Diterpene cyclases and the nature of the isoprene fold.

The structures and mechanism of action of many terpene cyclases are known, but no structures of diterpene cyclases have yet been reported. Here, we propose structural models based on bioinformatics, site-directed mutagenesis, domain swapping, enzyme inhibition, and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain approximately 20 alpha-helices and the same conserved "QW" and DxDD motifs as in triterpene cyclases, indicating the presence of a betagamma barrel structure. Plant diterpene cyclases have a similar catalytic motif and betagamma-domain structure together with a third, alpha-domain, forming an alphabetagamma structure, and in H(+)-initiated cyclases, there is an EDxxD-like Mg(2+)/diphosphate binding motif located in the gamma-domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (betagamma) bacterial triterpene cyclases to (betagamma) bacterial and thence to (alphabetagamma) plant diterpene cyclases.

Identifying unexpected therapeutic targets via chemical-protein interactome.

Drug medications inevitably affect not only their intended protein targets but also other proteins as well. In this study we examined the hypothesis that drugs that share the same therapeutic effect also share a common therapeutic mechanism by targeting not only known drug targets, but also by interacting unexpectedly on the same cryptic targets. By constructing and mining an Alzheimer's disease (AD) drug-oriented chemical-protein interactome (CPI) using a matrix of 10 drug molecules known to treat AD towards 401 human protein pockets, we found that such cryptic targets exist. We recovered from CPI the only validated therapeutic target of AD, acetylcholinesterase (ACHE), and highlighted several other putative targets. For example, we discovered that estrogen receptor (ER) and histone deacetylase (HDAC), which have recently been identified as two new therapeutic targets of AD, might already have been targeted by the marketed AD drugs. We further established that the CPI profile of a drug can reflect its interacting character towards multi-protein sets, and that drugs with the same therapeutic attribute will share a similar interacting profile. These findings indicate that the CPI could represent the landscape of chemical-protein interactions and uncover "behind-the-scenes" aspects of the therapeutic mechanisms of existing drugs, providing testable hypotheses of the key nodes for network pharmacology or brand new drug targets for one-target pharmacology paradigm.

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.

Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates: a crystallographic and computational investigation.

We report the X-ray structures of several bisphosphonate inhibitors of geranylgeranyl diphosphate synthase, a target for anticancer drugs. Bisphosphonates containing unbranched side chains bind to either the farnesyl diphosphate (FPP) substrate site, the geranylgeranyl diphosphate (GGPP) product site, and in one case, both sites, with the bisphosphonate moiety interacting with 3 Mg (2+) that occupy the same position as found in FPP synthase. However, each of three "V-shaped" bisphosphonates bind to both the FPP and GGPP sites. Using the Glide program, we reproduced the binding modes of 10 bisphosphonates with an rms error of 1.3 A. Activities of the bisphosphonates in GGPPS inhibition were predicted with an overall error of 2x by using a comparative molecular similarity analysis based on a docked-structure alignment. These results show that some GGPPS inhibitors can occupy both substrate and product site and that binding modes as well as activity can be accurately predicted, facilitating the further development of GGPPS inhibitors as anticancer agents.

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.

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.

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.

Solid-state NMR, crystallographic, and computational investigation of bisphosphonates and farnesyl diphosphate synthase-bisphosphonate complexes.

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.

Inhibition of Trypanosoma cruzi hexokinase by bisphosphonates.

Hexokinase is the first enzyme involved in glycolysis in most organisms, including the etiological agents of Chagas disease (Trypanosoma cruzi) and African sleeping sickness (Trypanosoma brucei). The T. cruzi enzyme is unusual since, unlike the human enzyme, it is inhibited by inorganic diphosphate (PPi). Here, we show that non-hydrolyzable analogues of PPi, bisphosphonates, are potent inhibitors of T. cruzi hexokinase (TcHK). We determined the activity of 42 bisphosphonates against TcHK, and the IC(50) values were used to construct pharmacophore and comparative molecular similarity indices analysis (CoMSIA) models for enzyme inhibition. Both models revealed the importance of electrostatic, hydrophobic, and steric interactions, and the IC(50) values for 17 active compounds were predicted with an average error of 2.4x by using the CoMSIA models. The compound most active against T. cruzi hexokinase was found to have a 2.2 microM IC(50) versus the clinically relevant intracellular amastigote form of T. cruzi, but only a approximately 1-2 mM IC(50) versus Dictyostelium discoideum and a human cell line, indicating selective activity versus T. cruzi.