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Volumn 18, Issue 12, 2008, Pages 3495-3499

Discovery of novel 4-amino-6-arylaminopyrimidine-5-carbaldehyde oximes as dual inhibitors of EGFR and ErbB-2 protein tyrosine kinases

Author keywords

Bioisostere; EGFR; ErbB 2; Oxime; Quinazoline; Receptor tyrosine kinase

Indexed keywords

EPIDERMAL GROWTH FACTOR RECEPTOR; EPIDERMAL GROWTH FACTOR RECEPTOR 2; EPIDERMAL GROWTH FACTOR RECEPTOR 3; OXIME DERIVATIVE;

EID: 44849136560     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2008.05.024     Document Type: Article
Times cited : (57)

References (31)
  • 21
    • 44849124311 scopus 로고    scopus 로고
    • 50 values were calculated with standard deviation from 2 to 8 separate experiments.
    • 50 values were calculated with standard deviation from 2 to 8 separate experiments.
  • 24
    • 4944227798 scopus 로고    scopus 로고
    • 14C-thymidine incorporation into cellular DNA as described except that total time in which cells were exposed to drug was 96 h
    • 14C-thymidine incorporation into cellular DNA as described. Emanuel S., et al. Mol. Pharm. 66 (2004) 635 except that total time in which cells were exposed to drug was 96 h
    • (2004) Mol. Pharm. , vol.66 , pp. 635
    • Emanuel, S.1
  • 25
    • 44849109456 scopus 로고    scopus 로고
    • Upstate Cell Signaling Solutions, Charlottesville, VA.
    • Upstate Cell Signaling Solutions, Charlottesville, VA.
  • 26
    • 44849124962 scopus 로고    scopus 로고
    • PDB code is 3BEL.
    • PDB code is 3BEL.
  • 27
    • 44849117091 scopus 로고    scopus 로고
    • S671-G998) which was confirmed by DNA sequencing. This expression construct was then integrated into bacmid DNA and a recombinant isolate used to tansfect SF9 insect cells as recommended by the vendor. The resulting baculovirus was then used to infect additional Sf9 cells to increase viral titer and volume.
    • S671-G998) which was confirmed by DNA sequencing. This expression construct was then integrated into bacmid DNA and a recombinant isolate used to tansfect SF9 insect cells as recommended by the vendor. The resulting baculovirus was then used to infect additional Sf9 cells to increase viral titer and volume.
  • 28
    • 44849104710 scopus 로고    scopus 로고
    • EGFR purification. All purification processes were carried out on an ÄKTA FPLC system (GE Healthcare) at 4 °C. The purification protocol was performed as described previously [J. Biol. Chem. (2002), 277, p. 46265-46272,]. Briefly, frozen cells were thawed and resuspended in 50 mM Tris pH 7.5, 200 mM NaCl, 1% glycerol, 5 mM BME, 1 X complete EDTA-free protease inhibitor cocktail (Roche). Resuspended cells were dounce homogenized and mechanically lysed with an Emulsiflex-C5 (Avestin) at 10,000-15,000 psi. The lysate was clarified by centrifugation at 40,000g for 1 h. The supernatant was filtered through a 0.8 μm vacuum filter and mixed to a TALON metal affinity resin (BD Biosciences) overnight at 4 °C. The lysate-TALON mixture was loaded into a column and washed with 10 column volumes of Buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole.)
    • EGFR purification. All purification processes were carried out on an ÄKTA FPLC system (GE Healthcare) at 4 °C. The purification protocol was performed as described previously [J. Biol. Chem. (2002), 277, p. 46265-46272,]. Briefly, frozen cells were thawed and resuspended in 50 mM Tris pH 7.5, 200 mM NaCl, 1% glycerol, 5 mM BME, 1 X complete EDTA-free protease inhibitor cocktail (Roche). Resuspended cells were dounce homogenized and mechanically lysed with an Emulsiflex-C5 (Avestin) at 10,000-15,000 psi. The lysate was clarified by centrifugation at 40,000g for 1 h. The supernatant was filtered through a 0.8 μm vacuum filter and mixed to a TALON metal affinity resin (BD Biosciences) overnight at 4 °C. The lysate-TALON mixture was loaded into a column and washed with 10 column volumes of Buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole.) EGFR was eluted using a 10 column volume linear gradient going from Buffer A to Buffer B (50 mM Tris pH 8.0, 300 mM NaCl, 250 mM Imidazole.) Fractions containing EGFR, as assayed by SDS-PAGE, were pooled. Thrombin was added to the pooled protein to remove the histidine tag (0.005 U of thrombin/μg EGFR). The reaction was dialyzed overnight against 50 mM Tris buffer pH 8.0, 250 mM NaCl, 1 mM DTT. Cleaved EGFR was filtered through a 0.2 μm SFCA cartridge filter, concentrated, and loaded onto a Superdex 200 HR 10/30 column (GE Healthcare,) preequilibrated with 50 mM Tris pH 8.0, 500 mM NaCl, 1 mM DTT. Fractions containing EGFR, as assayed by SDS-PAGE, were pooled and dialyzed against 10 mM Tris pH 8.0, 1 mM DTT, 1 mM sodium azide, 0.1 mM benzamidine.
  • 29
    • 44849101004 scopus 로고    scopus 로고
    • 16 X-ray diffraction data to a resolution of 2.0 Å were collected at the IMCA-CAT ID-17 beamline at the Argonne National Laboratory. Diffraction data were indexed, integrated, and scaled using the HKL2000 suite. The EGFR crystals belong to the P212121 space group, with unit cell parameters a = 45.50, b = 67.69, c = 103.25 Å. The structure was determined by molecular replacement with CNX [Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., Warren
    • 16 All model building was done using O [Jones, T. A., Zou, J. Y., Cowan, S. W., Kjeldgaard, M. (1991) Acta Crystallogr A47, 110-119] and refinement and map calculations were carried out using CNX [Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., Warren, G. L. (1998) Acta Crystallogr D 54, 905-921]. The final structure was refined to an Rfactor of 22.7 and Rfree of 26.7.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.