ABSTRACT
The profile of a series of triazine and pyrimidine based ROCK inhibitors is described. An initial binding mode was established based on a homology model and the proposed interactions are consistent with the observed SAR. Compounds from the series are potent in a cell migration assay and possess a favorable kinase selectivity. In vivo activity was demonstrated for compound 1A in a spontaneous hypertensive rat model.
Subject(s)
Antihypertensive Agents/chemistry , Hypertension/drug therapy , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemistry , Triazines/chemistry , rho-Associated Kinases/antagonists & inhibitors , Animals , Antihypertensive Agents/chemical synthesis , Antihypertensive Agents/pharmacology , Binding Sites , Computer Simulation , Disease Models, Animal , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Rats , Structure-Activity Relationship , Triazines/chemical synthesis , Triazines/pharmacology , rho-Associated Kinases/metabolismABSTRACT
IMAP is a non-separation-based, antibody-independent, FP assay that can be applied to many types of protein kinases and phosphatases. This technology is currently being used in many high-throughput screening campaigns throughout the industry. In this technology, a fluorescently labeled peptide substrate is phosphorylated and then captured on immobilized metal (M(III)) nanoparticles, an interaction that is enhanced at low pH (pH 5.5). The binding of the phosphorylated peptide to the nanoparticles is detected using FP. IMAP differs from other FP formats in that the polarization signal is antibody-independent and involves metal coordination complexes detected at low pH. Here, this technology is evaluated against a 4000000-member compound collection using a 1536-well assay design that is devoid of enzymes so that only interference of the compounds with the detection system is measured. Miniaturization of the assay to 1536-well plates is discussed. Compound interference due to inhibition of phosphopeptide binding to the M(III) nanoparticles is not observed. Additionally, it is concluded that the level of fluorescence compound interference is similar to typical FP formats for the majority of the compound collection.
