Background Hec1 (NDC80) is an integral part of the kinetochore and is overexpressed in a variety of human cancers making it an attractive molecular target for the design of novel anticancer therapeutics. blotting and immunofluorescent staining. The potency of TAI-1 was evaluated in three xenograft models in mice. Preliminary toxicity was evaluated in mice. Specificity to the target was tested with a kinase panel. Cardiac security was evaluated with hERG assay. Clinical correlation was performed with human gene database. Results TAI-1 showed strong potency across a broad spectrum of tumor cells. TAI-1 disrupted Hec1-Nek2 protein interaction led to Nek2 degradation induced significant chromosomal misalignment in metaphase and induced apoptotic cell death. TAI-1 was effective orally in animal models of triple unfavorable breast malignancy colon cancer and liver malignancy. Preliminary toxicity shows no effect on the body weights organ ATB 346 weights and blood indices at efficacious doses. TAI-1 shows high specificity to malignancy cells and to target and experienced no effect on the cardiac channel hERG. TAI-1 is usually synergistic with doxorubicin topotecan and paclitaxel in leukemia breast and liver malignancy cells. Sensitivity to TAI-1 was associated with the status of RB and P53 gene. Knockdown of RB and P53 in malignancy cells increased sensitivity to TAI-1. Hec1-overexpressing molecular subtypes of human lung cancer were identified. Conclusions The excellent potency security and synergistic profiles of this potent first-in-class Hec1-targeted small molecule TAI-1 ATB 346 show its potential for clinically power in anti-cancer treatment regimens. potency was improved to low nanomolar potency enabling possible clinical utility of the Hec1-targeted compound. This study explores the features and potential of the improved anticancer agent targeting Hec1 TAI-1 for preclinical development and clinical power. The and biological activity mechanism of action toxicity and security and translational implications are investigated. Methods Cell lines MDA-MB-231 MDA-MB-468 K562 HeLa MCF7 HCC1954 A549 COLO205 U2OS Huh-7 U937 HepG2 KG-1 PC3 BT474 MV4-11 RS4;11 MOLM-13 WI-38 HUVEC RPTEC and HAoSMC were from Development Center for Biotechnology New Taipei City Taiwan; MDA-MB-453 T47D ZR-75-1 ZR-75-30 MDA-MB-361 Hs578T NCI-H520 Hep3B PLC/PRF/5 were from Bioresource Collection and Research Center Hsinchu Taiwan. Cell lines were maintained in total 10% fetal bovine serum (Biowest Miami FL USA or Hyclone Thermo Scientific Rockford IL USA) and physiologic glucose (1?g/L) in DME ATB 346 (Sigma St. Louis MO USA). Studies conducted using cell lines RPMI8226 MOLT-4 and N87; drug-resistant cell lines MES-SA/Dx5 NCI/ADR-RES and K562R were from and tested by Xenobiotic Laboratories Plainsboro NJ USA. potency assay Cells were seeded in 96 well plates incubated for 24 hours compounds added and incubated for 96 hours. All testing points were ATB 346 tested in triplicate wells. Cell viability was determined by MTS assay using CellTiter 96? Aqueous Non-radioactive Cell Proliferation Assay system (Promega Madison WI USA) according to manufacturer’s instructions with MTS (Promega) and PMS (Sigma St. Louis MO). Data retrieved from spectrophotometer (BIO-TEK 340 BIOTEK VT USA) were processed in Excel and GraphPad Prism 5 (GraphPad Software CA USA) to calculate the concentration exhibiting 50% growth inhibition (GI50). All data represented the results of triplicate experiments. Immunoblot and co-immunoprecipitation analysis Western blotting and co-immunoprecipitation were done as described previously . Primary antibodies used: mouse anti-Nek2 and mouse anti-Mcl-1 (BD Pharmingen San Diego CA); rabbit anti-Hec1 (GeneTex Inc. Irvine CA); mouse anti-actin (Sigma); mouse anti-P84 and mouse anti-RB (Abcam Cambridge MA); rabbit anti-Cleaved Caspase3 rabbit-anti-Cleaved PARP rabbit anti-XIAP and mouse anti-P53 (Cell Signaling Technology Boston MA); mouse anti-Bcl-2 (Santa Cruz); mouse antikinase assay Inhibition of kinase activity by test compound was estimated by [33P] labeled radiometric assay. 20 kinase assays (Millipore) were adapted. The kinase reaction was performed according to individual manual CD5 with minor modification. In brief each test compound was evaluated at two concentrations (10?mM and 1?mM) in duplication. The kinase reaction were initiated by enzyme addition stopped at indicated time by the addition of 3% phosphoric acid harvested onto a filter plate by using a unifilter harvester (PerkinElmer) and counted by using TopCount (PerkinElmer). The results were the average of.