Novel bisphosphonate inhibitors of the human farnesyl pyrophosphate synthase

A structure-based approach was pursued in designing novel bisphosphonate inhibitors of the human farnesyl pyrophosphate synthase (hFPPS). Preliminary SAR and structural evidence for the simultaneous binding of these inhibitors into the isopentenyl pyrophosphate (IPP) and the geranyl pyrophosphate (GPP) substrate sub-pockets of the enzyme are presented.     

Bisphosphonates are potent inhibitors of Trypanosoma cruzi farnesyl pyrophosphate synthase.

We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase of Trypanosoma cruzi. The protein (T. cruzi farnesyl pyrophosphate synthase, TcFPPS) is an attractive target for drug development, since the growth of T. cruzi is inhibited by carbocation transition state/reactive intermediate analogs of its substrates, the nitrogen-containing bisphosphonates currently in use in bone resorption therapy. The protein predicted from the nucleotide sequence of the gene has 362 amino acids and a molecular mass of 41.2 kDa. Several sequence motifs found in other FPPSs are present in TcFPPS. Heterologous expression of TcFPPS in Escherichia coli produced a functional enzyme that was inhibited by the nitrogen-containing bisphosphonates alendronate, pamidronate, homorisedronate, and risedronate but was less sensitive to the non-nitrogen-containing bisphosphonate etidronate, which, unlike the nitrogen-containing bisphosphonates, does not affect parasite growth. The protein contains a unique 11-mer insertion located near the active site, together with other sequence differences that may facilitate the development of novel anti-Chagasic agents.     

Bisphosphonates derived from fatty acids are potent inhibitors of Trypanosoma cruzi farnesyl pyrophosphate synthase

Studies on the mode of action of a series of bisphosphonates derived from fatty acids, which had previously proved to be potent inhibitors against Trypanosoma cruzi proliferation in in vitro assays, have been performed. Some of these drugs proved to be potent inhibitors against the intracellular form of the parasite, exhibiting IC(50) values at the low micromolar level. As bisphosphonates are FDA clinically approved for treatment of bone resorption disorders, their potential innocuousness makes them good candidates to control tropical diseases.     

Evidence for differential folding of farnesyl pyrophosphate in the active site of aristolochene synthase: a single-point mutation converts aristolochene synthase into an (E)-beta-farnesene synthase

Sesquiterpene cyclases, many of which share significant structural similarity, catalyze the cyclization reactions of the universal alicyclic precursor farnesyl pyrophosphate to produce more than 300 different hydrocarbon skeletons with high regio- and stereospecificity. The molecular basis of this exquisite specificity is not well-understood, but the conformation adopted by FPP in the active site of a sesquiterpene cyclase is thought to be an important determinant of the reaction pathway. Aristolochene synthase (AS) from Penicillium roqueforti catalyzes the cyclization of farnesyl pyrophosphate to the bicyclic sesquiterpene aristolochene. The X-ray structure of AS suggested that the steric bulk of residue 92 was central in binding of FPP to the active site of AS in a quasi-cyclic conformation, thereby facilitating attack of C1 by the C10-C11 double bond to produce the cis-fused Decalin S-germacrene A. We demonstrate here that reduction of the size of the side chain of residue 92 leads to the production of the alicyclic sesquiterpenes (E)-beta- and (E,E)-alpha-farnesene. The relative amounts of linear products formed depended linearly on the size of the residues at position 92. ASY92A, in which Tyr92 had been replaced with Ala, produced almost 80% of alicyclic sesquiterpenes, suggesting an energetic separation of less than 0.8 kcal/mol between the cyclic and noncyclic reaction pathways. A mechanism by which FPP binds to the mutant enzymes in an extended conformation is proposed to explain the altered selectivity. The mutants also produced small amounts of additional hydrocarbons with a molecular weight of 204, namely, alpha-selinene, beta-selinene, selina-4,11-diene, (E,Z)-alpha-farnesene, and beta-bisabolene. The production of (E)-beta-farnesene and beta-bisabolene suggested that the initial cyclization of FPP to germacrene A in AS proceeded in a stepwise fashion through farnesyl cation.     

