GB2306646A - Assay for compounds which inhibit ras oncogene activity involving farnesyl-protein transferase - Google Patents

Abstract

An assay, for the identification of compounds which inhibit ras oncogene activity, comprises: (i) incubation of farnesyl pyrophosphate with farnesyl-protein transferase in the presence of a test substance; (ii) quenching the reaction with ethylenediaminetetraacetic acid (EPTA) or a lower alkanol (preferably methanol); (iii) detecting whether the farnesyl pyrophosphate is converted to farnesol, in which the ability of the test substance to inhibit ras oncogene activity is indicated by the rate of hydrolysis of the farnesyl pyrophosphate as compared to the said rate in the absence of the test compound. The pyrophosphate may be labelled with a signal generating moiety (preferably a radioisotope, fluorophore, enzyme or chromophore), or the conversion thereof to farnesol may be detected using an ion-exchange resin. The enzyme activity detected may be of a prenyl-protein transferase (especially geranylgeranyl-protein transferase type I).

Description

TITLE OF THE INVENTION A NOVEL ASSAY FOR DETERMINTNG FARNESYL-PROTEIN TRANSFERASE ACTIVITY BACKGROUND OF THE INVENTION The Ras protein is part of a signaling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation. Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein. In the inactive state, Ras is bound to GDP. Upon growth factor receptor activation Ras is induced to exchange GDP for GTP and undergoes a conformational change.

The GTP-bound form of Ras propagates the growth stimulatory signal until the signal is terminated by the intrinsic GTPase activity of Ras, which retums the protein to its inactive GDP bound form (D.R. Lowy and D.M. Willumsen, Ann. Rev. Biochem. 62:851-891 (1993)).

Mutated ras genes are found in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. The protein products of these genes are defective in their GTPase activity and constitutively transmit a growth stimulatory signal.

Ras must be localized to the plasma membrane for both normal and oncogenic functions. At least 3 post-translational modifications are involved with Ras membrane localization, and all 3 modifications occur at the C-terminus of Ras. The Ras C-terminus contains a sequence motif termed a "CAAX" or "Cys-Aaa1 -Aaa2-Xaa" box (Cys is cysteine, Aaa is an aliphatic amino acid, the Xaa is any amino acid) (Willumsen et al., Nature 310:583-586 (1984)).

Depending on the specific sequence, this motif serves as a recognition sequence for the enzymes farnesyl-protein transferase (FPTase) or geranylgeranyl-protein transferase type I (GGPTase I), which catalyze the alkylation of the cysteine residue of the CAAX motif with a C15 or C20 isoprenoid, respectively. (S. Clarke., Ann. Rev. Biochem. 61:355386 (1992); W.R. Schafer and J. Rine, Ann. Rev. Genetics 30:209-237 (1992)). The Ras protein is one of several proteins that are known to undergo post-translational farnesylation. Other farnesylated proteins include the Ras-related GTP-binding proteins such as Rap2a, fungal mating factors, the nuclear lamins, and the gamma subunit of transducin. James, et al., J. Biol. Chem. 269, 14182 (1994) have identified a peroxisome associated protein Pxf which is also farnesylated.James, et al., have also suggested that there are farnesylated proteins of unknown structure and function in addition to those listed above Inhibition of farnesyl-protein transferase has been shown to block the growth of Ras-transformed cells in soft agar and to modify other aspects of their transformed phenotype. It has also been demonstrated that certain inhibitors of FPTase selectively block the processing of the Ras oncoprotein intracellularly (N.E. Kohl et al., Science, 260:1934-1937 (1993) and G.L. James et al., Science, 260:1937-1942 (1993). Recently, it has been shown that an inhibitor of FPTase blocks the growth of ras-dependent tumors in nude mice (N.E.

Kohl et al., Proc. Natl. Acad. Sci USA., 91:9141-9145 (1994) and induces regression of mammary and salivary carcinomas in ras transgenic mice (N.E. Kohl et al., Nature Medicine, 1:792-797 (1995).

