title: High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules.
abstract: The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of 'lead-like' small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N⁶,N⁶-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K(+)-ATP channel opener pinacidil and show that it probably promotes cell survival, by 'off-target' inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.
authors.affiliation: University of Dundee, Scotland, UK. p.d.andrews@dundee.ac.uk
authors.forename: Paul D
authors.initials: PD
authors.lastname: Andrews
authors.forename: Melissa
authors.initials: M
authors.lastname: Becroft
authors.forename: Anders
authors.initials: A
authors.lastname: Aspegren
authors.forename: Jane
authors.initials: J
authors.lastname: Gilmour
authors.forename: Martyn J
authors.initials: MJ
authors.lastname: James
authors.forename: Scott
authors.initials: S
authors.lastname: McRae
authors.forename: Robert
authors.initials: R
authors.lastname: Kime
authors.forename: Robert W
authors.initials: RW
authors.lastname: Allcock
authors.forename: Achamma
authors.initials: A
authors.lastname: Abraham
authors.forename: Zhong
authors.initials: Z
authors.lastname: Jiang
authors.forename: Raimund
authors.initials: R
authors.lastname: Strehl
authors.forename: Joanne C
authors.initials: JC
authors.lastname: Mountford
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.forename: Miles D
authors.initials: MD
authors.lastname: Houslay
authors.forename: David R
authors.initials: DR
authors.lastname: Adams
authors.forename: Julie A
authors.initials: JA
authors.lastname: Frearson
mesh_terms: D000577:Amides / Q000494:pharmacology; D018929:Cell Culture Techniques; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006571:Heterocyclic Compounds / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007191:Indazoles / Q000737:chemistry / Q000494:pharmacology; D015394:Molecular Structure; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000577:Amides; D006571:Heterocyclic Compounds; D007191:Indazoles; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; C061570:protein kinase N; D054460:rho-Associated Kinases; D011493:Protein Kinase C