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.

P450 interaction with farnesyl-protein transferase inhibitors metabolic stability, inhibitory potency, and P450 binding spectra in human liver microsomes.

Methyl substitution at the 2-position of the imidazole ring greatly improved drug metabolism profiles, in human liver microsomes, of ras farnesyl-protein transferase inhibitor (FTI) candidates for drug development. Methyl substitution markedly reduced the P450 inhibitory potency of non-substituted FTIs for CYP3A4 (by a factor of 12-403) and 2C9 (by a factor of 4.2-28), while it had little effect on the CYP2D6 enzyme. An immunochemical inhibition study demonstrated that CYP3A4 plays a predominant role in the metabolism of both non-substituted and 2-methyl-substituted imidazole-containing FTI candidates. Very strong type II binding spectra with human liver microsomes were observed for all non-substituted FTIs, while methyl substitution markedly weakened type II spectra or shifted the type of spectra from II to I. This indicated that methyl substitution on the imidazole moiety interfered with the substrate-P450 heme interaction, likely due to a steric effect caused by the methyl group. A kinetics study revealed that the methyl substitution increased V(max) and K(m) values to the same extent. These studies suggested that the 2-methyl substitution on the imidazole ring improved its drug metabolism profile by reducing the potential to inhibit CYP3A4-mediated metabolism without affecting intrinsic metabolic clearance (V(max)/K(m)).

Diaryl ether inhibitors of farnesyl-protein transferase.

Imidazolemethyl diaryl ethers are potent inhibitors of farnesyl-protein transferase. The SNAr displacement reaction used to prepare these diaryl ethers was amenable to rapid parallel synthesis of FPTase inhibitors. The use of a broad range of commercially available phenols quickly identified compounds which proved active in cells.

N-Arylalkyl pseudopeptide inhibitors of farnesyltransferase.

Inhibitors of Ras protein farnesyltransferase are described which are reduced pseudopeptides related to the C-terminal tetrapeptide of the Ras protein that signals farnesylation. Reduction of the carbonyl groups linking the first three residues of the tetrapeptide leads to active inhibitors which are chemically unstable. Stability can be restored by alkylating the central amine of the tetrapeptide. Studies of the SAR of these alkylated pseudopeptides with concomitant modification of the side chain of the third residue led to 2(S)-(2(S)-¿[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)- methylpentyl]naphthalen-1-ylmethylamino¿acetylamino)-4 -methylsulfany lbutyric acid (11), a subnanomolar inhibitor. The methyl ester (10) of this compound exhibited submicromolar activity in the processing assay and selectively inhibited anchorage-independent growth of Rat1 cells transformed by v-ras at 2.5-5 microM.

Potent, non-thiol inhibitors of farnesyltransferase.

The structure-activity relationship of a series of non-thiol CaaX analogs, which are inhibitors of farnesyltransferase, is described. These inhibitors contain a substituted phenyl group at the N terminus, which may occupy a novel binding domain on the Ras protein.

Mutational analysis of conserved residues of the beta-subunit of human farnesyl:protein transferase.

The roles of 11 conserved amino acids of the beta-subunit of human farnesyl:protein transferase (FTase) were examined by performing kinetic and biochemical analyses of site-directed mutants. This biochemical information along with the x-ray crystal structure of rat FTase indicates that residues His-248, Arg-291, Lys-294, and Trp-303 are involved with binding and utilization of the substrate farnesyl diphosphate. Our data confirm structural evidence that amino acids Cys-299, Asp-297, and His-362 are ligands for the essential Zn2+ ion and suggest that Asp-359 may also play a role in Zn2+ binding. Additionally, we demonstrate that Arg-202 is important for binding the essential C-terminal carboxylate of the protein substrate.

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.

Farnesyltransferase inhibitors and anti-Ras therapy.

