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 GGPPS- and UPPS-inhibitor complexes, potentially important drug targets, in addition to revealing a remarkably broad spectrum of binding modes not seen in FPPS inhibition.

Fig. 1.

Chemistry and structures. (A) Illustration of H_R,S atom abstraction in E,Z prenyltransferases. (B) Structures of isoprenoid diphosphates. (C) Structure of GGPPS-Mg–FsPP-IPP complex. (D) Substrate-binding region in C. (E–I) Electron densities (green contoured at 1σ, red at 3σ) for IPP (E), GPP (F), FPP (G), FsPP (H), and GGPP (I), bound to GGPPS.

Fig. 2.

Bisphosphonates and GGPPS structures. (A) Structures of bisphosphonates investigated as GGPPS inhibitors. (B) GGPPS structure containing zoledronate (PDB ID code 2E91) showing dimer structure. (C) Stereoview of minodronate bound to GGPPS (PDB ID code 2E92). (D) Stereoview of zoledronate/GGPPS (PDB ID code 2E91) superimposed on zoledronate/IPP/FPPS structure (PDB ID code 2F8C).

Fig. 3.

Structures of hydrophobic bisphosphonates bound to GGPPS. (A) Stereoview of BPH-675 (PDB ID code 2E95). (B) Electron density (green contoured at 1σ, red at 3σ) for the two BPH-629 conformers (PDB ID code 2E93). (C) Structure of BPH-629 bound to GGPPS (monomer A model is shown).

Fig. 4.

Binding site motifs for GGPPS inhibitors. (A) FPP-GGPP site (in blue) occupied by BPH-364, BPH-675, and GGPP (human protein, PDB ID code 2FVI). (B) FPP-FPP site (in orange) occupied by zoledronate, minodronate, and BPH-629-1. (C) IPP-GGPP site (in pink) occupied by BPH-629-2. (D) IPP-FPP site (in green) occupied by GGPP in the yeast enzyme. (E) Superimposition of both BPH-629 conformers from chain A model. (F) Superimposition of yeast, human GGPP structures with BPH-675.

Fig. 5.

Bisphosphonate and UPPS structures. (A) Structures of additional UPPS inhibitors. (B) Structure of UPPS-BPH-629 complex (PDB ID code 2E98). (C) Structures of BPH-629 bound to four different sites (A and B molecules in the dimer are both shown).

Fig. 6.

Binding-site interactions and computational modeling. (A) BPH-629 protein–ligand interactions. (B) Diagram showing surface structure of all four BPH-629 molecules (from Fig. 5C Upper). (C) Diagram showing surface structure of all five inhibitors bound to UPPS (all crystal structures). (D) Graph showing correlation between experimental and 2D-QSAR predicted pIC₅₀ values for UPPS inhibition, (see SI Table 7). (E) Pharmacophores for UPPS, GGPPS (adapted from ref. 16), and FPPS (data from ref. 17).