Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice.

For Ras oncoproteins to transform mammalian cells, they must be post-translationally modified with a farnesyl group in a reaction catalysed by the enzyme farnesyl-protein transferase (FPTase). Inhibitors of FPTase have therefore been proposed as anti-cancer agents. We show that L-744,832, which mimics the CaaX motif to which the farnesyl group is added, is a potent and selective inhibitor of FPTase. In MMTV-v-Ha-ras mice bearing palpable tumours, daily administration of L-744,832 caused tumour regression. Following cessation of treatment, tumours reappeared, the majority of which regressed upon retreatment. No systemic toxicity was found upon necropsy of L-744,832-treated mice. This first demonstration of anti-FPTase-mediated tumour regression suggests that FPTase inhibitors may be safe and effective anti-tumour agents in some cancers.

Activated expression of the N-myc gene in human neuroblastomas and related tumors.

In neuroblastoma lines in which the N-myc gene is present as a single copy, the expression of N-myc as messenger RNA is increased relative to that in nonneuroblastoma cell lines and tumors. The increase of expression in neuroblastomas with amplified N-myc genes is the result of (i) an increase in the absolute amount of expression of each N-myc gene and (ii) an increase in the copy number of the N-myc gene. A second gene--which is amplified in many of the same lines as N-myc--is expressed to about the same degree in most human cell lines and primary tumors regardless of origin (when normalized to gene copy number). Thus, a change in the regulation of N-myc expression in neuroblastomas and certain other tumors results in greatly increased expression of each N-myc gene copy.

Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor.

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

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.

A farnesyltransferase inhibitor induces tumor regression in transgenic mice harboring multiple oncogenic mutations by mediating alterations in both cell cycle control and apoptosis.

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV-v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV-v-Ha-ras mice were interbred with p53(-/-) mice. Tumors in ras/p53(-/-) mice treated with L-744,832 regressed as efficiently as MMTV-v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV-v-Ha-ras mice were also interbred with MMTV-c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV-v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV-c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.

A peptidomimetic inhibitor of farnesyl:protein transferase blocks the anchorage-dependent and -independent growth of human tumor cell lines.

Farnesyl protein transferase (FPTase) catalyzes the first of a series of posttranslational modifications of Ras required for full biological activity. Peptidomimetic inhibitors of FPTase have been designed that selectively block farnesylation in vivo and in vitro. These inhibitors prevent Ras processing and membrane localization and are effective in reversing the transformed phenotype of Rat1-v-ras cells but not that of cells transformed by v-raf or v-mos. We have tested the effect of the FPTase inhibitor L-744,832 (FTI) on the anchorage-dependent and -independent growth of human tumor cell lines. The growth of over 70% of all tumor cell lines tested was inhibited by 2-20 microM of the FTI, whereas the anchorage-dependent growth of nontransformed epithelial cells was less sensitive to the effects of the compound. No correlation was observed between response to drug and the origin of the tumor cell or whether it contained mutationally activated ras. In fact, cell lines with wild-type ras and active protein tyrosine kinases in which the transformed phenotype may depend on upstream activation of the ras pathway were especially sensitive to the drug. To define the important targets of FTI action, the mechanism of cellular drug resistance was examined. It was not a function of altered drug accumulation or of FPTase insensitivity since, in all cell lines tested, FPTase activity was readily inhibited within 1 h of treatment with the inhibitor. Furthermore, the general pattern of inhibition of cellular protein farnesylation and the specific inhibition of lamin B processing were the same in sensitive and resistant cells. In addition, functional activation of Ras was inhibited to the same degree in sensitive and resistant cell lines. However, the FTI inhibited the epidermal growth factor-induced activation of mitogen-activated protein kinases in sensitive cells but not in two resistant cell lines. These data suggest that the drug does inhibit ras function and that resistance in some cells is associated with the presence of Ras-independent pathways for mitogen-activated protein kinase activation by tyrosine kinases. We conclude that FPTase inhibitors are potent antitumor agents with activity against many types of human cancer cell lines, including those with wild-type ras.

Resistance of a variant ras-transformed cell line to phenotypic reversion by farnesyl transferase inhibitors.

