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Activation and proliferation of resident cardiac progenitor cells has therapeutic potential to repair the heart after injury. However, research has been impeded by a lack of well-defined and characterized cell sources and difficulties in translation to screening platforms. Here, we describe the development, validation, and use of a 384-well phenotypic assay in primary human epicardium-derived cells (EPDCs) to identify compounds that induce proliferation while maintaining the progenitor phenotype. Using this assay, we screened 7400 structurally diverse compounds where greater than 90% are biologically annotated and known to modulate a broad range of biological targets. From the primary screen, we identified and validated hits and expanded upon the lead molecules of interest. A counterscreen was developed in human cardiac fibroblasts to filter out compounds with a general proliferative effect, after which the activity of selected molecules was confirmed across multiple EPDC donors. To further examine the mechanism of action of compounds with annotated targets, we performed knockdown experiments to understand whether a single known target was responsible for the proliferative effect, confirming results with protein expression and activity assays. Here, we were able to show that the annotated targets of compounds of interest were not responsible for the proliferative effect, which highlights potential differences in cell types and signaling pathways and possible polypharmacology. These studies demonstrate the feasibility of using relevant human primary cells in a phenotypic screen to identify compounds as novel biological tools and starting points for drug discovery projects, and we disclose the first small molecules to proliferate human primary EPDCs.

Chemical biology promises discovery of new and unexpected mechanistic pathways, protein functions and disease targets. Here, we probed the mechanism-of-action and protein targets of 3,5-disubstituted isoxazoles (Isx), cardiomyogenic small molecules that target Notch-activated epicardium-derived cells (NECs) in vivo and promote functional recovery after myocardial infarction (MI). Mechanistic studies in NECs led to an Isx-activated G(q) protein-coupled receptor (G(q)PCR) hypothesis tested in a cell-based functional target screen for GPCRs regulated by Isx. This screen identified one agonist hit, the extracellular proton/pH-sensing GPCR GPR68, confirmed through genetic gain- and loss-of-function. Overlooked until now, GPR68 expression and localization were highly regulated in early post-natal and adult post-infarct mouse heart, where GPR68-expressing cells accumulated subepicardially. Remarkably, GPR68-expressing cardiomyocytes established a proton-sensing cellular "buffer zone" surrounding the MI. Isx pharmacologically regulated gene expression (mRNAs and miRs) in this GPR68-enriched border zone, driving cardiomyogenic and pro-survival transcriptional programs in vivo. In conclusion, we tracked a (micromolar) bioactive small molecule's mechanism-of-action to a candidate target protein, GPR68, and validated this target as a previously unrecognized regulator of myocardial cellular responses to tissue acidosis, setting the stage for future (nanomolar) target-based drug lead discovery.