High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules

Paul D. Andrews, Melissa Becroft, Anders Aspegren, Jane Gilmour, Martyn J. James, Scott McRae, Robert Kime, Robert W. Allcock, Achamma Abraham, Zhong Jiang, Raimund Strehl, Joanne C. Mountford, Graeme Milligan, Miles D. Houslay, David R. Adams, Julie A. Frearson

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33 Citations (Scopus)

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. © The Authors Journal compilation © 2010 Biochemical Society.

Original languageEnglish
Pages (from-to)21-33
Number of pages13
JournalBiochemical Journal
Volume432
Issue number1
DOIs
Publication statusPublished - 2010

Keywords

  • Drug discovery
  • High-content screening
  • Human embryonic stem cell
  • Pinacidil
  • Protein kinase inhibitor
  • Rho-associated kinase (ROCK)
  • Y-27632

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    Andrews, P. D., Becroft, M., Aspegren, A., Gilmour, J., James, M. J., McRae, S., Kime, R., Allcock, R. W., Abraham, A., Jiang, Z., Strehl, R., Mountford, J. C., Milligan, G., Houslay, M. D., Adams, D. R., & Frearson, J. A. (2010). High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules. Biochemical Journal, 432(1), 21-33. https://doi.org/10.1042/BJ20101022