René Hemmig

Structural Basis for the Exceptional in vivo Efficacy of Bisphosphonate Drugs

To understand the structural basis for bisphosphonate therapy of bone diseases, we solved the crystal structures of human farnesyl pyrophosphate synthase (FPPS) in its unliganded state, in complex with the nitrogen‐containing bisphosphonate (N‐BP) drugs zoledronate, pamidronate, alendronate, and ibandronate, and in the ternary complex with zoledronate and the substrate isopentenyl pyrophosphate (IPP). By revealing three structural snapshots of the enzyme catalytic cycle, each associated with a distinct conformational state, and details about the interactions with N‐BPs, these structures provide a novel understanding of the mechanism of FPPS catalysis and inhibition. In particular, the accumulating substrate, IPP, was found to bind to and stabilize the FPPS–N‐BP complexes rather than to compete with and displace the N‐BP inhibitor. Stabilization of the FPPS–N‐BP complex through IPP binding is supported by differential scanning calorimetry analyses of a set of representative N‐BPs. Among other factors such as high binding affinity for bone mineral, this particular mode of FPPS inhibition contributes to the exceptional in vivo efficacy of N‐BP drugs. Moreover, our data form the basis for structure‐guided design of optimized N‐BPs with improved pharmacological properties.

Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Pagets disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.

Increased Mature Interleukin-1β (IL-1β) Secretion from THP-1 Cells Induced by Nigericin Is a Result of Activation of p45 IL-1β-converting Enzyme Processing

Perregaux and Gabel (Perregaux, D., and Gabel, C. A. (1994) J. Biol. Chem. 269, 15195–15203) reported that potassium depletion of lipopolysaccharide-stimulated mouse macrophages induced by the potassium ionophore, nigericin, leads to the rapid release of mature interleukin-1β (IL-1β). We have now shown a similar phenomenon in lipopolysaccharide-stimulated human monocytic leukemia THP-1 cells. Rapid secretion of mature, 17-kDa IL-1β occurred, in the presence of nigericin (4–16 μm). No effects on the release of tumor necrosis factor-α, IL-6, or proIL-1β were seen. Addition of the irreversible interleukin-1β-converting enzyme (ICE) inhibitor, Z-Val-Ala-Asp-dichlorobenzoate, or a radicicol analog, inhibited nigericin-induced mature IL-1β release and activation of p45 ICE precursor. The radicicol analog itself did not inhibit ICE, but markedly, and very rapidly depleted intracellular levels of 31-kDa proIL-1β. By contrast, dexamethasone, cycloheximide, and the Na+/H+ antiporter inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, had no effect on nigericin-induced release of IL-1β. We have therefore shown conclusively, for the first time, that nigericin-induced release of IL-1β is dependent upon activation of p45 ICE processing. So far, the mechanism by which reduced intracellular potassium ion concentration triggers p45 ICE processing is not known, but further investigation in this area could lead to the discovery of novel molecular targets whereby control of IL-1β production might be effected.

Ranking of High‐Affinity Ligands by NMR Spectroscopy

NMR spectroscopy is a powerful biophysical technique to detect and characterize molecular interactions. Its high sensitivity and robustness to detect weakly binding ligands makes it an attractive tool, particularly for the early phases of drug-discovery research that comprise hit finding and hit validation. Several methods are available to determine dissociation constants (KD) by NMR spectroscopy, [4–7] including the direct titration of protein target with increasing amounts of ligand and competition-based experiments, such as NMR reporter screening. However, these methods generally work only for weakly or moderately binding ligands, but not for tightly binding ligands. This situation precludes application of these methods to the later lead-optimization stage of drug-discovery projects. Potent compounds are generally evaluated in a functional assay, or by using different biophysical techniques, such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). In practice, however, data are sometimes conflicting, and ITC and SPR may not always be applicable. In these cases, application of an independent biophysical technique, such as NMR spectroscopy, would be desirable. Herein we describe a new NMR spectroscopic method that allows the precise determination of relative binding affinities of two tightly binding ligands. This approach is a valuable tool for the lead optimization process. Direct titration in NMR spectroscopy is not applicable for high-affinity ligands for two reasons: First, high-affinity ligands generally have slow dissociation kinetics (slow koff), so that upon titration of a ligand, a second signal set corresponding to the complexed state gradually appears while the signal set for the unbound state gradually disappears. In contrast, in the low-affinity case, fast koff rates generally lead to the shifting of signals as the fraction of complexed state increases, and the extent of chemical shift change can be conveniently plotted as a function of ligand concentration in order to determine the dissociation constant, KD. Second, it is a fundamental principle in biophysics that dissociation constants can be precisely measured only for protein concentrations in the range of KD. The high protein concentrations (typically double-digit micromolar) required for NMR spectroscopic measurements therefore allow the precise measurement of KD values in the micromolar or millimolar range, but not much lower. This is because the KD is encoded in the curvature of the titration curve, and the curvature is not precisely measurable for high affinities (Figure 1). Reporter screening extends this limit to the highnanomolar range, but not further since the dynamic range of quantification is about an order of magnitude around the reporter ligand, and the reporter ligand must show weak or intermediate binding.

A General Strategy for Targeting Drugs to Bone.

Targeting drugs to their desired site of action can increase their safety and efficacy. Bisphosphonates are prototypical examples of drugs targeted to bone. However, bisphosphonate bone affinity is often considered too strong and cannot be significantly modulated without losing activity on the enzymatic target, farnesyl pyrophosphate synthase (FPPS). Furthermore, bisphosphonate bone affinity comes at the expense of very low and variable oral bioavailability. FPPS inhibitors were developed with a monophosphonate as a bone-affinity tag that confers moderate affinity to bone, which can furthermore be tuned to the desired level, and the relationship between structure and bone affinity was evaluated by using an NMR-based bone-binding assay. The concept of targeting drugs to bone with moderate affinity, while retaining oral bioavailability, has broad application to a variety of other bone-targeted drugs.