Lipophilic bisphosphonates as dual farnesyl/geranylgeranyl diphosphate synthase inhibitors: an X-ray and NMR investigation.

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 anticancer 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 and 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.

Activity of sulfonium bisphosphonates on tumor cell lines.

We investigated three series of sulfonium bisphosphonates for their activity in inhibiting the growth of three human tumor cell lines. The first series consisted of 6 cyclic sulfonium bisphosphonates, the most active species having an (average) IC50 of 89 microM. The second consisted of 10 phenylalkyl and phenylalkoxy bisphosphonates, the most active species having an IC50 of 18 microM. The third series consisted of 17 n-alkyl sulfonium bisphosphonates, the most active species having an IC50 of approximately 240 nM. Three QSAR models showed that the experimental cell growth inhibition results could be well predicted. We also determined the structures of one sulfonium bisphosphonate bound to farnesyl diphosphate synthase, finding that it binds exclusively to the dimethylallyl diphosphate binding site. These results are of interest since they show that sulfonium bisphosphonates can have potent activity against a variety of tumor cell lines, the most active species having IC50 values much lower than conventional nitrogen-containing bisphosphonates.

Structural studies of Vgamma2Vdelta2 T cell phosphoantigens.

Human gammadelta T cells containing the Vgamma2Vdelta2 (Vgamma9Vdelta2) T cell receptor are stimulated by a broad variety of small, phosphorus-containing antigenic molecules called phosphoantigens. The structures of several species present in both Mycobacteria (TUBags1-4) and in Escherichia coli have been reported to contain a formyl-alkyl diphosphate core. Here we report the synthesis of the lead member of the series, 3-formyl-1-butyl diphosphate. This compound has low activity for gammadelta T cell stimulation, unlike its highly active isomer (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate, necessitating a revision of the structure of TUBag1. Likewise, the structure of the species identified as the pentyl analog (TUBag 2) is revised to 6-phosphogluconate. These results indicate that neither TUBag1 nor the m/e 275 species proposed for TUBag2 are 3-formyl-1-alkyl diphosphates, leading to the conclusion that none of the natural phosphoantigens (TUBags1-4) possess the structures reported previously.

Isoprenoid biosynthesis as a drug target: bisphosphonate inhibition of Escherichia coli K12 growth and synergistic effects of fosmidomycin.

We screened a library of 117 bisphosphonates for antibacterial activity against Escherichia coli. The most potent growth inhibitors where N-[methyl(4-phenylalkyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonates, known potent bone resorption inhibitors, and there was a generally good correlation between cell growth inhibition and E. coli farnesyl diphosphate synthase (FPPS) inhibition. However, some potent FPPS inhibitors had no activity in cell growth inhibition, and based on the result of Catalyst pharmacophore modeling, this could be attributed to the requirement of a large hydrophobic feature for cellular activity (due most likely to transport). The activity of the most potent compound, N-[methyl(4-phenylbutyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonate (13), was strongly potentiated by the drug fosmidomycin. The transcription profiles for 13 or fosmidomycin alone were different from those found with carbenicillin or ciprofloxacin alone, but there were many similarities between the combination (13-fosmidomycin) and carbenicillin or ciprofloxacin, reflecting the more potent bactericidal activity of the drug combination on bacterial growth.

Impact of immunization technology and assay application on antibody performance--a systematic comparative evaluation.

Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost ("DNA immunization"; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.

Phosphonosulfonates are potent, selective inhibitors of dehydrosqualene synthase and staphyloxanthin biosynthesis in Staphylococcus aureus.

Staphylococcus aureus produces a golden carotenoid virulence factor called staphyloxanthin (STX), and we report here the inhibition of the enzyme, dehydrosqualene synthase (CrtM), responsible for the first committed step in STX biosynthesis. The most active compounds are halogen-substituted phosphonosulfonates, with K(i) values as low as 5 nM against the enzyme and IC(50) values for STX inhibition in S. aureus as low as 11 nM. There is, however, only a poor correlation (R(2) = 0.27) between enzyme and cell pIC(50) (= -log(10) IC(50)) values. The ability to predict cell from enzyme data improves considerably (to R(2) = 0.72) with addition of two more descriptors. We also investigated the activity of these compounds against human squalene synthase (SQS), as a counterscreen, finding several potent STX biosynthesis inhibitors with essentially no squalene synthase activity. These results open up the way to developing potent and selective inhibitors of an important virulence factor in S. aureus, a major human pathogen.

A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulence.

