Abstract 2646: The unique binding mode of NTRC 0066-0, a novel inhibitor of the spindle assembly checkpoint kinase TTK (Mps1), leads to long target residence time and potent antitumor activity

Abstract

Abstract [purpose] An abnormal number of chromosomes, or ‘aneuploidy’, is a common feature of solid human tumors and a predictor of poor prognosis in breast, lung, brain and colorectal cancer. Aneuploidy is caused by malfunctioning of the Spindle Assembly Checkpoint (SAC), a surveillance mechanism that ensures the fidelity of chromosome segregation. The protein kinase TTK (commonly referred to as Mps1) is a component of the SAC. Inhibition of TTK gene expression by RNA interference and inhibition of TTK kinase activity by small molecule kinase inhibitors causes chromosome missegregation and cancer cell death. [experimental procedures] A novel class of compounds was identified that potently inhibits TTK enzyme activity and cancer cell line proliferation [1]. Its binding mode and that of reference inhibitors was characterized by protein crystallography. Binding kinetics and target residence time were determined by surface plasmon resonance using Biacore T200. Anti-proliferative activity was measured on a broad panel of cancer cell lines [2]. [results] The clinical candidate, NTRC 0066-0, inhibits TTK with subnanomolar potency (IC50) in a kinase enzyme assay and is more than 200 times selective over 276 kinases examined, including mitotic and cell cycle dependent kinases (CDKs). X-ray structures of the TTK kinase domain in complex with NTRC 0066-0 and analogs indicate that this class of compounds induces a large conformational shift in the glycine-rich loop, invoking an inactive kinase conformation. In surface plasmon resonance experiments, NTRC 0066-0 exhibited slow dissociation kinetics, resulting in a long target residence time. Parallel surface plasmon resonance experiments with mitotic kinases confirmed the exquisite selectivity of NTRC 0066-0 for TTK over Aurora and Polo-like kinases. NTRC 0066-0 potently inhibited the proliferation of a wide variety of human cancer cell lines with potencies in the same range as marketed cytotoxic agents. The crystal structure, binding kinetics and cellular potency of NTRC 0066-0 were compared to that of other TTK inhibitors such as Mps1-IN-2, AZD-3146, Mps-BAY2b and Bay 1161909 as well as analogs from the NTRC 0066-0 series. This suggest that the unique binding mode of NTRC 0066-0 results in long target residence time which contributes to its strong anti-tumor activity. In subsequent mouse xenograft models of human cancer cell lines, NTRC 0066-0 inhibited tumor growth as a single agent after oral administration at 20 mg per kg. [conclusions] NTRC 0066-0 is a novel TTK inhibitor with outstanding in vitro properties and potent anti-tumor activity in mouse xenograft models. Our data suggest that long target residence time corresponds with potent cellular activity for TTK inhibitors.