Patent Application: US-61571590-A

Abstract:
disclosed are methods and compositions for the identification , characterization and inhibition of farnesyl protein transferases , enzymes involved in the farnesylation of various cellular proteins , including cancer related ras proteins such as p21 ras . one farnesyl protein transferase which is disclosed herein exhibits a molecular weight of between about 70 , 000 and about 100 , 000 upon gel exclusion chromatography . the enzyme appears to comprise one or two subunits of approximately 50 kda each . methods are disclosed for assay and purification of the enzyme , as well as procedures for using the purified enzyme in screening protocols for the identification of possible anticancer agents which inhibit the enzyme and thereby prevent expression of proteins such as p21 ras . also disclosed is a families of compounds which act either as false substrates for the enzyme or as pure inhibitors and can therefore be employed for inhibition of the enzyme . the most potent inhibitors are ones in which phenylalanine occurs at the third position of a tetrapeptide whose amino terminus is cysteine .

Description:
the following examples illustrate techniques discovered by the inventors for the identification and purification of farnesyl protein transferase enzyme , as well as techniques for its assay and for the screening of new compounds which may be employed to inhibit this enzyme . these studies also demonstrate a variety of peptidyl compounds which themselves can be employed to inhibit this enzyme . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent laboratory techniques discovered by the inventors to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . peptides were obtained from peninsula laboratories or otherwise synthesized by standard techniques . all peptides were purified on hplc , and their identity was confirmed by amino acid analysis . just prior to use , each peptide was dissolved at a concentration of 0 . 8 mm in 10 mm dithiothreitol ( dtt ), and all dilutions were made in 10 mm dtt . unlabeled farnesyl pyrophosphate ( fpp ) was synthesized by the method of davisson , et al . ( 13 ). [ 1 - 3 h ] farnesyl pyrophosphate ( 20 ci / mmol ) was custom synthesized by new england nuclear . geraniol and farnesol ( both all - trans ) were obtained from aldrich chemical . all - trans geranylgeraniol was a gift of r . coates ( university of illinois ). recombinant wild type human p21 h - ras protein was produced in a bacterial expression system with pat - rash ( provided by channing j . der , la jolla cancer research foundation , la jolla , ca ), an expression vector based on pxvr ( 14 ). the plasmid was transformed into e coli jm105 , and the recombinant p21 h - ras protein was purified at 4 ° c . from a high speed supernatant of the bacterial extracts by sequential chromatography on deae - sephacel and sephadex g - 75 . purity was . sup .˜ 90 % as judged by coomassie blue staining of sds gels . purified p21 h - ras was concentrated to 15 mg / ml in 10 mm tris - chloride ( ph 7 . 5 ) containing 1 mm dtt , 1 mm edta , 3 mm mgcl 2 , and 30 μm gdp and stored in multiple aliquots at - 70 ° c . farnesyl : protein transferase activity was determined by measuring the amount of 3 h - farnesol transferred from 3 h ] farnesyl pyrophosphate ([ 3 h ] fpp ) to p21 h - ras protein . the standard reaction mixture contained the following concentrations of components in a final volume of 25 μl : 50 mm tris - chloride ( ph 7 . 5 ), 50 μm zncl 2 , 20 mm kcl , 1 mm dtt , and 40 μm p21 h - ras . the mixture also contained 10 pmoles of [ 3 h ] fpp (. sup .˜ 30 , 000 dpm / pmol ) and 1 . 8 - 3 . 5 μg of partially purified farnesyl : protein transferase ( see below ). after incubation for 1 h at 37 ° c . in 12 × 75 - mm borosilicate tubes , the reaction was stopped by addition of 0 . 5 ml of 4 % sds and then 0 . 5 ml of 30 % trichloroacetic acid ( tca ). the tubes were vortexed and left on ice for 45 - 60 min , after which 2 ml of a 6 % tca / 2 % sds solution were added . the mixture was filtered on a 2 . 