Discovery of a novel orally available microtubule-destabilization agent that targets the colchicine binding site and exhibits antitumor activity in KRAS -mutant models

Background: Microtubules are essential for regulating cell growth, cell division, protein trafficking, and key cell signaling events. Alterations to the microtubule network affect cell survival, thus making microtubule-targeting agents desirable as cancer therapeutic agents. Currently, several tubulin modulators are approved for cancer treatment. However, development of additional microtubule-targeting agents is still needed to improve treatment outcomes, mitigate side effects, and overcome resistance mechanisms. Here, we describe compound A, a novel, orally available microtubule-destabilizing agent that targets the colchicine binding site and exhibits single-agent antitumor activity in KRAS -mutant models. Methods: Inhibition of tubulin polymerization was measured using a recombinant polymerization assay and a cell-based microtubule sedimentation assay. Binding to the colchicine site of tubulin was determined by a binding competition assay using a BODIPY-colchicine probe. Disruption of tubulin formation was determined by immunofluorescence of microtubules in A549 cells. Compound binding to the tubulin complex was determined via X-ray crystallography. Cellular proliferation of KRAS -mutant and isogenic cell lines was measured using resazurin. KRAS -mutant mouse xenograft models were used to assess pharmacokinetics (PK), pharmacodynamics, and antitumor activity. Results: Compound A binds to the colchicine site and destabilizes tubulin polymerization. Treatment resulted in single-agent inhibition of proliferation of KRAS -mutant cancer cell lines. Immunofluorescence of microtubules in A549 cells revealed disruption of the microtubule network and dysregulated spindle formation. Compound A exhibited a favorable ADME and PK profile. Oral treatment of compound A as a single agent resulted in 56% tumor growth inhibition in a subcutaneously implanted KRAS -mutant lung xenograft model. Conclusions: Compound A is an orally bioavailable tubulin modulator that binds to the colchicine site and inhibits tumor growth in a KRAS -mutant model as a single agent. Our preclinical data provide proof of concept for the development of an orally available microtubule-destabilizing agent.