3710 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
O’Connor et al.
[3H]farnesyl pyrophosphate and a biotin-linked k-Ras(B) de-
capeptide (KKSKTKCVIM for FTase or CVLL decapeptide for
GGTase1). The radioactivity captured by the SPA beads were
counted by a Packard Topcount, and data were stored and
analyzed in an Oracle-based database.
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M. W.; Anthony, N. J .; Holtz, W. J .; Gomez, R. P.; Lee, T.-J .;
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W. C.; Yan, N.; Barbacid, M.; Hunt, J . T.; Meyers, C. A.;
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Potent Inhibitors of Ras Farnesyltransferase Possessing Cellular
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R.; Carr, D. M.; Kirschmeier, P.; Catino, J . J .; Bryant, M. S.;
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tives of 1-(8-Chloro-6,11-dihydro-5H-benzo[5,6]-cyclohepta[1,2-
b]pyridin-11-yl)piperazine and 1-(3-Bromo-8-chloro-6,11-dihydro-
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J . S.; Quin, J ., III; Bur, S.; Thomas, C. E.; Doherty, A. M.;
Scholten, J . D.; Zimmerman, K. K.; Gibbs, B. S.; Gowan, R. C.;
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Cellu la r Assa ys for In h ibition of Ha -Ra s P r ocessin g.
Subconfluent NIH3T3 ras-transformed cells were used for the
Ras processing assay. Briefly, cells were dosed with various
compounds, and lysates were prepared. They were boiled for
5 min in the Laemmli sample buffer, and proteins were
resolved on a 15% Tris-Gly gel (Bio-Rad, Richmond, CA).
Proteins were then transferred to nitrocellulose membranes.
The blots were probed with antibody Y13-238 to Ras purified
from hybridoma. Ras bands were visualized by ECL technique
(ECL kit, Amersham, Arlington Heights, IL), and signals were
quantified by densitometry using an image analysis program
Image-Pro Plus (Media Cybernetics, Silver Spring, MD).
Ack n ow led gm en t. We thank Prof. A. D. Hamilton
(Yale University) for fruitful discussions during the
course of this research. In addition, we thank Mr.
William Arnold for the large-scale preparation of several
key intermediates.
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