5 - cm glass fiber filter with a hoefer filtration unit ( fh 225 ). the tubes were rinsed twice with 2 ml of the same solution , and each filter was washed five times with 2 ml of 6 % tca , dried , and counted in a scintillation counter . one unit of activity is defined as the amount of enzyme that transfers 1 pmol of [ 3 h ] farnesol from [ 3 h ] fpp into acid - precipitable p21 h - ras per hour under the standard conditions . all steps were carried out at 4 ° c . except where indicated : brains from 50 male sprague - dawley rats ( 100 - 150 g ) were homogenized in 100 ml of ice - cold buffer containing 50 mm tris - chloride ( ph 7 . 5 ), 1 mm edta , 1 mm egta , 0 . 2 mm phenylmethylsulfonyl fluoride , and 0 . 1 mm leupeptin , and the extract was spun at 60 , 000 × g for 70 min . the supernantant was brought to 30 % saturation with solid ammonium sulfate , stirred for 30 minutes on ice , and centrifuged at 12 , 000 × g for 10 min to remove precipitated proteins , the resulting supernatant was adjusted to 50 % saturation with ammonium sulfate , and the resulting pellet was dissolved in . sup .˜ 20 ml of 20 mm tris - chloride ( ph 7 . 5 ) containing 1 mm dtt and 20 μm zncl 2 and dialyzed for 4 hours against 4 liters of the same buffer and then 4 liters of fresh buffer of the same composition for 12 h . the dialyzed material was divided into multiple aliquots and stored at - 70 ° c . a portion of the 30 - 50 % ammonium sulfate fraction ( 200 mg protein ) was chromatographed on a mono q 10 / 10 column using an fplc system ( pharmacia lkb biotechnology ). the column was run as described in the legend to fig5 . fractions eluting between 0 . 3 and 0 . 4 m nacl contained the majority of the transferase activity . these fractions were pooled , divided into multiple aliquots , and stored at - 70 ° c . an affinity column containing a peptide corresponding to the cooh - terminal six amino acids of p21 k - ras - b protein was prepared as follows . fifteen mg of the peptide tkcvim were coupled to 1 g of activated ch - sepharose 4b ( pharmacia lkb biotechnology ) according to the manufacturer &# 39 ; s instructions . the resulting 2 . 5 - ml slurry was poured into a column , and excess uncoupled peptide was removed by 10 cycles of alternating washes , each consisting of 40 column volumes of 0 . 1 m sodium acetate ( ph 4 . 0 ) and then 0 . 1 m tris - chloride ( ph 8 . 0 ). both buffers contained 1 m nacl and 10 mm dtt . the column was stored at 4 ° c . in 20 mm tris - chloride ( ph 7 . 2 ) and 0 . 02 % sodium azide . fifteen mg of mono q - purified material in 10 ml were applied to a 1 - ml peptide column equilibrated in 50 mm tris - chloride ( ph 7 . 5 ) containing 0 . 1 m nacl and 1 mm dtt ( buffer a ). the enzyme - containing solution was cycled through the column three times at room temperature . the column was washed with 20 ml of buffer a containing 0 . 2 % ( w / v ) octyl - β - d - glucopyranoside ( buffer b ). the enzyme was eluted with 20 ml of 50 mm tris - succinate ( ph 5 . 0 ) containing 1 mm dtt , 0 . 1 m nacl , and 0 . 2 % octyl - β - d - glucopyranoside . the ph 5 eluate was concentrated and washed twice with a 10 - fold excess of buffer b in a cf25 centriflo ultrafiltration cone ( amicon ) and brought to 1 ml ( 10 - fold concentration relative to the starting material ). affinity - purified farnesyl transferase (. sup .˜ 1 μg ) was chromatographed on a superose 12 column as described in the legend to fig7 . in the enzyme characterization experiments of fig1 - 4 , 8 , and 9 , a partially purified fraction of farnesyl : protein transferase was used . this enzyme was prepared by steps 1 and 2 as described above , after which 6 mg of the mono q - purified material was concentrated to 2 ml and then loaded onto a 1 . 6 × 50 - cm sephacryl s - 200 high resolution gel filtration column ( pharmacia lkb biotechnology ). the column was equilibrated with 50 mm tris - chloride ( ph 7 . 5 ) containing 1 mm dtt , 0 . 2 m nacl , 20 μm zncl 2 , and 0 . 2 % octyl - β - glucopyranoside and eluted with the same buffer at a flow rate of 15 ml / h . only the peak fraction , containing 1 mg protein and 40 % of initial activity , was used for studies . a modification of the procedure described by casey et al . ( ref . 