The force constants were kept constant for the rest of the simulated annealing

FlashPlate 384-well format using a library of 117 995 compounds screened at 10 mM was developed. A total of 1271 compounds from the screening were retested in duplicate and 591 showed confirmed activity. Cluster and physical property analyses of these hits highlighted 125 compounds as being suitable for IC50 evaluation and purity analysis. On the basis of doseresponse results and molecular similarity studies, a further 456 compounds were identified from the BioFocus DPI compound collection and order Aphrodine commercial sources for hit expansion IC50, and liquid chromatography and mass spectrometry purity measurements. From the results for compounds with a purity >70%, 123 compounds were identified with an IC50 o10 mM. Several hit series were identified in this exercise and three were selected for hit-to-lead studies. Two of these series, while maintaining Haspin kinase inhibition, performed poorly in cell-based assays; hence, a series of imidazopyridazines were chosen for further optimization. CHR-6494 – N-propylimidazopyridazin-6-amine) was identified as a compound having promising Haspin kinase and cell proliferation-inhibitory properties that merited further detailed biological and antitumor investigation. CHR-6494 is a potent inhibitor of histone H3T3 phosphorylation that has anti-proliferative effects in human cancer cells The next step was to show that the CHR-6494 compound was indeed an active Haspin inhibitor with important biological effects, such as inhibition of cell proliferation. To this end, we chose the initial biological model of human cancer cell lines, in which three of the best recognized examples, namely HeLa, HCT-116 and MDA-MB-231 were selected. CHR-6494 inhibited histone H3T3 phosphorylation in the three cancer cell lines in a dose-dependent manner as shown by western blot and immunofluorescence. The diminished H3T3 phosphorylation levels upon CHR-6494 treatment were not associated with downregulation of the total content of the Haspin protein, the enzyme targeted by the drug. The use of CHR-6494 did not modify H3S10 and H328 phosphorylation levels, two sites that are not phosphorylated by Haspin, emphasizing the enzyme specificity of the identified inhibitor. The specificity of CHR-6494 for Haspin was further confirmed by analysis of the enzymatic inhibitory capacity of the compound in a panel of 27 protein kinases, including Aurora B kinase. Using a FRET assay based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to protein cleavage, we observed that the IC50 value for Haspin was 2 nM, whereas for all other protein kinases, it was in the mM range. Once the biochemical effects of CHR-6494 as a Haspin inhibitor had been confirmed, we assessed its effect on cancer cell biology. Using the XTT -2H-tetrazolium-5-carboxanilide)) assay to measure cell viability in the three described cancer cell lines, we observed that CHR-6494 inhibited cancer cell growth dose dependently. The IC50 values were within the same range: 500 nM for HCT-116, 473 nM for HeLa and 752 nM for MDA-MB-231. Interestingly, IC50 sensitivity to this first-in-class Haspin inhibitor compound is in the same range as that of other anticancer compounds that target PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19858123 histone modifications, such as inhibitors of Aurora kinases A and B and histone deacetylase. To investigate whether Haspin inhibition by the CHR-6494 compound also caused misregulation of checkpoints, we analyzed the levels and nuclear localization of the key spindle assembly