TAI-1 is a highly potent, first-in-class inhibitor of Hec1 with IC50 of TAI-1 has a GI50 of 13.48 nM (K562 cells), which is close to 1000 times improvement in potency compared to INH1 (GI50 =11.7 μM). TAI-1 showed strong inhibition across a broad spectrum of tumor cells. TAI-1 interrupted Hec1-Nek2 protein interaction, led to Nek2 degradation, induced significant chromosomal misalignment in metaphase, and induced apoptotic cell death. TAI-1 was orally active in in vivo animal models of triple negative colon cancer, breast cancer and liver cancer. Preliminary toxicity shows no effect on the organ weights, body weights and blood indices at efficacious doses. TAI-1 shows high specificity to cancer cells and to target and had no effect on the cardiac channel hERG. TAI-1 is synergistic with topotecan, doxorubicin and paclitaxel in breast, leukemia and liver cancer cells. Sensitivity to TAI-1 was associated with the status of P53 and RB gene. Knockdown of P53 and RB in cancer cells increased sensitivity to TAI-1.