The molecular mechanism of nitrogen-containing bisphosphonates as antiosteoporosis drugs

Abstract

Osteoporosis and low bone mass are currently estimated to be a major public health risk affecting >50% of the female population over the age of 50. Because of their bone-selective pharmacokinetics, nitrogen-containing bisphosphonates (N-BPs), currently used as clinical inhibitors of bone-resorption diseases, target osteoclast farnesyl pyrophosphate synthase (FPPS) and inhibit protein prenylation. FPPS, a key branchpoint of the mevalonate pathway, catalyzes the successive condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. To understand the molecular events involved in inhibition of FPPS by N-BPs, we used protein crystallography, enzyme kinetics, and isothermal titration calorimetry. We report here high-resolution x-ray structures of the human enzyme in complexes with risedronate and zoledronate, two of the leading N-BPs in clinical use. These agents bind to the dimethylallyl/geranyl pyrophosphate ligand pocket and induce a conformational change. The interactions of the N-BP cyclic nitrogen with Thr-201 and Lys-200 suggest that these inhibitors achieve potency by positioning their nitrogen in the proposed carbocation-binding site. Kinetic analyses reveal that inhibition is competitive with geranyl pyrophosphate and is of a slow, tight binding character, indicating that isomerization of an initial enzyme-inhibitor complex occurs with inhibitor binding. Isothermal titration calorimetry indicates that binding of N-BPs to the apoenzyme is entropy-driven, presumably through desolvation entropy effects. These experiments reveal the molecular binding characteristics of an important pharmacological target and provide a route for further optimization of these important drugs.

Fig. 1.

Structure of human FPPS. (A) Stereoview of the superimposition of FPPS in complex with RIS and Mg²⁺ (blue) with the avian apo structure 1FPS (gray), made by superimposing the α-carbons in helices α1–α7. (B) Specific polar interactions of RIS and Mg²⁺. Amino acid numbering in this paper is offset by −14 residues compared with the Protein Data Bank depositions. (C) Stereoview detail of RIS binding to FPPS (blue). Three Mg²⁺ ions mediate the interaction between phosphates and conserved aspartate residues. A semitransparent avian apo structure is superimposed in gray to highlight the structural differences. (D) Chemical structures of RIS and ZOL.

Fig. 2.

Detail of the FPPS·ZOL·IPP ternary complex. (A) Close-up stereoview of the heterocyclic ring binding pocket. ZOL and residues within 4 Å of the BP side chain are shown in ball-and-stick format (lilac). The equivalent residues from the RIS complex are overlaid in blue, and the RIS is shown semitransparently. (B) Close-up stereoview of the IPP-binding site showing the network of electrostatic interactions involving the C terminus.

Fig. 3.

N-BP inhibition of human FPPS. (A) Competitive inhibition of RIS for the GPP site. Lineweaver–Burk plot of initial rate of FPPS; [IPP] = 10 μM. (B) Uncompetitive inhibition of RIS for the IPP site. Lineweaver–Burk plot of initial rate of FPPS; [GPP] = 5 μM. (C) Initial inhibition of FPPS by RIS and ZOL; [IPP] and [GPP] = 10 μM. Data were fitted to Eq. 1 by nonlinear regression. (D) FPPS preincubated with RIS. Enzyme was assayed after a 10-min preincubation with RIS; [IPP] and [GPP] were 10 μM. Fitting of data to Eq. 2 results in determination of an enzyme concentration of 8 nM.

Fig. 4.

Thermodynamics of ligand binding to human FPPS measured by ITC. (A) Binding of RIS to FPPS in the presence of 2 mM MgCl₂. (Upper) Data obtained for injections of RIS into buffer (lower trace) and into FPPS (upper trace). (Lower) Binding isotherm fit to a one-site binding model after subtraction of blank titration heats. (B) Titration of IPP into FPPS. Biphasic binding is observed, indicating occupation of both GPP and IPP sites. (C) ZOL binding to the FPPS·IPP complex. Displacement of IPP bound in the GPP site is accompanied by negative enthalpies. (D) Binding kinetics of ZOL in the presence of IPP. ZOL showed slow binding kinetics with IPP present (bold trace) but not in the absence of IPP (thin line).