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.
- SourceAvailable from: Jose MANUEL Martin Garcia
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- "Human CDK2 was expressed and purified as described . Briefly, the protein was expressed in E. coli BL21(DE3) cells with a His6-SUMO-1 N-terminal tag which, after immobilized metal affinity chromatography, was removed by the Senp2 protease. "
ABSTRACT: PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86 kDa by the symmetric association of three identical protomers. The ring encircles the DNA and acts as a docking platform for other proteins, most of them containing the PCNA Interaction Protein sequence (PIP-box). We have used NMR to characterize the interactions of PCNA with several other proteins and fragments in solution. The binding of the PIP-box peptide of the cell cycle inhibitor p21 to PCNA is consistent with the crystal structure of the complex. A shorter p21 peptide binds with reduced affinity but retains most of the molecular recognition determinants. However the binding of the corresponding peptide of the tumor suppressor ING1 is extremely weak, indicating that slight deviations from the consensus PIP-box sequence dramatically reduce the affinity for PCNA, in contrast with a proposed less stringent PIP-box sequence requirement. We could not detect any binding between PCNA and the MCL-1 or the CDK2 protein, reported to interact with PCNA in biochemical assays. This suggests that they do not bind directly to PCNA, or they do but very weakly, with additional unidentified factors stabilizing the interactions in the cell. Backbone dynamics measurements show three PCNA regions with high relative flexibility, including the interdomain connector loop (IDCL) and the C-terminus, both of them involved in the interaction with the PIP-box. Our work provides the basis for high resolution studies of direct ligand binding to PCNA in solution.PLoS ONE 11/2012; 7(11):e48390. DOI:10.1371/journal.pone.0048390 · 3.23 Impact Factor
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ABSTRACT: Inhibition of cyclin-dependent kinases (CDKs) with small molecules has been suggested as a strategy for treatment of cancer, based on deregulation of CDKs commonly found in many types of human tumors. Here, a new potent CDK2 inhibitor with pyrazolo[4,3-d]pyrimidine scaffold has been synthesized, characterized, and evaluated in cellular and biochemical assays. 7-Benzylamino-5(R)-[2-(hydroxymethyl)propyl]amino-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine, compound 7, was prepared as a bioisostere of the well-known CDK inhibitor roscovitine. An X-ray crystal structure of compound 7 bound to CDK2 has been determined, revealing a binding mode similar to that of roscovitine. Protein kinase selectivity profile of compound 7 and its biological effects (cell cycle arrest, dephosphorylation of the retinoblastoma protein, accumulation of the tumor suppressor protein p53, induction of apoptosis, inhibition of homologous recombination) are consistent with CDK inhibition as a primary mode of action. Importantly, as the anticancer activities of the pyrazolo[4,3-d]pyrimidine 7 exceed those of its bioisostere roscovitine, compound 7 reported here may be preferable for cancer therapy.Journal of Medicinal Chemistry 03/2011; 54(8):2980-93. DOI:10.1021/jm200064p · 5.45 Impact Factor
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ABSTRACT: Two novel pyrazolo[1,5-a]pyrimidine derivatives, 7-(2-[(18)F]fluoroethylamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile ([(18)F]FEMPPC, [(18)F]1) and N-(2-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-ylamino)ethyl)-2-[(18)F]fluoro-4-nitrobenzamide ([(18)F]FCMPPN, [(18)F]2), have been designed and successively labeled with (18)F by the nucleophilic substitution employing tosylate and nitryl as leaving groups, respectively. The radiochemical synthesis of both compounds was completed within 60min with final high-performance liquid chromatography purification included. The corresponding radiochemical yields (without decay correction) were approximately 35% and 30%, respectively. Meanwhile, we compared the uptake characteristics of [(18)F]1 and [(18)F]2 with those of [(18)F]FDG and L-[(18)F]FET in S180 tumor cells. Furthermore, the tumor uptake of [(18)F]1 and [(18)F]2 was assessed in mice bearing S180 tumor and compared with [(18)F]FDG and L-[(18)F]FET in the same animal model. In vitro cell uptake studies showed [(18)F]1 had higher uptake than [(18)F]FDG, [(18)F]2 and L-[(18)F]FET over the 2h period. In ex vivo biodistribution showed tumor/brain uptake ratios of [(18)F]2 were 12.35, 10.44, 8.69 and 5.13 at 15 min, 30 min, 60 min and 120 min post-injection, much higher than those of L-[(18)F]FET (2.43, 2.54, 2.93 and 2.95) and [(18)F]FDG (0.59, 0.61, 1.02 and 1.33) at the same time point. What's more, the uptake of [(18)F]1 in tumor was 1.88, 4.37, 5.51, 2.95 and 2.88 at 5 min, 15 min, 30 min, 60 min and 120 min post-injection, respectively. There was a remarkable increasing trend before 30 min. The same trend was present for L-[(18)F]FET before 30 min and [(18)F]FDG before 60 min. Additionally, the tumor/brain uptake ratios of [(18)F]1 were superior to those of [(18)F]FDG at all the selected time points, the tumor/muscle and tumor/blood uptake ratios of [(18)F]1 at 30 min were higher than those of L-[(18)F]FET at the same time point. MicroPET image of [(18)F]1 administered into S180 tumor-bearing mouse acquired at 30 min post-injection illustrated that the uptake in S180 tumor was obvious. These results suggest that compound [(18)F]1 could be a new probe for PET tumor imaging.Bioorganic & medicinal chemistry letters 08/2011; 21(16):4736-41. DOI:10.1016/j.bmcl.2011.06.072 · 2.42 Impact Factor