Advances in Bioanalytics and Biomarkers
Target and Mechanism Deconvolution After Phenotypic Screens
We have previously described a phenotypic signature associated with cardiovascular toxicity relevant to vascular calcification and atherosclerosis from a human primary cell-based coronary artery smooth muscle cell model of vascular inflammation (BioMAP® CASM3C system). The key biomarker activity in this signature is increased cell surface levels of serum amyloid A (SAA) protein. Analysis of a large reference database (BioMAP Phenotypic Profile Reference Database) of >3400 drugs and chemicals tested in this assay identified 147 compounds exhibiting the signature at one or more concentrations. For some of these compounds, specific mechanisms could be implicated and include MEK inhibition, HDAC inhibition, glucocorticoid (GR)/mineralocorticoid (MR) receptor agonism, IL-6 pathway agonism, as well as modulation of mitochondrial NAD+/NADH ratios.
To further characterize the mechanisms underlying this toxicity-associated signature, we took advantage of a second large reference database (BioPrint® Pharmacology Profile Database) comprised of in vitropharmacological profiles of drugs and chemicals screened against a broad range of targets (~148 receptors, ion channels, enzymes and transporters). We evaluated the in vitropharmacology profiles for compounds exhibiting the phenotypic signature associated with cardiovascular toxicity(data was available for 85 of 147 compounds). Target activities (in binding assays) enriched among the phenotypic actives include glucocorticoid receptor (GR), androgen receptor (AR), Chloride channel (Cl-channel), ML2 (MT3), (5-Hydroxytryptamine receptor 2B (5-HT2B), peripheral benzodiazepine receptor (BZD), MT1 and ML1. The identification of ML2 (MT3), also known as NAD(P)H quinone dehydrogenase 2 or NQO2, and MT1 receptors is interesting as these are receptors for melatonin. Melatonin has been reported to reduce blood pressure and also to reduce NAD+ levels through effects on NAMPT (nicotinamide phosphoribosyltransferase). Recent studies have suggested that NAMPT may play a role in the pathogenesis of atherosclerosis in experimental mouse models. In humans, serum concentrations of NAMPT have been shown to be an independent predictor of symptomatic carotid stenosis in patients undergoing carotid endarterectomy.
These results show how the combined analysis of phenotypic and pharmacology profiling data can confirm and extend our understanding of potential mechanisms associated with risk of cardiovascular toxicity. Pairing of target-based and phenotypic assays is an efficient and effective means to improve confidence in non-animal based screening of new drug leads for potential liabilities.