Normal progression through the cell cycle requires the sequential action of cyclin-dependent kinases CDK1, CDK2, CDK4, and CDK6. Direct or indirect deregulation of CDK activity is a feature of almost all cancers and has led to the development of CDK inhibitors as anticancer agents. The CDKactivating kinase (CAK) plays a critical role in regulating cell cycle by mediating the activating phosphorylation of CDK1, CDK2, CDK4, and CDK6. As such, CDK7, which also regulates transcription as part of the TFIIH basal transcription factor, is an attractive target for the development of anticancer drugs. Computer modeling of the CDK7 structure was used to design potential potent CDK7 inhibitors. Here, we show that a pyrazolo[1,5-a ]pyrimidine-derived compound, BS-181, inhibited CAK activity with an IC 50 of 21 nmol/L. Testing of other CDKs as well as another 69 kinases showed that BS-181 only inhibited CDK2 at concentrations lower than 1 Mmol/L, with CDK2 being inhibited 35-fold less potently (IC 50 880 nmol/L) than CDK7. In MCF-7 cells, BS-181 inhibited the phosphorylation of CDK7 substrates, promoted cell cycle arrest and apoptosis to inhibit the growth of cancer cell lines, and showed antitumor effects in vivo. The drug was stable in vivo with a plasma elimination half-life in mice of 405 minutes after i.p. administration of 10 mg/kg. The same dose of drug inhibited the growth of MCF-7 human xenografts in nude mice. BS-181 therefore provides the first example of a potent and selective CDK7 inhibitor with potential as an anticancer agent. [Cancer Res 2009;69(15):6208-15]
Recent reports indicate that some cancer types are especially sensitive to transcription inhibition, suggesting that targeting the transcriptional machinery provides new approaches to cancer treatment. Cyclin-dependent kinase (CDK)7 is necessary for transcription, and acts by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (PolII) to enable transcription initiation. CDK7 additionally regulates the activities of a number of transcription factors, including estrogen receptor (ER)-α. Here we describe a new, orally bioavailable CDK7 inhibitor, ICEC0942. It selectively inhibits CDK7, with an IC of 40 nmol/L; IC values for CDK1, CDK2, CDK5, and CDK9 were 45-, 15-, 230-, and 30-fold higher. studies show that a wide range of cancer types are sensitive to CDK7 inhibition with GI values ranging between 0.2 and 0.3 μmol/L. In xenografts of both breast and colorectal cancers, the drug has substantial antitumor effects. In addition, combination therapy with tamoxifen showed complete growth arrest of ER-positive tumor xenografts. Our findings reveal that CDK7 inhibition provides a new approach, especially for ER-positive breast cancer and identify ICEC0942 as a prototype drug with potential utility as a single agent or in combination with hormone therapies for breast cancer. ICEC0942 may also be effective in other cancers that display characteristics of transcription factor addiction, such as acute leukaemia and small-cell lung cancer. .
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.
Table of contents entryWe have solved crystal structures of CDK7--selective inhibitors bound to CDK2.We have used a combination of structural, biophysical and modelling approaches to model the binding of these inhibitors to CDK7, and have used this information to explain their selectivity. We identify specific CDK7 residues that contribute to these inhibitors' specificity. AbstractDeregulation of the cell cycle by mechanisms that lead to elevated activities of cyclin--dependent kinases (CDK) is a feature of many human diseases, in particular cancer. We have identified small molecule inhibitors that selectively inhibit CDK7, the kinase that phosphorylates cell cycle CDKs to promote their activities. To investigate the selectivity of these inhibitors we have used a combination of structural, biophysical and modelling approaches. We have determined crystal structures of the CDK7 selective compounds ICEC0942 and ICEC0943 bound to CDK2, and used these to build models of inhibitor binding to CDK7. Molecular dynamics simulations of inhibitors bound to CDK2 and CDK7 have generated possible models of inhibitor binding. In order to experimentally 3 validate these models, we measured isothermal titration calorimetry (ITC) binding data for recombinant wild type and binding site mutants of CDK7 and CDK2. We have identified specific residues of CDK7, notably Asp155, that are involved in determining inhibitor selectivity. Our molecular dynamics simulations also show that the flexibility of the G--rich and activation loops of CDK7 is likely an important determinant of inhibitor specificity similar to CDK2.4
Isoform selective inhibitors of the sirtuins (NAD + -dependent histone deacetylases) should enable an in depth study of the molecular biology underpinning these targets and how they are deregulated in diseases such as cancer and neurodegeneration. Herein, we present the discovery of structurally novel SIRT2 inhibitors. Hit molecule 8 was discovered through the chemical synthesis and biological characterization of a small-molecule compound library based around the 10,11-dihydro-5H-dibenz[b,f]azepine scaffold. In vitro screening assays revealed compound 8 to have an IC 50 of 18 μM against SIRT2 and to exhibit more than 30-fold selectivity compared to SIRT1. Cellular assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed hit 8 to have antiproliferative activity at a concentration of 30 μM. Computational studies were performed to predict the SIRT2 binding mode and to rationalise the observed selectivity.
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