6 ) was employed as follows : briefly , two standard transferase reactions of 25 - μl each were conducted for 1 hour at 37 ° c . the mixtures were then pooled , and a 25 - μl aliquot from the 50 - μl pooled sample was diluted to 250 μl with 2 % ( w / v ) sds . this mixture was precipitated with an equal volume of 30 % tca , filtered through nitrocellulose , ( 7 mm disc ), washed twice with 250 μl 6 % tca / 2 % sds followed by five washes with 5 % tca , digested with 8 μg trypsin , and subjected to cleavage with methyl iodide . the released 3 h - isoprenoids were extracted into chloroform / methanol and chromatographed on a reverse - phase hplc system as described in the legend to fig4 . sds polyacrylamide gel electrophoresis was carried out as described by laemmli ( 16 ). gels were calibrated with high range sds - page standards ( bio - rad ). protein content of extracts was measured by the method of lowry , et al . ( 17 ) except for that of the affinity - purified material , which was estimated by comparison to the bovine serum albumin marker ( m r 66 , 000 ) following sds gel electrophoresis and coomassie staining . as an initial attempt to identify a farnesyl protein transferase enzyme , rat brain cytosol was fractionated with ammonium sulfate and the active fraction subjected to ion exchange chromatography on a mono q column followed by gel filtration on sephacryl s - 200 . fig1 a shows that the active fraction from this column incorporated radioactivity from [ 3 h ] farnesol into trichloroacetic acid precipitable p21 h - ras in a time - dependent fashion at 37 ° c . the incorporated radioactivity could be visualized as a band of the expected molecular weight of . sup .˜ 21 kda on sds polyacrylamide gels ( fig1 b ). the concentration of [ 3 h ] farnesyl pyrophosphate that gave half - maximal reaction velocity was approximately 0 . 5 μm ( fig2 a ). the half - maximal concentration for p21 hras was approximately 5 μm , and there was no difference when the p21 h - ras was equilibrated with a nonhydrolyzable gtp or atp analogue or with gdp ( fig2 b ). with p21 h - ras as a substrate , the transferase reaction was inhibited by 0 . 15 mm edta , and this inhibition was reversed by 0 . 1 to 1 . 0 mm concentrations of zinc or magnesium chloride ( fig3 ). at higher concentrations of zinc chloride , inhibition was observed . to confirm that the transferred material was [ 3 h ] farnesol , the washed trichloracetic acid - precipitated material was digested with trypsin , the radioactivity released with methyl iodide , and the products subjected to reverse - phase hplc . the methyl iodide - released material comigrated with an authentic standard of all - trans farnesol ( c 15 ) ( fig4 a ). some radioactivity emerged from the column prior to the geranol standard ( c 10 ), but this was the same in the presence and absence of methyl iodide treatment . this early - eluting material was believed to represent some tryptic peptides whose radioactivity was not released by methyl iodide . fig5 shows the elution profile of farnesyl transferase activity from a mono q column . the activity appeared as a single sharp peak that eluted at approximately 0 . 35 m sodium chloride . the peak fractions from the mono q column were pooled and subjected to affinity chromatography on a column that contained a covalently - bound peptide corresponding to the carboxyl - terminal 6 - amino acids of p21 k - rasb . all of the farnesyl transferase activity was adsorbed to the column , and about 50 % of the applied activity was recovered when the column was eluted with a tris - succinate buffer at ph 5 . table i summarizes the results of a typical purification procedure that started with 50 rat brains . after ammonium sulfate precipitation , mono q chromatography , and affinity chromatography , the farnesyl transferase was purified approximately 61 , 000 - fold with a yield of 52 %. the final specific activity was about 600 , 000 units / mg . table i__________________________________________________________________________purification of franesyl : protein transferase from rat brain specific total purifi - protein activity activity cation recoveryfraction mg units / mg units fold % __________________________________________________________________________30 - 50 % ammonium sulfate 712 9 . 