Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor

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<ul><li><p>was modulating the response of the mice to theparasite.</p><p>11. S. Mellouk, S. J. Green, C. A. Nacy, S. L. Hoffman,J. Immunol. 146, 3971 (1991).</p><p>12. A. Nussler et al., Eur. J. Immunol 21, 227 (1991).13. K. A. Rockett, M. M. Awburn, W. B. Cowden, I. A.</p><p>Clark, Infect. Immun. 59, 3280 (1991).14. M. A. Marletta, P. S. Yoon, R. lyengar, C. D. Leaf,</p><p>J. S. Wishnok, Biochemistry27, 8706 (1988); D. J.Stuehr and C. F. Nathan, J. Exp. Med. 169,1543(1989).</p><p>15. R. G. Knowles and S. Moncada, Trends Biochem.Sci. 17, 399 (1992).</p><p>16. F. Y. Liew etal., Eur. J. Immunol. 21, 2489 (1991).17. T. R. Mosmann and R. L. Coftman, Immunol.</p><p>Today 8, 223 (1987).18. D. W. Taylor, in Malaria: Host Responses to Infec-</p><p>tion, M. M. Stevenson, Ed. (CRC Press, BocaRaton, FL, 1990), pp. 1-35.</p><p>19. S. A. McLean, C. D. Pearson, R. S. Phillips, Exp.Parasitol. 54, 296 (1982).</p><p>20. M. M. Stevenson and E. Skamene, Infect. Immun.54, 600 (1986).</p><p>21. K. A. Rockett, M. M. Awburn, B. B. Aggarwal, W.B. Cowden, I. A. Clark, ibid. 60, 3725 (1992).</p><p>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</p><p>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.</p><p>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.</p><p>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.</p><p>24 December 1992; accepted 4 May 1993</p><p>Selective Inhibition of ras-DependentTransformation by a Farnesyltransferase Inhibitor</p><p>Nancy E. Kohl, Scott D. Mosser, S. Jane deSolms,Elizabeth A. Giuliani, David L. Pompliano, Samuel L. Graham,</p><p>Robert L. Smith, Edward M. Scolnick, Allen Oliff,*Jackson B. Gibbs</p><p>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.</p><p>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-</p><p>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.</p><p>1934</p><p>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.</p><p>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</p><p>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.</p><p>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).</p><p>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</p><p>%88. QE 100 RIORM</p><p> on S</p><p>epte</p><p>mbe</p><p>r 27,</p><p> 201</p><p>2w</p><p>ww</p><p>.sci</p><p>ence</p><p>mag</p><p>.org</p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p></li><li><p>GGPTase-I, are relatively poor inhibitors ofFPTase (12, 18).</p><p>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-</p><p>L-731,734</p><p>H XHH</p><p>H2N - H- 0 &gt;</p><p>HOL-731.735</p><p>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.</p><p>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.</p><p>Com- IC. (nM)pound FPTase GGPTase-l GGPTase-ll</p><p>L-731,734 282 41 &gt;100,000 &gt;100,000L-731,735 18 6 &gt;100,000 &gt;100,000</p><p>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.</p><p>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</p><p>respect to FPP in the FPTase reaction.Although L-731,735 was a potent inhibitorof FPTase, it was a less effective (&gt;5000times) inhibitor of the type I and type IIGGPTases (Table 1).</p><p>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</p><p>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-</p><p>-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).</p><p>1935SCIENCE * VOL. 260 0 25 JUNE 1993</p><p> on S</p><p>epte</p><p>mbe</p><p>r 27,</p><p> 201</p><p>2w</p><p>ww</p><p>.sci</p><p>ence</p><p>mag</p><p>.org</p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p></li><li><p>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.</p><p>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...</p></li></ul>

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