©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Non-peptide Mimetic of Ras-CAAX: Selective Inhibition of Farnesyltransferase and Ras Processing (*)

(Received for publication, October 28, 1994)

Andreas Vogt (1) Yimin Qian (2) Michelle A. Blaskovich (1) Renae D. Fossum (2) Andrew D. Hamilton (2)(§) Said M. Sebti (1)(§)

From the  (1)Department of Pharmacology, School of Medicine, and the (2)Department of Chemistry, Faculty of Arts and Science, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Cysteine farnesylation of the carboxyl-terminal tetrapeptide CAAX (C = Cys, A = Leu, Ile, or Val, X = Met or Ser) of the oncogene product Ras is required for its malignant transformation activity. As a consequence farnesyltransferase (FTase), the enzyme responsible for this lipid modification, has become one of the most sought-after targets for anticancer drug development. We have recently designed peptide mimics of the COOH-terminal Cys-Val-Ile-Met of K(B)-Ras where the dipeptide Val-Ile was replaced by aminobenzoic acid derivatives. Although these peptidomimetics are potent inhibitors of FTase in vitro, they retain several undesirable peptide features that hamper their use in vivo. We report here the design, synthesis, and biological activity of the first non-peptide mimetics of CAAX where the tripeptide AAX was replaced by biphenyl derivatives. (R)-4-[N-(3-mercapto-2-aminopropyl)]amino-3`-carboxybiphenyl, where the cysteine is linked to the biphenyl derivative through a secondary amine, contains no amino acids, lacks peptidic features, and has no hydrolyzable bonds. This peptidomimetic is a potent inhibitor of FTase in vitro (IC = 50-150 nM) and disrupts Ras processing in whole cells. Furthermore, this non-peptide mimetic of CAAX is highly selective for FTase (666-fold) relative to the closely related geranylgeranyltransferase I. This selectivity is also respected in vivo since the processing of Ras but not the geranylgeranylated Rap1A was disrupted in whole cells. Structure activity relationship studies revealed that FTase recognition and inhibitory potency of CAAX peptidomimetics require free thiol and carboxylate groups separated by a hydrophobic moiety, and that precise positioning of these functional groups must correspond to that of the parent CAAX. The true CAAX peptidomimetic described in this manuscript has several desirable features for further development as a potential anticancer agent. It is not metabolically inactivated by FTase, does not require a prodrug strategy for inhibition in vivo, and is selective for farnesylation relative to geranylgeranylation.


INTRODUCTION

Ras is a small guanine nucleotide-binding GTPase that transduces biological information from the cell surface to the nucleus(1) . Its ability to transfer growth signals from receptor tyrosine kinases to a mitogen-activated protein kinase cascade puts it in the heart of signaling pathways that cause proliferation in normal cells and uncontrolled growth in cancer cells(2) . Indeed, mutations that lock Ras in its active, GTP-bound state lead to malignant transformation and are among the most frequently identified mutations in human cancers (1) . For example, 50% of colorectal and 95% of pancreatic human cancers have activated ras oncogenes.

Over the last decade several strategies have been investigated, with little success, to disrupt Ras function and, hence, to inhibit the growth of tumors with activated ras oncogenes. The search has recently intensified with the discovery that Ras requires lipid modification with a farnesyl group for localization to the plasma membrane, where it plays a pivotal role in growth signaling(3, 4, 5, 6, 7, 8, 9) . Because farnesylation is required as well as sufficient for Ras membrane association and transformation(10) , the enzyme that catalyzes this lipid modification, farnesyltransferase (FTase), (^1)has become a major target for the design of novel anticancer agents(11, 12) . FTase is an alpha and beta heterodimer that transfers farnesyl from farnesylpyrophosphate, a cholesterol biosynthesis intermediate, to the cysteine of proteins containing the carboxyl-terminal sequence CAAX (A = aliphatic and X = any amino acid except Leu or Ile)(13, 14) . A closely related prenyltransferase, geranylgeranyltransferase I (GGTase I) catalyzes cysteine geranylgeranylation of proteins ending in CAAX where X = Leu or Ile(15, 16) . Prenylation of CAAX sequences by FTase and GGTase I is followed by proteolysis of the tripeptide AAX and carboxymethylation of the resulting prenylated cysteine. Since the number of geranylgeranylated proteins in the cell far exceeds that of farnesylated proteins(15, 16) , it is critical that farnesylation inhibitors with potential anticancer activity be highly selective for FTase over GGTase I to minimize side effects.

