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• W2048010075 abstract "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 (PPi), suggesting a heterotropic modulator effect. In a previous study we identified a novel family of bisphosphonates, metabolically stable analogs of PPi, 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 PPi 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 IC50 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. 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 (PPi), suggesting a heterotropic modulator effect. In a previous study we identified a novel family of bisphosphonates, metabolically stable analogs of PPi, 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 PPi 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 IC50 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. Chagas disease remains the major parasitic disease burden in Latin America, despite recent advances in the control of its vectorial and transfusional transmission (1World Health OrganizationTech. Rep. Ser. 2002; 905: 1-109Google Scholar, 2Dias J.C. Silveira A.C. Schofield C.J. Mem. Inst. Oswaldo Cruz. 2002; 97: 603-612Crossref PubMed Scopus (551) Google Scholar). Specific chemotherapy against its etiological agent, the kinetoplastid parasite Trypanosoma cruzi, is unsatisfactory because current drugs have very limited efficacy in the prevalent, chronic phase of the disease, and there are frequent serious side effects (3Urbina J.A. Docampo R. Trends Parasitol. 2003; 19: 495-501Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). Thus, there is an urgent need for safer and more potent drugs to treat this condition, and several rational approaches are being developed, exploiting key biochemical differences between the parasite and its mammalian hosts (3Urbina J.A. Docampo R. Trends Parasitol. 2003; 19: 495-501Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 4Coura J.R. de Castro S.L. Mem. Inst. Oswaldo Cruz. 2002; 97: 3-24Crossref PubMed Scopus (782) Google Scholar). In this context, it is of note that T. cruzi, and several related kinetoplastid protozoa, have several unusual characteristics in their energy metabolism, which differentiates them from other eukaryotes. 1) Most of the glycolytic pathway is compartmentalized in peroxisome-like organelles termed glycosomes (5Taylor M.B. Gutteridge W.E. Exp. Parasitol. 1987; 63: 84-97Crossref PubMed Scopus (38) Google Scholar, 6Opperdoes F.R. Annu. Rev. Biochem. 1987; 41: 127-151Google Scholar). 2) The classic, allosteric modulators of the two key regulatory enzymes of the glycolytic pathway in mammalian, fungal, and bacterial organisms, hexokinase and phosphofructokinase, do not affect the kinetoplastid enzymes (7Urbina J.A. Crespo A. Mol. Biochem. Parasitol. 1984; 11: 225-239Crossref PubMed Scopus (26) Google Scholar, 8Taylor M. Gutteridge W.E. FEBS Lett. 1986; 201: 262-266Crossref PubMed Scopus (8) Google Scholar, 9Racagni G.E. Machado de Domenech E.E. Mol. Biochem. Parasitol. 1983; 9: 181-188Crossref PubMed Scopus (25) Google Scholar). This is associated with the absence of a Pasteur effect in these parasites, in addition to their flexibility in utilizing glucose or amino acids as carbon and energy sources (10Urbina J.A. Parasitol. Today. 1994; 10: 107-110Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 11Cazzulo J.J. FASEB J. 1992; 6: 3153-3161Crossref PubMed Scopus (175) Google Scholar, 12Cazzulo J.J. Subcell. Biochem. 1992; 18: 235-257Crossref PubMed Scopus (18) Google Scholar). 