Bisphosphonates target multiple sites in both cis - and trans -prenyltransferases

Abstract

Bisphosphonate drugs (e.g., Fosamax and Zometa) are thought to act primarily by inhibiting farnesyl diphosphate synthase (FPPS), resulting in decreased prenylation of small GTPases. Here, we show that some bisphosphonates can also inhibit geranylgeranyl diphosphate synthase (GGPPS), as well as undecaprenyl diphosphate synthase (UPPS), a cis-prenyltransferase of interest as a target for antibacterial therapy. Our results on GGPPS (10 structures) show that there are three bisphosphonate-binding sites, consisting of FPP or isopentenyl diphosphate substrate-binding sites together with a GGPP product-or inhibitor-binding site. In UPPS, there are a total of four binding sites (in five structures). These results are of general interest because they provide the first structures of GGPPSand UPPS-inhibitor complexes, potentially important drug targets, in addition to revealing a remarkably broad spectrum of binding modes not seen in FPPS inhibition.cell wall ͉ geranylgeranyl diphosphate synthase ͉ undecaprenyl diphosphate synthase ͉ x-ray structure I soprenoid biosynthesis involves the condensation of C 5 -diphosphates to form a very broad range of compounds used in cell membrane (cholesterol, ergosterol), cell wall (lipid I, II, peptidoglycan) and terpene biosynthesis, electron transfer (quinone, heme a, carotenoid, chlorophyll), and in many eukaryotes, cell signaling pathways (Ras, Rho, Rap, Rac). There has, therefore, been considerable interest in developing specific inhibitors of some of these pathways to modify cell function. For example, the bisphosphonate drugs used to treat bone resorption diseases such as osteoporosis (1) have been thought to function by targeting farnesyl diphosphate synthase (FPPS, EC 2.5.1.10) in osteoclasts, leading to dysregulation of cell-signaling pathways involving small GTPases, and in some parasitic protozoa, leading to inhibition of ergosterol biosynthesis (2). However, in recent work Goffinet et al. (3) proposed that the main biological activity of the most potent bisphosphonate zoledronate (Zometa) in humans cells is directed against protein geranylgeranylation. This opens up the intriguing possibility that it might be possible to enhance potency by developing drugs that work by inhibiting geranylgeranyl diphosphate synthase (GGPPS, EC 2.5.1.30), the enzyme that produces the geranylgeranyl diphosphate (GGPP) used to geranylgeranylate e.g., Rac, Rap, and Rho. Based on the recent observation of a previously uncharacterized (GGPP) inhibitor site in GGPPS (4), we reasoned that larger, more hydrophobic species than those in current use might bind to this site and exhibit enhanced activity, because of increased hydrophobic stabilization and, in cells, enhanced lipophilicity. Here, we thus report structures of a series of five bisphosphonates bound to GGPPS together with, for comparative purposes, the structures of five isoprenoid diphosphate-GGPPS complexes. We find three quite different binding modes, corresponding to FPP/GPP (substrate), IPP (substrate), and GGPP [product/ inhibitor (4)...

Lipophilic analogs of zoledronate and risedronate inhibit Plasmodium geranylgeranyl diphosphate synthase (GGPPS) and exhibit potent antimalarial activity

“…Moreover, the two sets of results are themselves quite close, with neither the extreme entropy or enthalpy driven behavior seen with FPPS (39), suggesting either that the charge center does not contribute to binding in GGPPS, or that the presence of the large neutral side chain simply reduces the effects of the cation center. The former view is supported by the observation in S. cerevisae GGPPS that the cation center is not essential for GGPPS inhibition (36), unlike the situation with FPPS (3).…”

“…As we and others reported previously (30,35,36), there are four main ligand (substrate or inhibitor) binding sites in human and S. cerevisae GGPPS, illustrated in (Fig. 3A).…”

“…The polar head group in this lipophilic bisphosphonate thus binds in a very similar manner as does its zoledronate parent. However, the alkyl chain present in the lipophilic species BPH-703 occupies the FPP substrate-hydrophobic site (b) rather than the GGPP product (or inhibitor) site (d) seen in other GGPPS structures (30,35,36).…”

Crystal Structure of Heterodimeric Hexaprenyl Diphosphate Synthase from Micrococcus luteus B-P 26 Reveals That the Small Subunit Is Directly Involved in the Product Chain Length Regulation

“…The size of the cleft is strongly involved in the regulation of the final product chain length (9,11,12,26,(33)(34)(35)(36)(37)(38)(39). The crystal structure of GGPP synthase from Saccharomyces cerevisiae (Sc-GGPPs) with its final product GGPP revealed that the hydrophobic tail of the product just fits the cleft in both of the two homodimeric subunits (18). Many mutagenesis investigations at the interior of the hydrophobic cleft have reported that the deeper cleft synthesizes the longer final product (12).…”

Deciphering the Metabolism of Undecaprenyl-Phosphate: The Bacterial Cell-Wall Unit Carrier at the Membrane Frontier

“…For instance, various bisphosphonate compounds mimicking diphosphate substrates have been found to inhibit UppS via a competition with C 15 -PP for enzyme binding. 36,127 Three dimensional structures of UppS-inhibitor complexes showed that several of these compounds occupied the enzyme active-site tunnel. High-throughput screening also provided several leads that were found to inhibit the growth of S. pneumoniae with satisfactory minimum inhibitory concentrations as a result of UppS activity inhibition.…”