A Quantitative Comparison of Wild‐Type and Gatekeeper Mutant Cdk2 for Chemical Genetic Studies with ATP Analogues

Elphick, Lucy M.; Lee, Sarah E.; Child, Emma S.; Prasad, Aarathi; Pignocchi, Cristina; Thibaudeau, Sébastien; Anderson, Alexandra; Bonnac, Laurent; Gouverneur, Véronique; Mann, David J.

doi:10.1002/cbic.200900052

Cited by 18 publications

(22 citation statements)

References 38 publications

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“…There are not currently enough CDK-selective agents available to comprehensively assess which of the many CDKs should be inhibited and in which combinations to block tumor growth. In this regard, the results of chemical-genetic screens (Bishop et al, 2000;Elphick et al, 2009;Enserink et al, 2009;Zimmermann et al, 2011;Horiuchi et al, 2012;Gravells et al, 2013) may be more informative than findings from knockout animals, since drug-inhibited CDKs may more closely resemble dominantnegative than null alleles because they likely still engage their cyclin partners and the rest of the CDK regulatory machinery. Additionally, future studies will be required to determine which combinations of subset-selective CDK inhibitors must be combined to overcome primary and acquired tumor resistance to these agents.…”

Section: Pan-cdk Inhibitorsmentioning

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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Section: Pan-cdk Inhibitorsmentioning

confidence: 99%

Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics

et al. 2015

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“…Accumulated experience so far indicates that AS-kinases have the same substrate specificity as their wild-type counterparts. 3,4,56 We have validated seven novel AS-ERK2 substrates biochemically and found that all seven are phosphorylated by wild-type ERK2. Despite these results, changes in enzymes specificity are still a possibility, and all substrates should be validated before beginning in-depth biological investigation into the function of a given PTM site.…”

Section: Challenges In Chemical Geneticsmentioning

confidence: 94%

Expanding applications of chemical genetics in signal transduction

Carlson

White

2012

Cell Cycle

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Chemical genetics represents an expanding collection of techniques applied to a variety of signaling processes. These techniques use a combination of chemical reporters and protein engineering to identify targets of a signaling enzyme in a global and non-directed manner without resorting to hypothesisdriven candidate approaches. In the last year, chemical genetics has been applied to a variety of kinases, revealing a much broader spectrum of substrates than had been appreciated. Here, we discuss recent developments in chemical genetics, including insights from our own proteomic screen for substrates of the kinase ERK2. These studies have revealed that many kinases have overlapping substrate specificity, and they often target several proteins in any particular downstream pathway. It remains to be determined whether this configuration exists to provide redundant control, or whether each target contributes a fraction of the total regulatory effect. From a general perspective, chemical genetics is applicable in principle to a broad range of posttranslational modifications (PTMs), most notably, methylation and acetylation, although many challenges remain in implementing this approach. Recent developments in chemical reporters and protein engineering suggest that chemical genetics will soon be a powerful tool for mapping signal transduction through these and other PTMs.

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“…One unit of cyclin E1/Cdk2 [27] in 5 μL was incubated for 10 mins at 30°C with either a compound or the appropriate solvent control (2 μL of 1:1 DMSO/H 2 O or kinase buffer). The enzyme activity was then assayed following addition of a substrate mix (1 μg histone H1 from Roche and 1 μCi γ-32P-ATP in kinase buffer; total reaction volume 17 μL) for 15 min at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…Sf9 cells were grown in Graces insect media (PAA) supplemented with 10% foetal bovine serum (PAA) and incubated at 27°C. Sf9 cells were infected with baculoviruses directing the expression of cyclin E1 and GST-Cdk2 [27]. Three days post-infection, the cells were harvested by centrifugation at 1,000 rpm at 4°C for 5 min.…”

Section: Methodsmentioning

confidence: 99%