3) Kinetoplastid parasites contain large stores of inorganic pyrophosphate (PPi) and other short chain polyphosphates, which are by far the most abundant high energy compounds in these cells (13Moreno B. Rodrigues C.O. Bailey B.N. Urbina J.A. Moreno S.N. Docampo R. Oldfield E. FEBS Lett. 2002; 523: 207-212Crossref PubMed Scopus (28) Google Scholar, 14Urbina J.A. Moreno B. Vierkotter S. Oldfield E. Payares G. Sanoja C. Bailey B.N. Yan W. Scott D.A. Moreno S.N.J. Docampo R. J. Biol. Chem. 1999; 274: 33609-33615Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 15Docampo R. de Souza W. Miranda K. Rohloff P. Moreno S.N. Nat. Rev. Microbiol. 2005; 3: 251-261Crossref PubMed Scopus (353) Google Scholar). Although earlier work had shown that T. cruzi ATP-dependent hexokinase (TcHK) 2The abbreviations used are: TcHK, Trypanosoma cruzi ATP-dependent hexokinase; FPPS, farnesyl diphosphate synthase; LIT, liver infusion tryptose; MIC, minimal inhibitory concentration; PEP, phosphoenolpyruvate. was not inhibited by its main regulator in vertebrates, d-glucose 6-phosphate (7Urbina J.A. Crespo A. Mol. Biochem. Parasitol. 1984; 11: 225-239Crossref PubMed Scopus (26) Google Scholar, 9Racagni G.E. Machado de Domenech E.E. Mol. Biochem. Parasitol. 1983; 9: 181-188Crossref PubMed Scopus (25) Google Scholar, 16Wilson J.E. Rev. Physiol. Biochem. Pharmacol. 1995; 126: 65-198Crossref PubMed Google Scholar), more recent studies have shown that this enzyme is inhibited in a non-competitive manner (with respect to ATP) by PPi, with a Ki of ∼500 μm, suggesting that PPi acts as an heterotropic, allosteric regulator (17Caceres A.J. Portillo R. Acosta H. Rosales D. Quinones W. Avilan L. Salazar L. Dubourdieu M. Michels P.A. Concepcion J.L. Mol. Biochem. Parasitol. 2003; 126: 251-262Crossref PubMed Scopus (69) Google Scholar). Subsequently, we identified a novel family of metabolically stable PPi analogs called bisphosphonates, which are potent and selective inhibitors of TcHK, as well as the proliferation of the clinically relevant intracellular amastigote form of the parasite (18Hudock 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. Oldfield E. J. Med. Chem. 2006; 49: 215-223Crossref PubMed Scopus (81) Google Scholar). We carried out a QSAR study of 42 compounds and were able to construct pharmacophore and comparative molecular similarity indices analysis (CoMSIA) models for enzyme inhibition (18Hudock 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. Oldfield E. J. Med. Chem. 2006; 49: 215-223Crossref PubMed Scopus (81) Google Scholar, 19Klebe G. Abraham U. Mietzner T. J. Med. Chem. 1994; 37: 4130-4146Crossref PubMed Scopus (1758) Google Scholar). However, at that point, no information was available on the mechanism of enzyme inhibition or on the biochemical and physiological effects of these bisphosphonates on the target cells. In the present work, we selected three compounds from the series (Fig. 1), including the two most potent ones, (9-ethyl-9H-3-carbazolyl)-aminomethylene-1,1-bisphosphonate (228), IC50 = 0.81 μm, and (3-bromo-phenyl)-aminomethylene-1,1-bisphosphonate (302), IC50 = 0.95 μm; and one of intermediate activity (2-(pyridin-4-yl)-1-hydroxyethane-1,1-bisphosphonate (3), IC50 = 12.7 μm, to carry out a detailed kinetic study of their inhibitory activity against TcHK, as well as to investigate their effects on glucose consumption, energy metabolism, and growth of the extracellular epimastigote form of the parasite. The results show that these compounds are very potent, non-competitive or mixed inhibitors of TcHK that block glucose consumption by intact parasites, leading to a metabolic shift in these cells to the use amino acids as carbon and energy sources. Furthermore, we found that growth inhibition induced by the 12 more potent bisphosphonates of the series was highly correlated with TcHK inhibition. Finally, in agreement