Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anti-cancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth, how cell activity can be predicted based on enzyme inhibition data, and, using x-ray diffraction, solid state NMR and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.

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We report the results of an investigation of the binding of a series of bisphosphonate drugs to human bone using 2H, 13C, 15N, and 31P nuclear magnetic resonance spectroscopy. The 31P NMR results show that the bisphosphonate groups bind irrotationally to bone, displacing orthophosphate from the bone mineral matrix. Binding of pamidronate is well described by a Langmuir-like isotherm, from which we deduce an approximately 30-38 A2 surface area per pamidronate molecule and a deltaG = -4.3 kcal mol(-1). TEDOR of [13C3, 15N] pamidronate on bone shows that the bisphosphonate binds in a gauche [N-C(1)] conformation. The results of 31P as well as 15N shift and cross-polarization measurements indicate that risedronate binds weakly, since it has a primarily neutral pyridine side chain, whereas zoledronate (with an imidazole ring) binds more strongly, since the ring is partially protonated. The results of 2H NMR measurements of side-chain 2H-labeled pamidronate, alendronate, zoledronate, and risedronate on bone show that all side chains undergo fast but restricted motions. In pamidronate, the motion is well simulated by a gauche+/gauche- hopping motion of the terminal -CH2-NH3(+) group, due to jumps from one anionic surface group to another. The results of double-cross polarization experiments indicate that the NH3(+)-terminus of pamidronate is close to the bone mineral surface, and a detailed model is proposed in which the gauche side-chain hops between two bone PO4(3-) sites.

Bisphosphonates are a class of molecules in widespread use in treating bone resorption diseases and are also of interest as immunomodulators and anti-infectives. They function by inhibiting the enzyme farnesyl diphosphate synthase (FPPS), but the details of how these molecules bind are not fully understood. Here, we report the results of a solid-state (13)C, (15)N, and (31)P magic-angle sample spinning (MAS) NMR and quantum chemical investigation of several bisphosphonates, both as pure compounds and when bound to FPPS, to provide information about side-chain and phosphonate backbone protonation states when bound to the enzyme. We then used computational docking methods (with the charges assigned by NMR) to predict how several bisphosphonates bind to FPPS. Finally, we used X-ray crystallography to determine the structures of two potent bisphosphonate inhibitors, finding good agreement with the computational results, opening up the possibility of using the combination of NMR, quantum chemistry and molecular docking to facilitate the design of other, novel prenytransferase inhibitors.

Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4⌬ cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

Clozapine is associated with non-neurological side effects that can be subjectively unpleasant and/or clinically serious. We sought to: (i) assess the nature and prevalence of side effects experienced by patients receiving maintenance treatment with clozapine and (ii) explore the relationship between clozapine plasma concentration and side effect burden. Patients were receiving clozapine maintenance treatment. Open questioning followed by systematic enquiry using the Antipsychotic Non-neurological Side Effects Rating Scale were used to assess side effects. Trough plasma clozapine and norclozapine concentrations were measured. One hundred and three patients participated. On open questioning, 61 patients reported a total of 117 side effects, whereas systematic enquiry identified an additional 649 side effects, with each patient reporting at least one. Clozapine plasma concentrations were significantly but weakly correlated with total Antipsychotic Non-neurological Side Effects Rating Scale score (Pearson correlation=0.29, P<0.004). Patients with a plasma clozapine concentration >0.25 mg/l were significantly more likely to have moderate/severe side effects than patients with lower plasma concentrations (63/76 vs. 12/23, chi=9.07, d.f.=1, P<0.01). The side-effect burden associated with maintenance clozapine treatment is high and the true extent can only be ascertained by systematic inquiry. The use of target plasma concentrations below those used for acute treatment should be explored as a strategy for minimizing side effects.

We have used isothermal titration calorimetry (ITC) to study the thermodynamics of binding of twelve bisphosphonates to human bone. The ITC results show that there are two binding sites. Site A is the weak, highly populated site seen by NMR and is characterized by an average ΔG of binding of −5.2 kcal. Site B is a strong binding site characterized by a ΔG of binding of −8.5 kcal. Binding to both sites is overwhelmingly entropy driven. Using a thermodynamic group approach and a linear regression method we predict the ΔG of binding of all twelve compounds with an R 2 =0.95 (a 0.19 kcal error variance estimate, about 3% of the total ΔG range), opening up the way to designing novel chemotherapy, immunotherapy and anti-infectious disease drugs having weak bone binding affinity.Bisphosphonates are the major drugs used to treat boneresorption diseases 1 . They act by preventing osteoclastic bone resorption, inhibiting the enzyme farnesyl diphosphate synthase (FPPS). Bisphosphonates also kill tumor cells 2 and many parasitic protozoa 3 and can activate γδ T cells of the immune system 4 to kill tumor cells 5 and bacteria 6 . There is thus interest in their use for immuno-chemotherapy of cancer 7 , and in the treatment of parasitic protozoan diseases 8 , where less avid bone binding might be advantageous. In earlier work 9 , we used NMR to probe how different bisphosphonates bind to bone. We found that the 31 P magic-angle sample-spinning NMR spectra of bound bisphosphonates exhibited a single broad peak, and that there was ~0.8 phosphate (Pi) released per bisphosphonate bound. These and other NMR results led to a model 9 in which a bisphosphonate -PO 3 2− group displaced Pi, while the cationic side-chains interacted electrostatically with anionic surface groups. However, a puzzling observation was that the free energy for binding was low (~−4.3 kcal, for pamidronate). Here, we investigate this topic further, by using isothermal titration calorimetry (ITC), which might yield information on any additional, tight binding site(s) that -if at low occupancy, would be difficult to detect via NMR.We investigated by ITC the interaction of the twelve bisphosphonates (1-12) shown above with human bone mineral, which enabled us to study the effects of having a 1-OH group removed (4,6), changing the position of the ring nitrogen in risedronate (7), removing the ring nitrogen in risedronate (8), and truncating the risedronate side-chain (9), in addition to studying several other bisphosphonates of interest (10-12) 11 . Representative ITC results for three compounds(1,2,4), together with their corresponding fitting curves, are shown in Figure 1A-B (all twelve fitting curves arse in Figure S1 in the Supporting Information), and the ΔG, ΔH and ΔS values so derived 10 are given in Table 1.There are several observations. First, there are only two types of ITC curve seen. Binding of half of the compounds (1-3, 5, 7 and 9) is characterized by both weak (Site A, Table 1) and strong (Site B, Table 1) interactions (two independent sites...