Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor

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  • was modulating the response of the mice to theparasite.

    11. S. Mellouk, S. J. Green, C. A. Nacy, S. L. Hoffman,J. Immunol. 146, 3971 (1991).

    12. A. Nussler et al., Eur. J. Immunol 21, 227 (1991).13. K. A. Rockett, M. M. Awburn, W. B. Cowden, I. A.

    Clark, Infect. Immun. 59, 3280 (1991).14. M. A. Marletta, P. S. Yoon, R. lyengar, C. D. Leaf,

    J. S. Wishnok, Biochemistry27, 8706 (1988); D. J.Stuehr and C. F. Nathan, J. Exp. Med. 169,1543(1989).

    15. R. G. Knowles and S. Moncada, Trends Biochem.Sci. 17, 399 (1992).

    16. F. Y. Liew etal., Eur. J. Immunol. 21, 2489 (1991).17. T. R. Mosmann and R. L. Coftman, Immunol.

    Today 8, 223 (1987).18. D. W. Taylor, in Malaria: Host Responses to Infec-

    tion, M. M. Stevenson, Ed. (CRC Press, BocaRaton, FL, 1990), pp. 1-35.

    19. S. A. McLean, C. D. Pearson, R. S. Phillips, Exp.Parasitol. 54, 296 (1982).

    20. M. M. Stevenson and E. Skamene, Infect. Immun.54, 600 (1986).

    21. K. A. Rockett, M. M. Awburn, B. B. Aggarwal, W.B. Cowden, I. A. Clark, ibid. 60, 3725 (1992).

    22. Mice (six per group) were CD4-depleted (8).Twelve days after the last antibody treatment, theywere injected intravenously with 4 x 107 cells permouse, or not reconstituted, and then infectedintravenously immediately with 105 P. c. chabaudipRBC. The preparation of the T cell clone has

    been described in detail (6). We enriched nalivesplenic T cells for CD4+ cells using a mouse T cellseparation kit (Pierce). Some of the mice recon-stituted with TH1 or TH2 were injected intraperito-neally with L-NMMA (250 mg/kg per day) dis-solved in sterile PBS. Courses of infection andnitrate levels in the sera were monitored as de-scribed (4, 5). Data shown are pooled from threeseparate experiments.

    23. We assayed parasite-specific antibody in the se-rum by an indirect fluorescent antibody test [S. A.McLean, C. D. Pearson, R. S. Phillips, Exp. Para-sitol. 54, 213 (1982)], using trophozoite-schizont-infected RBC as the target antigen. Isotype-spe-cific antibodies were obtained from serum sepa-rated by affinity chromatography on proteinA-Sepharose CL-4B into fractions containingIgGl, IgG2a, IgG2b, or IgG3 [I. Seppald, H.Sarvar, F. Peterfy, 0. Makela, Scand. J. Immunol.14, 335 (1981)]. Immunoglobulin M was isolatedby Sepharose 6B gel filtration from nonbindingmaterial that contained a pool of IgA and IgE. Weconfirmed the purity of each fraction by doubleradial immunodiffusion, using antibodies specificto each 1g isotype.

    24. We thank H. Hodson for L-NMMA and D-NMMAand E. Galloway and D. McLaughlin for technicalsupport. We thank The Wellcome Trust for finan-cial support.

    24 December 1992; accepted 4 May 1993

    Selective Inhibition of ras-DependentTransformation by a Farnesyltransferase Inhibitor

    Nancy E. Kohl, Scott D. Mosser, S. Jane deSolms,Elizabeth A. Giuliani, David L. Pompliano, Samuel L. Graham,

    Robert L. Smith, Edward M. Scolnick, Allen Oliff,*Jackson B. Gibbs

    To acquire transforming potential, the precursor of the Ras oncoprotein must undergofarnesylation of the cysteine residue located in a carboxyl-terminal tetrapeptide. Inhibitorsof the enzyme that catalyzes this modification, farnesyl protein transferase (FPTase), havetherefore been suggested as anticancer agents for tumors in which Ras contributes totransformation. The tetrapeptide analog L-731,735 is a potent and selective inhibitor ofFPTase in vitro. A prodrug of this compound, L-731,734, inhibited Ras processing in cellstransformed with v-ras. L-731,734 decreased the ability of v-ras-transformed cells to formcolonies in soft agar but had no effect on the efficiency of colony formation of cellstransformed by either the v-raf or v-mos oncogenes. The results demonstrate selectiveinhibition of ras-dependent cell transformation with a synthetic organic inhibitor of FPTase.

