A Novel Pyrazolo[1,5-a]pyrimidine Is a Potent Inhibitor of Cyclin-Dependent Protein Kinases 1, 2, and 9, Which Demonstrates Antitumor Effects in Human Tumor Xenografts Following Oral Administration

Abstract

Cyclin-dependent protein kinases (CDKs) are central to the appropriate regulation of cell proliferation, apoptosis, and gene expression. Abnormalities in CDK activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Here, we report the identification of a pyrazolo[1,5-a]pyrimidine derived compound, 4k (BS-194), as a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7, and CDK9 (IC₅₀= 3, 30, 30, 250, and 90 nmol/L, respectively). Cell-based studies showed inhibition of the phosphorylation of CDK substrates, Rb and the RNA polymerase II C-terminal domain, down-regulation of cyclins A, E, and D1, and cell cycle block in the S and G₂/M phases. Consistent with these findings, 4k demonstrated potent antiproliferative activity in 60 cancer cell lines tested (mean GI₅₀= 280 nmol/L). Pharmacokinetic studies showed that 4k is orally bioavailable, with an elimination half-life of 178 min following oral dosing in mice. When administered at a concentration of 25 mg/kg orally, 4k inhibited human tumor xenografts and suppressed CDK substrate phosphorylation. These findings identify 4k as a novel, potent CDK selective inhibitor with potential for oral delivery in cancer patients.

Proliferating Cell Nuclear Antigen (PCNA) Interactions in Solution Studied by NMR

“…Human CDK2 was expressed and purified as described [20]. Briefly, the protein was expressed in E. coli BL21(DE3) cells with a His 6 -SUMO-1 N-terminal tag which, after immobilized metal affinity chromatography, was removed by the Senp2 protease.…”

Development of a cyclin-dependent kinase inhibitor devoid of ABC transporter-dependent drug resistance

“…Collected HCT-116 cells were stained using the monoclonal antibody to ABCB1 (UlC2) and to ABCG2 (BXP-21) as previously described (Heathcote et al , 2010). Staining intensity was assessed by flow cytometry (FACS canto, Becton Dickinson, Mountain View, CA, USA) and analysed using the FlowJo software (Treestar Inc., Ashland, OR, USA).…”

“…Although the mechanisms responsible for such poor pharmacology/potency in the clinic are not entirely clear, potential reasons may include drug efflux in the case of flavopiridol or poor metabolic stability in the case of R-roscovitine (seliciclib; CYC202) (Robey et al , 2001; Nutley et al , 2005). We have previously designed and optimised a number of pyrazolo[1,5- a ]pyrimidine-based CDK inhibitors (Ali et al , 2009; Heathcote et al , 2010). Among these, BS-194 was found to be a very potent CDK inhibitor, with nanomolar activities against CDK1, 2, 7 and 9 (Heathcote et al , 2010; Figure 1A).…”

“…We have previously designed and optimised a number of pyrazolo[1,5- a ]pyrimidine-based CDK inhibitors (Ali et al , 2009; Heathcote et al , 2010). Among these, BS-194 was found to be a very potent CDK inhibitor, with nanomolar activities against CDK1, 2, 7 and 9 (Heathcote et al , 2010; Figure 1A). This compound was efficacious in vitro against a panel of cancer cell lines from different origins and demonstrated antitumour activity in human xenograft models of colon and breast cancer (Heathcote et al , 2010).…”

“…Among these, BS-194 was found to be a very potent CDK inhibitor, with nanomolar activities against CDK1, 2, 7 and 9 (Heathcote et al , 2010; Figure 1A). This compound was efficacious in vitro against a panel of cancer cell lines from different origins and demonstrated antitumour activity in human xenograft models of colon and breast cancer (Heathcote et al , 2010). In the present study, in cells exposed long term to a CDK inhibitor, we reveal that drug-induced overexpression of the ATP-binding cassette (ABC) transporters – ABCB1 (also known as P-glycoprotein, P-gp) and ABCG2 (breast cancer-resistant protein) – constitutes a major mechanism of drug efflux in vitro and in vivo .…”