Figure 6 - uploaded by Amy Barrett
Content may be subject to copyright.
Localization of GCK in cryopreserved hepatocytes. Overlay of GCK (FITC channel; green) localization with Hoechst nuclear stain (blue) in cryopreserved hepatocytes from images collected at 10X magnification. The contrast was held fixed for all images, and the full dynamic range of all the 12 bit images has been maintained. (A-C) GCK localization in male Sprague-Dawley rat hepatocytes at (A) 2.5 mM glucose, (B) 16.7 mM glucose, and (C) 2.5 mM glucose and 31.6 mM GKA-EMD. (D-H) GCK localization in cryopreserved human hepatocytes. (D) Translocation of GCK in GKRP-positive cells in two separate lots (TRZ: closed symbols; FOS: open symbols) of human hepatocytes at various concentrations of glucose. (E-F) Translocation of GCK in the presence of GKA-EMD at various concentrations of glucose for donor TRZ and FOS, respectively. (G-H) Representative images from donor TRZ at (G) 2.5 mM glucose and (H) 2.5 mM glucose with 31.6 mM GKA. Data points are mean 6 SEM for n = 4-8. doi:10.1371/journal.pone.0089335.g006
Source publication
Recent genetic and clinical evidence has implicated glucokinase regulatory protein (GKRP) in the pathogenesis of type 2 diabetes and related traits. The primary role of GKRP is to bind and inhibit hepatic glucokinase (GCK), a critically important protein in human health and disease that exerts a significant degree of control over glucose metabolism...
Citations
... The NADH produced is then detected using the diaphorase/resazurin system. 17 In each of the three HTS cases mentioned above, orthogonal assays with HTS hits screened against the diaphorase/resazurin alone were needed to triage false-positive compounds that inhibited/interfered with the reporter assay. While few compounds in a high-quality screening library would share resorufin's optical properties, this can be easily assessed when following up on hits by performing a ''preread'' after pintransferring compound, but before initiating the biochemical reaction. ...
Fluorescence is utilized as the output for a range of assay formats used in high-throughput screening (HTS). Interference with these assays from the compounds in libraries utilized in HTS is a well-recognized phenomenon, particularly for assays relying on UV excitation such as for direct detection of the oxidoreductase cofactors NADH or NADPH. In this study, we discuss these interference challenges and highlight the specific case of the diaphorase/resazurin system that can be coupled to enzymes utilizing NADH or NADPH. We review the utilization of this assay system in the literature and argue that the diaphorase/resazurin system is underutilized in assay development. It is the authors' hope that this Perspective and the accompanying Technical Brief in this issue will stimulate interest in a robust and sensitive coupling system to avoid assay fluorescence interference.
... The ADP-Glo™ technology was applied to three classes of kinases here indicating the broad utility of this assay methodology: a lipid kinase PI5P4Kα [ 18 ], a tyrosine kinase Yes1 [ 19 ], and a metabolic kinase glucokinase (GCK) [ 20 ] . The primary goal was to identify inhibitors of PI5P4Kα and Yes1 for the potential use as anticancer agents and activators of GCK for the potential treatment of diabetes. ...
... Fig. 3 Schematic of the ADP-Glo™ assay for glucokinase: This assay monitors glucokinase activity directly but also indirectly monitors the protein-protein interaction of glucokinase with glucokinase regulatory protein. A small molecule that disrupts the interaction of glucokinase with glucokinase regulatory protein would lead to an increase in observed luminescence [ 20 ] 13. Read the luminescence signal with a ViewLux with a 1-s exposure. ...
... 2. For the GCK assay, the average Z ′ = 0.7, CV = 4.2 %, and S/B = 4.2 [ 20 ]. Data were deposited in PubChem AID 743206. ...
Assays in which the detection of a biological phenomenon is coupled to the production of bioluminescence by luciferase have gained widespread use. As firefly luciferases (FLuc) and kinases share a common substrate (ATP), coupling of a kinase to FLuc allows for the amount of ATP remaining following a kinase reaction to be assessed by quantitating the amount of luminescence produced. Alternatively, the amount of ADP produced by the kinase reaction can be coupled to FLuc through a two-step process. This chapter describes the bioluminescent assays that were developed for three classes of kinases (lipid, protein, and metabolic kinases) and miniaturized to 1536-well format, enabling their use for quantitative high-throughput (qHTS) of small-molecule libraries.
... Our results suggest that indirect activation of hepatic GCK via loss-of-function GCKR mutations does not consistently lead to a hyperlipidemic phenotype, and are particularly relevant to the recent report of a selective small-molecule inhibitor of GKRP, which was shown to disrupt the GCK-GKRP interaction in vivo and reduced blood glucose levels in mouse models of obesity without apparent adverse effects on triglyceride levels (25). Continued exploration of the combination of genetic and clinical data with highly sensitive biochemical, cellbiological and animal model systems may provide further insight into the effects of pharmacological and genetic modulation of GKRP (25)(26)(27). ...
Significant resources have been invested in sequencing studies to investigate the role of rare variants in complex disease
etiology. However, the diagnostic interpretation of individual rare variants remains a major challenge, and may require accurate
variant functional classification and the collection of large numbers of variant carriers. Utilizing sequence data from 458
individuals with hypertriglyceridemia and 333 controls with normal plasma triglyceride levels, we investigated these issues
using GCKR, encoding glucokinase regulatory protein. Eighteen rare non-synonymous GCKR variants identified in these 791 individuals were comprehensively characterized by a range of biochemical and cell biological
assays, including a novel high-throughput-screening-based approach capable of measuring all variant proteins simultaneously.
