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.

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.

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.

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.

NMR studies of novel inhibitors bound to farnesyl-protein transferase.

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for the membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to inhibit ras-dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers. The FPTase-bound conformation of a tetrapeptide inhibitor, CVWM, and a novel pseudopeptide inhibitor, L-739,787, have been determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation. Structures were calculated with the combination of distance geometry and restrained energy minimization. Both peptide backbones are shown to adopt nonideal reverse-turn conformations most closely approximating a type III beta-turn. These results provide a basis for understanding the spatial arrangements necessary for inhibitor binding and selectivity and may aid in the design of therapeutic agents.

Phenobarbital and sulfonylurea-inducible operons encoding herbicide metabolizing cytochromes P-450 in Streptomyces griseolus.

We have identified the promoters for two inducible genes, in Streptomyces griseolus, that encode herbicide-metabolizing cytochromes P-450. They are in the class of promoters that have -35 and -10 sequences similar to those used in Escherichia coli by RNA polymerase E sigma 70. Transcription from either promoter was shown to be induced by sulfonylurea (chlorimuron ethyl) or phenobarbital. Mapping of mRNA showed that each cytochrome P450-encoding gene was transcribed on a separate multicistronic mRNA that encodes cytochrome P-450 (suaC or subC), ferredoxin (suaB or subB) and at least one other open reading frame. An inducible, site-specific DNA-binding activity was identified that bound to two similar 8-bp inverted repeat sequences within or near the sua promoter (suaP). A noninducible DNA-binding activity, distinct from that which bound to suaP, was found that bound to an 11-bp inverted repeat at the sub transcription start point.

Design and biological activity of (S)-4-(5-([1-(3-chlorobenzyl)-2-oxopyrrolidin-3-ylamino]methyl)imidazol-1-ylmethyl)benzonitrile, a 3-aminopyrrolidinone farnesyltransferase inhibitor with excellent cell potency.

The synthesis, structure-activity relationships, and biological properties of a novel series of imidazole-containing inhibitors of farnesyltransferase are described. Starting from a 3-aminopyrrolidinone core, a systematic series of modifications provided 5h, a non-thiol, non-peptide farnesyltransferase inhibitor with excellent bioavailability in dogs. Compound 5h was found to have an unusually favorable ratio of cell potency to intrinsic potency, compared with other known FTIs. It exhibited excellent potency against a range of tumor cell lines in vitro and showed full efficacy in the K-rasB transgenic mouse model.

Biochemical and biological analyses of farnesyl-protein transferase inhibitors.

The methods outlined in Subheading 3. provide a logical sequence of assays with which to evaluate the biochemical and biological properties of potential FPTase inhibitors. The clinical predictability of these assays must await the evaluation of one or more of these compounds in humans.

Farnesyl: proteintransferase inhibitors as agents to inhibit tumor growth.

Ras, a signal-transducing protein involved in mediating growth factor-stimulated proliferation, is mutationally activated in over 30% of human tumors. To be functional Ras must bind to the inner surface of the plasma membrane, with post-translational lipid modifications being necessary for this localization. The essential, first modification of Ras is farnesylation catalyzed by the enzyme farnesyl: proteintransferase (FPTase). Inhibitors of FPTase (FTIs) are currently being tested to determine if they are capable of tumor growth inhibition. Here we describe our efforts, along with those of other groups, in testing the biological and biochemical effects of FTIs.

Activation and detection of (pro)mutagenic chemicals using recombinant strains of Streptomyces griseus.

Two recombinant strains of Streptomyces griseus have been developed to report on the activation of promutagenic chemicals. This activation is monitored by reversion of the bacterial test strains to a kanamycin-resistant phenotype. Strain H69 detects point mutations and was reverted at an increased frequency by acetonitrile, 2-aminoanthracene, 1,2-benzanthracene, benzidine, benzo(a)pyrene, 9,10-dimethyl-1,2-benzanthracene, and glycine. The second strain, FS2, detects frame shift mutations and was reverted at an increased frequency by 1,2-benzanthracene, benzidine, and glycine. Compounds such as butylated hydroxytoluene, catechol, chlorobenzene, hydroquinone, potassium chloride, phenol, cis-stilbene, trans-stilbene, and toluene did not elicit positive responses in either strain. In addition, these strains are capable of detecting direct-acting mutagens such as N-methyl-N'-nitrosoguanidine and ICR-191, providing further evidence of their promise for detecting a wider range of mutagens. To our knowledge, this is the first report of bacterial strains capable of activating promutagenic compounds and detecting their mutagenic metabolites without the benefit of an exogenous activation system such as the rodent liver homogenate (S9).

Protein prenylation in eukaryotic microorganisms: genetics, biology and biochemistry.

Modification of proteins at C-terminal cysteine residue(s) by the isoprenoids farnesyl (C15) and geranylgeranyl (C20) is essential for the biological function of a number of eukaryotic proteins including fungal mating factors and the small, GTP-binding proteins of the Ras superfamily. Three distinct enzymes, conserved between yeast and mammals, have been identified that prenylate proteins: farnesyl protein transferase, geranylgeranyl protein transferase type I and geranylgeranyl protein transferase type II. Each prenyl protein transferase has its own protein substrate specificity. Much has been learned about the biology, genetics and biochemistry of protein prenylation and prenyl protein transferases through studies of eukaryotic microorganisms, particularly Saccharomyces cerevisiae. The functional importance of protein prenylation was first demonstrated with fungal mating factors. The initial genetic analysis of prenyl protein transferases was in S. cerevisiae with the isolation and subsequent characterization of mutations in the RAM1, RAM2, CDC43 and BET2 genes, each of which encodes a prenyl protein transferase subunit. We review here these and other studies on protein prenylation in eukaryotic microbes and how they relate to and have contributed to our knowledge about protein prenylation in all eukaryotic cells.