with our previous results (18Hudock 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. Oldfield E. J. Med. Chem. 2006; 49: 215-223Crossref PubMed Scopus (81) Google Scholar), it was found that the biochemical activity of 228 and 302 was highly selective: they do not affect the parasites sterol composition, which showed that they do not inhibit farnesyl diphosphate synthase (FPPS), a known target of other nitrogen-containing bisphosphonates in eukaryotes, including T. cruzi (20Gabelli S.B. McLellan J.S. Montalvetti A. Oldfield E. Docampo R. Amzel L.M. Proteins. 2006; 62: 80-88Crossref PubMed Scopus (126) Google Scholar, 21Rodan G.A. Martin T.J. Science. 2000; 289: 1508-1514Crossref PubMed Scopus (1479) Google Scholar, 22Martin M.B. Arnold W. Heath III, H.T. Urbina J.A. Oldfield E. Biochem. Biophys. Res. Commun. 1999; 263: 754-758Crossref PubMed Scopus (152) Google Scholar, 23Docampo R. Moreno S.N. Curr. Drug Targets Infect. Disord. 2001; 1: 51-61Crossref PubMed Scopus (75) Google Scholar). Parasite—The EP (24De Maio A. Urbina J.A. Acta Cient. Venez. 1984; 35: 136-141PubMed Google Scholar) stock of T. cruzi was used in this study. Handling of live T. cruzi was performed according to established guidelines (25Hudson L. Grover F. Gutteridge W.E. Klein R.A. Peters W. Neal R.A. Miles M.A. Scott M.T. Nourish R. Ager B.P. Trans. R. Soc. Trop. Med. Hyg. 1983; 77: 416-419Abstract Full Text PDF PubMed Scopus (38) Google Scholar). T. cruzi Cultivation and Antiproliferative Activities of Bisphosphonates—The epimastigote form of the parasite was cultivated in liver infusion tryptose (LIT) medium (24De Maio A. Urbina J.A. Acta Cient. Venez. 1984; 35: 136-141PubMed Google Scholar), supplemented with 10% newborn calf serum (Invitrogen) at 28 °C, with strong agitation (120 rpm). The cultures were initiated at a cell density of 2 × 106 epimastigotes ml–1 and cell densities were measured with an electronic particle counter (model ZBI; Coulter Electronics Inc., Hialeah, FL) and by direct counting with a hemocytometer. Cell viability was followed by trypan blue exclusion, using light microscopy. Experimental compounds were added as sterile, 100-fold stocks at a cell density of 1–2 × 107 epimastigotes ml–1. Growth inhibition was quantified by defining a percent growth factor: % GF = (GFdrug/GFcontrol) × 100, where GF = ((CD)96h – (CD)0h)/(CD)0h), and GFdrug is the growth factor in the presence of a given concentration of the drug, GFcontrol is the corresponding value for untreated cells, and (CD)xh are the cell densities of the cultures at x hours after the addition of the drug. IC50 values were calculated from the % GFs by non-linear correlation with the GraFit software package (Erithacus Software Ltd., Surrey, UK) and statistical analyses were carried out with the JMP 6.0 statistical package (SAS Institute Inc., Cary, NC). Glucose and ammonium concentrations in the growth medium were determined using enzymatic assays, as described (7Urbina J.A. Crespo A. Mol. Biochem. Parasitol. 1984; 11: 225-239Crossref PubMed Scopus (26) Google Scholar, 26Urbina J.A. Azavache V. Mol. Biochem. Parasitol. 1984; 11: 241-255Crossref PubMed Scopus (26) Google Scholar). TcHK Isolation, Purification, and Assay—T. cruzi hexokinase was purified to homogeneity from exponential phase epimastigotes as described by Caceres et al. (17Caceres A.J. Portillo R. Acosta H. Rosales D. Quinones W. Avilan L. Salazar L. Dubourdieu M. Michels P.A. Concepcion J.L. Mol. Biochem. Parasitol. 2003; 126: 251-262Crossref PubMed Scopus (69) Google Scholar); enzyme activity was determined by using a spectrophotometric assay, coupling its activity to that of d-glucose-6-phosphate dehydrogenase and following the reduction of NADP+ at 340 nm (7Urbina J.A. Crespo A. Mol. Biochem. Parasitol. 