Staphylococcus aureus produces hospital- and community-acquired infections, with methicillin-resistant S. aureus posing a serious public health threat. The golden carotenoid pigment of S. aureus, staphyloxanthin, promotes resistance to reactive oxygen species and host neutrophil-based killing, and early enzymatic steps in staphyloxanthin production resemble those for cholesterol biosynthesis. We determined the crystal structures of S. aureus dehydrosqualene synthase (CrtM) at 1.58 angstrom resolution, finding structural similarity to human squalene synthase (SQS). We screened nine SQS inhibitors and determined the structures of three, bound to CrtM. One, previously tested for cholesterol-lowering activity in humans, blocked staphyloxanthin biosynthesis in vitro (median inhibitory concentration approximately 100 nM), resulting in colorless bacteria with increased susceptibility to killing by human blood and to innate immune clearance in a mouse infection model. This finding represents proof of principle for a virulence factor-based therapy against S. aureus.

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

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.

Enthalpy versus entropy-driven binding of bisphosphonates to farnesyl diphosphate synthase.

We report the results of an ITC (isothermal titration calorimetry) investigation of the binding of six bisphosphonates to the enzyme farnesyl diphosphate synthase (FPPS; EC 2.5.1.10) from Trypanosoma brucei. The bisphosphonates investigated were zoledronate, risedronate, ibandronate, pamidronate, 2-phenyl-1-hydroxyethane-1,1-bisphosphonate, and 1-(2,2-bisphosphonoethyl)-3-iodo pyridinium. At pH = 7.4, both risedronate and the phenylethane bisphosphonate bind in an enthalpy-driven manner (DeltaH approximately -9 to 10 kcal mol-1), but the other four bisphosphonates bind in an entropy-driven manner (DeltaS varying from 31.2 to 55.1 cal K-1 mol-1). However, at pH = 8.5, zoledronate binding switches from entropy to enthalpy-driven. The DeltaG results are highly correlated with FPPS inhibition results obtained using a radiochemical assay (R2 = 0.85, N = 11, P < 0.001). The DeltaH and DeltaS results are interpreted in terms of a model in which bisphosphonates with charged side chains have positive DeltaH values, due to the enthalpic cost of desolvation (due to strong ion-dipole interactions) and, likewise, a positive DeltaS, due to an increase in water entropy (both ligand and protein associated) on ligand binding to FPPS: the hydrophobic effect. For the neutral side chains (risedronate at pH 7.4, 8.5 and zoledronate at pH 8.5, as well as the phenylethane bisphosphonate), binding is overwhelmingly enthalpy-driven, with the enhanced activity of the basic side chain containing species being attributable to their becoming protonated in the active site. Given the large size of the bisphosphonate market and the potential importance of the development of these compounds for cancer immunotherapy and anti-parasitic chemotherapy, these results are of broad general interest in the context of the development of new, potent, and selective FPPS inhibitors.

A crystallographic investigation of phosphoantigen binding to isopentenyl pyrophosphate/dimethylallyl pyrophosphate isomerase.

We report the crystallographic structures of the potent phosphoantigens Phosphostim (the bromohydrin of isopentenyl pyrophosphate) and E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate bound to the mevalonate pathway enzyme isopentenyl pyrophosphate/dimethylallyl pyrophosphate isomerase (IPPI). Racemic Phosphostim forms covalent complexes with IPPI: a 4-thioether with C67 and a 4-ester with E116. Only the E116 ester forms with the chiral species, S-Phosphostim, with the w.t. enzyme, while the C67 thioether forms with a mutant Y104F IPPI. The potent phosphoantigen HMBPP also binds to IPPI, but is only a weak ( approximately 50 muM) inhibitor. These results strongly support an SN2 reaction for inhibition of IPPI by Phosphostim, in contrast to the SN1 or concerted type of reaction found with epoxide inhibitors, which react at C-3, and are of general interest in the context of the development of novel mevalonate pathway inhibitors. They also provide clues as to the nature of the binding site of synthetic phosphoantigens in gammadelta T cell activation. In particular, both bromohydrin and epoxy phosphoantigens are potent, irreversible inhibitors of IPPI while HMBPP is only a weak inhibitor, ruling out an IPPI or IPPI-like target for HMBPP in gammadelta T cell activation.

Crystallographic structures of two bisphosphonate:1-deoxyxylulose-5-phosphate reductoisomerase complexes.

We have obtained the single-crystal X-ray crystallographic structures of the bisphosphonates [(1-isoquinolinylamino)methylene]-1,1-bisphosphonate and [[(5-chloro-2-pyridinyl)amino]methylene]-1,1-bisphosphonate, bound to the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase (DXR, EC 1.1.1.267, also known as 2-C-methyl-d-erythritol-4-phosphate synthase), an important target for the development of antimalarial drugs. Our results indicate that both bisphosphonates bind into the fosmidomycin binding site. The aromatic groups are in a shallow hydrophobic pocket, and the phosphonate groups are involved in electrostatic interactions with Mg2+ or a cluster of carboxylic acid groups and lysine while the fosmidomycin phosphonate-binding site is occupied by a sulfate ion (as also observed in the DXR/NADP+ structure). The availability of these two new crystal structures opens up the possibility of the further development of bisphosphonates and related systems as DXR inhibitors and, potentially, as antiinfective agents.