Developing Ras CAAX tetrapeptide mimics as anticancer drugs has been the focus of several laboratories over the last two years(11, 12) . This was prompted by the observation that FTase recognizes and farnesylates CAAX peptides which were also found to be potent competitive inhibitors of the enzyme (IC = 50-200 nM)(13, 17, 18, 19, 20, 21, 22) . Because of their peptidic nature, CAAX peptides do not inhibit Ras processing in whole cells. To enhance their poor cellular uptake and decrease their sensitivity to cellular proteases, several investigators have made CAAX pseudopeptides(23, 24, 25) . Reduction of the amino-terminal and central amide bonds of CAAX and neutralization of the free carboxylate resulted in greater activity in whole cells(23, 24, 25) . Although the FTase inhibitors discussed above are potent inhibitors in vitro, they retain several peptidic features. To avoid inherent problems of peptides, our strategy has been to design non-peptide CAAX mimetics. Working toward this goal, we initially made potent inhibitors of FTase where ``VI'' in CVIM, the carboxyl terminus of K(B)-Ras, was replaced by 4-aminobenzoic acid (4ABA) derivatives that served as hydrophobic spacers to link Cys to Met (i.e. Cys-4ABA-Met)(26, 27, 28) . Similarly, James et al.(29) used a benzodiazepine group between cysteine and methionine.

A critical and as yet unreported goal in this area has been to construct non-peptidic inhibitors with no amide bonds in their molecular backbone. Our approach to improving the in vivo potency and hydrolytic stability of our inhibitors involved extending the hydrophobic spacer strategy to include the terminal methionine as well as the central aliphatic residues of CVIM. Here we report the design, synthesis, and selective inhibition of FTase in vitro and in vivo by the first non-peptide CAAX mimetic where 4-amino-3`-carboxybiphenyl was designed as a VIM tripeptide mimetic.


EXPERIMENTAL PROCEDURES

Synthesis

The peptidomimetic 2 (Fig. 1) was synthesized as described previously(27) , and 3 was prepared by similar steps following reductive amination of 4-aminobenzoyl methionine methyl ester and N-tert-butoxycarbonyl-S-trityl cysteinal. The peptidomimetics 4a, 4b, and 4c were prepared from the corresponding 4-aminobiphenyl (prepared from Suzuki coupling (30) of 1-bromo-4-nitrobenzene and 1-bromo-3-(or 4-)methylbenzene and, for 4a and 4b, KMnO(4) oxidation, tert-butanol esterification, and hydrogenation) by reductive amination with N-tert-butoxycarbonyl-S-trityl cysteinal followed by deprotection. All compounds were more than 98% pure as determined by reverse phase high performance liquid chromatography, and their spectroscopic data were consistent with the assigned structures.


Figure 1: Ras CAAX peptidomimetics.



FTase and GGTase I Activity Assay

FTase and GGTase I activities from 60,000 times g supernatants of human Burkitt lymphoma (Daudi) cells (ATCC, Rockville, MD) were assayed as described previously for FTase(26, 27) . Briefly, 100 µg of the supernatants was incubated in 50 mM Tris, pH 7.5, 50 µM ZnCl(2), 20 mM KCl, 3 mM MgCl(2), and 1 mM dithiothreitol. The reaction was incubated at 37 °C for 30 min with recombinant Ha-Ras-CVLS (11 µM) and [^3H]FPP (625 nM; 16.3 Ci/mmol) for FTase, and recombinant Ha-Ras-CVLL (5 µM) and [^3H]geranylgeranylpyrophosphate (525 nM; 19.0 Ci/mmol) for GGTase I. The peptidomimetics were mixed with FTase and GGTase I before adding the reaction mixture. Recombinant Ha-Ras-CVLS was prepared as described previously (26) from bacteria obtained from Dr. Robert Crowl (Hoffman LaRoche Inc.)(31) . Recombinant Ha-Ras-CVLL was prepared from bacteria obtained from Drs. C. J. Der and A. D. Cox (University of North Carolina)(32) . The ability of human Burkitt lymphoma (Daudi) FTase to farnesylate peptides and peptidomimetics was determined as described previously(26, 27) .