Pharmacological inhibitors of the housekeeping enzyme farnesyl transferase (FT) inhibit the growth of ras-transformed cells in vitro and in vivo without antiproliferative effects on normal cells. In one direction to analyze the basis for this selectivity and to study modes of drug resistance that arise in animals, we characterized a variant ras-transformed cell line, 749r-1, which was resistant to phenotypic reversion with FT inhibitors. The transformed phenotype, growth potential, and actin cytoskeleton of 749r-1 cells were unaffected by treatment with the FT inhibitor 1-739,749 at concentrations up to 30-fold higher than those sufficient to revert ras-transformed cells. Resistance correlated with a reduced ability of L-739,749 to inhibit the farnesylation of Ras and lamin B and with a reduction in the susceptibility of endogenous FT to drug inhibition. These effects were not due to mutation of the FT subunits, changes in intracellular drug accumulation, or amplification of the multiple drug resistance gene (MDR). However, a similar reduction in the ability of L-739,749 to inhibit Ras farnesylation was also seen in ras-transformed cells rendered resistant by ectopic expression of farnesyl-independent RhoB, suggesting some mechanistic overlap. We concluded that 749r-1 cells sustained a stable alteration that conferred drug resistance by a novel mechanism.

Amplification of IMR-32 clones 8, G21, and N-myc in human neuroblastoma xenografts.

Amplification of clones 8, G21, and N-myc, which were derived from human neuroblastoma cell lines IMR-32 and NB-19, were studied in nine neuroblastoma xenografts. N-myc was amplified from 50- to 120-fold in eight of nine xenografts, clone 8 was amplified in five of the xenografts, and clone G21 was amplified in four of these five. Each of these clones was localized by in situ hybridization to homogeneously staining regions in metaphase spreads of xenograft chromosomes. In one xenograft a DNA rearrangement of clone 8 was observed, and only two of the sequences detected by G21 were amplified. Restriction enzyme mapping indicated that the rearrangement within clone 8 occurred at a position close to the rearrangement previously noted in neuroblastoma cell line NB-9.

Amplification and rearrangement of DNA sequences from the chromosomal region 2p24 in human neuroblastomas.

Seven DNA fragments which map to or very near human chromosome band 2p24 are shown to be differentially amplified in DNA from specific subsets of an enlarged series of human neuroblastoma cell lines and primary neuroblastomas. Of these DNA fragments, the probe NB-19-21 for the oncogene N-myc is the most frequently amplified, with a second expressed sequence (pG21) amplified in 9 of those 11 cell lines and 16 of those 25 tumors exhibiting amplification of N-myc. The remaining probes are in turn each amplified in progressively smaller, nested subsets of the cell lines and tumors in which both N-myc and pG21 are amplified. These data permit construction of models for the organization of a "neuroblastoma amplicon," i.e., an originally amplified DNA domain, with N-myc positioned most central and the other DNA fragments increasingly peripheral; comparable models result for the cell lines and the tumors. Five of the seven probes examined detect novel DNA fragments in these specimens, reinforcing previous observations that extensive DNA rearrangement can occur during DNA amplification in neuroblastoma cell lines and in primary neuroblastomas. Such rearrangements could contribute significantly to the evolution of the neuroblastoma amplicon in different specimens to progressively smaller units, preserving, in the limit, amplification of N-myc.

Antitumor effect of a farnesyl protein transferase inhibitor in mammary and lymphoid tumors overexpressing N-ras in transgenic mice.

We tested the antineoplastic effect of the farnesyltransferase inhibitor L-744,832 in mammary and lymphoid tumors overexpressing the N-ras proto-oncogene in transgenic mice. Mice bearing mammary tumors were randomly assigned to receive daily 40 mg/kg s.c. injections of this compound (experimental group, n = 6) or vehicle (control group, n = 6) per day for 5.5 weeks. Treatment with the compound significantly reduced the mammary tumor mean growth rate in the experimental group (-0.7 mm3/day), as compared with the control group (+28.2 mm3/day; P < 0.001). There was a significant difference in lymphoma incidence at the end of the treatment between the experimental (0 of 6) and the control (3 of 6) groups (P < 0.05). Therefore, this compound is effective in treating in vivo mammary carcinomas and lymphomas in which an activated N-Ras pathway drives tumorigenesis. The number of apoptotic figures in mammary tumors was significantly higher (P = 0.04) in the experimental (14.7 +/- 8.1) than it was in the control (5.7 +/- 3.5) group, indicating that apoptotic induction could contribute to the mechanism of antitumor activity of this compound. We analyzed the level of processing of N-Ras and H-Ras after immunoprecipitation and Western blotting of protein extracts obtained from mammary tumors treated with L-744,832 or vehicle, either in vivo or in vitro (after primary culture of the same tumors), and from several in vitro treated control cell lines. In all compound-treated mammary tumors and cell lines, H-Ras was mostly unprocessed (more so after in vitro than after in vivo treatment), whereas N-Ras remained mostly processed. Both H-Ras and N-Ras remained fully processed in all vehicle-treated samples. These findings are consistent with a less intense antineoplastic effect of the treatment with the compound in our N-ras model than the effect previously reported for the same compound in H-ras transgenics. In addition, the finding that, in compound-treated mammary tumors, the N-Ras protein remains mainly processed suggests that, in our model, other proteins in addition to Ras may be a target for the compound. Our results and the previous findings of frequent N-ras activation in human hematopoietic malignancies support a role for L-744,832 in the treatment of lymphomas and of mammary carcinomas with an activated N-Ras pathway, as well as the testing of a farnesyl protein transferase inhibitor in humans to establish its clinical relevance.