1984; 11: 225-239Crossref PubMed Scopus (26) Google Scholar, 17Caceres A.J. Portillo R. Acosta H. Rosales D. Quinones W. Avilan L. Salazar L. Dubourdieu M. Michels P.A. Concepcion J.L. Mol. Biochem. Parasitol. 2003; 126: 251-262Crossref PubMed Scopus (69) Google Scholar); the slope for the change in absorbance as a function of time was constant for at least 5 min and this value was taken to calculate the initial reaction velocities. Isolation and Purification of Glycosomes and Assay of TcHK Activity in Situ—Highly purified intact glycosomes were obtained from exponential phase epimastigotes as described (27Concepcion J.L. Gonzalez-Pacanowska D. Urbina J.A. Arch. Biochem. Biophys. 1998; 352: 114-120Crossref PubMed Scopus (60) Google Scholar); latencies, defined as: % latency = 100 × (TcHK activity of glycosomes in 0.2% Triton X-100 – TcHK activity of intact glycosomes/TcHK activity of glycosomes in 0.2% Triton X-100), were typically >95% (Triton X-100 concentration is v/v). Glycosomes were incubated in the presence of digitonin (25 μg mg–1 of protein) for 1 min, centrifuged at 35,000 × g for 2 min, suspended in buffer A (sucrose 225 mm, KCl 20 mm, KH2PO4 10 mm, Na2EDTA 5 mm, Tris-HCl 20 mm, MgCl2 1 mm, pH 7.2) and assayed for hexokinase activity, as described above. Analysis of Kinetic Data—Initial velocity data were obtained as a function of substrate (S) and inhibitor (I) concentrations, keeping the co-substrate concentration at saturating levels and fitted data to the following equation (28Urbina J.A. Osorno C.E. Rojas A. Arch. Biochem. Biophys. 1990; 282: 91-99Crossref PubMed Scopus (41) Google Scholar): 1/V = (1 + [I]/Kii)/Vm + (1 + [I]/Kis)Km/Vm[S]. Where Km and Vm are the apparent Michaelis-Menten constant and maximal velocity, respectively, and Kii = [E′S][I]/[E′SI]; Kis = [E′][I]/[E′], where [E′]is the concentration of the enzyme in the presence of fixed concentrations of co-substrate and co-factors. Glucose Consumption by Digitonin-permeabilized Epimastigotes—Exponential phase epimastigotes were collected by centrifugation (4,000 × g for 10 min), resuspended in buffer B (Tris-HCl 75 mm, NaCl 140 mm, KCl 11 mm, pH 7.4) at a final cell density of 108 cells ml–1 and incubated in the presence of digitonin at 30 μgmg–1 protein for 20 min, washed twice in the same buffer, and finally resuspended in buffer B, supplemented with phosphoenolpyruvate (PEP) 6 mm, ADP 6 mm, NaHCO3 7 mm, l-malate 1 mm. The parasites were preincubated at 28 °C with strong agitation (100 rpm) in the presence of varying concentrations of bisphosphonates for 60 min, and the experiment were started by addition of 3 mm d-glucose; 100-μl samples were taken every 15 min, centrifuged at 35,000 × g for 5 min, and the supernatants stored at –80 °C until assayed for d-glucose content, as described above (7Urbina J.A. Crespo A. Mol. Biochem. Parasitol. 1984; 11: 225-239Crossref PubMed Scopus (26) Google Scholar, 26Urbina J.A. Azavache V. Mol. Biochem. Parasitol. 1984; 11: 241-255Crossref PubMed Scopus (26) Google Scholar). 13C NMR Assay of Glucose Consumption by Intact Epimastigotes—These experiments were carried out as described (28Urbina J.A. Osorno C.E. Rojas A. Arch. Biochem. Biophys. 