7 . sup . a 6906 1 100mono q 30 275 8250 28 119affinity column ˜ 0 . 006 . sup . b 600 , 000 3600 61 , 855 52__________________________________________________________________________ the purification procedure was started with 50 rat brains . . sup . a one unit of enzyme activity is the amount of enzyme that transfers 1 pmol of [. sup . 3 h ] farnesol from [. sup . 3 h ] fpp into acidprecipitable p21 . sup . hras per h under the standard conditions . . sup . b protein concentration was estimated by coomassie blue staining of sds polyacrylamide gel using various amounts ( 0 . 5 to 2 μg ) of bovine serum albumin as a reference standard . fig6 a shows the sds gel electrophoretic profile of the proteins at each stage of this purification as visualized by silver staining . the peptide affinity column yielded a single protein band with an apparent subunit molecular weight of 50 , 000 . when the purified enzyme was subjected to sds gel electrophoresis under more sensitive conditions , the 50 - kda protein could be resolved into two closely spaced bands that were visualized in approximately equimolar amounts ( fig6 b ). to confirm that the 50 - kda band was the farnesyl transferase enzyme , the affinity column purified material was subjected to gel filtration . fig7 shows that the farnesyl transferase activity and the 50 - kda band co - eluted from this column at a position corresponding to an apparent molecular weight of 70 - 100 kda as determined from the behavior of markers of known molecular weight . the adherence of the farnesyl transferase to the peptide affinity column suggested that the enzyme was capable of recognizing short peptide sequences . to test for the specificity of this peptide recognition , the ability of various peptides to compete with p21 h - ras for the farnesyl transferase activity was measured . the peptide that was used for affinity chromatography corresponded to the carboxyl terminal six amino acids of p21 k - rasb ( tkcvim ) as expected , this peptide competitively inhibited farnesylation of p21 h - ras ( open circles in fig8 ). the terminal 4 - amino acids in this sequence ( cvim ) ( closed circles ) were sufficient for competition . these two short peptides were no less effective than a peptide that contained the final 10 - amino acids of the sequence ( kksktkcvim ) ( open triangles ). the simple transposition of the cysteine from the fourth to the third position from the cooh - terminus of the hexapeptide ( tkvcim ) ( closed triangles ) severely reduced inhibitory activity . an irrelevant peptide ( closed squares ) also did not inhibit . fig9 compares the inhibitory activities of four peptides of 10 - amino acids each , all of which contain a cysteine at the fourth position from the cooh - terminus . the peptides corresponding to the cooh - terminus of human p21 k - rasb and human lamin a and lamin b all inhibited farnesylation . all of these peptides are known to be prenylated in vivo ( 6 , 15 ). on the other hand , the peptide corresponding to the sequence of rat giαl , a 40 - kda g protein that does not appear to be farnesylated in vivo ( casey , p ., unpublished observations ), did not compete for the farnesyl transferase reaction . in data not shown it was found that the 10 - amino acid peptide corresponding to the cooh - terminus of p21 h - ras ( cvls ), p21 n - ras ( cvvm ), and p21 h - rasa ( ciim ) all competed for the farnesylation reaction . in the present example , a series of tetrapeptides were tested for their ability to bind to the rat brain p21 h - ras farnesyl : protein transferase as estimated by their ability to compete with p21 h - ras in a farnesyl transfer assay . peptides with the highest affinity had the structure cys - al - a2 - x , where a1 and a2 are aliphatic amino acids and x is a c - terminal methionine , serine , or phenylalanine . charged residues reduced affinity slightly at the a1 position and much more drastically at the a2 and x positions . effective inhibitors included tetrapeptides corresponding to the cooh - termini of all animal cell proteins known to be farnesylated . in contrast , the tetrapeptide cail , which corresponds to the