    The mammalian ras genes encode guano-sine triphosphate (GTP)-binding proteinsthat can acquire the potential to transformmammalian cells as a result of point muta-tions in codons 12, 13, or 61 (1). Mutated,oncogenic forms of ras are frequently foundin many human cancers, most notably inmore than 50% of colon and pancreaticcarcinomas (1, 2). These observations in-

    N. E. Kohl, S. D. Mosser, D. L. Pompliano, E. M.Scolnick, A. Oliff, J. B. Gibbs, Department of CancerResearch, Merck Research Laboratories, West Point,PA 19486.S. J. deSolms, E. A. Giuliani, S. L. Graham, R. L. Smith,Department of Medicinal Chemistry, Merck ResearchLaboratories, West Point, PA 19486.*To whom correspondence should be addressed.

    1934

    dicate that Ras functions in the pathogen-esis of human cancers and emphasize thepotential broad utility of anticancer agentsdirected against ras-induced cell transfor-mation.

    Ras is synthesized as a cytosolic precur-sor that ultimately localizes to the cytoplas-mic face of the plasma membrane after aseries of posttranslational modifications (3).The first and obligatory step in this series isthe addition of a farnesyl moiety to thecysteine residue of the COOH-terminalCAAX motif (C, cysteine; A, usually ali-phatic residue; X, any other amino acid) ina reaction catalyzed by farnesyl proteintransferase (FPTase). This modification isessential for Ras function, as demonstratedSCIENCE * VOL. 260 * 25 JUNE 1993

    by the inability of Ras mutants lacking theCOOH-terminal cysteine to be famesyl-ated, to localize to the plasma membrane,and to transform mammalian cells in cul-ture (4, 5). Moreover, strains of Saccharo-myces cerevisiae having a mutation inRAM1, a gene that encodes one of thestructural polypeptides of the yeast FPTase,are resistant to the biological effects ofoncogenic ras (6). The subsequent post-translational modifications-cleavage ofthe AAX residues, carboxyl methylation ofthe farnesylated cysteine, and palmitoyla-tion of cysteines located upstream of theCAAX motif-are not obligatory for Rasmembrane association or cell-transformingactivity (5, 7). Thus, FPTase appears to bean appropriate biochemical target for thedevelopment of inhibitors of posttransla-tional processing of Ras that might beexpected to interfere with Ras-mediatedcellular transformation.

    The substrates of the farnesylation reac-tion, famesyl diphosphate (FPP) and poly-peptides containing a CAAX motif, can beused as a starting point for the design ofFPTase inhibitors. Several analogs of FPPare potent and selective inhibitors ofFPTase in vitro (8, 9), and one has shownactivity in cells (9). The CAAX tetrapep-tide is the minimal sequence required forthe interaction of Ras with FPTase (10,11). Thus, tetrapeptides with amino acidsequences identical to the COOH-terminalsequences of protein substrates for FPTasecompete with Ras for farnesylation by act-ing as alternative substrates (12, 13).CAAX derivatives have also been identifiedthat are not substrates for farnesylation andtherefore behave as pure inhibitors ofFPTase (8, 13, 14).

    Although other cellular proteins besidesRas are in vivo substrates for farnesylation,most isoprenylated proteins are modified bythe 20-carbon geranylgeranyl moiety (15).Two classes of enzymes that catalyze theaddition of a geranylgeranyl group to pro-teins have been identified in mammaliancells. One class consists of geranylgeranylprotein transferase-I (GGPTase-I), whichmodifies proteins having a COOH-terminalCAAX sequence where X is a Leu residue(12, 16). The other class consists of one ormore enzymes (for example, GGPTase-II)that catalyze the modification of proteinsterminating in Cys-Cys or Cys-X-Cys (12,17). Comparison of the activity of variousCAAX tetrapeptides suggests that it may bepossible to design a specific inhibitor ofFPTase that does not affect the GGPTases.For example, CAAX tetrapeptides termi-nating in Ser or Met are potent inhibitorsof FPTase but are less active as inhibitors ofGGPTase-I or GOPTase-II (12). Con-versely, CAAX tetrapeptides terminating inLeu, which act as effective inhibitors of

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  • GGPTase-I, are relatively poor inhibitors ofFPTase (12, 18).

