Biomedical subjects

Eric Oldfield

Publications and source records attributed to Eric Oldfield.

At least 37 records · Page 2Linked to original sources

PubMed2005

Solid-state NMR and quantum chemical investigations of 13Calpha shielding tensor magnitudes and orientations in peptides: determining phi and psi torsion angles.

We report the experimental determination of the (13)C(alpha) chemical shift tensors of Ala, Leu, Val, Phe, and Met in a number of polycrystalline peptides with known X-ray or de novo solid-state NMR structures. The 700 Hz dipolar coupling between (13)C(alpha) and its directly bonded (14)N permits extraction of both the magnitude and the orientation of the shielding tensor with respect to the C(alpha)-N bond vector. The chemical shift anisotropy (CSA) is recoupled under magic-angle spinning using the SUPER technique (Liu et al., J. Magn. Reson. 2002, 155, 15-28) to yield quasi-static chemical shift powder patterns. The tensor orientation is extracted from the (13)C-(14)N dipolar modulation of the powder line shapes. The magnitudes and orientations of the experimental (13)C(alpha) chemical shift tensors are found to be in good accord with those predicted from quantum chemical calculations. Using these principal values and orientations, supplemented with previously measured tensor orientations from (13)C-(15)N and (13)C-(1)H dipolar experiments, we are able to predict the (phi, psi, chi(1)) angles of Ala and Val within 5.8 degrees of the crystallographic values. This opens up a route to accurate determination of torsion angles in proteins based on shielding tensor magnitude and orientation information using labeled compounds, as well as the structure elucidation of noncrystalline organic compounds using natural abundance (13)C NMR techniques.

Carbon Isotopes↗

PubMed2005

Pyridinium-1-yl bisphosphonates are potent inhibitors of farnesyl diphosphate synthase and bone resorption.

We report the design, synthesis and testing of a series of novel bisphosphonates, pyridinium-1-yl-hydroxy-bisphosphonates, based on the results of comparative molecular similarity indices analysis and pharmacophore modeling studies of farnesyl diphosphate synthase (FPPS) inhibition, human Vgamma2Vdelta2 T cell activation and bone resorption inhibition. The most potent molecules have high activity against an expressed FPPS from Leishmania major, in Dictyostelium discoideum growth inhibition, in gammadelta T cell activation and in an in vitro bone resorption assay. As such, they represent useful new leads for the discovery of new bone resorption, antiinfective and anticancer drugs.

Alkyl and Aryl Transferases↗

PubMed2005

Solid-state NMR fermi contact and dipolar shifts in organometallic complexes and metalloporphyrins.

We have used density functional theory methods to investigate the solid-state "magic-angle" spinning (MAS) NMR and single-crystal NMR/ENDOR spectra of paramagnetic organometallic complexes and metalloporphyrins. The solid-state MAS NMR chemical shifts (including both diamagnetic and hyperfine contributions) are predicted with a slope of 1.007 and an R2 = 0.967, corresponding to a 28 ppm (or 6.3%) error over the entire 442 ppm range. Single-crystal ENDOR hyperfine values, including both isotropic Fermi contact and dipolar couplings, are predicted with a slope of 1.009 and an R2 = 0.998, corresponding to a 0.93 MHz (or 1.2%) error over the entire 78.37 MHz range. In addition, single-crystal NMR shifts (including both hyperfine terms) are predicted with an R2 = 0.961. The ability to compute solid-state MAS NMR and single-crystal NMR/ENDOR data should facilitate the use of these techniques in investigating paramagnetic metal complexes and should be of particular use in studying paramagnetic metalloproteins, where structures are less accurately known.

Electron Spin Resonance Spectroscopy↗

PubMed2005

(67)Zn NMR chemical shifts and electric field gradients in zinc complexes: a quantum chemical investigation.

We have used quantum chemical methods to predict 67Zn NMR chemical shifts as well as quadrupole coupling constants (CQ) in a series of biomimetic and inorganic zinc complexes. The 67Zn chemical shifts are predicted with an R2 = 0.975, corresponding to a 24.3 ppm or 6.7% error over the entire 365 ppm 67Zn chemical shift range. The 67Zn CQ values are predicted with an R2 = 0.991, corresponding to a 1.17 MHz or 3.0% error over the entire 38.75 MHz range. The 67Zn NMR shifts in a series of complexes containing N,O ligands are, in general, highly correlated with the number of oxygen ligands. The ability to compute 67Zn NMR shifts as well as CQ values opens up the possibility of using both of these properties in structure determination or refinement in proteins.