cooh - terminus of the only known examples of geranylgeranylated proteins ( neural g protein γ subunits ) did not compete in the farnesyl transfer assay , suggesting that the two isoprenes are transferred by different enzymes . a biotinylated hexapeptide corresponding to the cooh - terminus of p21 k - rasb was farnesylated , suggesting that at least some of the peptides serve as substrates for the transferase . the data are consistent with a model in which a hydrophobic pocket in the farnesyl : protein transferase recognizes tetrapeptides through interactions with the cysteine and the last two amino acids . peptides were prepared by established procedures of solid - phase synthesis ( 18 ) tetrapeptides were synthesized on the milligen 9050 synthesizer using fmoc chemistry . after deprotection of the last residue , a portion of the resin was used to make the n - acetyl - modified version of cvim . this was done off - line in a solution of acetic anhydride and dimethylformamide at ph 8 ( adjusted with diisopropylethylamine ). the acetylated and unacetylated peptides were cleaved with 50 ml of trifluoroacetic acid : phenol ( 95 : 5 ) plus approximately 1 ml of ethanedithiol added as a scavenger . the n - octyl - modified version of cvim was synthesized on an applied biosystems model 430 synthesizer using tboc chemistry . the octyl group was added in an amino acid cycle using octanoic acid . the peptide was cleaved from the resin at 0 ° c . with a 10 : 1 : 1 ratio of hf ( mls ): resin ( g ): anisole ( ml ). the peptides were purified by high pressure liquid chromatography ( hplc ) on a beckman c18 reverse phase column ( 21 . 1 cm x 15 cm ), eluted with a water - acetonitrile gradient containing 0 . 1 % ( v / v ) trifluouroacetic acid . identity was confirmed for all peptides by fast atom bombardment ( fab ) mass spectrometry . just prior to use , each peptide was dissolved at a concentration of 0 . 8 mm in 10 mm dithiothreitol ( dtt ), and all dilutions were made in 10 mm dtt . biotinylated ktscvim was synthesized on an applied biosystems 430a synthesizer . the biotin group was added after removal of the n - terminal protecting group before cleavage of the peptide from the resin . specifically , a 4 - fold molar excess of biotin 4 - nitrophenyl ester was added to the 0 . 5 g resin in 75 ml dimethylformanide at ph 8 and reacted for 5 h at room temperature . cleavage , identification , and purification were carried out as described above . to synthesize s - acetoamido cvim , purified cvim was dissolved at a final concentration of 1 mm in 0 . 1 ml of 0 . 5 m tris - chloride ( ph 8 . 0 ) containing 15 mm dtt . the tube was flushed with nitrogen for 2 min , sealed , and incubated for 2 . 5 h at 37 ° c . to reduce the cysteine residue , after which iodoacetamide was added to achieve a final concentration of 35 mm . after incubation for 15 min at 37 ° c ., the reaction was stopped by addition of 10 mm dtt . complete alkylation of cvim was confirmed by fab spectrometry and hplc . the molecular weight of the product corresponded to the expected molecular mass of s - acetoamido cvim . the standard assay involved measuring the amount of [ 3 h ] farnesyl transferred from all - trans [ 3 h ] fpp to recombinant human p21 h - ras as described in example i . each reaction mixture contained the following concentrations of components in a final volume of 25μl : 50 mm tris - chloride ( ph 7 . 5 ), 50 μm zncl 2 , 0 mm kcl , 1 mm dtt , 30 or 40 μm p21 h - ras , 15 pmol [ 3 h ] fpp ( 12 - 23 , 000 dpm / pmol ), 4 to 7 . 5 μg of partially purified farnesyl : protein transferase ( mono q fraction , see example i ), and the indicated concentration of competitor peptide added in 3 μl of 10mm dtt . after incubation for 30 - 60 min at 37 ° c ., the amount of [ 3 h ] farnesyl present in trichloroacetic acid - precipitable p21 h - ras was measured by a filter assay as described in example i . a blank value (& lt ; 0 . 