ABSTRACT
Study of small molecule binding to live cells provides important information on the characterization of ligands pharmacologically. Here we developed and validated a label-free, liquid chromatography-mass spectrometry (LC-MS) based cell binding assay, using centrifugation to separate binders from non-binders. This assay was applied to various target classes, with particular emphasis on those for which protein-based binding assay can be difficult to achieve. In one example, to study a G protein coupled receptor (GPCR), we used one antagonist as probe and multiple other antagonists as competitor ligands. Binding of the probe was confirmed to be specific and saturable, reaching a fast equilibrium. Competition binding analysis by titration of five known ligands suggested a good correlation with their inhibition potency. In another example, this assay was applied to an ion channel target with its agonists, of which the determined binding affinity was consistent with functional assays. This versatile method allows quantitative characterization of ligand binding to cell surface expressed targets in a physiologically relevant environment.
Subject(s)
Receptors, G-Protein-Coupled , Tandem Mass Spectrometry , Chromatography, Liquid/methods , Ligands , Protein Binding , Receptors, G-Protein-Coupled/metabolismABSTRACT
We report the discovery and hit-to-lead optimization of a structurally novel indazole series of CYP11B2 inhibitors. Benchmark compound 34 from this series displays potent inhibition of CYP11B2, high selectivity versus related steroidal and hepatic CYP targets, and lead-like physical and pharmacokinetic properties. On the basis of these and other data, the indazole series was progressed to lead optimization for further refinement.
Subject(s)
Antihypertensive Agents/pharmacology , Cytochrome P-450 CYP11B2/antagonists & inhibitors , Hypertension/drug therapy , Indazoles/pharmacology , Animals , Antihypertensive Agents/chemical synthesis , Antihypertensive Agents/pharmacokinetics , Aromatase Inhibitors/chemical synthesis , Aromatase Inhibitors/pharmacokinetics , Aromatase Inhibitors/pharmacology , Cell Line , Cricetulus , Cytochrome P-450 CYP2D6 Inhibitors/chemical synthesis , Cytochrome P-450 CYP2D6 Inhibitors/pharmacokinetics , Cytochrome P-450 CYP2D6 Inhibitors/pharmacology , Humans , Indazoles/chemical synthesis , Indazoles/pharmacokinetics , Macaca mulatta , Male , Rats, Sprague-Dawley , Stereoisomerism , Steroid 11-beta-Hydroxylase/antagonists & inhibitorsABSTRACT
The inhibition of aldosterone synthase (CYP11B2) may be an effective treatment of hypertension and heart failure, among other ailments. Previously reported benzimidazole CYP11B2 inhibitors led the way for bioisosteric imidazopyridines that are both potent and selective over CYP11B1.
Subject(s)
Cytochrome P-450 CYP11B2/antagonists & inhibitors , Imidazoles/pharmacology , Pyridines/pharmacology , Animals , Cricetulus , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Imidazoles/pharmacokinetics , Macaca mulatta , Male , Microsomes, Liver/metabolism , Pyridines/chemical synthesis , Pyridines/chemistry , Pyridines/pharmacokinetics , Rats, Wistar , Steroid 11-beta-Hydroxylase/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
Hit-to-lead efforts resulted in the discovery of compound 19, a potent CYP11B2 inhibitor that displays high selectivity vs related CYPs, good pharmacokinetic properties in rat and rhesus, and lead-like physical properties. In a rhesus pharmacodynamic model, compound 19 displays robust, dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.
ABSTRACT
We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.
