Chemistry: molecular biology and microbiology – Vector – per se
Patent
1995-04-24
1998-10-13
Jacobson, Dian C.
Chemistry: molecular biology and microbiology
Vector, per se
435 15, 435193, 4352523, 43525233, 536 232, C12N 121, C12N 910, C12N 1554
Patent
active
058211187
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The ras gene is found activated in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein, since ras must be localized in the plasma membrane and must bind with GTP in order to transform cells (Gibbs, J. et al., Microbiol. Rev., 53, 171-286 (1989)). Forms of ras in cancer cells have mutations that distinguish the encoded protein from Ras in normal cells.
At least 3 post-translational modifications are involved with Ras membrane localization, and all 3-modifications occur at the C-terminus of Ras. The Ras C-terminus contains a sequence motif termed a "CAAX" or "Cys-Aaa1-Aaa2-Xaa" box (Aaa is an aliphatic amino acid and Xaa is any amino acid) (Willumsen et al., Nature, 310, 583-586(1984)). Other proteins having this motif include the Ras-related GTP-binding proteins such as Rho, fungal mating factors, the nuclear lamins, and the gamma subunit of transducin.
Farnesyl-protein transferase (FPTase) catalyzes the addition of the isoprenoid farnesyl, from farnesyl diphosphate, to a cysteine residue of such protein substrates having the CAAX terminus (Reiss, Y., Goldstein, J. L., Seabra, M. C., Casey, P. J. and Brown, M. S. (1990) Cell 62, 81-88; Schaber, M. D., O'Hara, M. B., Garsky, V. M., Moser, S. D., Bergstrom, J. D., Moores, S. L., Marshall. M. S., Friedman, P. A., Dixon, R. A. F. and Gibbs, J. B. (1990) J. Biol. Chem. 265, 14701-14704; Manne, V., Roberts, D., Tobin, A., O'Rourke, E., De Virgilio, M., Meyers, C., Ahmed, N., Kurz, B., Resh, M., Kung, H.-F. and Barbacid, M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7541-7545). Farnesylation of Ras facilitates its membrane binding which is essential for efficient cell transformation by oncogenic forms of Ras (Willumsen, B. M., Norris, K., Papageorge, A. G., Hubbert, N. L. and Lowy, D. R. (1984) EMBO J. 3, 2581-2584). Thus, inhibitors of FPTase may be antitumor agents. CAAX tetrapeptides are substrates for FPTase with kinetic properties similar to polypeptide substrates indicating that the critical determinants required for enzyme recognition are contained within the CAAX box (Reiss et al., Cell, supra; Schaber et al., J. Biol. Chem., supra; Pompliano, D. L., Rands, E., Schaber, M. d., Mosser, S. D., Anthony, N. J. and Gibbs, J. B. (1992) Biochem. 31, 3800-3807; Goldstein, J. L. Brown, M. S., Stradley, S. J., Reiss, Y. and Gierasch, L. M. (1991) J. Biol. Chem. 266, 15575-15578).
Mammalian FPTase is an .alpha..beta. heterodimeric protein. Complementary DNAs encoding part or all of the .alpha. subunit of FPTase from bovine and rat brain have been isolated (Kohl, N. E. et al., (1991) J. Biol. Chem. 266, 18884-18888; Chen et al. (1991) Cell, 66, 327-334; Chen et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11368-11372). The proteins that they encode share >95% amino acid sequence identity with one another and 30% identity with the RAM2-encoded subunit of Saccharomyces cerevisiae FPTase (He, B. et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11373-11377; Kohl et al., supra). A cDNA encoding the .beta. subunit of rat brain FPTase has been isolated and the protein it encodes shares 37% amino acid sequence identity with the DPR1/RAM1 (RAM1) encoded subunit of S. cerevisiae FPTase (Goodman, L. E. et al. (1988) Yeast, 4, 271-281; Chen et al. (1991) Cell, 66, 327-334) However these cDNAs have not been used for expression of the complete FPTase that is readily isolatable in large quantities.
Previously, amounts of FPTase useful in assessing the inhibitory activity of pharmaceutical agents have been isolated from animal tissue such as rat or bovine brain. However, relatively low amounts of FPTase are present in rat and bovine brain and purification of the enzyme involves a number of separation steps.
Human FPTase, the ideal enzyme to employ in an assay directed at discovering a human anticancer agent, has not been employed previously because of the limited amounts of human FPTase available fr
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Diehl Ronald E.
Gibbs Jackson B.
Kohl Nancy E.
Omer Charles A.
Daniel Mark R.
Jacobson Dian C.
Merck & Co. , Inc.
Muthard David A.
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