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. .

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

Background:Cyclin-dependent kinases (CDKs) control cell cycle progression, RNA transcription and apoptosis, making them attractive targets for anticancer drug development. Unfortunately, CDK inhibitors developed to date have demonstrated variable efficacy.Methods:We generated drug-resistant cells by continuous low-dose exposure to a model pyrazolo[1,5-a]pyrimidine CDK inhibitor and investigated potential structural alterations for optimal efficacy.Results:We identified induction of the ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2, in resistant cells. Assessment of features involved in the ABC transporter substrate specificity from a compound library revealed high polar surface area (>100 Å2) as a key determinant of transporter interaction. We developed ICEC-0782 that preferentially inhibited CDK2, CDK7 and CDK9 in the nanomolar range. The compound inhibited phosphorylation of CDK substrates and downregulated the short-lived proteins, Mcl-1 and cyclin D1. ICEC-0782 induced G2/M arrest and apoptosis. The permeability and cytotoxicity of ICEC-0782 were unaffected by ABC transporter expression. Following daily oral dosing, the compound inhibited growth of human colon HCT-116 and human breast MCF7 tumour xenografts in vivo by 84% and 94%, respectively.Conclusion:We identified a promising pyrazolo[1,5-a]pyrimidine compound devoid of ABC transporter interaction, highly suitable for further preclinical and clinical evaluation for the treatment of cancer.

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<p>Pharmacokinetic determination of ICEC0942 plasma and tumor concentrations at different doses and over time.</p>

<p>Analysis of ICEC0942 treatment of MCF7 cells.</p>

<p>Cell Cycle analysis of MCF7 cells treated with ICEC0942.</p>