Biological characterization of 2-aminothiazole-derived Cdk4/6 selective inhibitor in vitro and in vivo.

Abnormalities in the p16INK4a/ cyclin-dependent kinase (Cdk)4, 6/ Retinoblastoma (Rb) pathway frequently occur in various human cancers. Thus, Cdk4/6 is an attractive target for cancer therapy. Here we report the biological characterization of a 2-aminothiazole-derived Cdk4/6 selective inhibitor, named Compound A in vitro and in vivo. Compound A potently inhibits Cdk4 and Cdk6 with high selectivity (more than 57-fold) against other Cdks and 45 serine/threonine and tyrosine kinases. Compound A inhibits Rb protein (pRb) phosphorylation at Ser780, inhibits E2F-dependent transcription, and induces cell-cycle arrest at G1 in the T98G human glioma cell line. Among 82 human cells derived from various tissues, cell lines derived from hematological cancers (leukemia/lymphoma) tended to be more sensitive to Compound A in cell proliferation assay. Rb-negative cells tended to be insensitive to Compound A, as we had expected. In a nude rat xenograft model, Compound A inhibited pRb phosphorylation and bromodeoxyuridine (BrdU) incorporation in Eol-1 xenograft tumor at plasma concentration of 510 nM. Interestingly Compound A only moderately inhibited those pharmacodynamic and cell cycle parameters of normal crypt cells in small intestine even at 5 times higher plasma concentration. In F344 rats, Compound A did not cause immunosuppression even at 17 times higher plasma conc. These results suggest that Cdk4/6 selective inhibitors only moderately affects on the cell cycle of normal proliferating tissues and has a safer profile than pan-Cdk inhibitor in vivo.

Imidazopyridine derivatives as potent and selective Polo-like kinase (PLK) inhibitors.

A novel class of imidazopyridine derivatives was designed as PLK1 inhibitors. Extensive SAR studies supported by molecular modeling afforded a highly potent and selective compound 36. Compound 36 demonstrated good antitumor efficacy in xenograft nude rat model.

Identification of beta-catenin as a novel substrate of Polo-like kinase 1.

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays an important role in M phase progression by regulating various downstream substrates via phosphorylation. Here, we identified beta-catenin as a novel substrate of Plk1 and determined that Ser-718 is a phosphorylation site for Plk1 by using a phospho-specific antibody that cross-reacts with Plk1-dependent phosphorylation sites. Ser-718 of beta-catenin was directly phosphorylated by recombinant Plk1 in vitro, with the phosphorylation signal in cells increasing with overexpression of Plk1 and decreasing when endogenous Plk1 was depleted by small interfering RNA. The phosphorylation at Ser-718 was correlated with the cell cycle-dependent expression of Plk1 which reached a maximum in M phase. We also confirmed that there is a physical interaction between beta-catenin and Plk1 using coimmunoprecipitation and a GST pull-down assay. These results demonstrate that beta-catenin is a physiological substrate of Plk1 in cells, which may provide a novel insight into the role of beta-catenin in M phase.

A serine protease inhibitor prevents endoplasmic reticulum stress-induced cleavage but not transport of the membrane-bound transcription factor ATF6.

Mammalian cells express several transcription factors embedded in the endoplasmic reticulum (ER) as transmembrane proteins that are activated by proteolysis, and two types of these proteins have been extensively investigated. One type comprises the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2). The other type comprises the activating transcription factors 6 (ATF6alpha and ATF6beta), which are activated in response to ER stress. It was shown previously that both SREBP and ATF6 are cleaved sequentially first by the Site-1 protease (serine protease) and then by the Site-2 protease (metalloprotease) (Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 1355-1364). In this study, we examined various protease inhibitors and found that 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, prevented ER stress-induced cleavage of ATF6alpha and ATF6beta, resulting in inhibition of transcriptional induction of ATF6-target genes. AEBSF also inhibited production of the mature form of SREBP-2 that was induced in response to sterol depletion, and appeared to directly prevent cleavage of ATF6alpha and ATF6beta by inhibiting Site-1 protease. As the Site-1 protease is localized in the Golgi apparatus, both SREBP and ATF6 must relocate to the Golgi apparatus to be cleaved. We showed here that AEBSF treatment had little effect on ER stress-induced translocation of ATF6 from the ER to the Golgi apparatus, but blocked nuclear localization of ATF6. These results indicate that the transport of ATF6 from the ER to the Golgi apparatus and that from the Golgi apparatus to the nucleus are distinct steps that can be distinguished by treatment with AEBSF.