ABSTRACT
An uHTS campaign was performed to identify selective inhibitors of PKC-theta. Initial triaging of the hit set based on selectivity and historical analysis led to the identification of 2,4-diamino-5-nitropyrimidines as potent and selective PKC-theta inhibitors. A homology model and initial SAR is presented demonstrating that a 2-arylalkylamino substituent in conjunction with suitable 4-diamino substituent are essential for achieving selectivity over many kinases. Additional hit to lead profiling is presented on selected compounds.
Subject(s)
Isoenzymes/antagonists & inhibitors , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , Humans , Interleukin-2/metabolism , Models, Molecular , Molecular Structure , Protein Conformation , Protein Kinase C-theta , Structure-Activity RelationshipABSTRACT
Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.
Subject(s)
Adenosine Triphosphatases/metabolism , Biological Assay/methods , Protein Kinase Inhibitors/analysis , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Binding Sites/drug effects , Fluorescent Dyes/chemistry , Humans , KineticsABSTRACT
The discovery of novel 5,7-disubstituted[1,6]naphthyridines as potent inhibitors of Spleen Tyrosine Kinase (SYK) is discussed. The SAR reveals the necessity for a 7-aryl group with preference towards para substitution and that this in combination with 5-aminoalkylamino substituents further improved the potency of the compounds. The initial SAR as well as a survey of the other positions is discussed in detail.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Precursors/antagonists & inhibitors , Naphthyridines/chemistry , Naphthyridines/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Spleen/enzymology , Animals , Humans , Inhibitory Concentration 50 , Intracellular Signaling Peptides and Proteins , Structure-Activity Relationship , Syk KinaseABSTRACT
The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.
Subject(s)
Benzimidazoles/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Immunosuppressive Agents/chemical synthesis , Isoquinolines/chemical synthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Administration, Oral , Animals , Antibodies, Monoclonal/pharmacology , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , CD3 Complex/immunology , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Female , Humans , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Interleukin-2/blood , Isoquinolines/chemistry , Isoquinolines/pharmacology , Jurkat Cells , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Conformation , Protein Binding , Structure-Activity RelationshipABSTRACT
An imidazo[4,5-h]isoquinolin-7,9-dione (1) was identified as an adenosine 5'-triphosphate competitive inhibitor of lck by high throughput screening. Initial structure-activity relationship studies identified the dichlorophenyl ring and the imide NH as important pharmacophores. A binding model was constructed to understand how 1 binds to a related kinase, hck. These results suggested that removing the gem-dimethyl group and flattening the ring would enhance activity. This was realized by converting 1 to the imidazo[4,5-h]isoquinolin-9-one (20), resulting in an 18-fold improvement in potency against lck and a 50-fold increase in potency in a cellular assay.