Evaluation of farnesyl:protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models.

Farnesyl:protein transferase (FPTase) inhibitors (FTIs) were originally developed as potential anticancer agents targeting the ras oncogene and are currently in clinical trials. Whereas FTIs inhibit the farnesylation of Ha-Ras, they do not completely inhibit the prenylation of Ki-Ras, the allele most frequently mutated in human cancers. Whereas farnesylation of Ki-Ras is blocked by FTIs, Ki-Ras remains prenylated in FTI-treated cells because of its modification by the related prenyltransferase, geranylgeranyl:protein transferase type I (GGPTase-I). Hence, cells transformed with Ki-ras tend to be more resistant to FTIs than Ha-ras-transformed cells. To determine whether Ki-ras-transformed cells can be targeted by combining an FTI with a GGPTase-I inhibitor (GGTI), we evaluated potent, selective FTIs, GGTIs, and dual prenylation inhibitors (DPIs) that have both FTI and GGTI activity. We find that in human PSN-1 pancreatic tumor cells, which harbor oncogenic Ki-ras, and in other tumor lines having either wild-type or oncogenic Ki-ras, treatment with an FTI/GGTI combination or with a DPI blocks Ki-Ras prenylation and induces markedly higher levels of apoptosis relative to FTI or GGTI alone. We demonstrate that these compounds can inhibit their enzyme targets in mice by monitoring pancreatic and tumor tissues from treated animals for inhibition of prenylation of Ki-Ras, HDJ2, a substrate specific for FPTase, and Rap1A, a substrate specific for GGPTase-I. Continuous infusion (72 h) of varying doses of GGTI in conjunction with a high, fixed dose of FTI causes a dose-dependent inhibition of Ki-Ras prenylation. However, a 72-h infusion of a GGTI, at a dose sufficient to inhibit Ki-Ras prenylation in the presence of an FTI, causes death within 2 weeks of the infusion when administered either as monotherapy or in combination with an FTI. DPIs are also lethal after a 72-h infusion at doses that inhibit Ki-Ras prenylation. Because 24 h infusion of a high dose of DPI is tolerated and inhibits Ki-Ras prenylation, we compared the antitumor efficacy from a 24-h FTI infusion to that of a DPI in a nude mouse/PSN-1 tumor cell xenograft model and in Ki-ras transgenic mice with mammary tumors. The FTI and DPI were dosed at a level that provided comparable inhibition of FPTase. The FTI and the DPI displayed comparable efficacy, causing a decrease in growth rate of the PSN-1 xenograft tumors and tumor regression in the transgenic model, but neither treatment regimen induced a statistically significant increase in tumor cell apoptosis. Although FTI/GGTI combinations elicit a greater apoptotic response than either agent alone in vitro, the toxicity associated with GGTI treatment in vivo limits the duration of treatment and, thus, may limit the therapeutic benefit that might be gained by inhibiting oncogenic Ki-Ras through dual prenyltransferase inhibitor therapy.

Mouse mammary tumor virus-Ki-rasB transgenic mice develop mammary carcinomas that can be growth-inhibited by a farnesyl:protein transferase inhibitor.

For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These compounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regression in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher concentrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predominant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki-rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI.

Role of c-myc in the transformation of REF52 cells by viral and cellular oncogenes.

