Phenotypic Screening for Inhibitors of a Mutant Thrombopoietin Receptor
An inhibitor for the thrombopoietin receptor (TpoR) would be more specific for the treatment of myeloproliferative neoplasms (MPNs) due to constitutively active mutant TpoR compared to the current treatment approach of inhibiting Janus kinase 2 (JAK2). We describe a cell-based high-throughput phenotypic screening approach to identify inhibitors for constitutively active mutant TpoR. A stepwise elimination process is used to differentiate generally cytotoxic compounds from compounds that specifically inhibit growth of cells expressing wild-type TpoR and/or mutant TpoR. We have systematically optimized the phenotypic screening assay and documented in this chapter critical parameters for a successful phenotypic screen, such as cell growth and seeding optimization, plate reproducibility and uniformity studies, and an assay robustness analysis with a pilot screen.
Key wordsMyeloproliferative neoplasms Thrombopoietin receptor Phenotypic screening Cell-based assay Cell viability ATP
There is no financial conflict of interest among the authors. Support for A.N., A.K., and M.L.C was from an A*STAR core funding to the Experimental Therapeutics Centre. Support for C.C.D. was from UEFISCDI PCCA PN II 133/2012 and OPERATIONAL COMPETITIVITATY PROGRAM 2014-2020 POC-A1-A1.1.4-E-2015/P_37_798/149/2016. S.N.C. and A.K.S. received funding from the Ludwig Institute for Cancer Research. S.N.C. also received supports from FRS-FNRS, Salus Sanguinis Foundation, the Action de Recherche Concertée project ARC10/15-027 of the Université catholique de Louvain, the Fondation contre le Cancer, the PAI Programs BCHM61B5, and Belgian Medical Genetics Initiative.
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