High-resolution cryo-EM of the human CDK-activating kinase for structure-based drug design.

Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.

ICEC0942, an Orally Bioavailable Selective Inhibitor of CDK7 for Cancer Treatment.

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 IC50 of 40 nmol/L; IC50 values for CDK1, CDK2, CDK5, and CDK9 were 45-, 15-, 230-, and 30-fold higher. In vitro studies show that a wide range of cancer types are sensitive to CDK7 inhibition with GI50 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. Mol Cancer Ther; 17(6); 1156-66. ©2018 AACR.

Inhibitor Selectivity for Cyclin-Dependent Kinase†7: A†Structural, Thermodynamic, and Modelling Study.

Deregulation of the cell cycle by mechanisms that lead to elevated activities of cyclin-dependent kinases (CDK) is a feature of many human diseases, cancer in particular. We 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 used a combination of structural, biophysical, and modelling approaches. We determined the 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 (MD) simulations of inhibitors bound to CDK2 and CDK7 generated possible models of inhibitor binding. To experimentally validate these models, we gathered isothermal titration calorimetry (ITC) binding data for recombinant wild-type and binding site mutants of CDK7 and CDK2. We identified specific residues of CDK7, notably Asp155, that are involved in determining inhibitor selectivity. Our MD 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.

Dynamic kinetic resolution of dehydrocoronamic acid.

Dehydrocoronamic acid can be racemised by dehydration of an N-acyl derivative to an azlactone, which undergoes facile racemisation. For the N-trifluoroacetyl derivative, the racemisation process was combined with an enzymatic resolution, to achieve a dynamic kinetic resolution process by which the racemate can be converted to either enantiomer.

The Discovery of Novel 10,11-Dihydro-5H-dibenz[b,f]azepine SIRT2 Inhibitors.

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 IC50 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.

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.

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.

The development of a selective cyclin-dependent kinase inhibitor that shows antitumor activity.

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 CDK-activating 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 micromol/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.