6 % of input [ 3 h ] fpp ) was determined in parallel incubations containing no enzyme . this blank value was subtracted before calculating &# 34 ;% of control &# 34 ; values . c . transfer of [ 3 h ] farnesyl from [ 3 h ] fpp to biotinylated ktscvlm peptide this assay takes advantage of the fact that peptides containing the cys - aax motif of ras proteins can serve as substrates for prenylation by farnesyl transferase . a heptapeptide containing the terminal four amino acids of p21 k - rasb was chosen as a model substrate since it has a 20 to 40 - fold higher affinity for the enzyme than dces the cooh - terminal peptide corresponding to p21 h - ras . a tiotinylated peptide is used as substrate so that the reaction product , [ 3 ] farnesylated peptide , can be trapped on a solid support such as streptavidinagarose . the bound [ 3 h ] farnesylated peptide can then be washed , separated from unincorporated [ 3 h ] fpp , and subjected to scintillation counting . the biotin - modified ktscvim is synthesized on an applied biosystems 430a synthesizer using established procedures of solid phase peptide synthesis . the biotin group is added after deprotection of lysine and before cleavage of the peptide from the resin . the identity and purity of the biotinylated peptide is confirmed by quantitative amino acid analysis and fast atom bombardment ( fab ) mass spectrometry . an aliquot of biotinylated ktscvim ( 0 . 4 mg ) is dissolved in 0 . 6 ml of 10 mm sodium acetate ( ph 3 ) buffer containing 1 mm dtt and 50 % ethanol to give a final concentration of 0 . 67 mg / ml or 601 μm . this solution can be stored at 4 ° c . for at least 1 month . immediately prior to use , the peptide solution is diluted with 1 mm dtt to achieve a peptide concentration of 18 μm . the standard reaction mixture contains the following components in a final volume of 25 μl : 50 mm tris - chloride ( ph 7 . 5 ), 50 μm zncl 2 , 20 mm kcl , 1 mm dtt , 0 . 2 % ( v / v ) octyl - β - glucopryranoside , 10 - 15 pmol of [ 3 h ] fpp ( 15 - 50 , 000 dpm / pmol ), 3 . 6 μm biotinylated ktscvim , and 2 - 4 units of enzyme . after incubation at 37 ° c . for 30 - 60 min in 0 . 5 - ml siliconized microfuge tubes , the reaction is stopped by addition of 200 μl of 20 mm tris - chloride ( ph 7 . 5 ) buffer containing 2 mg / ml bovine serum albumin , 2 % sds , and 150 mm nacl . a 25 - μl aliquot of well mixed streptavidin - agarose ( bethesda research laboratories , cat . no . 5942sa ) is then added , and the mixture is gently shaken for 30 min at room temperature to allow maximal binding of the [ 3 h ] farnesylated peptide to the beads . the beads are then collected by spinning the mixture for 1 min in a microfuge ( 12 , 500 rpm ). the supernatant is removed , and the beads are washed three times with 0 . 5 ml of 20 mm tris - chloride ( ph 7 . 5 ) buffer containing 2 mg / ml bovine serum albumin , 4 % sds , and 150 mm nacl . the pellet is resuspended in 50 μl of the same buffer and transferred to a scintillation vial using a 200 - μl pipettor in which the tip end has been cut off at an angle . the beads remaining in the tube are collected by rinsing the tube with 25 μl of the above buffer and adding it plus the pipettor to the vial . a blank value , which consists of the radioactivity adhering to the beads in parallel incubations containing no enzyme , should be less than 0 . 5 % of the input [ 3 h ] fpp . to screen peptides for their affinity for the farnesyl : protein transferase , studies were conducted wherein the ability of the peptides to compete with p21 h - ras for acceptance of [ 3 h ] farnesyl from [ 3 h ] fpp as catalyzed by a partially purified rat brain farnesyl : protein transferase was tested . as a reference point for the peptides , the tetrapeptide cvim corresponding to the cooh - terminal sequenoe of p21 k - rasb was employed . fig1 shows a series of typical experiments in which alanine ( fig1 a ), lysine ( fig1 b ), or leucine ( fig1 c ) was systematically substituted at each of the three positions following cysteine in cvim . in each experiment the results were compared with those obtained with cvim . alanine and lysine were tolerated only at the a1 position . insertion of these amino acids at the a2 or x positions decreased the affinity for the enzyme by more than 30 - fold as estimated by the concentration required for 50 % inhibition . leucine was tolerated at the a2 position , but it decreased the affinity when inserted at the x