1990; 282: 91-99Crossref PubMed Scopus (41) Google Scholar), with minor modifications. Exponential phase epimastigotes were collected by centrifugation and washed in buffer B as described above, resuspended at a cell density of 1010 cells ml–1 in the same buffer, preincubated with strong agitation (100 rpm) at 28 °C in the presence of 50 μm 228 or 302 for 6 h, and the experiment started by the addition of 10 mm 1-d-[13C]glucose (Sigma); 1-ml samples were collected every 30 min, centrifuged at 35,000 × g for 5 min, and the supernatants stored at –80 °C until assayed by 13C NMR. Perchloric acid extracts of the sediments (whole cells) were prepared as described before (14Urbina J.A. Moreno B. Vierkotter S. Oldfield E. Payares G. Sanoja C. Bailey B.N. Yan W. Scott D.A. Moreno S.N.J. Docampo R. J. Biol. Chem. 1999; 274: 33609-33615Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and also stored at –80 °C. 13C NMR spectra were obtained using a 11.744 tesla Bruker ADVANCE 500 NMR spectrometer, which operates at 125.77 MHz for 13C (500.13 MHz for 1H); chemical shifts are reported with respect to external p-dioxane (66.5 ppm). 1H decoupled 13C NMR free induction decays were acquired with 6.4 μs (45°) excitation pulses, a 250 ppm (31.447 Hz) spectral width, composite pulse 1H decoupling, and a 2-s recycle delay; 1024 free induction decays were acquired and averaged for each spectrum and processed with a 1-Hz exponential filter before Fourier transformation. Three independent experiments were carried out, with essentially identical results. Studies of Lipid Composition—For the analysis of the effects of the experimental compounds on epimastigote lipid composition, total lipids from control and drug-treated cells were extracted and fractionated into neutral and polar lipid fractions by silicic acid column chromatography and gas-liquid chromatography (29Urbina J.A. Payares G. Sanoja C. Molina J. Lira R. Brener Z. Romanha A.J. Intern. J. Antimicrob. Agents. 2003; 21: 39-48Crossref PubMed Scopus (86) Google Scholar, 30Urbina J.A. Payares G. Sanoja C. Lira R. Romanha A.J. Int. J. Antimicrob. Agents. 2003; 21: 27-38Crossref PubMed Scopus (138) Google Scholar). The neutral lipid fractions were first analyzed by thin layer chromatography (on Merck 5721 silica gel plates with heptane/isopropyl ether/glacial acetic acid (60:40:4) as developing solvent) and conventional gas-liquid chromatography (isothermal separation in a 4-m glass column packed with 3% OV-1 on Chromosorb 100/200 mesh, with nitrogen as carrier gas at 24 ml min–1 and flame ionization detection using a Varian 3700 gas chromatograph). For quantitative analysis and structural assignments, the neutral lipids were separated in a high-resolution capillary column (25 m × 0.20-mm inner diameter Ultra-2 column, 5% phenylmethylsiloxane, 0.33 μm film thickness) using a Hewlett-Packard 6890 Plus gas chromatograph equipped with a HP5973N mass sensitive detector. The lipids were dissolved in chloroform and injected; the column was kept a 50 °C for 1 min, then the temperature was increased to 270 °C at a rate of 25 °C min–1 and finally to 300 °C at a rate of 1 °C min–1. The carrier gas (He) flow was kept constant at 0.5 ml min–1. Injector temperature was 250 °C and the detector was kept at 280 °C. Drugs—The bisphosphonates used in this work (Fig. 1) were synthesized, purified, and characterized as described before (18Hudock 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. Oldfield E. J. Med. Chem. 2006; 49: 215-223Crossref PubMed Scopus (81) Google Scholar). Kinetics of Inhibition of TcHK by Bisphosphonates—The chemical structures of the 12 most potent bisphosphonate inhibitors of TcHK identified in our previous study (18Hudock 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. Oldfield E. J. Med. Chem. 2006; 49: 215-223Crossref PubMed Scopus (81) Google Scholar) are shown in Fig. 1. We have now performed a detailed kinetic study to clarify the inhibition mechanism of three of these compounds (228, 302, and 3) against the pure enzyme, as well as the enzyme in digitonin-permeabilized but otherwise intact