Functionally deleterious variants were collectively associated with hypertriglyceridemia, but a range of in silico prediction algorithms showed little consistency between algorithms and poor agreement with functional data. We extended our
study by obtaining sequence data on family members; however, functional variants did not co-segregate with triglyceride levels.
Therefore, despite evidence for their collective functional and clinical relevance, our results emphasize the low predictive
value of rare GCKR variants in individuals and the complex heritability of lipid traits.
Monogenic diabetes includes several clinical conditions generally characterized by early-onset diabetes, such as neonatal diabetes, maturity-onset diabetes of the young (MODY) and various diabetes-associated syndromes. However, patients with apparent type 2 diabetes mellitus may actually have monogenic diabetes. Indeed, the same monogenic diabetes gene can contribute to different forms of diabetes with early or late onset, depending on the functional impact of the variant, and the same pathogenic variant can produce variable diabetes phenotypes, even in the same family. Monogenic diabetes is mostly caused by impaired function or development of pancreatic islets, with defective insulin secretion in the absence of obesity. The most prevalent form of monogenic diabetes is MODY, which may account for 0.5–5% of patients diagnosed with non-autoimmune diabetes but is probably underdiagnosed owing to insufficient genetic testing. Most patients with neonatal diabetes or MODY have autosomal dominant diabetes. More than 40 subtypes of monogenic diabetes have been identified to date, the most prevalent being deficiencies of GCK and HNF1A. Precision medicine approaches (including specific treatments for hyperglycaemia, monitoring associated extra-pancreatic phenotypes and/or following up clinical trajectories, especially during pregnancy) are available for some forms of monogenic diabetes (including GCK- and HNF1A-diabetes) and increase patients’ quality of life. Next-generation sequencing has made genetic diagnosis affordable, enabling effective genomic medicine in monogenic diabetes. Monogenic diabetes encompasses forms of diabetes that result from a single pathogenic genetic alteration and usually have an early onset. This Primer gives an overview of the epidemiology, pathophysiology, diagnosis and treatment of these conditions, as well as patient quality of life and open research questions.
Genome-wide association studies (GWAS) have generated considerable interest in glucokinase regulatory protein (GKRP; gene name GCKR) which is an inhibitor of hepatic glucokinase (GCK), an enzyme that plays a critical role in glucose update and disposal in liver. From the initial discovery of GCKR variants associated with triglyceride and glucose levels through the identification of pleiotropic associations with a wide variety of metabolic phenotypes, we have learned a great deal about the importance of GKRP as a critical node in hepatic metabolism. GKRP remains one of the few well-studied GWAS loci where attempts have been made to understand the functional as well as the phenotypic impact of genetic variants across the allelic spectrum. Given the interest in developing liver-specific glucokinase activators and small molecules which disrupt the GKRP:GCK interaction for the treatment of type 2 diabetes, these genetic insights provide a wealth of information regarding efficacy and potential adverse on-target effects in humans.
The cell cycle is a fundamental process of cell biology, and its progression is highly regulated. A critical mode of regulation for proper advancement of the cell cycle is the activation of the CDKs by the CDC25 family of dual specificity phosphatases. The CDC25 proteins are often overexpressed or misregulated in cancer, resulting in dysregulated cell growth, genomic instability and evasion of apoptosis. The oncogenic role of the CDC25 proteins has inspired over two decades of drug discovery efforts to inhibit their enzymatic activity. Despite these efforts, no therapeutic agents targeting family of CDC25 phosphatases emerged. In order to identify new classes of CDC25B inhibitors, new approaches to target CDC25 are needed. We have employed a novel approach to inhibit the CDC25 family member CDC25B by targeting its interaction with its native substrate, the CDK2/Cyclin A complex. We used two different methods, fragment-based drug discovery and ???gray-box??? high-throughput screening, to identify inhibitors of the CDC25B-CDK2/Cyclin A protein-protein interaction. Using NMR- based fragment based screening, we identified a small molecule ligand of the CDC25B catalytic domain. We solved the co-crystal structure with this ligand bound to CDC25B, and used this structure to develop more potent analogs. We have shown that fragment-derived compounds can disrupt the CDC25B-CDK2/Cyclin A interaction and inhibit CDC25B catalytic activity. To our knowledge, our inhibitor-bound crystal structure of CDC25B is the first crystal structure with CDC25B bound to a small molecule ligand. We have also developed several protein-protein interaction assays to quantify the interaction between CDC25B and CDK2/Cyclin A. We employed these assays in three high- throughput screens to identify several classes of CDC25B-CDK2/Cyclin A protein-protein interaction inhibitors. The inhibitors we identified do not target CDC25B, but disrupt the protein- protein interaction by targeting CDK2/Cyclin A. Importantly, we have developed a high quality screening assay for the identification of CDC25B-CDK2/Cyclin A interaction inhibitors. This assay will be useful for future drug discovery efforts targeting the CDC25B-CDK2/Cyclin A interaction. In summary, we have developed two new approaches to inhibit CDC25B. These results pave the way towards the development of new chemical probes and potential therapeutic agents targeting CDC25B.