Imidazoles↗

PubMed2005

Metallic nature and surface diffusion of CO adsorbed on Ru nanoparticles in aqueous media: A 13C NMR study.

We report the first observation of the 13C nuclear magnetic resonance spectroscopy (NMR) of 13CO, adsorbed from 13CO saturated 0.5 M sulfuric acid solutions, onto the surfaces of commercial Ru-black nanoparticles. The 13C NMR spectra consist of a symmetrically broadened peak having a large isotropic shift as compared to CO adsorbed onto supported Ru catalysts. The variation of the spin-lattice relaxation rate follows Korringa behavior, indicating the metallic nature of adsorbed CO, in addition to varying across the spectrum in a Korringa-like manner. Motional narrowing of the NMR spectrum at higher temperatures, together with an additional contribution to the spin-lattice relaxation rate, indicate that adsorbed CO undergoes rapid diffusion on the particle surfaces. A two-band model analysis of the NMR results indicates that the CO adsorption bond is weaker on Ru as compared to either Pt or Pd. This is also supported by a reduction in the activation energy for CO diffusion on Ru vs either Pt or Pd nanoparticles.

Adsorption↗

PubMed2005

A crystallographic investigation of phosphoantigen binding to isopentenyl pyrophosphate/dimethylallyl pyrophosphate isomerase.

We report the crystallographic structures of the potent phosphoantigens Phosphostim (the bromohydrin of isopentenyl pyrophosphate) and E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate bound to the mevalonate pathway enzyme isopentenyl pyrophosphate/dimethylallyl pyrophosphate isomerase (IPPI). Racemic Phosphostim forms covalent complexes with IPPI: a 4-thioether with C67 and a 4-ester with E116. Only the E116 ester forms with the chiral species, S-Phosphostim, with the w.t. enzyme, while the C67 thioether forms with a mutant Y104F IPPI. The potent phosphoantigen HMBPP also binds to IPPI, but is only a weak ( approximately 50 muM) inhibitor. These results strongly support an SN2 reaction for inhibition of IPPI by Phosphostim, in contrast to the SN1 or concerted type of reaction found with epoxide inhibitors, which react at C-3, and are of general interest in the context of the development of novel mevalonate pathway inhibitors. They also provide clues as to the nature of the binding site of synthetic phosphoantigens in gammadelta T cell activation. In particular, both bromohydrin and epoxy phosphoantigens are potent, irreversible inhibitors of IPPI while HMBPP is only a weak inhibitor, ruling out an IPPI or IPPI-like target for HMBPP in gammadelta T cell activation.

Antigens↗

PubMed2004

Inhibition of isoprene biosynthesis pathway enzymes by phosphonates, bisphosphonates, and diphosphates.

We have investigated the docking of a variety of inhibitors and substrates to the isoprene biosynthesis pathway enzymes farnesyl diphosphate synthase (FPPS), isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IPPI) and deoxyxylulose-5-phosphate reductoisomerase (DXR) using the Lamarckian genetic alogorithm program, AutoDock. The docked ligand structures are predicted with a approximately 0.8 A rms deviation from the structures determined crystallographically. The errors found are a function of the number of atoms in the ligand (R = 0.91, p < 0.0001) and, to a lesser extent, on the resolution of the crystallographic structure (R = 0.70, p < 0.008). The structures of three isoprenoid diphosphates docked to the FPPS enzyme reveal strong electrostatic interactions with Mg(2+), lysine and arginine active site residues. Similar results are obtained with the docking of four IPPI inhibitors to the IPPI enzyme. The DXR substrate, deoxyxylulose-5-phosphate, is found to dock to Mn(2+)-NADPH-DXR in an almost identical manner as does the inhibitor fosimdomycin to Mn(2+)-DXR (ligand heavy atom rms deviation = 0.90 A) and is poised to interact with NADPH. Bisphosphonate inhibitors are found to bind to the allylic binding sites in both eukaryotic and prokaryotic FPPSs, in good accord with recent crystallographic results (a 0.4 A rms deviation from the X-ray structure with the E. coli enzyme). Overall, these results show for the first time that the geometries of a broad variety of phosphorus-containing inhibitors and substrates of isoprene biosynthesis pathway enzymes can be well predicted by using computational methods, which can be expected to facilitate the design of novel inhibitors of these enzymes.