The oncoprotein encoded by mutant ras genes is initially synthesized as a cytoplasmic precursor which requires posttranslational processing to attain biological activity; farnesylation of the cysteine residue present in the CaaX motif located at the carboxy-terminus of all Ras proteins is the critical modification. Once farnesylated and further modified, the mature Ras protein is inserted into the cell's plasma membrane where it participates in the signal transduction pathways that control cell growth and differentiation. The farnesylation reaction that modifies Ras and other cellular proteins having an appropriate CaaX motif is catalyzed by a housekeeping enzyme termed farnesyl-protein transferase (FPTase). Inhibitors of this enzyme have been prepared by several laboratories in an effort to identify compounds that would block Ras-induced cell transformation and thereby function as Ras-specific anticancer agents. A variety of natural products and synthetic organic compounds were found to block farnesylation of Ras proteins in vitro. Some of these compounds exhibit antiproliferative activity in cell culture, block the morphological alterations associated with Ras-transformation, and can block the growth of Ras-transformed cell lines in tumor colony-forming assays. By contrast, these compounds do not affect the growth or morphology of cells transformed by the Raf or Mos oncoproteins, which do not require farnesylation to achieve biological activity. The efficacy and lack of toxicity observed with FPTase inhibitors in an animal tumor model suggest that specific FPTase inhibitors may be useful for the treatment of some types of cancer.

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.

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.

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.

A priori prediction of activity for HIV-1 protease inhibitors employing energy minimization in the active site.

We have observed a high correlation between the intermolecular interaction energy (Einter) calculated for HIV-1 protease inhibitor complexes and the observed in vitro enzyme inhibition. A training set of 33 inhibitors containing modifications in the P1' and P2' positions was used to develop a regression equation which relates Einter and pIC50. This correlation was subsequently employed to successfully predict the activity of proposed HIV-1 protease inhibitors in advance of synthesis in a structure-based design program. This included a precursor, 47, to the current phase II clinical candidate, L-735,524 (51). The development of the correlation, its applications, and its limitations are discussed, and the force field (MM2X) and host molecular mechanics program (OPTIMOL) used in this work are described.

Protein farnesyltransferase inhibitors block the growth of ras-dependent tumors in nude mice.

The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its transforming activity. Cell-active inhibitors of the enzyme that catalyzes this reaction, protein farnesyltransferase, have been shown to selectively block ras-dependent transformation of cells in culture. Here we describe the protein farnesyltransferase inhibitor 2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl] pentyloxy-3-phenylpropionylmethioninesulfone methyl ester (L-739,749), which suppressed the anchorage-independent growth of Rat1 cells transformed with viral H-ras and the human pancreatic adenocarcinoma cell line PSN-1, which harbors altered K-ras, myc, and p53 genes. This compound also suppressed the growth of tumors arising from ras-transformed Rat1 cells in nude mice by 66%. Under the same conditions, doxorubicin inhibited tumor growth by 33%. Control tumors formed by v-raf- or v-mos-transformed Rat1 cells were unaffected by L-739,749. Furthermore, mice treated with L-739,749 exhibited no evidence of systemic toxicity. This is a demonstration of antitumor activity in vivo using a synthetic small molecule inhibitor of protein farnesyltransferase.

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.

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.

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.

Design and synthesis of HIV protease inhibitors. Variations of the carboxy terminus of the HIV protease inhibitor L-682,679.

A series of tetrapeptide analogues of 1 (L-682,679), in which the carboxy terminus has been shortened and modified, was prepared and their inhibitory activity measured against the HIV protease in a peptide cleavage assay. Selected examples were tested as inhibitors of virus spread in cell culture. Compound 12 was a 10-fold more potent enzyme inhibitor than 1 in vitro and 30-fold more potent in inhibiting the viral spread in cells.

Topically active carbonic anhydrase inhibitors. 1. O-acyl derivatives of 6-hydroxybenzothiazole-2-sulfonamide.

A series of O-acyl derivatives of 6-hydroxybenzothiazole-2-sulfonamide (4, L-643,799) was prepared and the potential utility of each series member as a topically active inhibitor of ocular carbonic anhydrase was determined. In vitro studies showed these esters to be substrates for ocular esterases which liberate 4 during corneal translocation. The most interesting series member, 2-sulfamoyl-6-benzothiazolyl 2,2-dimethylpropionate (22, L-645,151), acting as a prodrug form of 4, was found to enhance delivery through the isolated albino rabbit cornea by 40-fold when compared to the parent phenol 4. Studies in rabbits revealed that 22 is a potent topically active ocular hypotensive carbonic anhydrase inhibitor.