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Updated: Mar 13, 2026
Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
A Phenotypic Screen for Small-Molecule Inhibitors of Constitutively Active Mutant Thrombopoietin Receptor Implicated
Anna Ngo, Ann Zhufang Koay, Christian Pecquet
1Experimental Therapeutics Centre, 31 Biopolis Way, #03-01 Nanos, Singapore 138669, Singapore.. Singapore.
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.
Conclusion:
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.
Insights
Researchers identified a novel monoamine receptor inhibitor that targets cells with active thrombopoietin receptor (TpoR) or erythropoietin receptor (EpoR) signaling. This compound shows synergistic effects with ruxolitinib, offering a potential new treatment for myeloproliferative neoplasms.
Area of Science:
- Pharmacology
- Molecular Biology
- Oncology
Background:
- Myeloproliferative neoplasms (MPNs) are often driven by mutations in Janus Kinase 2 (JAK2) or the thrombopoietin receptor (TpoR).
- Current treatments like ruxolitinib target JAK2, but specific TpoR inhibitors could offer greater precision for TpoR-mutated MPNs.
Purpose of the Study:
- To identify specific inhibitors of TpoR signaling using a cell-based phenotypic screen.
- To evaluate the efficacy and potential synergistic effects of identified compounds in preclinical models.
Main Methods:
- Screened a library of 505,483 small molecules for inhibitory activity against cells expressing TpoR mutants.
- Utilized Ba/F3 cell lines engineered to express specific TpoR mutations (e.g., W515L) and wild-type receptors.
- Assessed compound effects on cell viability, colony formation in primary bone marrow cells, and drug-drug interactions.
Main Results:
- Identified indole analogs with preferential inhibition of cell viability in TpoR W515L mutant cells.
- Observed preferential inhibition in cells expressing erythropoietin receptor (EpoR) under specific stimulation conditions.
- Demonstrated inhibition of ex vivo colony formation in JAK2 V617F mouse bone marrow cells.
- Showcased synergistic effects when the identified indole analog was combined with ruxolitinib, particularly when stimulating both IL-3 and TpoR pathways.
Conclusions:
- A novel monoamine receptor inhibitor was discovered, capable of inhibiting cell viability in TpoR- and EpoR-driven signaling pathways.
- The identified compound exhibits drug synergism with ruxolitinib, suggesting a potential combination therapy strategy for MPNs.