Ras and Rap1A Processing Assay

Human Ha-Ras oncogene-transformed Balb/c 3T3 cells (EJ3 cells) (33) were treated with peptidomimetics or vehicle for 20 h. Cells were lysed in lysis buffer (10 mM Na(2)HPO(4), pH 7.25, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 12 mM sodium deoxycholate, 1 mM NaF, 0.2% NaN(3), 2 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin). Ras proteins were immunoprecipitated from cleared lysate, with anti-Ras antibody Y13-259 along with 30 µl of Protein A-agarose goat anti-rat IgG complex. Immunoprecipitates were washed and electrophoresed on a 12.5% SDS-PAGE, transferred onto nitrocellulose, and probed with Y13-259. Positive antibody reactions were visualized using peroxidase-conjugated goat anti-rat IgG and an enhanced chemiluminescence detection system (ECL; Amersham Corp.).

For Rap1A processing assays, 50 µg of cell lysates were electrophoresed as described above for Ras processing and transferred to nitrocellulose. The membranes were then probed with anti-Rap1A (Santa Cruz Biotechnology, Santa Cruz, CA). Antibody reactions were visualized using peroxidase-conjugated goat anti-rabbit IgG and ECL chemiluminescence as described above.


RESULTS AND DISCUSSION

Design and Synthesis of Non-peptide CAAX Mimetics

Over the past 2 years, we (26, 27, 28) and others (23, 24, 25, 29) have succeeded in synthesizing CAAX peptidomimetics that are potent inhibitors of FTase, a novel target of anticancer drug development. However, these peptidomimetics still retain several amide bonds and other peptide features and are thus vulnerable to proteolytic degradation inside cells. A desirable goal in using these molecules as therapeutic agents is to eliminate these features and, hence, make true non-peptide CAAX mimetics. Initially, we designed a family of mimetics of tetrapeptide CVIM 1 (from K(B)-Ras) where VI was replaced by aminobenzoic acid (ABA) derivatives(26, 27, 28) . Cys-4ABA-Met 2 is a CVIM peptidomimetic (Fig. 1) that potently inhibits FTase (IC = 50-200 nM). Although this peptidomimetic lacks one peptide bond and is made of 2 instead of 4 amino acids, it still retains several peptide features. We have therefore extended our hydrophobic spacer strategy and have designed a 4-amino-3`-carboxybiphenyl derivative as a ``VIM'' tripeptide mimic with restricted conformational flexibility. Attachment of this group to cysteinal via reductive amination leads to (R)-4-[N-(3-mercapto-2-aminopropyl)]amino-3`-carboxybiphenyl 4a (Fig. 1). This derivative contains no amide bonds and thus is a true non-peptide mimetic of the CAAX tetrapeptide. Although 4a and its parent compounds 1, 2, and 3 are structurally quite distinct, they share several features that are key to FTase recognition. They all have free cysteine thiol, terminal amine, and carboxylate groups that are separated by a hydrophobic moiety. Furthermore, molecular modeling studies gave energy-minimized structures that show similar distances between the cysteine thiol and the free carboxylate in both 4a and CVIM in its extended conformation (Fig. 2). We have also made several analogues of 4a to further probe the FTase binding site. Among these, analogue 4b (Fig. 1) contains the free carboxylate in the 4`- rather than the 3`-position of the biphenyl. In peptidomimetic 4b the separation between the cysteine thiol and carboxylate no longer corresponds to that of CVIM. Another analogue with a methyl group in the 3` position (4c) was also synthesized to determine whether a negatively charged carboxylate at the 3` position is critical to FTase inhibition.


Figure 2: Energy-minimized structures for CVIM and 4a.



