Discovery and Characterization of Selective, First-in-Class Inhibitors of Citron Kinase

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.