It has recently been shown that FPTase inhibitors are inhibitors of proliferation of vascular smooth muscle cells and are therefore useful in the prevention and therapy of arteriosclerosis and diabetic disturbance of blood vessels (JP H7-1 12930).

Indirect inhibition of farnesyl-protein transferase in vivo has been demonstrated with lovastatin (Merck & Co., Rahway, NJ) and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et al., Science 245:379 (1 989)). These drugs inhibit HMG-CoA reductase, the rate limiting enzyme for the production of polyisoprenoids including farnesyl pyrophosphate. FPTase utilizes farnesyl pyrophosphate to covalently modify the Cys thiol group of the Ras CAAX box with a farnesyl group (Reiss et awl., Cell, 62:81-88 (1990); Schaber et al., J.

Biol. Chem., 265:14701-14704 (1990); Schafer etal., Science, 249:1133-1139(1990); Manne et al., Proc. Natl. Acad. Sci USA, 87:7541-7545 (1990)). Inhibition of farnesyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane localization in cultured cells. However, direct inhibition of FPTase would be more specific and attended by fewer side effects than would occur with the required dose of a general inhibitor of isoprene biosynthesis.

Inhibitors of FPTase have been described in two general classes. The first are analogs of farnesyl diphosphate (FPP), while the second class of inhibitors is related to the protein substrates (e.g., Ras) for the enzyme. The peptide derived inhibitors that have been described are generally cysteine containing molecules that are related to the CAAX motif that is the signal for protein prenylation. (Schaber et al., ibid; Reiss et. al., ibid; Reiss et al., PNAS, 88:732-736 (1991)). Such inhibitors may inhibit protein prenylation while serving as alternate substrates for the FPTase enzyme, or may be purely competitive inhibitors (U.S. Patent 5,141,851, University of Texas; N.E. Kohl et al., Science, 260:1934-1937 (1993); Graham, et al., J. Med. Chem., 37, 725 (1994)).

Assays useful in determining FPTase activity of compositions have been previously described (see for example U.S.

Patent No. 5,185,248). However, such assays incorporate a CAAXsubstrate, which is farnesylated in the presence of farnesyl pyrophosphate and FPTase.

it is therefore the object of the instant invention to provide a rapid assay to determine farnesyl-protein transferase activity which does not require a CAAX-substrate and therefore has a simplified concentration dependency equation. Such an assay could also be used to detect activity of other prenyl-protein transferases such as geranylgeranyl-protein transferase type I.

SUMMARY OF THE INVENTION The instant invention provides assays designed to detect activity of compositions that contain mammalian farnesyl-protein transferase (FPTase), especially human FPTase, by measuring the enzymatically mediated hydrolysis of farnesyl pyrophosphate. These assays can also be used to identify substances that inhibit farnesylprotein transferase and the famesylation of ras proteins.In particular, the invention describes an assay for identifying compounds that inhibit ras oncogene activity, comprising: (a) incubating farnesyl pyrophosphate with farnesyl-protein transferase in the presence of a test substance; (b) quenching the reaction with a substance selected from EDTA or a lower alkanol; and (c) detecting whether the farnesyl pyrophosphate is converted to farnesol, in which the ability of the test substance to inhibit ras oncogene activity is indicated by the rate of hydrolysis of the farnesyl pyrophosphate as compared to the rate of hydrolysis in the absence of the test substance.

DETAILED DESCRIPTION OF THE INVENTION In the assay of the instant invention, farnesylpyrophosphate (FPP) is reacted with a composition that comprises FPTase. Hydrolysis of the farnesyl pyrophosphate to farnesol is an indication of FPTase activity and inhibition of such hydrolysis under the reaction conditions in the presence of a test substance is an indication of the ability of the test substance to inhibit FPTase activity.

The farnesylation of ras proteins is required for their biological activity, i.e., the anchoring of the ras protein to the innerside of the plasma membrane and its role in signal transduction.