Non-peptide CAAX Mimetic Potently Inhibits FTase

Cys-4ABA-Met 2 (1-10 µM) inhibited FTase in a concentration-dependent manner with an IC of 150 nM (Table 1). This value is similar to our previously reported IC values (26, 27) . Reduction of the amide bond between cysteine and aminobenzoic acid resulted in reduced Cys-4ABA-Met 3 which had an IC of 300 nM. Replacing the methionine and the COOH-terminal amide bond in 3 by another aromatic ring to obtain the biphenyl-based peptidomimetic 4a (Fig. 1) improved potency (Table 1). Peptidomimetic 4a had an IC of 150 nM toward partially purified FTase from human Burkitt lymphoma cells and 50 nM toward rat brain FTase purified to homogeneity (a gift from Dr. Patrick Casey, Duke University). Thus, despite major structural differences between the parent compound CVIM 1 and 4a, the second generation CAAX peptidomimetic 4a retained the potent FTase inhibitory activity of its parent tetrapeptide CVIM 1 and the first generation CAAX peptide mimetics 2 and 3 (Table 1). The position of the free carboxylate group is critical to FTase inhibition since placing this group at the 4`- position as in 4b (Fig. 1) dramatically decreases its affinity toward FTase (Fig. 3A, Table 1). Peptidomimetic 4b (IC = 6650 nM) is 45-fold less potent than its isomer 4a. This strongly suggests that within the active site of FTase, there must be a positively charged residue such as Lys to interact with the negative charge of the free carboxylate. Interestingly, however, replacing the 3`-carboxylate with a methyl group as in 4c decreased potency by only 5-fold (IC = 765 nM). This shows that the FTase binding site prefers a neutral group at the 3`-position to a negative charge at the 4`-position, suggesting either steric hindrance or repulsive interactions from the microenvironment surrounding the 4`-region of the biphenyl.




Figure 3: FTase and GGTase I inhibition studies. Partially purified FTase and GGTase I were incubated with Ras CAAX peptidomimetics and their ability to transfer [^3H]farnesyl to Ha-Ras-CVLS (FTase) and [^3H]geranylgeranyl to Ha-Ras-CVLL (GGTase I) was determined as described under ``Experimental Procedures.'' A, FTase inhibition by: 4a (box) and 4b (); B, FTase (box) and GGTase I () inhibition by 4a. Each curve is representative of at least four independent experiments.



An earlier proposal (29, 34) suggested that potent inhibitory activity toward FTase might require inhibitors to take up a beta turn conformation bringing the cysteine thiol and free carboxylate in close proximity to form a bidentate complex with Zn. Recently, we have provided evidence that argues against this model(27) . Consistent with this, the biphenyl-based non-peptide CAAX mimetic described here cannot take up this structure (Fig. 2), leading us to conclude that potent inhibition of FTase does not require a beta turn conformation.

Non-peptide CAAX Mimetics Are Not Farnesylated

Besides having poor cellular uptake and being rapidly degraded, a third disadvantage of natural CAAX peptides is that they are farnesylated by FTase. This results in metabolic inactivation since farnesylated CAAX derivatives are no longer inhibitors of FTase(26) . Fig. 4shows that the natural peptide CVLS (carboxyl-terminal CAAX of Ha-Ras) is farnesylated by FTase from Burkitt lymphoma cells. Replacing the tripeptide VLS with 4-amino-3`-carboxybiphenyl, as in 4a, did not affect potency toward FTase inhibition (Table 1) but prevented farnesylation of the cysteine thiol (Fig. 4). None of our peptidomimetics are metabolically inactivated by FTase (Fig. 4).


Figure 4: Ras CAAX peptide and peptidomimetic farnesylation. The transfer of [^3H]farnesyl to peptides and peptidomimetic by FTase was determined by silica G TLC as described under ``Experimental Procedures.'' FPP and F-peptide designate farnesyl pyrophosphate and farnesylated peptide, respectively. Lane 1, FPP only; lane 2, FPP and CVLS but no FTase; lane 3, FPP and FTase but no peptide; lanes 4-9, all contained FTase and FPP. Lane4, VCIM; lane5, CVLS; lane6, 3; lane7, 4a; lane 8, 4b, lane 9, 4c. Data are representative of two independent experiments.



