title: Phenotypic Screen for Cardiac Regeneration Identifies Molecules with Differential Activity in Human Epicardium-Derived Cells versus Cardiac Fibroblasts.
abstract: 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.
authors.forename: Amalia I
authors.initials: AI
authors.lastname: Paunovic
authors.forename: Lauren
authors.initials: L
authors.lastname: Drowley
authors.forename: Anneli
authors.initials: A
authors.lastname: Nordqvist
authors.forename: Elke
authors.initials: E
authors.lastname: Ericson
authors.affiliation: Discovery Sciences, AstraZeneca, Mereside , Alderley Park, Macclesfield SK10 4TG, Cheshire, United Kingdom.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Mouchet
authors.forename: Anna
authors.initials: A
authors.lastname: Jonebring
authors.forename: Gunnar
authors.initials: G
authors.lastname: Grönberg
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Kvist
authors.forename: Ola
authors.initials: O
authors.lastname: Engkvist
authors.affiliation: Discovery Sciences, AstraZeneca R&D Darwin , 310 Milton Science Park, Milton Rd., Cambridge, CB4 0WG, United Kingdom.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Brown
authors.forename: Karin
authors.initials: K
authors.lastname: Gedda
authors.affiliation: Molecular Cell Biology, Leiden University Medical Center , Leiden, The Netherlands.
authors.forename: Marie-José
authors.initials: MJ
authors.lastname: Goumans
authors.forename: Qing-Dong
authors.initials: QD
authors.lastname: Wang
authors.forename: Alleyn T
authors.initials: AT
authors.lastname: Plowright
mesh_terms: D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D055808:Drug Discovery; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006321:Heart / Q000187:drug effects / Q000502:physiology; D006801:Humans; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D010496:Pericardium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010641:Phenotype; D012038:Regeneration / Q000187:drug effects