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  • The exact mechanism by which


    The exact mechanism by which N-BPs inhibit FPPS remains unclear. Computer modeling [10] suggests that N-BPs mimic the structure of the enzyme’s natural isoprenoid pyrophosphate substrates, geranyl pyrophosphate (GPP)/dimethylallyl pyrophosphate (DMAPP) or act as carbocation transition state analogs [13]. We have previously noted that modeling in the GPP pocket of compounds with dramatic differences in potency does not adequately explain binding differences [14]. Recent X-ray crystallographic studies of E. coli FPPS [15] demonstrate binding of risedronate to the GPP/DMAPP substrate pocket with the side chain positioned in the hydrophobic cleft that normally accommodates an isoprenopid lipid, and the phosphonate groups bound to three magnesium ions. However, the conclusions based on these studies must be tempered by three considerations. Firstly, the authors incubated FPPS with an equimolar solution of IPP and risedronate, which may not be representative of pharmacological concentrations in vivo. Secondly, the positively charged, N-methyl analog of risedronate [16], [17] cannot undergo equivalent stabilization at the GPP site because its nitrogen is not a prostaglandin e1 acceptor, yet is more potent (LED=0.0001 mg/P/kg) than risedronate. Thirdly, extrapolation of the structural results from the E. coli enzyme to the human enzyme must be interpreted with caution pending an X-ray crystal structure of the human enzyme.
    Results and discussion
    Experimental methodology
    The Isoprenoid Biosynthetic Pathway
    Nitrogenous Bisphosphonates
    GGDPS Inhibitors Given the recognition that the basis for the effects of the NBPs to inhibit osteoclast function is now understood to be primarily related to the depletion of GGPP as compared to FPP, efforts have been undertaken to develop agents with specificity for inhibiting GGDPS, as compared to FDPS. The first inhibitors identified were bisphosphonates and azaprenyl diphosphates with IC50 values against purified enzyme of 150–700nM [14]. After an extensive synthetic program, our group has also reported that diprenyl bisphosphonates have striking inhibitory activities against GGDPS with the most active compounds having IC50s in the 150–200nM range [110]. Shown in Figure 13.3 are some of the reported compounds tested in enzyme assay. Structure–activity relationships have established that, with regard to the alkyl substituents on the carbon central to the phosphates, there is a requirement for at least one geranyl chain [111]. Crystallographic studies of GGDPS with inhibitors demonstrate the binding of the inhibitors to substrate and product domains enabling prediction for small molecule structures with even greater activity [113]. We have developed two classes of bisphosphonates; mono- and disubstituted series 110, 111. The biochemical mechanism for one of the most potent agents, digeranyl bisphosphonate (DGBP), to inhibit GGDPS is competitive with regard to FPP as indicated in the double reciprocal plot shown in Figure 13.4 (unpublished data-from A. Wiemer and R. Hohl). While both classes display inhibition in enzyme assays, in general the monosubstituted compounds are less active in intact cellular assays to deplete cells of GGPP and result in the accumulation of small GTPases that are not geranylgeranylated [111]. We have hypothesized that this is because of the high charge-to-mass ratio that the monosubstituted bisphosphonates would have at physiological pH. This characteristic would be predicted to impair intracellular distribution of these compounds. Strategies to improve cellular penetration of both the mono- and dialkylated bisphosphonates include a prodrug approach such as pivaloyloxymethyl modification [114]. The investigational use of GGDPIs has been to deplete cells of GGPP and diminish protein geranylgeranylation. Methods have been developed to measure both FPP and GGPP in cells treated with prenyl synthase inhibitors in tissue culture and in tissues from animals treated with such inhibitors 115, 116. These approaches have afforded the ability to measure the expected biochemical changes that accompany GGDPS inhibition, namely the reduction of GGPP and increase in FPP levels.