Non-peptide CAAX Mimetics Are Highly Selective for FTase Relative to GGTase I

Geranylgeranylation is a more common protein prenylation than farnesylation(15) . It is therefore critical to design CAAX peptidomimetics with high selectivity toward inhibiting FTase to minimize side effects. In the parent CAAX tetrapeptides, the X position determines whether the cysteine thiol will be farnesylated by FTase or geranylgeranylated by GGTase I. Those proteins or peptides with Leu or Ile at the X position are geranylgeranylated. The peptidomimetics described here no longer contain amino acids that would dictate this selectivity, and therefore it is important to evaluate their ability to inhibit GGTase I prior to further development. Fig. 3B shows that our most potent FTase inhibitor is a very poor GGTase I inhibitor. The ability of 4a to inhibit the transfer of geranylgeranyl to Ras-CVLL (IC = 100,000 nM) was 666-fold less than that of 4a to inhibit the transfer of farnesyl to Ras-CVLS (IC = 150 nM) (Table 1). This selectivity was much more pronounced than in the first generation peptidomimetics 2 and 3, which were more selective for FTase relative to GGTase I by only 10 and 15-fold, respectively (Table 1). Furthermore, the free carboxylate is not responsible for this selectivity since replacement of this group by a methyl (4c) did not increase affinity toward GGTase I (Table 1). These results suggest that FTase and GGTase I binding sites are quite different and that differences among Leu, Ile, and Met side chains cannot be the only predictors of selectivity.

Non-peptide CAAX Mimetics Inhibit Ras but Not Rap1A Processing

A key factor that will determine further development of these molecules as anticancer drugs is whether they inhibit Ras processing in whole cells and whether the selectivity toward FTase is retained in vivo. To determine the efficiency of our CAAX peptidomimetics to disrupt Ras processing, we treated ras oncogene-transformed cells with these inhibitors, immunoprecipitated processed (P) and unprocessed (U) Ras proteins from lysates, separated them by SDS-PAGE, and immunoblotted with anti-Ras antibody that recognizes both forms of Ras. Farnesylated Ras runs faster than unprocessed Ras on SDS-PAGE(23, 24, 25, 28, 29) . Fig. 5A (lane1) shows that cells treated with vehicle contain only processed Ras, whereas cells treated with lovastatin (lane2) contained both processed and unprocessed Ras, indicating that lovastatin inhibited Ras processing. Lovastatin, an HMG-CoA reductase inhibitor that inhibits the biosynthesis of farnesylpyrophosphate and geranylgeranylpyrophosphate, is used routinely as a positive control for inhibition of processing of both geranylgeranylated and farnesylated proteins(23, 24, 25, 28, 29) . Cells treated with reduced Cys-4ABA-Met 3 in its free carboxylate forms did not inhibit Ras processing (data not shown). In contrast, the corresponding methyl ester of 3 (200 µM) inhibited FTase (Fig. 5A, lane3). This is consistent with previous work showing that neutralization of the carboxylate of CAAX peptides enhances their ability to inhibit Ras processing(23, 25, 29) . This prodrug strategy relies on cellular esterases to activate the peptidomimetics. Although peptidomimetic 4a has a free carboxylate negative charge, it was able to enter cells and potently inhibit Ras processing (lane 4, 100 µM4a). Peptidomimetic 4a inhibited Ras processing with concentrations as low as 50 µM (lane 5), whereas its corresponding parent compound 3 did not inhibit Ras processing at concentrations as high as 200 µM (data not shown). Peptidomimetic 4a was as potent as the methylester of its parent compound 3 (Fig. 5A, lane3). Furthermore, 4a is the first CAAX peptidomimeticthat effectively inhibits Ras processing in whole cells directly without relying on cellular enzymes for activation. The hydrophobic character of the biphenyl group may compensate for the free carboxylate negative charge hence allowing the peptidomimetic to penetrate membranes and promoting its cellular uptake. Peptidomimetics 4b and 4c were not able to inhibit Ras processing (Fig. 5A, lanes7 and 8).


Figure 5: Ras and Rap1A processing. A, Ras-transformed Balb/c 3T3 cells were treated with inhibitors, the lysate was immunoprecipitated and blotted with anti-Ras antibody. B, lysates were blotted with anti-Rap1A antibody as described under ``Experimental Procedures.'' Lane 1, control; lane 2, lovastatin; lane 3, methyl ester of 3 (200 µM); lane4, 4a (100 µM); lane5, 4a (50 µM); lane6, 4a (25 µM); lane7, 4b; lane8, 4c. Data are representative of three independent experiments.