position . the substitution of phenylalanine for iscleucine at the a2 position increased the affinity for the enzyme by 6 - fold , with half - maximal inhibition occurring at 25 nm ( fig1 ). no such effect was observed when phenylalanine was inserted at either of the other two positions . in addition to performing assays with p21 h - ras as a substrate , assays were also performed in which the substrate was a biotinylated heptapeptide , ktscvim , which contains the cooh - terminal four amino acids of p21 h - rasb ( 2 ). the biotin was attached to the nh 2 - terminus by coupling to the resin - attached peptide . the [ 3 h ] farnesylated product : was isolated by allowing it to bind to beads coated with streptavidin as described in section c . above . fig2 shows that the peptide cvfm was more potent than cvim when either p21 h - ras or the biotinylated heptapeptide was used as acceptor ( fig1 a and 12b , respectively ). in contrast to the other studies , which were conducted with a partially purified enzyme , the studies of fig1 were carried out with a homogeneous preparation of affinity - purified farnesyl : protein transferase . the free sulfhydryl group for the cysteine is likely required for tetrapeptide inhibition , as indicted by the finding that derivitization with iodoacetamide abolished inhibitory activity ( fig1 a ). a blocked nh 2 - terminus is not required , as indicated by similar inhibitory activity of n - acetyl cvim and n - octyl cvim ( fig1 b ) as compared to that of cvim ( fig1 a ). fig1 summarizes the results of all competition assays in which substitutions in the cvim sequence were made . the results are presented in terms of the peptide concentration required for 50 % inhibition . table ii summarizes the results of other experiments in which tetrapeptides corresponding to the cooh - termini of 19 proteins were studied , many of which are known to be farnesylated . the implications of these studies are discussed below in section 3 . table ii______________________________________inhibition of rat farnesyl : protein transferase bycooh - terminal tetrapeptides corresponding to known proteins concentration cooh - for 50 % terminal inhibitionprotein species tetrapeptide μm______________________________________ * p21 . sup . k - rasb human , cvim 0 . 15 mouse * p21 . sup . k - rasa human ciim 0 . 15p21 . sup . n - ras human cvvm 0 . 15p21 . sup . n - ras mouse cvlm 0 . 15 * lamin b human , caim 0 . 15 xenopus laevislamin a human , csim 0 . 20 xenopus laevisretinal cgmp bovine ccvq 0 . 35phosphodies - terase , α - subunit * ras1 s . cerevisciae ciic 0 . 35 * ras2 s . cerevisciae ciis 0 . 35 * γ - subunit of bovine cvis 1 . 0transducinp21 . sup . h - ras chicken cvis 1 . 0p21 . sup . h - ras human , rat cvls 3 . 0 * a - mating s . cerevisciae cvia 5 . 0factorrap2b human cvil 11dras dictostlelium clil 17rapla / krevl human clll 22 * mating factor r . toruloides ctva 30γ - subunit of bovine cail 100g proteinhmg coa s . cerevisciae ciks & gt ; 100reductase - 1______________________________________ enzyme activity was measured in the presence of the indicated tetrapeptid as described in the legend to fig1 . each tetrapeptide was tested at seven different concentrations ranging from 0 . 03 to 100 μm . the concentration giving 50 % inhibition was calculated from the inhibition curve . * shown to be farnesylated in vivo . the current data extend the observations on the p21 ras farnesyl : protein transferase set forth in example i , and further indicate that the recognition site for this enzyme is restricted to four amino acids of the cys - a1 - a2 - x type . as a reference sequence for these studies , the peptide cvim was used . this peptide inhibited the farnesyl : protein transferase by 50 % at a concentration of 0 . 15 μm . substitution of various amino acids into this framework yielded peptides that gave 50 % inhibitions at a spectrum of concentrations ranging from 0 . 025 μm ( cvfm ) to greater than 50 μm ( fig1 ). in general , the highest inhibitory activities were achieved when the a1 and a2 positions were occupied with nonpolar aliphatic or aromatic amino acids . this stringency was more severe at the a2 than at the a1 position . thus , peptides containing lysine or glutamic acid at the a1 position gave 50 % inhibition at 0 . 