Structural analysis of plasmid and chromosomal loci involved in site-specific excision and integration of the SLP1 element of Streptomyces coelicolor.

SLP1int (integrated [int] form of Streptomyces lividans plasmid 1 [SLP1]) is a Streptomyces coelicolor A3(2) transmissible sequence capable of autonomous replication as well as site-specific integration into and excision from the S. coelicolor chromosome. We report here that the plasmid and chromosomal loci involved in the integration of SLP1 and the two loci at which the recombination occurs during excision all share at least 111 base pairs of a 112-base-pair DNA sequence. Recombinational cross-over during integration or excision occurred nonrandomly within the common att sequence at or near a 25-base-pair inverted repeat. We suggest that chromosomally integrated plasmidogenic segments such as SLP1int may be involved in the acquisition and structural organization of genes encoding the diverse metabolic capabilities observed in different streptomycetes.

Plasmid formation in Streptomyces: excision and integration of the SLP1 replicon at a specific chromosomal site.

We present data showing that the SLP1 plasmids found in Streptomyces lividans after mating with S. coelicolor strain A3(2) originate as deletion mutants of a 17 kb segment of the S. coelicolor chromosome. Excision of the entire 17 kb segment yields a transiently existing plasmid containing a site for integration into the chromosome of recipient SLP1- S. lividans strains at a unique locus that corresponds to the original chromosomal location of SLP1 in S. coelicolor. The deletion mutants of SLP1 lack the attachment site and/or other regions required for its integration, and thus persist in the recipient as autonomously replicating plasmids. Plasmids that contain the complete 17 kb sequence of the chromosomally integrated SLP1 segment were constructed in vitro by circularization of restriction endonuclease-generated fragments of chromosomal DNA carrying a tandemly-duplicated integrant of SLP1. Transformation of an SLP1- S. lividans strain with such plasmids results in chromosomal integration of the SLP1 sequence at the same site at which it is integrated in S. lividans cells that acquire the sequence by mating with S. coelicolor. A model for the site-specific excision and integration of SLP1 is presented.

Mechanism of release of the avian rotavirus tRNATrp primer molecule from viral DNA by ribonuclease H during reverse transcription.

Employing enzymatic reactions containing reverse transcriptase and appropriately defined substrates, we have demonstrated that the tRNATrp primer molecule required for the initiation of DNA synthesis is cleaved from viral DNA by an enzymatic activity associated with the reverse transcriptase molecule. Since the alpha subunit of reverse transcriptase facilitates release of the tRNATrp primer from viral DNA and this activity is inhibited by a known inhibitor of reverse-transcriptase-associated RNAase H, it appears that the RNAase H activity, rather than the DNA endonuclease activity, is involved in this reaction. The cleavage site for RNAase H-mediated removal of the tRNATrp primer from viral DNA is located at or near the tRNATrp-viral DNA junction, and transcription of most, if not all, of the tRNATrp-binding site into (+) polarity DNA occurs before RNAase-H-mediated cleavage takes place. These studies indicate that an additional function can be ascribed to the reverse-transcriptase-associated RNAase H activity, which in this instance acts like an endonuclease, not requiring the unblocked termini of an RNA-DNA hybrid molecule for its activity.

Photoreactive analogues of prenyl diphosphates as inhibitors and probes of human protein farnesyltransferase and geranylgeranyltransferase type I.

Photoreactive analogues of prenyl diphosphates have been useful in studying prenyltransferases. The effectiveness of analogues with different chain lengths as probes of recombinant human protein prenyltransferases is established here. A putative geranylgeranyl diphosphate analogue, 2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate (DATFP-FPP), was the best inhibitor of both protein farnesyltransferase (PFT) and protein geranylgeranyltransferase-I (PFFT-I). Shorter photoreactive isprenyl diphosphate analogues with geranyl and dimethylallyl moieties and the DATFP-derivative of farnesyl monophosphate were much poorer inhibitors. DATFP-FPP was a competitive inhibitor of both PFT and PGGT-I with Ki values of 100 and 18 nM, respectively. [32P]DATFP-FPP specifically photoradiolabelled the beta-subunits of both PFT and PGGT-I. Photoradiolabelling of PGGT-I was inhibited more effectively by geranylgeranyl diphosphate than farnesyl diphosphate, whereas photoradiolabelling of PFT was inhibited better by farnesyl diphosphate than geranylgeranyl diphosphate. These results lead to the conclusions that DATFP-FPP is an effective probe of the prenyl diphosphate binding domains of PFT and PGGT-I. Furthermore, the beta-subunits of protein prenyltransferases must contribute significantly to the recognition and binding of the isoprenoid substrate.

Site-specific insertion of biologically functional adventitious genes into the Streptomyces lividans chromosome.

We report that transformation of Streptomyces lividans with cloned DNA of the SLP1 genetic element results in integration of the element at the same chromosomal locus (attB) normally occupied by SLP1 in its original host, Streptomyces coelicolor, and in S. lividans that has received SLP1 by mating. We constructed SLP1 derivatives that can integrate foreign DNA at the attB site and used these to introduce adventitious DNA sequences into the S. lividans chromosome. We also identified three regions of SLP1 essential for its integration and demonstrated that integration of the SLP1 element does not require expression of functions necessary for stable maintenance or transfer of extrachromosomal forms of SLP1.