We then investigated the selectivity of our Ras farnesylation inhibitors by determining their ability to inhibit processing of Rap1A, a small G-protein that is geranylgeranylated(15, 16) . Cells were treated with lovastatin or peptidomimetics exactly as described for Ras processing experiments. Lysates were then separated by SDS-PAGE and immunoblotted with anti-Rap1A antibody as described under ``Experimental Procedures.'' Control cells contained only the geranylgeranylated Rap 1A (Fig. 5B, lane1), whereas lovastatin-treated cells contained both processed and unprocessed forms of Rap 1A, indicating, as expected, that lovastatin inhibited the processing of Rap 1A (Fig. 5B, lane2). Peptidomimetic 4a, which inhibited Ras processing, was not able to inhibit Rap 1A geranylgeranylation (Fig. 5B, lanes4-6). Peptidomimetics 4b and 4c did not inhibit Rap 1A processing (Fig. 5B, lanes7 and 8).

Thus, we have designed the first non-peptide CAAX mimetic that contains no hydrolyzable groups and lacks other peptidic features. Although we have made major structural alterations in the parent CAAX tetrapeptide, the peptidomimetic retained high potency toward inhibiting FTase. Structure activity relationship studies further revealed that the crucial structural features for FTase recognition and potent inhibition are free thiol and carboxylate groups maintained at a critical distance and orientation by a hydrophobic spacer. Furthermore, these true CAAX peptidomimetics have several desirable features as potential therapeutic agents. They are selective inhibitors for farnesylation relative to geranylgeranylation both in vitro and in vivo, they do not require a prodrug strategy for whole cell activity, and they are not metabolically inactivated by FTase. We are presently evaluating their selectivity toward antagonizing oncogenic Ras signaling and their efficacy as anticancer agents against tumors with activated ras oncogenes.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed.

(^1)
The abbreviations used are: FTase, farnesyltransferase; GGTase, geranylgeranyltransferase; PAGE, polyacrylamide gel electrophoresis; CAAX, tetrapeptides where C = cysteine, A = aliphatic amino acid, and X = any amino acid; 4ABA, 4-aminobenzoic acid; FPP, farnesylpyrophosphate.