7 and 1 . 5 μm , respectively . when these two residues were inserted at the a2 position , the affinity for the enzyme declined by more than 50 - fold . glycine and proline lowered inhibitory activity moderately at the a1 position ( 50 % inhibition at 4 and 8 μm ) and somewhat more severely at the a2 position ( 8 and 20 μm ). the x position showed the highest stringency . in the context of cvix , methionine was the preferred residue but phenylalanine and serine were tolerated with only modest losses in activity ( 0 . 5 and 1 μm , respectively ). aliphatic resides and proline were disruptive at this position , with 50 % inhibitions in the range of 5 - 11 μm . glutamic acid , lysine , and glycine were not tolerated at all ; 50 % inhibition required concentrations above 40 μm . a study of tetrapeptides corresponding to the cooh - termini of known proteins ( table ii ) gave results that were generally in keeping with those obtained with the substituted cvim peptides . they provided the additional information that glutamine and cysteine are well tolerated at the x position ( ccvq and ciic ). all of the proteins that are known to be farnesylated in intact cells ( indicated by the asterisks in table ii ) followed the rules outlined above , and all inhibited farnesylation at relatively low concentrations ( 5 μm or below ) with the exception of the ctva sequence , which is found in the mating factor of r . toruloides ( 19 ). this peptide inhibited the rat brain farnesyl : protein transferase by 50 % only at the high concentrations of 30 μm . it is likely that the farnesyl : protein transferase in this fungal species has a different specificity than that of the rat brain . the peptide cail , which corresponds to the cooh - terminus of the γ - subunit of bovine brain g proteins ( 20 , 21 ), did not compete efficiently with p21 h - ras for farnesylation ( table ii ). a 50 % inhibition at the hiqhest concentration tested ( 100 μm ) was observed . the inhibitory activity was lower than that of cvil ( 12 μm ) or caim ( 0 . 15 μm ). thus , the combination of alanine at the al position and leucine at the x position is more detrimental than either single substitution . this finding is particularly relevant since the gamma subunit of g proteins from human brain ( 22 ) and rat pc12 cells ( 23 ) have been shown to contain a geranylgeranyl rather than a farnesyl . these findings suggest the existence of a separate geranylgeranyl transferase that favors cail and perhaps other related sequences . the studies with the biotinyated heptapeptide ( fig1 b ) confirm that at least some of the short peptides act as substrates for the enzyme . the saturation curves relating reaction velocity to the concentration of either p21 h - ras or the biotinylated heptapeptide are complex and sigmoidal . the inhibition curves with the various peptides differ from classic competitive inhibition curves . finally , as mentioned in example i , the maximal velocity of the purified enzyme is relatively low . these findings suggest that the binding of the peptides to the enzyme is not a simple equilibrium reaction . rather , there may be a slow binding that requires conformational change . the observation that the a1 position shows a relaxed amino acid specificity suggests that the residue at this position may not contact the farnesyl : transferase directly . rather , the contacts may involve only the cysteine and the residues at the a2 and x positions . a working model for the active site of the farnesyl : protein transferase places the peptide substrate in an extended conformation with a largely hydrophobic pocket of the enzyme interacting with the x group of the caax - containing substrate . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the composition , : methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . the references listed below are incorporated herein by reference to the extent that they supplement , explain , provide a background for or teach methodology , techniques and / or compositions employed herein . 1 . bos , j . 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