About ∼90% of the solid tumors and ∼75% of the hematologic malignancies are aneuploidy. The process that gives rise to aneuploidy is chromosomal instability (CIN), characterized by chromosome mis-segregation and other mitotic aberrations. KIF18A (kinesin family member 18A), part of the kinesin family of motor proteins, is expressed in the G2/M phase of the cell cycle and regulates chromosome positioning during cell division. Genetic depletion of KIF18A impacts the chromosome alignment, elongation of mitotic spindles, and traps chromosomally unstable tumor cells in mitosis, while having negligible impact on normal cells. KIF18A and CIN or whole genome doubling (WGD+, or aneuploidy) have been tested and are synthetic lethal in tumor cells. Thus, KIF18A inhibition has become an attractive approach for the treatment of the vulnerable CIN/WGD+ related tumors. Here we report the discovery and development of small-molecule KIF18A inhibitors, leading to the nomination of a preclinical candidate compound (PCC). The PCC molecule had superior in vitro potency, in vivo efficacy in cancer models, and good druggability. Thereafter we completed IND-enabling studies and submitted the pre-IND application in November 2024, with a scheduled IND filing in early 2025.The PCC molecule exhibited potent target engagement both in enzymes and cell-based assays with IC50 in double digit nM in multiple cancer cell lines including breast cancer, ovarian cancer, NSCLC, and colorectal cancer. The enzyme activity is highly selective within the kinesin superfamily members (CENP-E, Eg5, Chromokinesin, KIF3C, KIFC3, MCAK and MKLP) with IC50 all >30 μM. In the 435-kinase panel screening, 1 μM of the PCC molecule did not significantly inhibit any of the 435 kinases (<25% inhibition in all tested kinases), demonstrating great selectivity in the kinase panel. The PCC molecule also exhibited a good in vitro safety profile in hERG assay and in the 44-enzyme/receptor/ion-channel safety panel profiling. The PCC molecule proved desirable in vitro ADME characteristics and excellent in vivo pharmacokinetic profile with low clearance, long half-life, as well as good oral bioavailability in mice, rats, beagle dogs and non-human primates. Furthermore, The PCC molecule exhibited excellent in vivo efficacy in CIN+ ovarian cancer OVCAR3 CDX model, and two CIN+ triple negative breast cancer (MDA-MB-231 and HCC1806) models, without body weight loss and other safety issues observed. These anti-tumor efficacies were dose-dependent and well correlated with a robust pharmacodynamic (PD) western blot detection using phosphor-H3, a mitotic marker. In non-GLP and GLP-tox studies in rats and beagle dogs, the PCC molecule was well tolerated, providing a potential good therapeutic window. Overall, a novel and highly potent KIF18A inhibitor is ready to enter phase 1 clinical trials in 2025 for the treatment of CIN/WGD+ cancers.Citation Format:Congcong Cai, Shiya Zhu, Chunlin Chen, Jian Liu, Hongkun Li, Shu Cai, Linjun Wang, Min Wen, Mingxin Qian, Shifeng Liu, Bin Shi, Hancheng Zhang. A fully characterized KIF18A inhibitor with superior potency and good druggability ready for IND application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1756.