ABSTRACT
Elucidating the role of secreted frizzled-related protein 5 (SFRP5) in metabolism and obesity has been complicated by contradictory findings when knockout mice were used to determine metabolic phenotypes. By overexpressing SFRP5 in obese, prediabetic mice we consistently observed elevated hyperglycemia and glucose intolerance, supporting SFRP5 as a negative regulator of glucose metabolism. Accordingly, Sfrp5 mRNA expression analysis of both epididymal and subcutaneous adipose depots of mice indicated a correlation with obesity. Thus, we generated a monoclonal antibody (mAb) against SFRP5 to ascertain the effect of SFRP5 inhibition in vivo. Congruent with SFRP5 overexpression worsening blood glucose levels and glucose intolerance, anti-SFRP5 mAb therapy improved these phenotypes in vivo. The results from both the overexpression and mAb inhibition studies suggest a role for SFRP5 in glucose metabolism and pancreatic ß-cell function and thus establish the use of an anti-SFRP5 mAb as a potential approach to treat type 2 diabetes.
Subject(s)
Glucose/metabolism , Insulin-Secreting Cells/metabolism , Intercellular Signaling Peptides and Proteins/physiology , Adaptor Proteins, Signal Transducing , Animals , Antibodies, Monoclonal/immunology , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Immunoglobulin G/immunology , Insulin-Secreting Cells/drug effects , Intercellular Signaling Peptides and Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Obesity/complications , Obesity/genetics , Obesity/metabolismABSTRACT
OBJECTIVE: Although the human genome encodes â¼ 20,000 protein-coding genes, only a very small fraction of these have been explored as potential targets for therapeutic development. The challenge of identifying and validating new protein targets has contributed to the significant reduction in the productivity of the pharmaceutical industry in the recent decade, highlighting the continued need to find new therapeutic targets. RESEARCH DESIGN AND METHODS: The traditional methods to discover new targets are expensive, low throughput and time consuming, usually taking years to validate or invalidate a target. To address these limitations, as a proof of concept, we explored the hydrodynamic tail vein (HTV) injection as a gene delivery method for direct in vivo phenotypic screening of novel secreted factor targets for Type II diabetes therapeutics. RESULTS: High levels and sustained expression of target proteins were observed in diabetic mouse models tested, allowing us to identify multiple novel hormones that may regulate glucose metabolism. CONCLUSIONS: These results suggest that HTV is a low-cost, high-throughput method for direct in vivo phenotypic drug screening in metabolic disorders and could be applicable to many other disease areas as well. This method if combined with other approaches such as human genetic studies could provide a significant value to future drug discovery.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Hypoglycemic Agents/pharmacology , Proteome , Animals , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 2/physiopathology , Drug Discovery/methods , Gene Transfer Techniques , Glucose/metabolism , High-Throughput Screening Assays/methods , Humans , Hydrodynamics , Injections, Intravenous , Male , Mice , Mice, Inbred C57BL , Phenotype , Tail/blood supplyABSTRACT
Organic small molecules generally act by perturbing the function of one or more cellular target proteins, the identification of which is essential to an understanding of the molecular basis of drug action. Here we describe the application of methotrexate-linked small molecule ligands to a mammalian three-hybrid interaction trap for proteome-wide identification of small molecule targets, quantification of the targeting potency of unmodified small molecules for such targets in intact cells, and screening for inhibitors of small molecule-protein interactions. During the course of this study we also identified the pyrido[2,3-d]pyrimidine PD173955, a known SRC kinase inhibitor, as a potent inhibitor of several ephrin receptor tyrosine kinases. This finding could perhaps be exploited in the design of inhibitors for this kinase subfamily, members of which have been implicated in the pathogenesis of various diseases, including cancer.