New insights into human farnesyl pyrophosphate synthase inhibition by second-generation bisphosphonate drugs

Pamidronate, alendronate, APHBP and neridronate are a group of drugs, known as second-generation bisphosphonates (2G-BPs), commonly used in the treatment of bone-resorption disorders, and recently their use has been related to some collateral side effects. The therapeutic activity of 2G-BPs is related to the inhibition of the human Farnesyl Pyrophosphate Synthase (hFPPS). Available inhibitory activity values show that 2G-BPs act time-dependently, showing big differences in their initial inhibitory activities but similar final IC₅₀ values. However, there is a lack of information explaining this similar final inhibitory potency. Although different residues have been identified in the stabilization of the R₂ side chain of 2G-BPs into the active site, similar free binding energies were obtained that highlighted a similar stability of the ternary complexes, which in turns justified the similar IC₅₀ values reported. Free binding energy calculations also demonstrated that the union of 2G-BPs to the active site were 38 to 54 kcal mol^?1 energetically more favourable than the union of the natural substrate, which is the basis of the inhibition potency of the hFPPS activity.     

Molecular interactions of nitrogen-containing bisphosphonates within farnesyl diphosphate synthase

Bisphosphonates, known for their effectiveness in the treatment of osteoporosis, inhibit bone resorption via mechanisms that involve binding to bone mineral and cellular effects on osteoclasts. The major molecular target of nitrogen-containing bisphosphonates (N-BPs) in osteoclasts is farnesyl diphosphate synthase (FPPS). N-BPs likely inhibit this enzyme by mimicking one or more of the natural isoprenoid lipid substrates (GPP/DMAPP and IPP) but the mode of inhibition is not established. The active site of FPPS comprises a subsite for each substrate. Kinetic studies with recombinant human FPPS indicate that both potent (risedronate) and weak (NE-58051) enzyme inhibitors compete with GPP for binding to FPPS, however, binding to this site does not completely explain the difference in potency of the two inhibitors, suggesting that a second binding site may also be a target of bisphosphonate inhibition. Using the docking software suite Autodock, we explored a dual inhibitor binding mode for recombinant human FPPS. Experimental support for dual binding is suggested by Dixon plots for the inhibitors. N-BPs may inhibit by binding to both the GPP and a second site with differences in potency at least partly arising from inhibition at the second site.     

Investigation of the catalytic mechanism of farnesyl pyrophosphate synthase by computer simulation

Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways, farnesyl pyrophosphate, by the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP). Recently, FPPS has been shown to represent an important target for the treatment of parasitic diseases such as Chagas disease and African trypanosomiasis. Bisphosphonates, pyrophosphate analogues in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with different side chains, are able to inhibit the FPPS enzyme. Moreover, nitrogen-containing bisphosphonates have been proposed as carbocation transition state analogues of FPPS. On the basis of structural and kinetic data, different catalytic mechanisms have been proposed for FPPS. By analyzing different reaction coordinates we propose that the reaction occurs in one step through a carbocationic transition state and the subsequent transfer of a hydrogen atom from IPP to the pyrophosphate moiety of DMAPP. Moreover, we have analyzed the role of the active site amino acids on the activation barrier and the reaction mechanism. The structure of the active site is well conserved in the isoprenyl diphosphate synthase family; thus, our results are relevant for the understanding of this important class of enzymes and for the design of more potent and specific inhibitors for the treatment of parasitic diseases.     

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.     

Glycogen synthase kinase-3 inhibition by 3-anilino-4-phenylmaleimides: insights from 3D-QSAR and docking

Glycogen synthase kinase-3, a serine/threonine kinase, has been implicated in a wide variety of pathological conditions such as diabetes, Alzheimer’s disease, stroke, bipolar disorder, malaria and cancer. Herein we report 3D-QSAR analyses using CoMFA and CoMSIA and molecular docking studies on 3-anilino-4-phenylmaleimides as GSK-3α inhibitors, in order to better understand the mechanism of action and structure-activity relationship of these compounds. Comparison of the active site residues of GSK-3α and GSK-3β isoforms shows that all the key amino acids involved in polar interactions with the maleimides for the β isoform are the same in the α isoform, except that Asp133 in the β isoform is replaced by Glu196 in the α isoform. We prepared a homology model for GSK-3α, and showed that the change from Asp to Glu should not affect maleimide binding significantly. Docking studies revealed the binding poses of three subclasses of these ligands, namely anilino, N-methylanilino and indoline derivatives, within the active site of the β isoform, and helped to explain the difference in their inhibitory activity.     