    To obtain selective antagonists ofFPTase, we examined CAAX tetrapeptidesas models for the development of inhibi-tors. The COOH-terminal tetrapeptide ofhuman K4A-Ras, CIM, was used as atemplate to design the tetrapeptide analogsL-731,734, which is N-{2(S)-[2(R)-amino-3-mercaptopropylamino]-3 (S )-methyl-pentyl}isoleucyl-homoserine lactone, andL-731,735, which is N-{2(S )-[2(R)-amino-3-mercaptopropylaminoJ-3 (S ) -methyl-pentyl}isoleucyl-homoserine (Fig. 1). Thesecompounds differ from CIIM in two respects.First, in both compounds, the two NH2-terminal peptide bonds were reduced. Sec-ond, either homoserine lactone or homo-serine was substituted for methionine. TheNH2-terminal peptide bonds were reducedin order to confer resistance to hydrolysis byaminopeptidases, enzymes commonly foundin mammalian cell extracts (19). Homo-

    L-731,734

    H XHH

    H2N - H- 0 >

    HOL-731.735

    Fig. 1. Structure of L-731,734, which is N-{2(S)-[2(R)-amino-3-mercaptopropylamino]-3(S)-methylpentyl}isoleucyl-homoserine lac-tone, and L-731,735, which is N{2(S)-[2(R)-ami-no-3-mercaptopropylamino]-3(S)-methylpentyl}isoleucyl-homoserine.

    Table 1. Selective inhibition of farnesyl proteintransferase. Prenyl protein transferase assayswere done essentially as described (12) andcontained the following: FPTase, 100 nM[3H]FPP and 650 nM E. col/iproduced Ras-CVLS; GGPTase-l, 100 nM [3H]GGPP and 500nM E. co/-produced Ras-CAIL (I, lie);GGPTase-ll, 100 nM [3H]GGPP and 500 nM E.col-produced YPT1. All enzymes were partiallypurified from bovine brain cytosol as described(12). FPTase values are the average of fourseparate determinations + SEM. GGPTase val-ues represent three determinations. IC., 50%inhibitory concentration.

    Com- IC. (nM)pound FPTase GGPTase-l GGPTase-ll

    L-731,734 282 41 >100,000 >100,000L-731,735 18 6 >100,000 >100,000

    serine was anticipated to be tolerated as areplacement for methionine because of itsstructural similarity to serine, which is foundin the X position of the Ha-Ras (20) and S.cerevisiae RAS2 (21) CAAX motifs. Al-though this compound (homoserine in the Xposition) was anticipated to be an effectiveinhibitor in vitro, the charge on the COOH-terminal carboxylate was perceived as a pos-sible impediment to the entry of CAAXtetrapeptides into whole cells. Cyclization ofhomoserine to a lactone might facilitate cellpenetrability by masking the anionic carbox-ylate and increasing the solubility of thecompound in lipid.

    L-731,735 exhibited potent inhibitionof FPTase in vitro. In assays containing[3Hjfamesyl diphosphate and Ras producedin Escherichia coli (Ras-CVLS: V, Val; L,Leu; S, Ser), 50% inhibition (IC50) ofpartially purified FPTase from bovine brainwas observed at a concentration of 18 nML-731,735 (Table 1). The related lactonecompound, L-731,734, was less potent ininhibiting FPTase (IC50 = 282 nM). Theseresults suggest that the COOH-terminalcarboxylate is an important determinant ofintrinsic FPTase inhibitory potency and areconsistent with our observation that amida-tion of the COOH-terminal carboxyl groupof the tetrapeptide CVLS similarly de-creased FPTase inhibitory activity (8).When evaluated as inhibitors of homoge-neous recombinant human FPTase, IC50values for L-731,734 and L-731,735 werecomparable to those observed with the bo-vine enzyme (22). L-731,735 is a nonsub-strate inhibitor that is competitive withrespect to Ras [inhibition constant (K,) =20 6 nM] and noncompetitive with

    respect to FPP in the FPTase reaction.Although L-731,735 was a potent inhibitorof FPTase, it was a less effective (>5000times) inhibitor of the type I and type IIGGPTases (Table 1).