Aldose-Ketose Isomerases↗

PubMed2004

Crystallization and preliminary X-ray diffraction study of the farnesyl diphosphate synthase from Trypanosoma brucei.

Farnesyl diphosphate synthase (FPPS) catalyses the formation of farnesyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate and is an RNAi-validated drug target in Trypanosoma brucei, the causative agent of African sleeping sickness. A T. brucei FPPS (390 amino acids) has been expressed in Escherichia coli and the recombinant protein has been crystallized in the absence and presence of the bisphosphonate inhibitor minodronate. Diffraction data were collected at 100 K using synchrotron radiation from both crystal types. Crystals obtained in the absence of minodronate belong to space group I222, with unit-cell parameters a = 61.43, b = 118.12, c = 120.04 A, while crystals grown in the presence of minodronate belong to space group C2, with unit-cell parameters a = 131.98, b = 118.10, c = 63.25 A, beta = 112.48 degrees. An initial model of the drug-free protein has been built using a homology model with the molecular-replacement method and refined to 3.3 A resolution. It shows mostly helical structure and resembles the structure of avian farnesyl diphosphate synthase, but with the addition of two loop regions.

Alkyl and Aryl Transferases↗

PubMed2004

The H(+)-pyrophosphatase of Rhodospirillum rubrum is predominantly located in polyphosphate-rich acidocalcisomes.

Acidocalcisomes are acidic, calcium storage compartments with a H(+) pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens. In this work, we report that the H(+)-pyrophosphatase (H(+)-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H(+)-PPase with pyrophosphate (PPi) and short and long chain polyphosphates (polyPs) but a lack of markers of the plasma membrane. polyP was also localized to the acidocalcisomes by using 4',6'-diamino-2-phenylindole staining and identified by using 31P NMR and biochemical methods. Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations. The number of acidocalcisomes and chromatophore membranes as well as the amounts of PPi and polyP increased when bacteria were grown in the light. Taken together, these results suggest that the H(+)-PPase of R. rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PPi synthesis in the chromatophore membranes.

Bacterial Chromatophores↗

PubMed2004

Crystallographic structures of two bisphosphonate:1-deoxyxylulose-5-phosphate reductoisomerase complexes.

We have obtained the single-crystal X-ray crystallographic structures of the bisphosphonates [(1-isoquinolinylamino)methylene]-1,1-bisphosphonate and [[(5-chloro-2-pyridinyl)amino]methylene]-1,1-bisphosphonate, bound to the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase (DXR, EC 1.1.1.267, also known as 2-C-methyl-d-erythritol-4-phosphate synthase), an important target for the development of antimalarial drugs. Our results indicate that both bisphosphonates bind into the fosmidomycin binding site. The aromatic groups are in a shallow hydrophobic pocket, and the phosphonate groups are involved in electrostatic interactions with Mg2+ or a cluster of carboxylic acid groups and lysine while the fosmidomycin phosphonate-binding site is occupied by a sulfate ion (as also observed in the DXR/NADP+ structure). The availability of these two new crystal structures opens up the possibility of the further development of bisphosphonates and related systems as DXR inhibitors and, potentially, as antiinfective agents.

Aldose-Ketose Isomerases↗

PubMed2004

Synthesis of chiral phosphoantigens and their activity in gamma delta T cell stimulation.

Gammadelta T cells expressing Vgamma2Vdelta2 T cell receptors are activated by a broad range of phosphorus-containing small molecules, termed phosphoantigens, and are of interest in the context of the chemotherapy of B cell malignancies. Here, we report the synthesis of four pairs of chiral phosphoantigens: the bromohydrins of isopentenyl diphosphate (Phosphostim), the epoxides of isopentenyl diphosphate (EIPP); and the corresponding bromohydrin and epoxide analogs of but-3-enyl diphosphate. The ability of each compound to stimulate human Vgamma2Vdelta2 T cells was determined by TNF-alpha release and cell proliferation. In these assays, the (R)-bromohydrin diphosphates were, on average, about twice as active as the (S)-bromohydrin diphosphates. In contrast, the (S)-form of EIPP was about twice as active as (R)-EIPP. The activities of the epoxy but-3-enyl diphosphates were both very low. These results suggest that chiral phosphoantigens, as opposed to racemic mixtures, may have utility in immunotherapy.

