Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle. The cyclins, their activating agents, and endogenous CDK inhibitors are frequently mutated in human cancers, making CDKs interesting targets for cancer chemotherapy. Our aim is the discovery of selective CDK4/cyclin D1 inhibitors. An ATP-competitive pyrazolopyrimidinone CDK inhibitor was identified by HTS and docked into a CDK4 homology model. The resulting binding model was consistent with available SAR and was validated by a subsequent CDK2/inhibitor crystal structure. An iterative cycle of chemistry and modeling led to a 70-fold improvement in potency. Small substituent changes resulted in large CDK4/CDK2 selectivity changes. The modeling revealed that selectivity is largely due to hydrogen-bonded interactions with only two kinase residues. This demonstrates that small differences between enzymes can efficiently be exploited in the design of selective inhibitors.

Drug discovery operations have been transformed over the past 20 yr by a series of technological innovations and scientific advances that the pharmaceutical industry has been quick to exploit. The trend has been to embrace automation and high-throughput techniques and to integrate research functions into a consolidated framework for drug discovery.

Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-ones was identified that inhibit the cyclin-dependent kinase (CDK) 4/cyclin D1 complex-mediated phosphorylation of a protein substrate with IC(50)s in the low micromolar range. On the basis of preliminary structure-activity relationships (SAR), a model was proposed in which these inhibitors occupy the ATP-binding site of the enzyme, forming critical hydrogen bonds to the same residue (Val96) to which the amino group in ATP is presumed to bind. X-ray diffraction studies on a later derivative bound to CDK2 support this binding mode. Iterative cycles of synthesis and screening lead to a novel series of potent, CDK2-selective 6-(arylmethyl)pyrazolopyrimidinones. Placement of a hydrogen-bond donor in the meta-position on the 6-arylmethyl group resulted in approximately 100-fold increases in CDK4 affinity, giving ligands that were equipotent inhibitors of CDK4 and CDK2. These compounds exhibit antiproliferative effects in the NCI HCT116 and other cell lines. The potency of these antiproliferative effects is enhanced in anilide derivatives and translates into tumor growth inhibition in a mouse xenograft model.

Quinazolines have been identified as inhibitors of CDK4/D1 and CDK2/E. Aspects of the SAR were investigated using solution-phase, parallel synthesis. An X-ray crystal structure was obtained of quinazoline 51 bound in CDK2 and key interactions within the ATP binding pocket are defined.

ADVERTISEMENT RETURN TO ISSUEPREVLetterNEXTIndenopyrazoles as Novel Cyclin Dependent Kinase (CDK) InhibitorsDavid A. Nugiel, Ana-Marie Etzkorn, Anup Vidwans, Pamela A. Benfield, Michael Boisclair, Catherine R. Burton, Sarah Cox, Phillip M. Czerniak, Deborah Doleniak, and Steven P. SeitzView Author Information The DuPont Pharmaceuticals Company, Box 80336, Wilmington, Delaware 19880-0336 Cite this: J. Med. Chem. 2001, 44, 9, 1334–1336Publication Date (Web):April 19, 2001Publication History Received4 January 2001Published online19 April 2001Published inissue 1 April 2001https://pubs.acs.org/doi/10.1021/jm0100032https://doi.org/10.1021/jm0100032rapid-communicationACS PublicationsCopyright © 2001 American Chemical SocietyRequest reuse permissionsArticle Views934Altmetric-Citations72LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Cells,Drug discovery,Inhibition,Peptides and proteins,Tumors Get e-Alerts

A new structural type of kinase inhibitor, containing a benzocarbazole nucleus, has been identified. Members of the series are selective for inhibition of the cyclin dependent kinase family of enzymes. Although the cdks are highly homologous, representatives of the series showed intra-cdk selectivities, especially for cdk4. SAR studies elucidated the important features of the molecules for inhibition.

NF kappa B is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NF kappa B is anchored in the cytoplasm via the inhibitory protein I kappa B alpha. Following exposure to diverse pro-inflammatory signals (e.g. TNF alpha, IL1, LPS) various signal transduction cascades are initiated converging on the I kappa B kinase (IKK). IKK phosphorylates I kappa B alpha on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCF beta-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of I kappa B alpha. Here we reconstitute phosphorylation dependent ubiquitination of I kappa B alpha using recombinant components. Our results suggest that the cullin specificity of the SCF complex may reflect its ability to associate with Rbx1. We demonstrate specific ubiquitination of I kappa B alpha by Ubc3 and Ubc4 in a phosphorylation and SCF beta-TRCP dependent manner and that both are capable of associating with the SCF beta-TRCP complex isolated from human cells. Finally, we show that Ubc4 is in excess to Ubc3 in THP.1 cells and 19 times more efficient in catalyzing the reaction, suggesting that Ubc4 is the preferentially used Ubc in this reaction in vivo. Our results also suggest that ubiquitin is transferred directly from the Ubc to phospho-I kappa B alpha in a SCF beta-TRCP dependent reaction. Oncogene (2000) 19, 3529 - 3536