REFERENCES

  1. Barbacid, M. (1987) Annu. Rev. Biochem. 56, 779-828 [CrossRef][Medline] [Order article via Infotrieve]
  2. McCormick, F. (1993) Nature 363, 15-16 [CrossRef][Medline] [Order article via Infotrieve]
  3. Willumsen, B. M., Christensen, A., Hubbert, N. C., Papageorge, A. C., and Lowy, D. R. (1984) Nature 310, 583-586 [Medline] [Order article via Infotrieve]
  4. Willumsen, B. M., Norris, K., Papageorge, A. G., Hubbert, N. C., and Lowy, D. R. (1984) EMBO J. 3, 2581-2585 [Abstract]
  5. Hancock, J. F., Magee, A. I., Childs, J. E., and Marshall, C. J. (1989) Cell57, 1167-1177 [Medline] [Order article via Infotrieve]
  6. Gutierrez, L., Magee, A. I., Marshall, C. J., and Hancock, J. F. (1989) EMBO J. 8, 1093-1098 [Abstract]
  7. Casey, P. J., Solski, P. A., Der, C. J., and Buss, J. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8323-8327 [Abstract]
  8. Jackson, J. H., Cochrane, C. G., Bourne, J. R., Solski, P. A., Buss, J. E., and Der, C. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3042-3046 [Abstract]
  9. Hancock, J. F., Paterson, H., and Marshall, J. C. (1990) Cell 63, 133-139 [Medline] [Order article via Infotrieve]
  10. Kato, K., Cox, A. D., Hisaka, M. M., Graham, S. M., Buss, J. E., and Der, C. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6403-6407 [Abstract]
  11. Gibbs, J. B. (1991) Cell 65, 1-4 [Medline] [Order article via Infotrieve]
  12. Gibbs, J. B., Oliff, A., and Kohl, N. E. (1994) Cell 77, 175-178 [Medline] [Order article via Infotrieve]
  13. Reiss, Y., Goldstein, J. L., Seabra, M. C., Casey, P. J., and Brown, M. S. (1990) Cell 62, 81-88 [Medline] [Order article via Infotrieve]
  14. Reiss, Y., Seabra, M. C., Armstrong, S. A., Slaughter, C. A., Goldstein, J. L., and Brown, M. S. (1991) J. Biol. Chem. 266, 10672-10877 [Abstract/Free Full Text]
  15. Casey, P. (1992) J. Lipid Res. 33, 1731-1740 [Medline] [Order article via Infotrieve]
  16. Cox, A. D., and Der, C. J. (1992) Curr. Opin. Cell Biol. 4, 1008-1016 [Medline] [Order article via Infotrieve]
  17. Reiss, Y., Stradley, S. J., Gierasch, L. M., Brown, M. S., and Goldstein, J. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 732-736 [Abstract]
  18. Manne, V., Roberts, D., Tobin, A., O'Rourke, E., DeVirgillio, 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 [Abstract]
  19. Moores, S. L., Schaber, M. D., Mosser, S. D., Rands, E., O'Hara, M. B., Garsky, V. M., Marshall, M. S., Pompliano, D. L., and Gibbs, J. B. (1991) J. Biol. Chem. 266, 14603-14610 [Abstract/Free Full Text]
  20. Goldstein, J. L., Brown, M. S., Stradley, S. J., Reiss, Y., and Gierasch, L. M. (1991) J. Biol. Chem. 266, 15575-15578 [Abstract/Free Full Text]
  21. Brown, M. S., Goldstein, J. L., Paris, K. J., Burnier, J. P., and Marsters, J. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8313-8316 [Abstract]
  22. Pompliano, D. L., Rands, E., Schaber, M. D., Mosser, S. D., Neville, J. A., and Gibbs, J. B. (1992) Biochemistry 31, 3800-3807 [Medline] [Order article via Infotrieve]
  23. Kohl, N. E., Mosser, S. D., deSolms, S. J., Guiliani, E. A., Pompliano, D. L., Graham, S. L., Smith, R. L., Scolnick, E. M., Oliff, A., and Gibbs, J. B. (1993) Science 260, 1934-1937 [Medline] [Order article via Infotrieve]
  24. Graham, S. L., deSolms, S. J., Guiliani, E. A., Kohl, N. E., Mosser, S. D., Oliff, A. I., Pompliano, D. L., Rands, E., Breslin, M. J., Deana, A. A., Garsky, V. M., Scholz, T. H., Gibbs, J. B., and Smith, R. L. (1994) J. Med. Chem. 37, 725-732 [Medline] [Order article via Infotrieve]
  25. Garcia, A. M., Rowell, C., Ackerman, K., Kowalczyk, J. J., and Lewis, M. D. (1993) J. Biol. Chem. 268, 18415-18418 [Abstract/Free Full Text]
  26. Nigam, M., Seong, C.-M., Qian, Y., Hamilton, A. D., and Sebti, S. M. (1993) J. Biol. Chem. 268, 20695-20698 [Abstract/Free Full Text]
  27. Qian, Y., Blaskovich, M. A., Saleem, M., Seong, C.-M., Wathen, S. P., Hamilton, A. D., and Sebti, S. M. (1994) J. Biol. Chem. 269, 12410-12413 [Abstract/Free Full Text]
  28. Qian, Y., Blaskovich, M. A., Seong, C.-M., Vogt, A., Hamilton, A. D., and Sebti, S. M. (1994) Bioorg. Med. Chem. Lett., 4, 2579-2584 [CrossRef]
  29. James, J. L., Goldstein, J. L., Brown, M. S., Rawson, T. E., Somers, T. C., McDowell, R. S., Crowley, C. W., Lucas, B. K., Levinson, A. D., and Marsters, J. C., Jr. (1993) Science 260, 1937-1942 [Medline] [Order article via Infotrieve]
  30. Watanabe, T., Miyaura, N., and Suzuki, A. (1992) Syn. Lett. 3, 207-210
  31. Lacal, J. C., Santos, E., Notario, V., Yamaski, S., Kung, H. F., Seamans, C., McAndrew, S., and Crowl, R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 5305-5309 [Abstract]
  32. Cox, A. D., Hisaka, M. M., Buss, J. E., and Der, C. J. (1992) Mol. Cell. Biol. 12, 2606-2615 [Abstract]
  33. Sebti, S. M., Tkalcevic, G. T., and Jani, J. P. (1991) Cancer Commun. 3, 141-147 [Medline] [Order article via Infotrieve]
  34. Stradley, S. J., Rizo, J., and Gierasch, L. M. (1993) Biochemistry 32, 12586-12590 [Medline] [Order article via Infotrieve]

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