Transcriptional regulation of farnesyl pyrophosphate synthase by liver X receptors

Liver X receptors (LXRs) are members of the nuclear receptor superfamily that are involved in cholesterol and lipid metabolism. In addition to liver, the brain is another site where LXRs may control cholesterol homeostasis. In the brain, the regulation of cholesterol homeostasis is independent from other parts of the body, and its disturbance is associated with neurodegenerative disorders, such as Alzheimer's disease. We have used PCR-based suppressive subtractive cloning to identify new LXR target genes in brain cells. In this report, we show that farnesyl pyrophosphate synthase (FPPS) is a new target gene for LXR in astrocytes and neurons. Farnesyl pyrophosphate is an obligate intermediate for de novo cholesterol synthesis and a substrate for protein farnesylation. Stimulation of FPPS mRNA synthesis by an LXR agonist, Hypocholamide, was observed in several cell lines from the central nervous system. We identified a single putative direct repeat 4 (DR4) LXR response element in the FPPS promoter. In a reporter gene assay, LXR transactivated a reporter gene bearing a truncated FPPS promoter containing this DR4 cis-element but not if the DR4 element was mutated. Using gel-mobility shift assay, we further demonstrated the direct interaction between the LXR/retinoid X receptor (RXR) heterodimer and the response element. Taken together, our results indicate that LXRs directly regulate FPPS gene expression, and thus may play a role in modulating cholesterol synthesis in the brain.     

Purification and characterization of farnesyl pyrophosphate synthase from Capsicum annuum

Farnesyl pyrophosphate synthase (FPP) displaying dimethylallyl transferase activity (EC 2.5.1.1) and geranyl transferase activity (EC 2.5.1.10) was purified from Capsicum fruits. This prenyltransferase has a molecular mass of 89,000 +/- 5000 Da resulting from the association of two apparently identical subunits having a molecular mass of 43,000 +/- 2000 Da. Antibodies raised against Capsicum FPP synthase selectively blocked the transferase activity. Analysis of the immunological relationships between FPP synthase and geranylgeranyl pyrophosphate synthase (EC 2.5.1.1, EC 2.5.1.10 and EC 2.5.1.30) revealed that these two enzymes though performing the same mechanism of catalysis and accepting identical substrates have different antigenic determinants. Thus, in connection to previous work, this immunological study suggests that Capsicum FPP is strictly located in the extraplastidial compartment.     

Design of more potent squalene synthase inhibitors with multiple activities

With the increasing realization that modulating a multiplicity of targets can be an asset in the treatment of multifactorial disorders, we hereby report the synthesis and evaluation of the first compounds in which antioxidant, anti-inflammatory as well as squalene synthase (SQS) inhibitory activities are combined by design, in a series of simple molecules, extending their potential range of activities against the multifactorial disease of atherosclerosis. The activity of the initially synthesized antihyperlipidemic morpholine derivatives (1-6), in which we combined several pharmacophore moieties, was evaluated in vitro (antioxidant, inhibition of SQS and lipoxygenase) and in vivo (anti-dyslipidemic and anti-inflammatory effect). We further compared the in vitro SQS inhibitory action of these derivatives with theoretically derived molecular interactions by performing an in silico docking study using the X-ray crystal structure of human SQS. Based on low energy preferred binding modes, we designed potentially more potent SQS ligands. We proceeded with synthesizing and evaluating these new structures (7-12) in vitro and in vivo, to show that the new derivatives were significantly more active than formerly developed congeners, both as SQS inhibitors (20-70-fold increase in activity) and antioxidants (4-30-fold increase in activity). A significant correlation between experimental activity [Log(1/IC(50))] and the corresponding binding free energy (ΔG(b)) of the docked compounds was shown. These results, taken together, show a promising alternative and novel approach for the design and development of multifunctional antiatherosclerosis agents.     

Cooperative inhibition of human thymidylate synthase by mixtures of active site binding and allosteric inhibitors

Thymidylate synthase (TS) is a target in the chemotherapy of colorectal cancer and some other neoplasms. It catalyzes the transfer of a methyl group from methylenetetrahydrofolate to dUMP to form dTMP. On the basis of structural considerations, we have introduced 1,3-propanediphosphonic acid (PDPA) as an allosteric inhibitor of human TS (hTS); it is proposed that PDPA acts by stabilizing an inactive conformer of loop 181-197. Kinetic studies showed that PDPA is a mixed (noncompetitive) inhibitor versus dUMP. In contrast, versus methylenetrahydrofolate at concentrations lower than 0.25 microM, PDPA is an uncompetitive inhibitor, while at PDPA concentrations higher than 1 microM the inhibiton is noncompetive, as expected. At the concentrations corresponding to uncompetitive inhibition, PDPA shows positive cooperativity with an antifolate inhibitor, ZD9331, which binds to the active conformer. PDPA binding leads to the formation of hTS tetramers, but not higher oligomers. These data are consistent with a model in which hTS exists preferably as an asymmetric dimer with one subunit in the active conformation of loop 181-197 and the other in the inactive conformation.     

Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue

The prenyl transferase undecaprenyl pyrophosphate synthetase was partially purified from the cytosolic fraction of Escherichia coli. Its enzymic products were characterized as a family of cis-polyprenyl phosphates, which ranged in carbon number from C55 to C25. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [3H]2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate, was shown to label Lactobacillus plantarum and E. coli undecaprenyl pyrophosphate synthetase on UV irradiation in the presence of isopentenyl pyrophosphate and divalent cation. The only labeled polypeptide migrated on electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel at a molecular weight of approximately 30,000. No protein was radiolabeled when the natural substrate, t,t-farnesyl pyrophosphate was included in the irradiation mixture. Irradiation in the presence of MgCl2 without isopentenyl pyrophosphate gave less labeling of the polypeptide. Irradiation with only isopentenyl pyrophosphate gave little labeling of the polypeptide. When the enzyme was irradiated with 3H-photoprobe, [14C]isopentenyl pyrophosphate, and MgCl2, the labeled polypeptide gave a ratio of 14C/3H that indicated the product must also bind to the enzyme on irradiation. These results demonstrate the ability to radiolabel the allylic pyrophosphate binding site and possibly product binding site of undecaprenyl pyrophosphate synthetase by a process which is favored when both cosubstrate and divalent cation are present.     

Probing the active site of rat porphobilinogen synthase using newly developed inhibitors

The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Porphobilinogen synthase catalyzes a rate-limiting step for the biosyntheses of tetrapyrrolic natural products. In the present study, a variety of new substrate analogs and reaction intermediate analogs were synthesized, which were used as probes for studying the active site of rat porphobilinogen synthase. The compounds 1, 3, 6, 9, 14, 16, and 28 were found to be competitive inhibitors of rat porphobilinogen synthase with inhibition constants ranging from 0.96 to 73.04 mM. Compounds 7, 10, 12, 13, 15, 17, 18, and 26 were found to be irreversible enzyme inhibitors. For irreversible inhibitors, loose-binding inhibitors were found to give stronger inactivation. The amino group and carboxyl group of the analogs were found to be important for their binding to the enzyme. This study increased our understanding of the active site of porphobilinogen synthase.     

Dispersed family of human genes with sequence similarity to farnesyl pyrophosphate synthetase

Prenyltransferases are a group of enzymes involved in the biosynthesis of both sterol and nonsterol isoprene compounds. Somatic cell hybrid studies and in situ hybridization show that the human genome contains five distinct loci that hybridize to the cDNA for the enzyme farnesyl pyrophosphate synthetase (FPS), a prenyltransferase that catalyzes the synthesis of an intermediate common to both the sterol and the nonsterol branches of the isoprene biosynthetic pathway. The loci identified in this report may correspond to unique prenyltransferase genes related to FPS or to pseudogenes. The loci mapped have been identified as farnesyl pyrophosphate synthetase-"like"-1 (FPSL-1) on chromosome 1q24-31, FPSL-2 on chromosome 7, FPSL-3 on chromosome 14, FPSL-4 on chromosome 15q14-q21, and FPSL-5 on chromosome Xq21-22. Multiple copies of sequences similar to those of FPS are also present in both the mouse and the rat.     

Structures of a potent phenylalkyl bisphosphonate inhibitor bound to farnesyl and geranylgeranyl diphosphate synthases

We report the X-ray crystallographic structures of the bisphosphonate N-[methyl(4-phenylbutyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonate (BPH-210), a potent analog of pamidronate (Aredia), bound to farnesyl diphosphate synthase (FPPS) from Trypanosoma brucei as well as to geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae. BPH-210 binds to FPPS, together with 3 Mg(2+), with its long, hydrophobic phenylbutyl side-chain being located in the same binding pocket that is occupied by allylic diphosphates and other bisphosphonates. Binding is overwhelmingly entropy driven, as determined by isothermal titration calorimetry. The structure is of interest since it explains the lack of potency of longer chain analogs against FPPS, since these would be expected to have a steric clash with an aromatic ring at the distal end of the binding site. Unlike shorter chain FPPS inhibitors, such as pamidronate, BPH-210 is also found to be a potent inhibitor of human geranylgeranyl diphosphate synthase. In this case, the bisphosphonate binds only to the GGPP product inhibitory site, with only 1 (chain A) or 0 (chain B) Mg(2+), and DeltaS is much smaller and DeltaH is approximately 6 k cal more negative than in the case of FPPS binding. Overall, these results are of general interest since they show that some bisphosphonates can bind to more than one trans-prenyl synthase enzyme which, in some cases, can be expected to enhance their overall activity in vitro and in vivo.     

