A Novel Approach to the Discovery of Small‐Molecule Ligands of CDK2

Martin, Mathew P.; Alam, R.; Betzi, S.; Ingles, Donna J.; Zhu, J.; Schönbrunn, E.

doi:10.1002/cbic.201200316

Cited by 70 publications

(70 citation statements)

References 37 publications

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“…The CDK2/ANS assay is based on the fluorescence emitted from the interaction of ANS within the allosteric pocket of CDK2 [40]. For the assays, the previously recommended concentrations of ANS and CDK2 at 50 μM and 1.6 μM (0.5mg/ml) respectively, was used.…”

Section: Methodsmentioning

confidence: 99%

Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid: A Potential Role in Cancer Prevention

Dachineni

Kumar

et al. 2016

Molecular Cancer Research

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Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin as a chemopreventive agent; however, the mechanisms leading to its anti-cancer effects are still being elucidated. We hypothesized that aspirin’s chemopreventive actions may involve cell cycle regulation through modulation of the levels or activity of cyclin A2/cyclin dependent kinase-2 (CDK2). In this study, HT-29 and other diverse panel of cancer cells were used to demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2 (CCNA2) and CDK2 protein and mRNA levels. The down regulatory effect of either drugs on cyclin A2 levels was prevented by pretreatment with lactacystin, an inhibitor of proteasomes, suggesting the involvement of 26S proteasomes. In-vitro kinase assays showed that lysates from cells treated with salicylic acid had lower levels of CDK2 activity. Importantly, three independent experiments revealed that salicylic acid directly binds to CDK2. Firstly, inclusion of salicylic acid in naïve cell lysates, or in recombinant CDK2 preparations, increased the ability of the anti-CDK2 antibody to immunoprecipitate CDK2, suggesting that salicylic acid may directly bind and alter its conformation. Secondly, in 8-anilino-1-naphthalene-sulfonate (ANS)-CDK2 fluorescence assays, pre-incubation of CDK2 with salicylic acid, dose-dependently quenched the fluorescence due to ANS. Thirdly, computational analysis using molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. These results demonstrate that aspirin and salicylic acid down-regulate cyclin A2/CDK2 proteins in multiple cancer cell lines, suggesting a novel target and mechanism of action in chemoprevention. Implications Biochemical and structural studies indicate that the anti-proliferative actions of aspirin are mediated through cyclin A2/CDK2.

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Section: Methodsmentioning

confidence: 99%

Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid: A Potential Role in Cancer Prevention

Dachineni

Kumar

et al. 2016

Molecular Cancer Research

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“…3C, ATP competitors such as roscovitine partially or fully occupy the ATP binding pocket (De Azevedo et al, 1997). Allosteric inhibitors have been discovered that bind adjacent to the ATP binding pocket, but do not engage the GEGTYG motif (Martin et al, 2012) (Fig. 3D) and represent a second distinct class of CDK inhibitors.…”

Section: Creative Approaches To Cdk Inhibitionmentioning

confidence: 99%

Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics

et al. 2015

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Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATPcompetitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non-ATPcompetitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non-ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer.

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“…3 Recently, crystal structures of CDK2 bound with the extrinsic fluorophore 8-anilino-1-naphtalene sulfonate (ANS) have been reported. 15,16 In the crystals, 2 molecules of ANS bind a cavity formed by αC and the nearby strands β4 and β5 and induce a remarkable outward displacement of the αC helix, ultimately resulting in disruption of the recognition and binding site of Cyclin A. Ternary crystal structures of CDK2 in complex with ANS and type I inhibitors (JWS648, SU9516, and staurosporine) confirmed the truly allosteric nature of this fluorophore and resulted in CDK2 conformations nearly identical to that of the binary CDK2-ANS complex. 15,16 This is the first time that a completely allosteric ligand proved to be able to displace the αC helix and to inactivate a CDK kinase via an allosteric mechanism, prospecting a new strategy to design inhibitors with potentially improved selectivity.…”

Section: Introductionmentioning

confidence: 99%

“…ANS displacement assay was performed as described by Martin et al 16 The assays were performed in 96-well plates in a total volume of 50 microliters of 40 mM Hepes pH 7.5 ANS (final concentration 50 μM), and different concentrations of the different compounds (ranging from 0.01 to 50 μM) were added and the fluorescence (excitation 360 nM, emission 460 nM) measured using an Infinite M200 Microplate Reader (Tecan) to determine the intrinsic fluorescence of the compounds. Recombinant CDK2 (final concentrations 1.5 μM) was then added and a second measurement taken.…”

mentioning

confidence: 99%