ABSTRACT
Although it is increasingly being recognized that drug-target interaction networks can be powerful tools for the interrogation of systems biology and the rational design of multitargeted drugs, there is no generalized, statistically validated approach to harmonizing sequence-dependent and pharmacology-dependent networks. Here we demonstrate the creation of a comprehensive kinome interaction network based not only on sequence comparisons but also on multiple pharmacology parameters derived from activity profiling data. The framework described for statistical interpretation of these network connections also enables rigorous investigation of chemotype-specific interaction networks, which is critical for multitargeted drug design.
Subject(s)
Pharmacogenetics/methods , Protein Kinases/metabolism , Proteome/antagonists & inhibitors , Proteome/metabolism , Drug Design , Proteome/analysis , Systems Biology/methodsABSTRACT
AMP-activated protein kinase (AMPK) is a key sensor and regulator of intracellular and whole-body energy metabolism. We have identified a thienopyridone family of AMPK activators. A-769662 directly stimulated partially purified rat liver AMPK (EC50 = 0.8 microM) and inhibited fatty acid synthesis in primary rat hepatocytes (IC50 = 3.2 microM). Short-term treatment of normal Sprague Dawley rats with A-769662 decreased liver malonyl CoA levels and the respiratory exchange ratio, VCO2/VO2, indicating an increased rate of whole-body fatty acid oxidation. Treatment of ob/ob mice with 30 mg/kg b.i.d. A-769662 decreased hepatic expression of PEPCK, G6Pase, and FAS, lowered plasma glucose by 40%, reduced body weight gain and significantly decreased both plasma and liver triglyceride levels. These results demonstrate that small molecule-mediated activation of AMPK in vivo is feasible and represents a promising approach for the treatment of type 2 diabetes and the metabolic syndrome.
Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Enzyme Activators/chemistry , Enzyme Activators/therapeutic use , Metabolic Syndrome/drug therapy , Multienzyme Complexes/metabolism , Protein Serine-Threonine Kinases/metabolism , Pyrones/chemistry , Pyrones/therapeutic use , Thiophenes/chemistry , Thiophenes/therapeutic use , AMP-Activated Protein Kinases , Animals , Biphenyl Compounds , Cell Line , Diabetes Mellitus, Type 2/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Activators/pharmacology , Fatty Acid Synthases/drug effects , Fatty Acid Synthases/genetics , Fatty Acid Synthases/metabolism , Glucose-6-Phosphatase/drug effects , Glucose-6-Phosphatase/genetics , Glucose-6-Phosphatase/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , In Vitro Techniques , Metabolic Syndrome/metabolism , Metformin/chemistry , Metformin/pharmacology , Metformin/therapeutic use , Mice , Mice, Obese , Molecular Weight , Multienzyme Complexes/drug effects , Phosphoenolpyruvate Carboxykinase (GTP)/drug effects , Phosphoenolpyruvate Carboxykinase (GTP)/genetics , Phosphoenolpyruvate Carboxykinase (GTP)/metabolism , Protein Serine-Threonine Kinases/drug effects , Pyrones/pharmacology , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Thiophenes/pharmacologyABSTRACT
Herein we describe the identification and characterization of a class of molecules that are believed to extend into a region of p38 known as the 'switch pocket'. Although these molecules lack a canonical hinge binding motif, they show K(i) values as low as 100 nM against p38. We show that molecules that interact with this region of the protein demonstrate different binding kinetics than a canonical ATP mimetic, as well as a wide range of kinome profiles. Thus, the switch pocket presents new opportunities for kinome selectivity which could result in unique biochemical responses and offer new opportunities in the field of kinase drug discovery.
Subject(s)
Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Adenosine Triphosphate/chemistry , Binding Sites , Computer Simulation , Crystallography, X-Ray , Fluorescence Resonance Energy Transfer , Kinetics , Mitogen-Activated Protein Kinase 14/metabolism , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
A novel and innovative high-throughput screening assay was developed to identify both activators and inhibitors of AMP-activated protein kinase (AMPK) using microarrayed compound screening (microARCS) technology. Test compounds were arrayed at a density of 8640 on a polystyrene sheet, and the enzyme and peptide substrate were introduced into the assay by incorporating them into an agarose gel followed by placement of the gels onto the compound sheet. Adenosine triphosphate (ATP) was delivered via a membrane, and the phosphorylated biotinylated substrate was captured onto a streptavidin affinity membrane (SAM trade mark ). For detection, the SAM trade mark was removed, washed, and imaged on a phosphor screen overnight. A library of more than 700,000 compounds was screened using this format to identify novel activators and inhibitors of AMPK.
Subject(s)
Multienzyme Complexes/antagonists & inhibitors , Oligonucleotide Array Sequence Analysis/methods , Protein Serine-Threonine Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases , Adenosine Triphosphate/analysis , Animals , Miniaturization , Multienzyme Complexes/analysis , Protein Serine-Threonine Kinases/analysis , Rats , Sensitivity and Specificity , Time FactorsABSTRACT
5-Aminopyrazole-4-carboxamide was used as an alternative scaffold to substitute for the pyrazolopyrimidine of a known "bumped kinase inhibitor" to create selective inhibitors of calcium-dependent protein kinase-1 from both Toxoplasma gondii and Cryptosporidium parvum. Compounds with low nanomolar inhibitory potencies against the target enzymes were obtained. The most selective inhibitors also exhibited submicromolar activities in T. gondii cell proliferation assays and were shown to be non-toxic to mammalian cells.
ABSTRACT
AMP-activated protein kinase (AMPK) is well established as a sensor and regulator of intracellular and whole-body energy metabolism. A high-throughput screen was performed in order to identify chemotypes that are bound by AMPK. A novel thienopyridone compound (1) was identified and subsequently optimized. The structure-activity relationships that emerged from this effort are described.