Tyrosine 92 of aristolochene synthase directs cyclisation of farnesyl pyrophosphate

A mutant of Aristolochene Synthase (AS), in which Tyr 92 was replaced by Val, produced the alicyclic beta-(E)-farnesene as the major product, indicating that cyclisation of FPP is controlled by Tyr 92 in AS.     

Photoaffinity analogues of farnesyl pyrophosphate transferable by protein farnesyl transferase

Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized analogues (3-6) of farnesyl pyrophosphate (FPP) to probe the range of modifications possible to the FPP skeleton which allow for efficient transfer by FTase. Photoaffinity analogues of FPP (5, 6) were prepared by substituting perfluorophenyl azide functional groups for the omega-terminal isoprene of FPP. Substituted anilines replace the omega-terminal isoprene in analogues 3 and 4. Compounds 3-5 were prepared by reductive amination of the appropriate anilines with 8-oxo-geranyl acetate, followed by ester hydrolysis, chlorination, and pyrophosphorylation. Additional substitution of three methylenes for the beta-isoprene of FPP gave photoprobe 6 in nine steps. Preparation of the analogues required TiCl(4)-mediated imine formation prior to NaBH(OAc)(3) reduction for anilines with a pK(a) < 1. The azide moiety was not affected by Ph(3)PCl(2) conversion of allylic alcohols 13-16 into corresponding chlorides 17-20. Analogues 3-6 are efficiently transferred to target N-dansyl-GCVLS peptide substrate by mammalian FTase. Comparison of analogue structures and kinetics of transfer to those of FPP reveals that ring fluorination and para substituents have little effect on the affinity of the analogue pyrophosphate for FTase and its transfer efficiency. These results are also supported with models of the analogue binding modes in the active site of FTase. The transferable azide photoprobe 5 photoinactivates FTase. Transferable analogues 5 and 6 allow the formation of appropriately posttranslationally modified photoreactive peptide probes of isoprene function.     

Competitive inhibition of aristolochene synthase by phenyl-substituted farnesyl diphosphates: evidence of active site plasticity

Analogues of farnesyl diphosphate (FPP, ) containing phenyl substituents in place of methyl groups have been prepared in syntheses that feature use of a Suzuki-Miyaura reaction as a key step. These analogues were found not to act as substrates of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti (AS). However, they were potent competitive inhibitors of AS with K(I)-values ranging from 0.8 to 1.2 microM. These results indicate that the diphosphate group contributes the largest part to the binding of the substrate to AS and that the active sites of terpene synthases are sufficiently flexible to accommodate even substrate analogues with large substituents suggesting a potential way for the generation of non-natural terpenoids. Molecular mechanics simulations of the enzyme bound inhibitors suggested that small changes in orientations of active site residues and subtle alterations of the conformation of the backbones of the inhibitors are sufficient to accommodate the phenyl-farnesyl-diphosphates.     

Design and synthesis of a transferable farnesyl pyrophosphate analogue to Ras by protein farnesyltransferase

The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its membrane localization and is required for both its biological activity and ability to induce malignant transformation. We describe the design and synthesis of a farnesyl pyrophosphate (FPP) analogue, 8-anilinogeranyl pyrophosphate 3 (AGPP), in which the omega-terminal isoprene unit of the farnesyl group has been replaced with an aniline functionality. The key steps in the synthesis are the reductive amination of the alpha,beta-unsaturated aldehyde 5 to form the lipid analogue 6, and the subsequent conversion of the allylic alcohol 7 to the chloride 8 via Ph(3)PCl(2) followed by displacement with [(n-Bu)(4)N](3)HP(2)O(7) to give AGPP (3). AGPP is a substrate for protein farnesyltransferase (FTase) and is transferred to Ras by FTase with the same kinetics as the natural substrate, FPP. AGPP is highly selective, showing little inhibitory activity against either geranylgeranyl-protein transferase type I (GGTase I) (K(i) = 0.06 microM, IC(50) = 20 microM) or squalene synthase (IC(50) = 1000 microM). AGPP is the first efficiently transferable analogue of FPP to be modified at the omega-terminus that provides a platform from which additional analogues can be made to probe the biological function of protein farnesylation. AGPP is the first example of a class of compounds that are alternate substrates for protein isoprenylation that are not inhibitors of squalene synthase.     