    NIH 3T3 cells transformed by v-ras wereused to evaluate the effect of these com-pounds on the posttranslational processingof Ras in intact cells. The cells were incu-bated in the presence of the indicatedcompound for 24 hours and were labeledwith [35Slmethionine during the final 20hours. Ras was immunoprecipitated fromdetergent lysates of cell extracts with themonoclonal antibody to Ras, Y13-259 (23).Viral Ha-Ras contains a threonine at aminoacid position 59 that is a substrate forautophosphorylation (24). The viral Ras inthe transfected cells consists of a mixture ofphosphorylated and unphosphorylated pro-tein with approximately 25% being phos-phorylated (25). Farnesylation and phos-phorylation both alter the migration of viralHa-Ras during SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) (Fig. 2A). Inthe presence of lovastatin, a compound thatblocks Ras processing in cells by inhibitinga rate-limiting step in the isoprenoid bio-synthetic pathway (5, 26, 27), the phos-phorylated and unphosphorylated forms ofRas migrated more slowly (Fig. 2A), indi-cating a lack of posttranslational process-ing. A similar pattern of migration wasobserved for cells incubated in the presenceof 250 pM L-731,734 (Fig. 2A), demon-strating that this compound similarlyblocked processing of Ras in cells. Furtherinhibition of Ras processing was not ob-served at higher concentrations ofL-731,734 (up to 1 mM) (28). Titration of

    Fig. 2. Inhibition of Ras pro- A L-731,734 B L-731,735cessing by L-731,734. Meta-bolic labeling and immuno- I =L =Lprecipitation of Ras protein 0 0 8 o 0 o 8gwas done essentially as de- 5' '0 (- cmscribed (26). Briefly, NIH3T3 cells transformed by vi-ral H-ras were incubatedwith the indicated concen-tration of L-731,734 (A) or -45 -45L-731,735 (B) (dissolved inmethanol; the final concen-tration of methanol in the as-

    -29say was 0.1 %) for 4 hours, at UPwhich time fresh compound p -29was added together with UP[35S]methionine (133 ,uCi/ml) p -(Amersham). After incuba- -18 p23 4tion for another 20 hours,Ras was immunoprecipi- 1 2 3 4 5tated from detergent lysatesof cell extracts with monoclonal antibody to Ras Y13-259 (10 ,g), resolved by SDS-PAGE (13%gels), and detected by fluorography. MeOH, 0.1% methanol; Lovastatin, lovastatin (15 [iM);Peptide, competition of the antibody with an excess of the peptide spanning the epitope (Ras60-76; -100-fold molar excess); P, processed, unphosphorylated Ras; U, unprocessed, unphos-phorylated Ras; PP, processed, phosphorylated Ras; and UP, unprocessed, phosphorylated Ras.Molecular sizes of the protein standards are indicated on the right (in kilodaltons).

    1935SCIENCE * VOL. 260 0 25 JUNE 1993

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  • L-731,734 in this assay indicated that thecompound inhibited Ras processing with anIC50 of -100 FM (Fig. 2A). L-731,734 (upto 1 mM) did not inhibit the processing ofa chimeric Ras protein (Ras-CVLL) that isprenylated by a geranylgeranyl moiety (28,29). This result indicates the specificity ofL-731,734 for the FPTase reaction in cells.

    To demonstrate that the change in Rasmobility on SDS-PAGE correlated with aninability of Ras to localize to the plasmamembrane, we subjected v-ras cells treatedwith solvent alone or L-731,734 to cellfractionation. Most of the Ras in the sol-vent-treated cells was associated with themembrane, whereas the unprocessed Ras i...

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