrated greater translocation in response to sorbitol and GKA-EMD . BAY-41-2272 site Interestingly, when the same glucose starvation and compound treatment protocol was utilized for freshly isolated human hepatocytes from a single obese female donor, GKRP expression was undetectable across all conditions tested, and GCK localization was exclusively cytoplasmic. While these results are preliminary, significant heterogeneity of GCK localization between individuals is consistent with previous reports of variation in human GCK 
localization and activity due to factors including genetic variation in GCKR and metabolic conditions such as obesity or T2D. Discussion During the past decade, human genetics studies have emphasized the critical importance of both GCK and GKRP in health and disease. Additionally, significant kinetic and structural efforts have greatly increased understanding of the properties of the interaction between GCK and GKRP, particularly in the context of pharmacological modulation of GCK. However, to date, small molecule screening efforts have primarily focused on GCK enzymatic activity, with testing of GKRP inhibition of GCK restricted to secondary validation experiments. We focused primarily on GKRP, a potentially attractive target because of its unique tissue specificity, distinct regulation from GCK, and lack of known small molecule probes other than physiological regulators such as F1P and S6P. Here, we describe three unique assays to measure the interaction between recombinant human GCK and GKRP that could be further used to characterize small molecules of interest, as we demonstrate for S6P, F1P, and previously described GKAs. All three GCK-GKRP assays developed were robust in 1536-well format, indicating they could be useful for further small-molecule high-throughput screens or as validation assays. In our screens of the LOPAC1280 library, we included S6P at PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639073 its EC80 value in all assays to bias us towards detecting potential competitors of S6P; however, each assay can also be performed in the absence of S6P. The overall hit rate was very low for these three assays, which is consistent with a recent publication by Amgen in a GCKGKRP screen in which two weak hits were identified from the library screen and subsequently optimized. Suramin and ATA were identified as inhibitors of the GCK-GKRP interaction in all three assays described herein. These compounds, however, are known to be promiscuous and were not PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19637192 pursued further. This orthogonal set of assays detects luciferase-driven luminescence, fluorescence emission at 590 nm, or time-resolved fluorescence with emission at 615 and 665 nm, minimizing detection modality overlap and the potential for false positives. Each assay type also has additional features that may be useful for specific applications. For example, the diaphorase-coupled and ADPGl