Farnesylation of ras oncogene products is required for ras mediated transformation of normal cells to cancer cells. The transfer of the farnesyl group from FPP, the donor molecule, to ras proteins is mediated by an enzyme FPTase. Peptides that are analogous to the ras protein and shorter peptides that contain the CAAX box motif are effective substrates of the enzyme and have been utilized in previously described assays to determine activity and inhibition of FPTase.

Applicants have surprisingly found that, in the absence of a peptidyl substrate containing the CAAX motif, water acts as a substrate of the FPTase enzyme, and FPP present in a non-anhydrous mixture containing the enzyme is converted to famesol. Applicants have also found the enzymatic hydrolysis of FPP to farnesol may be selectively inhibited with compounds that have been shown to inhibit the enzymatic famesylation of the ras protein. Since the inhibition of the enzymatic hydrolysis is dependent on the concentration of an inhibitor compound and on the relative inhibitory activity of the inhibitor, an assay which detects the extent of hydrolysis of FPP can be utilized to determine relative inhibitory activity of novel compounds.

It has previously been shown that the lipid prenyltransferase FPP synthase can hydrolyze its substrate geranyl diphosphate to geraniol and pyrophosphate (Poulter et al., Biochemistry, 15:1079-1083 (1976)). However, the protein-prenyl transferases show no amino acid sequence similarity to FPP synthase and the hydrolysis of geranyl diphosphate by FPP synthase is strongly dependent on inorganic pyrophosphate. Hydrolysis of FPP to farnesol by FPTase does not require pyrophosphate.

in the assays of the invention, hydrolysis of FPP by FPTase may be detected by a variety of methods. For example, hydrolysis of FPP can be detected by a change in mobility of the reaction product as determined by chromatographic methods, including but not limited to TLC (thin layer chromatography), HPLC (high perfonnance liquid chromatography), etc. Another example of detection of FPP hydrolysis is the differential binding of the FPP substrate and farnesol product to ion exchange resins. The assay of the invention and its components are described in more detail herein below.

The FPTase used in the assay may be obtained from a variety of sources. For example, FPTase used in the assay may be isolated from any of a variety of mammalian cells, tissues or organs using purification techniques well known in the art. For example, purification of FPTase from natural sources is described in detail in U.S. Pat. No. 5,141,851 (Brown et al.).

The FPTase utilized in the instant assays may also be obtained by expression of the enzyme in a suitable host cell which has been transformed with a plasmid operably containing cDNAs encoding an a subunit and ss subunit of a mammalian FPTase as described in WIPO publication WO 94/10184 and by Omer et al., Biochemistry, 32:5167-5176 (1993). For example, the transformed E. coli culture that has been deposited with the American Type Culture Collection and which has been designated ATCC 69091 may be utilized to express the enzyme. Purification of such a recombinantly expressed FPTase may be performed as described in WO 94/10184. Techniques useful for the cloning and expression of FPTase are also described in U.S. Pat. No.

5,185,248.

Further methods of preparing the FPTase used in the instant invention is to synthetically prepare the enzyme, or chemically synthesizing the cDNA encoding the enzyme followed by expression and purification of the enzyme. Such alternative preparations of FPTase are noted in U.S. Pat. No. 5,185,248.

FPP used in the instant invention may be obtained from a variety of commercial sources (e.g., Sigma Chemical Co.; Aldrich Chemical Co.; etc.). The FPP that is utilized may be labeled with any of a variety of signal generating compounds including radiolabels, fluorogenic compounds, colorimetric compounds, enzymes, etc., using standard metabolic labeling techniques. Indeed, radiolabeled FPP ([3H]FPP and [14C]FPP) are commercially available (e.g., Amersham; New England Nuclear, American Radiochemicals).

The reaction conditions used in the FPTase assay may be adjusted to optimize the enzymatic hydrolysis of FPP in vitro. For example, using the reaction conditions described in the examples herein, appropriate concentrations of cations such as Mg2+, Mn2+ or Cd2+ may be added to the reaction buffer. Although FPTase is active at a wide range of pHs, optimal activity may be achieved by adjusting the pH between 6.8 and 8.0. Because the FPTase enzyme is heat labile, high temperatures (e.g., 65" C for 30 mins) should be avoided during the reaction.