Cells of the established REF52 line completely resist stable transformation by activated ras oncogenes, apparently because expression of ras p21 above a low threshold level inhibits cell proliferation. Adenovirus E1A and simian virus 40 (SV40) large T antigen enable ras oncogenes to transform REF52 cells and therefore protect REF52 cells from ras-induced growth arrest. The present study investigated the role of c-myc in regulating the responses of REF52 cells to ras oncogenes. We report that transcriptionally activated c-myc oncogenes enabled ras to transform REF52 cells but the efficiency of transformation was 20- to 30-fold lower than with E1A. In contrast, myc and E1A were similarly active as ras collaborators when assayed on primary baby rat kidney (BRK) cells. Relative difficulties transforming REF52 celis by myc and ras did not result from a requirement to express either gene at higher levels in REF52 as compared with BRK transformants. Steady state levels of endogenous c-myc RNA were unaltered in REF52 cells transformed by ras together with c-myc, E1A or SV40 large T antigen. Furthermore, ras-induced growth arrest was not accompanied by a decline in c-myc RNA levels. These results suggest that transcriptional control of c-myc is not affected either by the anti-proliferative effects of ras or by the collaborating activities of E1A and SV40 large T antigen.

Massive accumulation of neutral lipids in cells conditionally transformed by an activated H-ras oncogene.

Phenotypic consequences of ras oncogene expression were studied in cells conditionally transformed by T24 H-ras and a temperature-sensitive SV40 large T antigen (tsA58). Previous studies have demonstrated that transformation of REF52 cells by ras and SV40 large T antigen requires continuous T antigen expression. Thus, tsA58/T24 H-ras transformants ceased growing when transferred to a restrictive temperature for T antigen expression. Inhibition of cell growth was accompanied by massive accumulations of cholesterol esters, triglycerides and a third lipid species, identified as glycerol ethers on the basis of mobility on TLC. Cholesterol esters were derived from serum lipoproteins, and appeared to accumulate because LDL receptor expression and activity did not decline in growth arrested cells. Triglycerides and glycerol ethers were products of cell metabolism. The process lacked features characteristic of adipocyte differentiation, but may suggest mechanisms important in diseases, such as atherosclerosis, that involve abnormal accumulations of neutral lipids. Accumulating lipid species may also include metabolites induced by ras that accumulate in growth-arrested cells.

CA1A2X-competitive inhibitors of farnesyltransferase as anti-cancer agents.

For Ras oncoproteins to transform mammalian cells, they must be post-translationally farnesylated in a reaction catalysed by the enzyme farnesyl-protein transferase (FPTase). Inhibitors of FPTase have therefore been proposed as anti-cancer agents. In this review Charles Omer and Nancy Kohl discuss the development of FPTase inhibitors that are kinetically competitive with the protein substrate in the farnesylation reaction. These compounds are potent and selective inhibitors of the enzyme that block the tumourigenic phenotypes of ras-transformed cells and human tumour cells in cell culture and in animal models.

Farnesyltransferase inhibitors versus Ras inhibitors.

Over the past few years, the idea that farnesyl-protein transferase (FPTase) inhibitors might be effective antiproliferative/antitumor agents has been realized in studies of cultured cells and in rodent models of cancer. Most of the studies with FPTase inhibitors have focused on inhibiting the growth of ras-transformed cells in vitro or the growth of ras-dependent tumors in mice. More recently, it has been recognized that the antiproliferative effect of FPTase inhibitors may extend beyond ras-driven tumors. It now seems likely that the ability of FPTase inhibitors to reverse the malignant phenotype results, at least in part, from inhibiting the farnesylation of proteins other than Ras.

Treatment with farnesyl-protein transferase inhibitor induces regression of mammary tumors in transforming growth factor (TGF) alpha and TGF alpha/neu transgenic mice by inhibition of mitogenic activity and induction of apoptosis.

Mouse mammary tumor virus-transforming growth factor alpha (MMTV-TGF alpha) and MMTV-TGF alpha/neu transgenic mice develop mammary tumors after a long latency and therefore provide useful model systems for breast cancer with its recognized activation of receptor tyrosine kinase signaling. We used these mice to study the antitumor effect of L-744,832 (FTI), a potent and selective inhibitor of farnesyl-protein transferase, and hence of Ras function. A total of 55 mice were assigned randomly to treatment with FTI or vehicle, and one-half of the mice were crossed over after initial treatment to the opposite group. L-744,832 induced reversible regression of mammary tumors that was paralleled by a decrease in serum levels of TGF alpha secreted by the tumor cells. There was no difference in response to treatment with FTI between MMTV-TGF alpha mice, in which tumorigenesis was accelerated by multiparity or the chemical carcinogen 7,12-dimethylbenzanthracene, and MMTV-TGF alpha/neu mice. The tumor histological type had no impact on FTI sensitivity. For mechanistic analyses, tumor excision biopsies were obtained from 12 mice before and after treatment with L-744,832. In these samples, tumor regression was paralleled biochemically by inhibition of mitogen-activated protein kinase activity and biologically by an increase in G1-phase and decrease in S-phase fractions, as well as induction of apoptosis. These results suggest that the potential clinical use of FTI could be expanded to include cancers harboring activated receptor tyrosine kinases as well as those containing activated Ras.