Intersubunit location of the active site of farnesyl diphosphate synthase: reconstruction of active enzymes by hybrid-type heteromeric dimers of site-directed mutants

Farnesyl diphosphate synthase is a homodimer of subunits having typically two aspartate-rich motifs with two sets of substrate binding sites for an allylic diphosphate and isopentenyl diphosphate per molecule of a homodimeric enzyme. To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interaction, we constructed several expression plasmids that overproduce hybrid-type heterodimers of Bacillus stearothermophilus FPP synthases constituting different types of mutated monomers, which exhibit little catalytic activity as homodimers, by combining two tandem fps genes for the manipulated monomer subunit with a highly efficient promoter trc within an overexpression pTrc99A plasmid. A heterodimer of a combination of subunits of the wild type and of R98E, a mutant subunit which exhibits little enzymatic activity as a dimer form (R98E)(2), exhibited 78% of the activity of the wild-type homodimer enzyme, (WT)(2). Moreover, when a hybrid-type heterodimeric dimer of FPP synthase mutant subunits (R98E/F220A) was prepared, the FPP synthase activity was 18- and 390-fold of that of each of the almost inactive mutants as a dimeric enzymes, (R98E)(2) and (F220A)(2) [Koyama, T., et al. (1995) Biochem. Biophys. Res. Commun. 212, 681-686], respectively. These results suggest that the subunits of the FPP synthase interact with each other to form a shared active site in the homodimer structure rather than an independent active site in each subunit.     

Probing the active site of Pseudomonas aeruginosa porphobilinogen synthase using newly developed inhibitors

Porphobilinogen synthase catalyzes the first committed step of the tetrapyrrole biosynthesis pathway. In an aldol-like condensation, two molecules of 5-aminolevulinic acid (ALA) form the first pyrrole, porphobilinogen. Newly synthesized analogues of a reaction intermediate of porphobilinogen synthase have been employed in studying the active site and the catalytic mechanism of this early enzyme of tetrapyrrole biosynthesis. This study combines structural and kinetic evaluation of the inhibition potency of these inhibitors. In addition, one of the determined protein structures provides for the first time structural evidence of a magnesium ion in the active site. From these results, we can corroborate an earlier postulated enzymatic mechanism that starts with formation of a C-C bond, linking C3 of the A-side ALA to C4 of the P-side ALA through an aldole addition. The obtained data are discussed with respect to the current literature.     

Design, synthesis and evaluation of potent thymidylate synthase X inhibitors

Three synthesized series of compounds based on a thiazolidine core allowed identification of potent inhibitors of thymidylate synthase X. The evaluation of the catalytic activity of the enzyme in the presence of these molecules revealed two distinct classes of compounds that inhibit ThyX with submicromolar concentrations, which could lead, after optimization, to effective inhibitors with potential biomedical interest.     

Analysis of interactions between potent inhibitors of ATP sulfurylase via molecular dynamics

The dynamic behaviour of adenosine triphosphate sulfurylase (ATPs) is analysed to investigate its energetic interactions with inhibitors recently studied theoretically and tested experimentally. The interactions between ATPs and three inhibitors namely, 2,2′-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), butylated hydroxytoluene (BHT), and 3-tert-butyl-4-hydroxyanisole (BHA) in aqueous solution were studied via molecular dynamics simulations. The results of the absolute and relative free energies reveal the existence of synergism in the system. Additionally, the system demonstrates strong steric effect between ABTS and BHA that is not only due to the size of these inhibitors but also their surroundings.     

Imidazolinones: potent inhibitors of acetohydroxyacid synthase

The imidazolinones, a new chemical class of herbicides, were shown to be uncompetitive inhibitors of acetohydroxyacid synthase from corn. This is the first common enzyme in the biosynthetic pathway for valine, leucine, and isoleucine. The K_i for the imidazolinones tested ranged from 2 to 12 micromolar. These results may explain the mechanism of action of these new herbicides.     

Substituted piperidines--highly potent renin inhibitors due to induced fit adaptation of the active site.

The identification, synthesis and activity of a novel class of piperidine renin inhibitors is presented. The most active compounds show activities in the picomolar range and are among the most potent renin inhibitors ever identified.     

Trihalobenzocycloheptapyridine analogues of Sch 66336 as potent inhibitors of farnesyl protein transferase

SCH 66336 is a trihalo tricyclic compound that is currently undergoing Phase II clinical trials for the treatment of solid tumors. Modifications of SCH 66336 by incorporating such groups as amides, acids, esters, ureas and lactams off the first or the distal piperidine (from the tricycle) provided potent FPT inhibitors some of which exhibited good cellular activity. A number of these compounds incorporate properties that might improve pharmacokinetic stability of these inhibitors by virtue of their increased solubility or by their change in log P.