glycosomes, and these results are presented in Figs. 2 and 3 and Tables 1 and 2. Against the pure enzyme (Fig. 2 and Table 1) all three compounds behaved as mixed to non-competitive inhibitors against ATP, as indicated by the similar values of Kii and Kis. Against d-glucose, 228 and 302 exhibited competitive behavior (data not shown), but compound 3 was again a non-competitive inhibitor (Table 1). In all cases, the values of the inhibitory constants were 100–1000-fold lower that the Ki values obtained for PPi in our previous study (500 μm, see Ref. (17Caceres A.J. Portillo R. Acosta H. Rosales D. Quinones W. Avilan L. Salazar L. Dubourdieu M. Michels P.A. Concepcion J.L. Mol. Biochem. Parasitol. 2003; 126: 251-262Crossref PubMed Scopus (69) Google Scholar)).FIGURE 3Kinetics of inhibition of in situ glycosomal TcHK by bisphosphonates. Lineweaver-Burk plots of the effects of 228 (A), 302 (B), and 3 (C) on glycosomal TcHK, isolated and purified from T. cruzi epimastigotes as described under “Materials and Methods.” The inhibitor concentrations were 0, 0.2, 0.5, 0.6, 1, 1.2, and 1.5μm for 228; 0, 0.5, 1, 1.5, 2, 2.5, and 3μm for 302; and 0, 1, 2, 3, 6, and 8 for 3. Inset, secondary plots of intercepts and slopes versus inhibitor concentrations, used to calculate Kii and Kis, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Kinetics of inhibition of pure T. cruzi hexokinase by bisphosphonatesInhibitor/substrateKiiaInhibition constants given in micromolar.KisaInhibition constants given in micromolar.Mechanism228/ATPbIn the presence of 2 mm d-glucose and 3 mm MgCl2.2.81.7Mixed228/d-glucosecIn the presence of 1 mm ATP and 3 mm MgCl2.0.5Competitive302/ATPbIn the presence of 2 mm d-glucose and 3 mm MgCl2.1.20.6Mixed302/d-glucosecIn the presence of 1 mm ATP and 3 mm MgCl2.0.4Competitive3/ATPbIn the presence of 2 mm d-glucose and 3 mm MgCl2.4.32.8Mixed3/d-glucosecIn the presence of 1 mm ATP and 3 mm MgCl2.6.03.3Non-competitivea Inhibition constants given in micromolar.b In the presence of 2 mm d-glucose and 3 mm MgCl2.c In the presence of 1 mm ATP and 3 mm MgCl2. Open table in a new tab TABLE 2Kinetics of inhibition of glycosomal T. cruzi hexokinase in situ by bisphosphonatesInhibitor/substrateKiiaInhibition constants given in micromolar.KisaInhibition constants given in micromolar.Mechanism228/ATPbIn the presence of 2 mm d-glucose and 3 mm MgCl2.1.71.2Non-competitive228/d-glucosecIn the presence of 1 mm ATP and 3 mm MgCl2.4.32.4Non-competitive302/ATPbIn the presence of 2 mm d-glucose and 3 mm MgCl2.2.6Competitive302/d-glucosecIn the presence of 1 mm ATP and 3 mm MgCl2.10.62.9Mixed3/ATPbIn the presence of 2 mm d-glucose and 3 mm MgCl2.10.511.4Non-competitive3/d-glucosecIn the presence of 1 mm ATP and 3 mm MgCl2.43.93.4MixedPPi/ATPbIn the presence of 2 mm d-glucose and 3 mm MgCl2.819819Non-competitivePPi/d-glucosecIn the presence of 1 mm ATP and 3 mm MgCl2.3,0401,720Mixeda Inhibition constants given in micromolar.b In the presence of 2 mm d-glucose and 3 mm MgCl2.c In the presence of 1 mm ATP and 3 mm MgCl2. Open table in a new tab TcHK, as well as the five subsequent enzymes of the glycolytic pathway in T. cruzi and related organisms, functions in vivo in the matrix of the glycosomes, membrane bound peroxisome-like organelles in which protein concentrations can be as high as 350 mg ml–1 (5Taylor M.B. Gutteridge W.E. Exp. Parasitol. 1987; 63: 84-97Crossref PubMed Scopus (38) Google Scholar, 6Opperdoes F.R. Annu. Rev. Biochem. 