FPTase inhibitors

In recent years several FPTase inhibitors were developed as peptidomimetics of the key tetrapeptide fragment for use as potential anticancer agents. Miller at... 

To function, Ras must be attached to the plasma membrane. Translocation from the cytoplasm to membrane requires a series of posttranslational modifications that begin with farnesylation of the cysteine residue, the fourth amino acid residue from the C terminus of the protein, by famesyl protein transferase (FPTase) (64). Attachment of the hydrophobic 15-carbon lipid farnesyl group allows Ras molecule insertion into the plasma membrane and is crucial for Ras signaling activity and transformation properties. As farnesylation is required for oncogenic Ras function, FPTase inhibitors (FTIs) are obvious candidate antineoplastic agents. Several drugs that inhibit Ras farnesylation are at various stages of clinical development (65). 

Several non-peptide small molecule compoimds from Merck, Schering-Plough, Bristol-Myers Squibb, and Janssen are in phase I clinical trials for the treatment of cancer. This has bolstered the significance of this approach of cancer chemotherapy. Other organizations that have ongoing active FPTase inhibitor programs include Abbott, Rhone Poulenc Rorer, Parke- Davis, Pfizer, Zeneca, Eisai and Biomeasure. While several review articles have recently been published that cover various aspects of FPTase inhibitors [25-28,31,60,61], the discussion of FPTase inhibitors derived from natural products is limited. Therefore, this article will not be an extensive review of the literature dealing with chemically derived inhibitors of farnesyl-protein transferase. Rather, it will focus on inhibitors of famesyl-protein transferase that have been identified from natural product sources discovered by Merck or other institutions. 

To identify FPTase inhibitors traditional sources of samples (chemical sample collections, natural product and combinatorial chemistry libraries) were randomly screened with varying degrees of success. Tetrapeptides that were structurally similar to the CaaX-box motif were almost immediately discovered as being both relatively potent inhibitors and efficient substrates of the enzyme. These initial peptide leads led to the formation of a number of medicinal chemistry teams whose primary objectives were to convert these tetrapeptides into non-peptidic... 

Cembranolide, a diterpene, is unique in that it is the only FPTase inhibitor isolated from a marine specimen, Lobophytum cristagalli (Order Alcyonacea, Family Alcyoniidae), that was collected at a 15m depth from Seychelles Island. The compound was isolated by silica gel chromatography followed by reverse phase HPLC and the structure of this 14/5-bicyclic diterpenoid (Figure 24) was elucidated by NMR and confirmed by X-ray crystallography [115]. 

Similar to cembranolide, clavaric acid was competitive with respect to the Ras-peptide substrate Kj = 1.4 jM) and non- and/or un-competitive with FPP despite having a negatively charged carboxyl group. This is only the second example of a non-nitrogenous FPTase inhibitor that is competitive with peptide substrate. These two (cembranolide and clavaric acid) structurally distinct compounds show remarkably similar profiles of the mechanism of FPTase inhibition [117]. 

Patulin (Figure 42) is a small polyketide-derived mycotoxin, originally isolated as an antibiotic in the 1940 s. It was isolated from fungal strains of Byssochlamys nivea [149] as a FPTase inhibitor. 

Modified peptides containing reduced amide bonds, i. e. compounds L-731,735 and L-731,734, were designed by the Merck groups [7] (Fig. 1), and have shown to be potent inhibitors of partially purified FPTase, the homoserine compound being the more active inhibitor in vitro. Subsequent inhibitors include L-739,749 and L-739,750 [8] and even trancated versions of the C-terminal tetrapeptide CAAX motif were prepared which do not have a C-terminal carboxyl... 

While inhibition of formation of biosynthetic precursors upstream of FPP is possible, the j>otential deleterious effects are great given the widespread use of FPP in other biological processes within cells. However, selective inhibition of FPTase should be feasible and has the added implied advantage of having more beneficial and fewer harmful downstream consequences. This rationale has been the major impetus for identifying specific inhibitors of FPTase and has been the focus of many pharmaceutical companies and academic institutions. 

Since FPTase is an enzyme that requires two substrates (FPP and Ras) and a divalent cation (zinc) for optimal activity, inhibitors of the enzyme would be expected to distribute into several categories. For example, inhibitors would be expected to be i) competitive with either or both substrates, ii) non-competitive with either or both substrates, and iii) uncompetitive with either or both substrates. In addition, inhibitors could be either reversible or irreversible of enzymatic activity. 

Initial drug discovery efforts focused on exploiting the Ras peptide sequence, and in particular, the sequence at the carboxyl terminus. CaaX box tetrapeptides such as CVIM and CVFM (Figure 5) are potent inhibitors of FPTase activity [29]. CVIM, in contrast to CVFM, is a good substrate for the en2yme and is prenylated efficiently [29]. 

An alternative strategy was to focus on the FPP substrate that resulted in the synthesis of several phosphinic acid derivatives those were competitive inhibitors of FPTase activity. For example, phosphinic acid A (Figure 7) showed potent activity in in vitro assays (IC50 = 30 nM) but was not active in the cell-based assays. This inactivity is most likely due to the lack of cell penetration of phosphinic acid [44]. 

Banyu Pharmaceutical Company reported that J-104134 (Figure 7) was a potent inhibitor of rat FPTase activity (IC50 = 5 nM). This compound, though structurally similar to many FPP competitive natural product inhibitors (vide infra), contain multiple free carboxyl groups but, surprisingly, unlike the natural product inhibitors, was active in cell-based assays (IC50 = 4.3 jiM) [63]. 

Sixteen consensus sequences were re-synthesized, tested and all sixteen were active exhibiting varying degrees of potency. The most active peptide contained the sequence D-tryptophan-D-methionine-D-4-chlorophenyl alanine-L-y-carboxyglutamic acid (Figure 10). This compound was a potent (IC50 42 nM), specific inhibitor of FPTase, and most intriguingly, unlike other Ca -based inhibitors, was competitive with... 

Using assay conditions where both substrates were used at km levels chaetomellic, actinoplanic, oreganic and zaragozic acids and alkyl citrates (for example, viridiofungins) were discovered from different microbial extracts. These compounds were potent selective inhibitors of FPTase activity and did not inhibit GGPTase or squalene synthase. All five classes of compounds were competitive with respect to FPP and were reversible inhibitors of FPTase activity. Three additional reports have appeared during the intervening period that described the isolation of CP 225917, manumycin, and RPR 113228 as FPP competitive inhibitors. These inhibitors, their in vitro activity and their corporate sources are summarized in Table 1. 

Actinoplanic acids A and B exhibited IC50 values of 230 and 50 nM, respectively, against rHFPTase. Both compounds are competitive with FPP and displayed Kj values of 98 and 8 nM, respectively. The inhibition profile of these compounds was uncompetitive with respective to the Ras peptide substrate. The inhibition of FPTase by actinoplanic acids is selective and reversible and these compounds did not inhibit the human squalene synthase and bovine brain GGPTase (ICso s > 1 jjM) [87,88]. Similar to other examples of inhibitors that are competitive with respect to FPP, esterification of the carboxy groups of actinoplanic acids completely eliminated inhibitory activity. The increased potency of actinoplanic acid B is associated with the increased number of negative charges when compared with acid A [87,88]. 

A large number of compounds representing this family were subsequently isolated from a variety of fungal sources. Because the discovery, isolation and biological activity of these compounds have been extensively reviewed [91], the discussion here will be limited to those that are reported to be inhibitors of FPTase. 

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