Sebastian H. B. Kroll

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.

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 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.

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

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 (>100u2009Å2) 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.

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.

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.

Abstract 700: Gene expression profiling of cyclin-dependent kinase (CDK) inhibition in cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnCyclin-dependent kinases (CDKs) form complexes with partner cyclins to regulate cell cycle progression and transcription. CDK1, CDK2, CDK4 and CDK6 all regulate cell cycle progression and CDK8 and CDK9 are regulators of transcription. CDK7 is a unique member of this family because it can regulate the cell cycle CDKs as part of the CDK-activating kinase (CAK) and also transcription in conjunction with the TFIIH transcription factor by phosphorylating the COOH-terminal domain of RNA polymerase II (Pol II CTD). A hallmark of cancer is deregulated cell cycle progression and many cancers have abnormal CDK activity which makes CDKs a good target for cancer therapy. Here we show BS-194, a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7 and CDK9 (IC50 = 3, 30, 30, 250 and 90nmol/L, respectively). In vitro cell based assays have shown that BS-194 inhibits the phosphorylation of CDK substrates, Rb and RNA Poll II, down regulation of cyclins A, E and D1 and cell cycle arrest in the S and G2/M phases. BS-194 also has potent anti-proliferative activity in 60 cancer cell lines tested (mean GI50 = 28nmol/L). BS-194 is orally bioavailable and shows potent in vivo tumour growth inhibition of xenograft models of MCF7 and HCT116 cells.nnWe have used BS-194 as a potent inhibitor of CDK activity in HCT116 cells and performed gene microarray analysis to determine novel pathways and identify novel biomarkers associated with CDK inhibition. In summary, the gene microarray has identified genes differentially expressed in response to BS-194 treatment that are primarily involved in cell cycle, apoptosis and transcription pathways.nnCitation Format: Silvia Ottaviani, Sean Delaney, Hetal Patel, Manikandan Periyasamy, Alexander Bondke, Brian Slafer, Richard Starkey, Sebastian H.B. Kroll, Matthew J. Fuchter, Anthony G.M. Barrett, R. Charles Coombes, Simak Ali. Gene expression profiling of cyclin-dependent kinase (CDK) inhibition in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 700. doi:10.1158/1538-7445.AM2013-700

Abstract 694: Development of selective and potent CDK7 inhibitors for breast cancer therapy.

Cyclin dependent kinases (CDK) function as either regulators of cell cycle progression or transcription. CDK7 is unique from other CDKs because it has a dual role in the regulation of cell cycle and transcription making it a desirable target for drug development. It forms a complex with Cyclin H and MAT1 to form the CDK-activating kinase (CAK) that regulates CDK1, CDK2, CDK4 and CDK6, which are important in cell cycle progression. The CAK complex can also act as a transcriptional regulator as part of the TFIIH transcription factor by phosphorylating the COOH-terminal domain of RNA polymerase II (Pol II CTD). In addition, CDK7 as part of the CAK complex can inhibit the phosphorylation of serine 118 on the estrogen receptor (ER), a key player in breast cancer pathogenesis. Our target validation studies using siRNA have shown that CDK7 reduces growth of the human colorectal cancer cell line, HCT116, induces apoptosis and reduces phosphorylation of key biomarkers (RNA Poll II, CDK1 and CDK2). Using computer modelling of the CDK7 structure, we have designed CDK7 inhibitors, which were selected from a library of pyrazolo [1,5-a]pyrimidine-derived compounds. BS-181, is our first CDK7 inhibitor which inhibits CAK activity with an IC50 of 21 nmol/L. BS-181 has poor pharmacokinetic properties and is not orally bioavailable therefore after extensive studies and chemical modifications we have developed a more potent orally bioavailable CDK7 selective inhibitor, BS-181-L. Extensive in vitro ADME studies have highlighted that BS-181-L has good aqueous solubility, plasma proteins binding and no hERG liability. BS-181-L inhibits in vivo tumour cell growth in two cancer cell line models, MCF7 and HCT116 cells. In these studies BS-181-L shows no organ and haematological toxicity. We show BS-181-L down regulates key biomarkers in both the peripheral blood mononuclear cells (PBMCs) by flow cytometry and tumour tissue by immunohistochemistry (IHC) and western blotting. In addition, we show that BS-181-L reduces ER activity in reporter assays and ER (serine 118) phosphorylation. Furthermore, combination studies of BS-181-L and anti-estrogens show additive growth inhibitory effects on breast cancer cells. In summary, here we present data for a potent CDK7 inhibitor, BS-181-L, which has improved CDK7 selectivity, potency and bioavailability from our previously identified CDK7 inhibitor, BS-181. Furthermore, dual inhibition of CDK7 and ER has an additive effect in breast cancer cell growth. Taken together, this data highlights a potential new therapeutic approach for breast cancer. Citation Format: Hetal Patel, Silvia Ottaviani, Manikandan Periyasamy, Alexander Bondke, Brian Slafer, Richard Starkey, Sebastian H.B Kroll, Sean Delaney, Zahida Zahoor, Matthew J. Fuchter, Anthony G.M Barrett, R. Charles Coombes, Simak Ali. Development of selective and potent CDK7 inhibitors for breast cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 694. doi:10.1158/1538-7445.AM2013-694