title: A Phenotypic Screen for Small-Molecule Inhibitors of Constitutively Active Mutant Thrombopoietin Receptor Implicated in Myeloproliferative Neoplasms.
abstract: BACKGROUND
  Rather than a Janus Kinase 2 inhibitor (ruxolitinib), a specific thrombopoietin receptor (TpoR) inhibitor would be more specific for the treatment of myeloproliferative neoplasms due to TpoR mutations.
  
  
  OBJECTIVE
  A cell-based phenotypic approach to identify specific TpoR inhibitors was implemented and a library of 505,483 small molecules was screened for inhibitory effects on cells transformed by TpoR mutants.
  
  
  RESULTS
  Among the identified hits are two analogs of 3-(4-piperidinyl) indole. The analogs showed about five-fold preferential inhibition of cell viability towards Ba/F3 cells expressing the TpoR W515L mutation compared to the parental cells. There was no significant difference in inhibition of cell viability between the TpoR wild type and the TpoR W515L mutant cells. Preferential inhibition of viability was observed in Ba/F3 cells expressing erythropoietin receptor (EpoR) when stimulated with Epo compared to stimulation with interleukin-3 (IL3). The indole analog inhibited ex vivo colony formations of primary bone marrow cells from heterozygous JAK2 V617F knock-in mice. Drug combination treatment study was performed using ruxolitinib and the indole analog. Drug synergistic effects were observed when cells were stimulated to proliferate through both the IL3 and TpoR pathways. Our compound specifically targets monoamine receptors in the rhodopsin-like receptor family of G protein-coupled receptor.
  
  
  CONCLUSIONS
  This screen has identified a monoamine receptor inhibitor that can inhibit viability of cells with active TpoR or EpoR signalings. Drug synergism with ruxolitib is demonstrated.
authors.forename: Anna
authors.initials: A
authors.lastname: Ngo
authors.forename: Ann Zhufang
authors.initials: AZ
authors.lastname: Koay
authors.forename: Christian
authors.initials: C
authors.lastname: Pecquet
authors.forename: Carmen C
authors.initials: CC
authors.lastname: Diaconu
authors.forename: Yasmine
authors.initials: Y
authors.lastname: Ould-Amer
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Huang
authors.forename: Congbao
authors.initials: C
authors.lastname: Kang
authors.forename: Anders
authors.initials: A
authors.lastname: Poulsen
authors.forename: May Ann
authors.initials: MA
authors.lastname: Lee
authors.forename: David
authors.initials: D
authors.lastname: Jenkins
authors.forename: Andrew
authors.initials: A
authors.lastname: Shiau
authors.forename: Stefan N
authors.initials: SN
authors.lastname: Constantinescu
authors.affiliation: Experimental Therapeutics Centre, 31 Biopolis Way, #03-01 Nanos, Singapore 138669, Singapore.. Singapore.
authors.forename: Meng Ling
authors.initials: ML
authors.lastname: Choong
mesh_terms: D001853:Bone Marrow / Q000473:pathology; D002460:Cell Line; D004357:Drug Synergism; D057166:High-Throughput Screening Assays; D006801:Humans; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000503:physiopathology*; D009570:Nitriles; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines; D053628:Receptors, Thrombopoietin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries
chemical_list: D009570:Nitriles; D011720:Pyrazoles; D011743:Pyrimidines; D053628:Receptors, Thrombopoietin; D054852:Small Molecule Libraries; C540383:ruxolitinib
keywords: G protein-coupled receptor; Janus Kinase 2.; Myeloproliferative neoplasms; drug
  combination study; phenotypic screening; thrombopoietin receptor