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Discovery and Characterization of Selective, First-in-Class Inhibitors of Citron Kinase
Joshua J Maw1, Jesse A Coker1, Tarun Arya1
1Center for Therapeutics Discovery, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, United States.
Abstract:
Citron kinase (CITK) is an AGC-family serine/threonine kinase that regulates cytokinesis. Despite knockdown experiments implicating CITK as an anticancer target, no selective CITK inhibitors exist. We transformed a previously reported kinase inhibitor with weak off-target CITK activity into a first-in-class CITK chemical probe, C3TD879. C3TD879 is a Type I kinase inhibitor which potently inhibits CITK catalytic activity (biochemical IC50 = 12 nM), binds directly to full-length human CITK in cells (NanoBRET Kd < 10 nM), and demonstrates favorable DMPK properties for in vivo evaluation. We engineered exquisite selectivity for CITK (>17-fold versus 373 other human kinases), making C3TD879 the first chemical probe suitable for interrogating the complex biology of CITK. Our small-molecule CITK inhibitors could not phenocopy the effects of CITK knockdown in cell proliferation, cell cycle progression, or cytokinesis assays, providing preliminary evidence that the structural roles of CITK may be more important than its kinase activity.
Insights
Researchers developed C3TD879, the first selective chemical probe for Citron kinase (CITK). This probe inhibits CITK activity, offering a tool to study its role in cancer and cell division.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Citron kinase (CITK) is an AGC-family kinase regulating cytokinesis.
- CITK is implicated as an anticancer target, but selective inhibitors are lacking.
Purpose of the Study:
- To develop the first selective chemical probe for Citron kinase (CITK).
- To investigate the role of CITK's kinase activity versus its structural functions.
Main Methods:
- Transformation of a weak off-target inhibitor into a potent and selective CITK probe (C3TD879).
- Biochemical assays (IC50), cellular target engagement (NanoBRET Kd), and selectivity profiling (>373 kinases).
- Comparison of small-molecule inhibitor effects with CITK knockdown in cell proliferation, cell cycle, and cytokinesis assays.
Main Results:
- C3TD879 potently inhibits CITK (IC50 = 12 nM) and binds directly in cells (Kd < 10 nM).
- Engineered exquisite selectivity for CITK (>17-fold over 373 kinases) with favorable DMPK properties.
- Small-molecule CITK inhibitors failed to phenocopy CITK knockdown effects on cell proliferation, cell cycle, or cytokinesis.
Conclusions:
- C3TD879 is the first chemical probe for interrogating CITK biology.
- Preliminary evidence suggests CITK's structural roles may be more critical than its kinase activity in cellular processes.
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