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.     

Bisphosphonates derived from fatty acids are potent growth inhibitors of Trypanosoma cruzi

We have investigated the effect of a series of bisphosphonates derived from fatty acids against Trypanosoma cruzi proliferation in in vitro assays. Some of these drugs proved to be potent inhibitors against the intracellular form of the parasite exhibiting IC50 values at the low micromolar level. As bisphosphonates are FDA clinically approved for treatment of bone resorption, their potential innocuousness makes them good candidates to control tropical diseases.     

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.     

Synthesis and biological evaluation of 1-amino-1,1-bisphosphonates derived from fatty acids against Trypanosoma cruzi targeting farnesyl pyrophosphate synthase

We have investigated the effect of a series of 1-amino-1,1-bisphosphonates derived from fatty acids against proliferation of the clinically more relevant form of Trypanosoma cruzi, the causative agent of American trypanosomiasis (Chagas' disease). Some of these drugs were potent inhibitors against the intracellular form of the parasite, exhibiting IC50 values at low micromolar level. Cellular activity was associated with the inhibition of enzymatic activity of T. cruzi farnesyl pyrophosphate synthase. As bisphosphonate-containing drugs are FDA-approved for the treatment of bone resorption disorders, their potential innocuousness makes them good candidates to control tropical diseases.     

Fluorine-containing aryloxyethyl thiocyanate derivatives are potent inhibitors of Trypanosoma cruzi and Toxoplasma gondii proliferation

As a part of our project aimed at developing new safe chemotherapeutic and chemoprophylactic agents against tropical diseases, fluorine-containing drugs structurally related to 4-phenoxyphenoxyethyl thiocyanate (1) were designed, synthesized, and evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas' disease), and Toxoplasma gondii, the etiological agent of toxoplasmosis. This thiocyanate derivative had previously proven to be an effective agent against T. cruzi proliferation. Fluorine-containing thiocyanate derivatives 2 and 3 were threefold more potent than our lead drug 1 against intracellular T. cruzi. The biological evaluation against T. gondii was also very promising. The IC(50) values corresponding to 2 and 3 were at the very low micromolar level against tachyzoites of T. gondii. Both of these drugs are interesting examples of effective antiparasitic agents that have outstanding potential not only as lead drugs but also to be used for further in vivo studies.     

Bisphosphonates as inhibitors of Trypanosoma cruzi hexokinase: kinetic and metabolic studies

Trypanosoma cruzi, the etiologic agent of Chagas disease, has an unusual ATP-dependent hexokinase (TcHK) that is not affected by D-glucose 6-phosphate, but is non-competitively inhibited by inorganic pyrophosphate (PP(i)), suggesting a heterotropic modulator effect. In a previous study we identified a novel family of bisphosphonates, metabolically stable analogs of PP(i), which are potent and selective inhibitors of TcHK as well as the proliferation of the clinically relevant intracellular amastigote form of the parasite in vitro (Hudock, M. P., Sanz-Rodriguez, C. E., Song, Y., Chan, J. M., Zhang, Y., Odeh, S., Kosztowski, T., Leon-Rossell, A., Concepcion, J. L., Yardley, V., Croft, S. L., Urbina, J. A., and Oldfield, E. (2006) J. Med. Chem. 49, 215-223). In this work, we report a detailed kinetic analysis of the effects of three of these bisphosphonates on homogeneous TcHK, as well as on the enzyme in purified intact glycosomes, peroxisome-like organelles that contain most of the glycolytic pathway enzymes in this organism. We also investigated the effects of the same compounds on glucose consumption by intact and digitonin-permeabilized T. cruzi epimastigotes, and on the growth of such cells in liver-infusion tryptose medium. The bisphosphonates investigated were several orders of magnitude more active than PP(i) as non-competitive or mixed inhibitors of TcHK and blocked the use of glucose by the epimastigotes, inducing a metabolic shift toward the use of amino acids as carbon and energy sources. Furthermore, there was a significant correlation between the IC(50) values for TcHK inhibition and those for epimastigote growth inhibition for the 12 most potent compounds of this series. Finally, these bisphosphonates did not affect the sterol composition of the treated cells, indicating that they do not act as inhibitors of farnesyl diphosphate synthase. Taken together, our results suggest that these novel bisphosphonates act primarily as specific inhibitors of TcHK and may represent a novel class of selective anti-T. cruzi agents.     

Design of potent bisphosphonate inhibitors of the human farnesyl pyrophosphate synthase via targeted interactions with the active site 'capping' phenyls

Nitrogen-containing bisphosphonates (N-BPs) are potent active site inhibitors of the human farnesyl pyrophosphate synthase (hFPPS) and valuable human therapeutics for the treatment of bone-related malignancies. N-BPs are also useful in combination chemotherapy for patients with breast, prostate and multiple myeloma cancers. A structure-based approach was employed in order to design inhibitors that exhibit higher lipophilicity and better occupancy for the GPP sub-pocket of hFPPS than the current therapeutic drugs. These novel analogs were designed to bind deeper into the GPP sub-pocket by displacing the side chains of the 'capping' residue Phe 113 and engaging in favorable π-interactions with the side chain of Phe112.     

Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas’ disease. We have undertaken a detailed structure–activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme–ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60–70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.     

Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents

Intracellular protozoan parasites are potent stimulators of cell-mediated immunity. The induction of macrophage proinflammatory cytokines by Trypanosoma cruzi is considered to be important in controlling the infection and the outcome of Chagas' disease. Here we show that the potent tumour necrosis factor-alpha-, interleukin-12- and nitric oxide-inducing activities of T.cruzi trypomastigote mucins were recovered quantitatively in a highly purified and characterized glycosylphosphatidylinositol (GPI) anchor fraction of this material. The bioactive trypomastigote GPI fraction was compared with a relatively inactive GPI fraction prepared from T. cruzi epimastigote mucins. The trypomastigote GPI structures were found to contain additional galactose residues and unsaturated, instead of saturated, fatty acids in the sn-2 position of the alkylacylglycerolipid component. The latter feature is essential for the extreme potency of the trypomastigote GPI fraction, which is at least as active as bacterial endotoxin and Mycoplasma lipopeptide and, therefore, one of the most potent microbial proinflammatory agents known.     

Structure and mechanism of the farnesyl diphosphate synthase from Trypanosoma cruzi: implications for drug design

Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C5 alcohols (isopentenyl and dimethylallyl) to form C10 and C15 diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.     

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.     

Expression of farnesyl pyrophosphate synthase is increased in diabetic cardiomyopathy

Farnesyl pyrophosphate synthase (FPPS)-catalyzed isoprenoid intermediates are involved in diabetic cardiomyopathy. This study investigated the specific role of FPPS in the development of diabetic cardiomyopathy. We demonstrated that FPPS expression was elevated in both in vivo and in vitro models of diabetic cardiomyopathy. FPPS inhibition decreased the expression of proteins related to cardiac fibrosis and cardiomyocytic hypertrophy, including collagen I, collagen III, connective tissue growth factor, natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Furthermore, FPPS inhibition and knockdown prevented phosphorylated c-Jun N-terminal kinase 1/2 (JNK1/2) activation in vitro. In addition, a JNK1/2 inhibitor downregulated high-glucose-induced responses to diabetic cardiomyopathy. Finally, immunofluorescence revealed that cardiomyocytic size was elevated by high glucose and was decreased by zoledronate, small-interfering farnesyl pyrophosphate synthase (siFPPS), and a JNK1/2 inhibitor. Taken together, our findings indicate that FPPS and JNK1/2 may be part of a signaling pathway that plays an important role in diabetic cardiomyopathy.     

Synthesis and biological evaluation of 2-alkylaminoethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii targeting farnesyl diphosphate synthase

The effect of a series of 2-alkylaminoethyl-1,1-bisphosphonic acids against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii has been studied. Most of these drugs exhibited an extremely potent inhibitory action against the intracellular form of T. cruzi, exhibiting IC(50) values at the low micromolar level. This cellular activity was associated with a strong inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas' disease chemotherapy. Compound 17 was an effective agent against amastigotes exhibiting an IC(50) value of 0.84 microM, while this compound showed an IC(50) value of 0.49 microM against the target enzyme TcFPPS. Interestingly, compound 19 was very effective against both T. cruzi and T. gondii exhibiting IC(50) values of 4.1 microM and 2.6 microM, respectively. In this case, 19 inhibited at least two different enzymes of T. cruzi (TcFPPS and solanesyl diphosphate synthase (TcSPPS); 1.01 microM and 0.25 microM, respectively), while it inhibited TgFPPS in T. gondii. In general, this family of drugs was less effective against the activity of T. cruzi SPPS and against T. gondii growth in vitro. As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their potential low toxicity makes them good candidates to control tropical diseases.     

New potent 5-substituted benzofuroxans as inhibitors of Trypanosoma cruzi growth: quantitative structure-activity relationship studies

Benzofuroxan derivatives have been shown to inhibit the growth of Trypanosoma cruzi, the etiological agent of Chagas' disease. Therefore, 2D- and 3D-QSAR models of their in vitro antichagasic activity were developed. Six new derivatives were synthesized to complete a final set of 26 structurally diverse benzofuroxans. The 2D-QSAR model (r = 0.939, r(adj)(2) = 0.849) was generated using multiple regression analysis of tabulated substituents' physicochemical properties and indicator variables. In addition, a 3D-QSAR model (r(2) = 0.997, q(2) = 0.802) was obtained using a comparative molecular field analysis (CoMFA). Due to the well-known benzofuroxan tautomerism, in both approaches (2D- and 3D-QSAR) it was necessary to include an indicator variable to consider the N-oxide position (I(6)). This parameter was established using low-temperature NMR experiments. Both QSAR models identified the electrophilic character of the substituent alpha-atom as a requirement for activity. Further support was found using a density functional theory (DFT) approach.