Synthesis of vinyl pyrophosphonate analogues of farnesyl pyrophosphate: New potential inhibitors of farnesyl protein transferase

The synthesis of new bioisosteric analogues of farnesyl pyrophosphate where a vinyl pyrophosphonate replaced the pyrophosphate moiety is described. These compounds have been prepared using a Horner–Wadsworth–Emmons procedure and a modified Michelson reaction. They have been evaluated for the inhibition of farnesyl protein transferase. © 2002 Wiley Periodicals, Inc. Heteroatom Chem 13:654–661, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.10081     

含氟农药的比较分子场分析研究

用比较分子场分析(CoMFA)方法对112种含氟农药分子的生物活性及毒性同时进行了定量构效关系研究。用78个化合物作为训练集,以距离比较方法(DISCO)确认的药效团为叠合规则构建CoMFA模型,发现影响活性的立体场与静电场的贡献分别为60.4%和39.6%,影响毒性的立体场与静电场的贡献分别为59.2%和40.8%。药效模型与毒效模型在交叉验证时的相关系数平方(R^2)分别为0.652和0.611,非交叉验证的R^2分别为0.982和0.977,方差比F(8,69)值分别为463.6及362.9,活性和毒性的标准偏差-极差比s/△γ值分别为3.6%和2.9%,表明模型具有较好的自预测能力。对测试组34个化合物进行了活性和毒性的预测,活性与毒性预测的标准偏差-极差比s/△γ值分别为10.4%和6.4%。最后,还建立了一个由97个化合物构建的扩大的模型,各种统计量得到了进一步提高。并预计了一个活性较高且毒性很低的新化合物。     

咪唑啉酮类除草剂的三维构效关系研究

从三维角度出发,采取不同的构象搜索方法得到了咪唑酮类化合物分子的活性构象。利用比较分子场分析方法进一步证实了模板分子构象的正确性。并从静电场、立体场及活性关系等方面进行了三维定量构效关系研究,得到了具有较强预测能力的QSAR模型。     

3D-QSAR study of hallucinogenic phenylalkylamines by using CoMFA approach

The three-dimensional quantitative structure–activity relationship (3D-QSAR) has been studied on 90 hallucinogenic phenylalkylamines by the comparative molecular field analysis (CoMFA). Two conformations were compared during the modeling. Conformation I referred to the amino group close to ring position 6 and conformation II related to the amino group trans to the phenyl ring. Satisfactory results were obtained by using both conformations. There were still differences between the two models. The model based on conformation I got better statistical results than the one about conformation II. And this may suggest that conformation I be preponderant when the hallucinogenic phenylalkylamines interact with the receptor. To further confirm the predictive capability of the CoMFA model, 18 compounds with conformation I were randomly selected as a test set and the remaining ones as training set. The best CoMFA model based on the training set had a cross-validation coefficient q ² of 0.549 at five components and non cross-validation coefficient R ² of 0.835, the standard error of estimation was 0.219. The model showed good predictive ability in the external test with a coefficient R _pre² of 0.611. The CoMFA coefficient contour maps suggested that both steric and electrostatic interactions play an important role. The contributions from the steric and electrostatic fields were 0.450 and 0.550, respectively.     

Alpha-phosphono lactone analogues of farnesyl pyrophosphate: an asymmetric synthesis via ring-closing metathesis

An alpha-phosphono lactone derivative of farnesol has been prepared, in both racemic and nonracemic forms, to provide a new type of farnesyl pyrophosphate analogue. Attempted preparation of the racemic alpha-phosphono lactone through rearrangement of a vinyl phosphate derived from the parent lactone resulted in both rearrangement and lactone ring opening, revealing that the farnesyl lactone was not stable to the excess of strong base required for the rearrangement. A procedure for C-P bond formation based on generation of the lactone enolate, reaction with a P(III) reagent, and oxidation was successful in providing the racemic alpha-phosphono lactone, in part, because only 1 equiv of strong base was required. The same strategy for phosphonate synthesis then was applied to the nonracemic farnesyl lactone, prepared through a sequence including allylation of farnesal with a nonracemic borane reagent, reaction of the product alcohol with acryloyl chloride, and formation of an unsaturated lactone through ring-closing metathesis. A similar strategy gave the corresponding racemic alpha-phosphono lactam through a six-step sequence from farnesal.     

Probing the structures of leishmanial farnesyl pyrophosphate synthases: homology modeling and docking studies

Leishmania donovani and Leishmania major farnesyl pyrophosphate synthase ( LdFPPS and LmFPPS) are potential targets for the development of antileishmanial therapy. The protein sequence for LdFPPS was recently elucidated in our laboratory. Highly refined homology models were generated using the protein sequences of LdFPPS and the closely related LmFPPS enzyme. A ligand-refined model of LmFPPS with a bound bisphosphonate ligand was generated using restraint-guided molecular mechanics followed by quantum mechanics/molecular mechanics refinement. The ligand-refined model of LmFPPS was further validated through extensive pose validation, enrichment, and other docking studies involving known bisphosphonate inhibitors. The model was able to explain the critical binding site interactions and site-directed mutagenesis data obtained from experimental studies on related FPPS enzymes. The ligand-refined model in conjunction with the validated docking protocol could be utilized in the future for structure-based virtual screening and rational drug design studies against these targets.     

Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene-producing plants, Scoparia dulcis and Croton sublyratus, have been isolated using the homology-based polymerase chain reaction (PCR) method. Both clones contained highly conserved aspartate-rich motifs (DDXX(XX)D) and their N-terminal residues exhibited the characteristics of chloroplast targeting sequence. When expressed in Escherichia coli, both the full-length and truncated proteins in which the putative targeting sequence was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to produce geranylgeranyl diphosphate (GGPP). The structural factors determining the product length in plant GGPPSs were investigated by constructing S. dulcis GGPPS mutants on the basis of sequence comparison with the first aspartate-rich motif (FARM) of plant farnesyl diphosphate synthase. The result indicated that in plant GGPPSs small amino acids, Met and Ser, at the fourth and fifth positions before FARM and Pro and Cys insertion in FARM play essential roles in determination of product length. Further, when a chimeric gene comprised of the putative transit peptide of the S. dulcis GGPPS gene and a green fluorescent protein was introduced into Arabidopsis leaves by particle gun bombardment, the chimeric protein was localized in chloroplasts, indicating that the cloned S. dulcis GGPPS is a chloroplast protein.     

Biosynthesis of isoprenoids in Escherichia coli: stereochemistry of the reaction catalyzed by farnesyl diphosphate synthase

[formula: see text] Farnesyl diphosphate (FPP) synthase from Escherichia coli catalyzes the condensation of isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP) with selective removal of the pro-R hydrogen at C2 of IPP, the same stereochemistry observed for the pig liver, yeast, and avian enzymes.     

Slow-onset feedback inhibition: inhibition of Mycobacterium tuberculosis alpha-isopropylmalate synthase by L-leucine

This report describes the first demonstration of slow-onset feedback inhibition of an enzyme that catalyzes the first committed step in a biosynthetic pathway. alpha-Isopropylmalate synthase (IPMS) catalyzes the first committed step of the l-leucine biosynthetic pathway and is feedback-inhibited by l-leucine. Initial velocity experiments on the Mycobacterium tuberculosis IPMS indicate that inhibition by l-leucine is linearly noncompetitive versus alpha-ketoisovalerate. Time-courses displayed a burst of product formation followed by a linear steady-state rate when reactions were initiated by the addition of enzyme. The burst rate showed a hyperbolic dependence on the concentration of l-leucine indicating that inhibition proceeds in two steps, an initial rapid binding step followed by slow isomerization to a more tightly bound complex.     

Synthesis and application of a fluorescent substrate analogue to study ligand interactions for undecaprenyl pyrophosphate synthase

Farnesyl pyrophosphate (FPP) serves as a common substrate for many prenyltransferases involved in the biosynthesis of isoprenoid compounds. Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the chain elongation of FPP to C(55) undecaprenyl pyrophosphate (UPP) which acts as a lipid carrier in bacterial peptidoglycan synthesis. In this study, 7-(2,6-dimethyl-8-diphospho-2,6-octadienyloxy)-8-methyl-4-trifluoromethyl-chromen-2-one geranyl pyrophosphate, a fluorescent analogue of FPP, was prepared and utilized to study ligand interactions with E. coli UPPs. This compound displays an absorbance maximum at 336 nm and emission maximum at 460 nm without interference from protein autofluorescence. It is a competitive inhibitor with respect to FPP (K(i) = 0.57 microM) and also serves as an alternative substrate (K(m) = 0.69 microM and k(cat) = 0.02 s(-)(1)), but mainly reacts with one isopentenyl pyrophosphate (IPP) probably due to unfavorable product translocation. Fluorescence intensity of this compound is reduced when bound to the enzyme (1:1 stoichiometry), and is recovered by FPP replacement. Using stopped-flow apparatus, the interaction of enzyme with the compound was measured (k(on) = 55.3 microM(-)(1) s(-)(1) and k(off) = 31.6 s(-)(1)). The product dissociation rate constant (0.5 s(-)(1)) determined from the competition experiments is consistent with our previous prediction from kinetic simulation. Unlike several other prenyltransferase reactions in which FPP dissociates slowly, UPPs binds FPP in a rapid equilibrium manner with a fast release rate constant of 30 s(-)(1). The fluorescent analogue of FPP presented here may provide a tool to investigate the ligand interactions for a broad class of FPP-binding proteins.