The assay of the invention is accomplished by exposing FPP to non-anhydrous conditions in the presence of the FPTase enzyme.

Preferably, the reaction is conducted using reaction conditions which favor the enzymatic hydrolysis of FPP; e.g., when using the reaction conditions described in the examples herein below, the assay may be conducted in the presence of appropriate concentrations of cations such as Mg2+, Mn2+ or Cd2+; and at an appropriate pH, such as between 6.S and 8.0, and at am apppropriate temperature, such as between 30 and 37 C. Detection of the amount of hydrolysis of FPP is a direct indication of FPTase activity. Substances that inhibit FPTase activity and which therefore inhibit ras mediated transformation of normal cells to cancer cells may be identified by their ability to inhibit the enzymatic hydrolysis of FPP when added into the assay mixture.

The assay of the invention may be conducted in a liquid phase or a solid-liquid phase. Hydrolysis of the FPP may be assessed in a variety of ways depending upon the reaction format that is utilized.

Where the assay is conducted in a liquid phase, the hydrolyzed product may be separated from the reaction mixture at the completion of assay by a number of techniques, including but not limited to, chromatographic separation (e.g., thin layer chromatography, high performance chromatography, etc.). Where a labeled FPP is used, the reaction product may be isolated and the amount of label incorporated into the isolated reaction product may then be measured as a direct indication of the degree of hydrolysis.

The assay of the instant invention is also useful in determining the activity of compositions comprising other known prenyl-protein transferases and for determining the relative activity of compounds that inhibit such prenyl-protein transferases. Thus, the enzymatic activity of a composition that comprises GGPTase I may be assessed by the ability of the composition to selectively catalyze the hydrolysis of geranylgeranylpyrophosphate.

The invention is further defined by reference to the following examples, which are illustrative and not limiting. All temperatures are in degrees Celsius.

MATERIALS AND METHODS [3H]FPP (100-500 nM) (15-60 Ci/mmol, from American Radiochemicals, St. Louis, MO or DuPont-NEN, Boston, MA) was incubated with FPTase in buffer A which consists of 50 mM Hepes pH 7.5, 5 mM MgC12, 5 mM Dithiothreitol (DTT), 100 CM ZI1CI2 Aliquots of the reaction were quenched with 10-50 volumes of methanol. 100-500 mg of AG-IXS (BioRad, Hercules, CA) was added to each quenched sample and the sample was vortexed. The samples were then processed using the following methods.

Method 1 The methanol quenched sample, to which AG-1X8 had been added, was put into a glass pipette plugged with glass wool. The methanol flow through was collected along with two 1 ml aliquots of methanol that were used to rinse the resin in the plugged pipette and added to scintillation fluid. The radioactivity was counted in a ss counter.

Method 2 The methanol quenched sample, to which AG-1X8 had been added, was put into a microcentrifuge tube and vortexed. The sample was then centrifuged for 30 seconds at 5-15,000 x G. 0.8 ml of the methanolic liquid was collected and 0.5 ml of fresh methanol was added to the resign. The sample was vortexed, resin repelleted and 0.5 ml of the methanolic liquid collected. Both methanolic samples were added to scintillation fluid and the radioactivity was counted in a ss counter.

In both methods, the famesol product was contained in the methanol while the unreacted FPP was bound to the AG-1X8 resin.

Thus, the progress of the reaction was followed by monitoring the increase in radioactivity in the methanol fraction with time.

EXAMPLE 1 Six 700 1ll reactions were prepared. All reactions contained Buffer A and 100 nM [3H]-FPP. Reaction 1 contained no FPTase. Reaction 2 contained no FPTase and 100 nM of a-hydroxy famesyl phosphonic acid. Reaction 3 contained 10 nM FPTase.

Reaction 4 contained 10 nM FPTase and 100 nM of a-hydroxy farnesyl phosphonic acid. Reaction 5 contained 50 nM FPTase. Reaction 6 contained 50 nM FPTase and 100 nM of a-hydroxy farnesyl phosphonic acid.

