Solid-State NMR, Crystallographic, and Computational Investigation of Bisphosphonates and Farnesyl Diphosphate Synthase−Bisphosphonate Complexes

Abstract

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.

NMR Hyperfine Shifts in Blue Copper Proteins: A Quantum Chemical Investigation

“…Comparisons with the results from Calc8 models, which have the same size as the Calc7 models, but without geometry optimization (Table 1), clearly indicate that geometry optimization is still important for good predictions of NMR hyperfine shifts in BCPs, even when very large structural units are used in the calculations, since the R 2 value degrades to 0.65 for Calc8. This is consistent with the results of previous investigations, 22,[47][48][49][50] showing that QM refinement of protein X-ray structures is generally needed to provide the best property predictions, at least in the case of metal-binding sites.…”

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

“…In fact, the UPPS 2D-QSAR descriptors involving positive charge actually indicate a slight reduction in activity with positive charge (SI Table 6). Although perhaps at first surprising, it now seems clear that FPPS inhibition requires a positive-charge feature, as deduced by QSAR (17) and observed by NMR (13), but GGPPS inhibition with the compounds investigated so far does not because, especially for the larger inhibitors, the combination of a large hydrophobic feature together with, in some cases, Mg 2ϩ binding, provides good potency. That is, because the more potent GGPPS inhibitors are not transition state/reactive intermediate analogs, there is no charge required.…”

“…For example, geranyl (C 10 ) diphosphate synthase and FPPS are potently inhibited by bisphosphonates (9)(10)(11)(12)(13)19). Moreover, other long-chain prenyltransferases are also potently inhibited by bisphosphonates, such as zoledronate, and we show in SI Table 11 a compilation of IC 50 values for GGPPS, hexaprenyl diphosphate synthase (HPPS, from Sulfolobus solfataricus) and octaprenyl diphosphate synthase (OPPS, from E. coli) for each of the inhibitors investigated here.…”

New Experimental Therapies Targetting Breast Cancer Cell

“…Interestingly, these compounds exhibit potent antiproliferative activities in vitro on human epidermoid A431 cells (Guenin et al, 2005). In parallel, recently crystallographic and computational investigation reveal that the presence of phenyl ring in the side chain permitted non N-BPs to interact with farnesyl enzyme (Mao et al, 2006). Based on these data, we have synthesized a class of BPs that contains bromobenzyl in their side chains (BP7033Br, Fig.…”

“…Inhibition of Ras processing using non bromo-containing BP7033 is also reached (Hamma-kourbali et al, 2003). Also, the addition of phenyl function in the side chain of BPs rendered the catalytic pocket of geranyl and/or farnesyl synthase enzymes of the mevalonate pathway more accessible (Mao et al, 2006;Steeg et al, 2006). BP7033Br ALK reduces MDA-MB-231 and D3H2LN cell viabilities about 90% with a concentration 4-fold inferior to that of BP7033Br.…”