Expression profiling by high throughput sequencing
Summary
Today, novel candidate therapeutics are identified in an environment which is intrinsically different from the clinical context in which they are ultimately evaluated. We present a strategy that allows biological relevance to be assessed in the early stages of drug discovery. Using molecular phenotyping and an in vitro model of diabetic cardiomyopathy, we show that by quantifying pathway reporter gene expression, molecular phenotyping can cluster compounds based on pathway profiles and dissect associations between pathway activities and disease phenotypes simultaneously. Molecular phenotyping identified a class of calcium-signaling modulators that can reverse disease-regulated pathways and phenotypes, which was validated by structurally distinct compounds of relevant classes. The technique was applicable to compounds with a range of binding specificities and detected false-positive hits missed by classical phenotypic assays. Our results advocate application of molecular phenotyping in drug discovery, promoting biological relevance as a key selection criterion early in the drug development cascade.
Overall design
Molecular Phenotyping after 12h of compound treatment; A total number of 80 compounds were profiled with molecular phenotyping, along with four control conditions: media control, which represents the healthy status; GEC, which mimicks the stress condition in T2D patients; BM, a cocktail of bosentan, an enthothelin receptor antagonist, and mifepristone, a progesterone receptor antagonist, which is supposed to reverse the disease phenotype; and GEC+BM, which mimicks a successful reversal of the phenotype from the stress condtion. The compounds were screened on six plates (A-F), with one replicate for each compound and one replicate for each control on each plate. Some compounds that failed the first screening run were tested in two cherry-picking replication plates (R1 and R2). Calcium channel inhibitor hits were then tested in phenotypic high-content microscopy assays with two or three biological replicates each.
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