1987; 41: 127-151Google Scholar). In an effort to study the activity of this enzyme under more natural conditions, we next investigated its kinetic properties and inhibition by bisphosphonates in highly purified glycosomes, made permeable to small molecules by briefly incubating with digitonin. The use of this plant glycoside detergent to permeabilize the glycosomal membrane was suggested by previous studies from our group, which showed that it was rich in the endogenous sterols of the parasite (31Quinones W. Urbina J.A. Dubourdieu M. Concepcion J.L. Exp. Parasitol. 2004; 106: 135-149Crossref PubMed Scopus (37) Google Scholar), plus that the glycosome is one the organelles involved in de novo sterol biosynthesis in this parasite (27Concepcion J.L. Gonzalez-Pacanowska D. Urbina J.A. Arch. Biochem. Biophys. 1998; 352: 114-120Crossref PubMed Scopus (60) Google Scholar). Preliminary data indicated that incubation of glycosomes in the presence of 25 μg of digitonin/mg of protein for 1 min allowed full expression of TcHK activity with <5% release of the enzyme (or other glycosomal markers, such as phosphoglucose isomerase) from the matrix of the organelle. Because the in situ activity of this enzyme had not been reported previously, a detailed kinetic study was carried out; the results (not shown) indicated classical Michaelis-Menten behavior, with Km values of 463 and 30 μm for ATP and d-glucose, respectively, and a Vm value of 11.5 μmol min–1 mg of protein–1. We also investigated the inhibitory effects of PPi on the in situ TcHK activity and found that it was purely non-competitive toward ATP (Kii = Kis = 819 μm), but mixed toward d-glucose (Table 2). The values of Km for ATP and the Ki for PPi for the glycosomal enzyme were significantly higher than those previously obtained for the pure enzyme (17Caceres A.J. Portillo R. Acosta H. Rosales D. Quinones W. Avilan L. Salazar L. Dubourdieu M. Michels P.A. Concepcion J.L. Mol. Biochem. Parasitol. 2003; 126: 251-262Crossref PubMed Scopus (69) Google Scholar). When we investigated the kinetics of inhibition of the enzyme in glycosomes (Fig. 3 and Table 2), we found that 228 and 3 were again mixed to non-competitive inhibitors toward ATP, but 302 produced an apparently competitive inhibition; all compounds were non-competitive or mixed inhibitors against d-glucose (data not shown, Table 2). Again, the values of the inhibitory constants for bisphosphonates against this form of the enzyme were 2–3 orders of magnitude lower than were those for PPi (Table 2). Effects of Bisphosphonates on Glucose Consumption by Digitonin-permeabilized and Intact T. cruzi Epimastigotes—Next, we investigated the effect of bisphosphonates on the glucose consumption by whole, digitonin-permeabilized T. cruzi epimastigotes. Previous work had shown that incubation of these cells with digitonin at 30 μgmg–1 protein for 20 min render their plasma membranes permeable to both low and high molecular weight compounds, but no glycosomal proteins were released (27Concepcion J.L. Gonzalez-Pacanowska D. Urbina J.A. Arch. Biochem. Biophys. 1998; 352: 114-120Crossref PubMed Scopus (60) Google Scholar); similar conditions have been used in the same cells to characterize the in situ activities of other organelles, such as mitochondria (32Rodrigues C.O. Catisti R. Uyemura S.A. Vercesi A.E. Lira R. Rodriguez C. Urbina J.A. Docampo R. J. Eukaryot. Microbiol. 2001; 48: 588-594Crossref PubMed Scopus (54) Google Scholar) and acidocalcisomes (15Docampo R. de Souza W. Miranda K. Rohloff P. Moreno S.N. Nat. Rev. Microbiol. 2005; 3: 251-261Crossref PubMed Scopus (353) Google Scholar, 33Docampo R. Moreno S.N.J. Parasitol. Today. 1999; 15: 443-448Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 34Docampo R. Scott D.A. Vercesi A.E. Moreno S.N.J. Biochem. J. 1995; 310: 1005-1012Crossref PubMed Scopus (183) Google Schola" @default.
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