100 ,uL aliquots were taken from each reaction 5, 10, 30 and 60 min and 0 min time points were taken from reaction 1 and 2.

The samples were processed via Method 1. FPP hydrolysis was seen to be dependent upon the amount of FPTase added to the reaction and was inhibited by the FPP analog, a-hydroxy farnesyl phosphonic acid. The rate of FPP hydrolysis was approximately 3 x 1 0-4/sec which is about 2% of rate of FPP transfer to a CAAX substrate.

EXAMPLE 2 Two reactions were prepared. Reaction 1 was 650 l and contained Buffer A, 1 % polyethylene glycol (av. MW 10,000), 100 nM [3H]FPP and 25 nM FPTase. Reaction 2 was 300 1 and contained Buffer A, 1% polyethylene glycol (av. MW 10,000), 100 nM [3H]FPP, 25 nM FPTase and 1 I1M { ((2(S)-[2(S)-[2(R)-amino-3-mercapto]- propylamino-3 (S) -methyl] pentyl oxy-3 -phenylpropionyl-methi onine- sulphone) 1. The sulphone is an inhibitor of FPTase-mediated FPP addition to Ras protein and is competitive with Ras protein substrate.

50 1 aliquots of each reaction were taken at 0, 5, 10, 20, 30 and 60 minutes and quenched in 1 ml of methanol. The samples were processed using Method 2.

The results indicate that the hydrolysis of FPP was inhibited by ((2(S)-[2(S)- [2(R)-amino-3-mercapto] -propylamino-3 (S)- methyl]pentyloxy-3-phenylpropionyl-methionine-sulphone) ) .

EXAMPLE 3 Two reactions were prepared. Each reaction was 315 Fl and contained Buffer A, 1% polyethylene glycol (av. MW 10,000) and 100 nM [3H]FPP. Reaction 1 contained no FPTase and reaction 2 contained 50 nM FPTase. Two 50 Crl aliquots of each reaction were taken at 0, 10 and 60 min. One of the two aliquots was quenched in 1 ml of methanol and the other aliquot was quenched on ice in 2.5 1 of 0.5 M EDTA. The methanol quenched samples were processed by Method 2. The EDTA quenched samples were separated by thin layer chromatography using the method described in Moores et al. for separating farnesylated peptides. As a standard for the thin layer chromatogram, FPP was hydrolyzed to famesol with approximately 0.2 M HCI at 37"C for 20 min.

The results showed that time dependent increase in radioactivity in the methanolic fraction depended on FPTase. The appearance of a radioactive spot that co-migrated with famesol generated by acid hydrolysis of FPP occurred concomitantly with the appearance of radioactivity in the methanolic fraction.

Claims (9)

WHAT IS CLAIMED IS:

1. An assay for identifying compounds that inhibit ras oncogene activity, comprising: (a) incubating farnesyl pyrophosphate with farnesyl-protein transferase in the presence of a test substance; (b) quenching the reaction with a substance selected from EDTA or a lower alkanol; and (c) detecting whether the farnesyl pyrophosphate is converted to famesol, in which the ability of the test substance to inhibit ras oncogene activity is indicated by the rate of hydrolysis of the farnesyl pyrophosphate as compared to the rate of hydrolysis in the absence of the test substance.

2. The assay according to Claim 1 in which the farnesyl pyrophosphate is labeled with a signal generating compound.

3. The assay according to Claim 2 in which the signalgenerating compound comprises a radiolabel, a fluor, an enzyme or a colorometric signal-generating compound.

4. The assay according to Claim 1 in which the farnesyl-protein transferase employed is one which has been at least partially purified.

5. The assay of Claim 1 in which the quenching substance is a lower alkanol.

6. The assay in Claim 5 in which the quenching substance is methanol.

7. The assay of Claim 1 wherein the conversion of famesyl protein transferase to farnesol is detected using an ion exchange resin.

8. The assay of Claim 1 wherein the enzyme activity being determined is a prenyl-protein transferase.

9. The assay of Claim 8 wherein the prenyl-protein transferase is geranylgeranyl-protein transferase type I.