Pseudodipeptide inhibitors of protein farnesyltransferase.

A series of pseudodipeptide amides are described that inhibit Ras protein farnesyltransferase (PFTase). These inhibitors are truncated versions of the C-terminal tetrapeptide (CAAX motif) of Ras that serves as the signal sequence for PFTase-catalyzed protein farnesylation. In contrast to CAAX peptidomimetics previously reported, these inhibitors do not have a C-terminal carboxyl moiety, yet they inhibit farnesylation in vitro at < 100 nM. Despite the absence of the X residue in the CAAX motif, which normally directs prenylation specificity, these pseudodipeptides are greater than 100-fold selective for PFTase over type 1 protein geranylgeranyltransferase.

Pseudopeptide inhibitors of Ras farnesyl-protein transferase.

Inhibitors of Ras farnesyl-protein transferase are described. These are reduced pseudopeptides related to the C-terminal tetrapeptide of the Ras protein that signals farnesylation. Deletion of the carbonyl groups between the first two residues of the tetrapeptides either preserves or improves activity, depending on the peptide sequence. The most potent in vitro enzyme inhibitor described (IC50 = 5 nM) is Cys [psi CH2NH]Ile[psi CH2NH]Phe-Met (3). To obtain compounds able to suppress Ras farnesylation in cell culture, further structural modification to include a homoserine lactone prodrug was required. Compound 18 (Cys[psi CH2NH]Ile[psi CH2NH]Ile-homoserine lactone) reduced the extent of Ras farnesylation by 50% in NIH3T3 fibroblasts in culture at a concentration of 50 microM. Structure-activity studies also led to 12 (Cys[psi CH2NH]Val-Ile-Leu), a potent and selective inhibitor of a related enzyme, the type-I geranylgeranyl protein transferase.

Clavaric acid and steroidal analogues as Ras- and FPP-directed inhibitors of human farnesyl-protein transferase.

We have identified a novel fungal metabolite that is an inhibitor of human farnesyl-protein transferase (FPTase) by randomly screening natural product extracts using a high-throughput biochemical assay. Clavaric acid [24, 25-dihydroxy-2-(3-hydroxy-3-methylglutaryl)lanostan-3-one] was isolated from Clavariadelphus truncatus; it specifically inhibits human FPTase (IC50 = 1.3 microM) and does not inhibit geranylgeranyl-protein transferase-I (GGPTase-I) or squalene synthase activity. It is competitive with respect to Ras and is a reversible inhibitor of FPTase. An alkaline hydrolysis product of clavaric acid, clavarinone [2,24,25-trihydroxylanostan-3-one], lacking the 3-hydroxy-3-methylglutaric acid side chain is less active as a FPTase inhibitor. Similarly, a methyl ester derivative of clavaric acid is also inactive. In Rat1 ras-transformed cells clavaric acid and lovastatin inhibited Ras processing without being overtly cytotoxic. Excess mevalonate reversed the effects of lovastatin but not of clavaric acid suggesting that the block on Ras processing by clavaric acid was due to inhibition of FPTase and not due to inhibition of HMG-CoA reductase. Despite these results, the possibility existed that clavaric acid inhibited Ras processing by directly inhibiting HMG-CoA reductase. To directly examine the effects of clavaric acid and clavarinone on HMG-CoA reductase, cholesterol synthesis was measured in HepG2 cells. No inhibition of HMG-CoA reductase was observed indicating that the inhibition of Ras processing by this class of compounds is due to inhibition of FPTase. To date, clavaric acid is the second reported nitrogen-free compound that competes with Ras to inhibit FPTase activity. A series of related compounds derived from computer-based similarity searches and subsequent rational chemical synthetic design provided compounds that exhibited a range of activity (0.04 --> 100 microM) against FPTase. Modest changes in the structures of these inhibitors dramatically change the inhibitory activity of these inhibitors.