Competitive with FPP

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]. 

Eight classes of natural products that are inhibitors of FPTase have been described from a variety of microbial sources. Mechanistically, these compounds are competitive with FPP. All compounds, with the exception of manumycins, possess negative charges in the form of one or more carboxylic, sulfuric or phosphoric acids, and are not active in cell-based assays. The lack of cell-based activities for these classes of natural products is linked to the negative charge present in these compounds that appears to be detrimental for cell wall penetration. Manumycin, in contrast, is not only active in cell-based assays but is also active in animal models. 

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]. 

To date, a total of 29 classes of natural products isolated from a variety of sources have been reported to inhibit FPTase activity. These include poly-carboxylic acids containing fatty chains that are competitive with FPP,... 

The development status of these molecules is not known. It will be interesting to note whether any differences emerge from the CAAX competitive versus FPP competitive molecules as more data become available for these compounds. Since FPP itself contributes to the CAAX peptide binding pocket, the interaction of FPP competitive FTIs with CAAX peptide competitive FTIs will be of interest. The selectivity of FPP competitive FTIs for the FTase pathway versus other biochemical pathways utilizing FPP, such as ubiquinone synthesis and the heme farnesyltransferase, has also not been reported. These other FPP reactions have important roles in mitochondrial function, which presents some risk for adverse events or possibly opportimities for modulating early apoptotic events. 

The FPP analog HFP (ot-hydroxyfarnesylphosphonate. Figure 5.3), which was one of the first potent FTIs to be reported [47], is competitive with respect to FPP, and binds to FTase with a nearly equal affinity to its... 

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. 

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. 

CP-225917 and CP-263114 were weak inhibitors of rat brain FPTase (IC50 values of 6 and 20 jUM, respectively) and rat liver microsomal squalene synthase activities (IC50 values of 43 and 160 jUM, respectively) [97]. The relatively weak activities of these compounds against both enzymes is consistent with the SAR of a later series of compoimds. These compounds show competitive binding with respect to FPP with squalene synthase. While mechanistic data relating to FPTase have not been reported, it is reasonable to assume that these compounds compete with FPP for their inhibition in this enzyme as well and can be classified within the group of FPP competitors. 

In summary, the inhibition of FPTase by manumycin A is i) selective (it is 36-fold less active against GGPTase-I), ii) surprisingly competitive with respect to FPP with a Kj value of 1.2 jUM and iii) noncompetitive with respect to the peptide substrate [99]. Furthermore, manumycin A is active in decreasing the growth of Ki-m -transformed fibroblast cells and is apparently effective against both Ki- and N-ra -dependent fibrosarcomas in tumorigenesis mouse models [110]. 

This compound i) inhibited recombinant human FPTase (IC50 = 2.1 jUM), ii) was competitive with respect to FPP (Kj = 0.4 /JM), iii) was 30-fold selective for FPTase over GGPTase I (IC50 = 59 jUM) and iv) was completely inactive against squalene synthase [111]. 

Pepticinnamin C (whose structure was not disclosed) was the most active member of this family (IC50= 100 nM) against partially purified human FPTase, while pepticinnamin E was 3-fold less active (IC50 = 300 nM) [112]. Pepticinnamin E was competitive with the Ras peptide Kj = 1.8 jiM) and noncompetitive with FPP Kj = 1.9 /iM) [114]. 

After FPP synthesis, the biochemical reactions and enzymes involved have not been fully understood and well characterized. Akhila et al. [21] proposed a complete biosynthetic pathway for artemisinin, starting from mevalonic acid and IPP. The pathway branches at FPP. FPP is converted in to squalene by the enzyme squalene synthase (SQS) and subsequently into sterol. SQS is the key enzyme catalyzing the first step of the sterol biosynthetic pathway, a pathway in competition with that of artemisinin biosynthesis [26]. 

One approach to develop FPP-competitive/FPP mimetic FTIs is to utilize FPP analogs with modified isoprenoid moieties that are potent FTIs. The Gibbs and Spielmann laboratories have discovered that certain... 

Fig. 13.4. DGBP competitively inhibits GGDPS with respect to FPP. In vitro GGDPS assays were performed utilizing various concentrations of FPP substrate or DGBP using an established method [110]. A double reciprocal plot liV vs. lA) is shown.

FPP Ki = 2 nM) and uncompetitive with the Ras substrate Ki = 32 nM) [69]. While numerous reports of FPP competitive inhibitors derived from microbial sources are available none are peptidic vide infra). The... 

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