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Volumn 281, Issue 5376, 1998, Pages 533-538

Exploiting chemical libraries, struture, and genomics in the search for kinase inhibitors

Author keywords

[No Author keywords available]

Indexed keywords

CYCLIC AMP DEPENDENT PROTEIN KINASE; CYCLIC GMP DEPENDENT PROTEIN KINASE; CYCLIN A; CYCLIN DEPENDENT KINASE; CYCLIN DEPENDENT KINASE INHIBITOR; FLAVOPIRIDOL; FUNGAL PROTEIN; OLOMOUCINE; PROTEIN KINASE C; PROTEIN TYROSINE KINASE; PURINE DERIVATIVE; PURVALANOL; ROSCOVITINE; UNCLASSIFIED DRUG;

EID: 0032563315     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5376.533     Document Type: Article
Times cited : (858)

References (42)
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    • note
    • Starfish is the major source for cdc2-cyclin B kinase. The recombinant human cdc2-cyclin B is likely to contain inactive monomers and dimers that would interfere with CDK inhibition assays [see (11)].
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    • Supplemental material is available at www. sciencemag.org/feature/data/976815.shl.
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    • note
    • 2. Observed deviations: root means square (rms) bond lengths = 0.008 Å; rms bond angles = 1.31°. The final model includes 279 residues of CDK2 (residues 36 to 43 and 153 to 163 are not included because of weak or missing electron density), purvalanol B, 91 water molecules, and one molecule of ethyleneglycol. Efforts to crystallize the CDK2-purvalanol A complex resulted crystals of poor quality.
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    • note
    • 50 for 52 and flavopiridol at concentrations of 30 and 7 μM, respectively. Three cultures [110 ml, in yeast extract, peptone, and dextrose (YPD)] were inoculated with single colonies of YRP1 (MATα, Δerg6::LEU2, Δpdr5::TRP1, Δsnq2::HIS6) and grown at 30°C with constant agitation in a water bath incubator. When the cell density reached an optical density (OD) of 0.9 (at a wavelength of 600 nm), 27.5 μl of a 100 mM dimethyl sulfoxide (DMSO) stock solution of 52 or flavopiridol or DMSO alone was added. After 2 hours the cells were harvested by centrifugation and flash frozen with liquid nitrogen. For the temperature-sensitive cdc28 mutants, three cultures (75 ml, YPD) of AFS199 (cdc28-73), AA104 (cdc28-4), and their isogenic background strain AFS34 (MATa, ade2-1, his3-22, leu2-3, trp1-1, ura3) were grown from single colonies to an OD of 0.9 (600 nm) and harvested as described. Frozen cells were stored at -80°C.
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    • note
    • The diminished growth inhibitory activity of compound 52Me is unlikely to result from poorer bioavailability because a similar N6 methylation is observed to increase the in vivo potency of a related series of purine-based inhibitors. The residual growth inhibitory activity of 52Me likely reflects activity against other cellular targets. Compounds 52, 52Me, and flavopiridol failed to cause a uniform arrest morphology in yeast. FACS analysis also did not reveal synchronization of yeast cells after treatment with 52 or 52Me, which may be due to inhibition of a variety CDKs responsible for different cell cycle transitions (as is observed in FACS experiments on mammalian cells) or activity against other targets not specifically examined in vitro.
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    • note
    • Transcripts that showed a significant and reproducible change in concentration (greater than twofold) in cells treated with the compounds between triplicate hybridizations for each of at least two independent experiments were examined further.
  • 28
    • 2642654309 scopus 로고    scopus 로고
    • note
    • Because the PHO85 gene is nonessential it should be possible to determine if these inductions are a direct consequence of Pho85p kinase inhibition by determining if the same inductions are seen after treating with inhibitor in a strain lacking the kinase.
  • 31
    • 2642698935 scopus 로고    scopus 로고
    • note
    • Transcript profiles were also measured for the cdc28 temperature-sensitive allele cdc28-13. The cdc28-13 strain contains an arginine to asparagine mutation at residue 283 near the COOH-terminus, which does not significantly affect kinase activity at the permissive temperature but does cause cell cycle arrest when switched to the nonpermissive temperature (32). The cdc28-13 strain showed very few changes in mRNA transcripts when compared with wild type at the permissive temperature. The levels of only 11 mRNAs changed by more than twofold, consistent with the observation that this mutant has essentially wild-type kinase activity at 25°C. In addition, the nearly identical gene expression patterns obtained for the cdc28-13 and isogenic wild-type CDC28 strain demonstrate the reproducibility of these experiments.
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    • note
    • 2-M defect could also serve as a mimic of CDK inhibition by 52 or flavopiridol.
  • 38
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    • U.S. Patent Application 1368.002
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    • 2642659464 scopus 로고    scopus 로고
    • note
    • For example, using a screen of our purine libraries, to be described elsewhere, we have identified a compound that causes extensive depolymerization of microtubules and condensation of DNA.
  • 41
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    • J. Rine, W. Hansen, E. Hardeman, R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 80, 6750 (1983); M. Schena, D. Shalon, R.W. Davis, P. O. Brown, Science 270, 467 (1995).
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  • 42
    • 2642596125 scopus 로고    scopus 로고
    • note
    • We thank A. Murray for providing the CDC28 temperature-sensitive strains and for helpful discussions, C. Mimotopes for providing derivatized pins, D. Drubin and members of his lab, the National Cancer Institute for performing cellular screens, and M.-H. Ho for help with expression data analysis. Supported by the Director, Office of Health Effects Research of the U.S. Department of Energy (to S.-H. K. and P.G.S.), the Association pour la Recherche sur le Cancer (ARC9157 to L.M.), the Counsil Regional de Bretagne (to L.M.), and the CaP Cure Foundation. N.S.G. is supported by a NSF predoctoral fellowship and A.-M.T. by a long-term fellowship from the Human Frontier Science Program. The coordinates have been deposited with Protein Data Bank with ID code 1CKP.


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