Alcohols↗

PubMed2004

On the Mössbauer spectra of isopenicillin N synthase and a model (FeNO)7 (S = 3/2) system.

We have carried out a series of density functional theory (DFT) calculations to predict the 57Fe Mössbauer quadrupole splittings (DeltaEQ) and isomer shifts (deltaFe) for the nitrosyl complex of isopenicillin N synthase with the substrate delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (IPNS.ACV.NO) and an {FeNO}7 (S = 3/2) model system, FeL(NO)(N3)2 (L = N,N',N' '-trimethyl-1,4,7-triazacyclononane). B3LYP predictions on the model compound are in almost exact agreement with experiment. The same DFT methods did not enable the prediction of the experimental DeltaEQ and deltaFe results for IPNS.ACV.NO when using the experimental protein crystal structure but did permit good predictions of DeltaEQ, deltaFe, and the asymmetry parameter (eta) when using a fully optimized structure. This optimized structure also enabled good predictions of the Mössbauer spectra of the photodissociation product of IPNS.ACV.NO. Mulliken and natural bonding orbital (NBO) spin density analyses indicate an electronic configuration of FeII (S = 2) anti-ferromagnetically coupled to NO (S = 1/2) in the protein as well as in the model system and the geometry optimized structure helps explain part of the enzyme reaction.

Binding Sites↗

PubMed2004

Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes.

Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of Pi residues) of short chain polyP were found. The presence of polyP of approximately 70-75 phosphate units was identified by 31P NMR and by urea-polyacrylamide gel electrophoresis of platelet extracts. An analysis of human platelet dense granules, purified using metrizamide gradient centrifugation, indicated that polyP was preferentially located in these organelles. This was confirmed by visualization of polyP in the dense granules using 4',6-diamidino-2-phenylindole and by its release together with pyrophosphate and serotonin upon thrombin stimulation of intact platelets. Dense granules were also shown to contain large amounts of calcium and potassium and both bafilomycin A1-sensitive ATPase and pyrophosphatase activities. In agreement with these results, when human platelets were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester to measure their intracellular Ca2+ concentration ([Ca2+]i), they were shown to possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by the following: 1) the increase in [Ca2+]i induced by nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2 and 2) the effect of ionomycin, which could not take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin, monensin, or NH4Cl. All of these characteristics of the platelet dense granules, together with their known acidity and high density (both by weight and by electron microscopy), are similar to those of acidocalcisomes (volutin granules, polyP bodies) of bacteria and unicellular eukaryotes. The results suggest that acidocalcisomes have been conserved during evolution from bacteria to humans.

Adenosine Triphosphatases↗

PubMed2004

Tryptophan chemical shift in peptides and proteins: a solid state carbon-13 nuclear magnetic resonance spectroscopic and quantum chemical investigation.

We have obtained the carbon-13 nuclear magnetic resonance spectra of a series of tryptophan-containing peptides and model systems, together with their X-ray crystallographic structures, and used quantum chemical methods to predict the (13)C NMR shifts (or shieldings) of all nonprotonated aromatic carbons (C(gamma), C(delta 2) and C(epsilon 2). Overall, there is generally good accord between theory and experiment. The chemical shifts of Trp C(gamma) in several proteins, hen egg white lysozyme, horse myoglobin, horse heart cytochrome c, and four carbonmonoxyhemoglobins, are also well predicted. The overall Trp C(gamma) shift range seen in the peptides and proteins is 11.4 ppm, and individual shifts (or shieldings) are predicted with an rms error of approximately 1.4 ppm (R value = 0.86). Unlike C(alpha) and N(H) chemical shifts, which are primarily a function of the backbone phi,psi torsion angles, the Trp C(gamma) shifts are shown to be correlated with the side-chain torsion angles chi(1) and chi(2) and appear to arise, at least in part, from gamma-gauche interactions with the backbone C' and N(H) atoms. This work helps solve the problem of the chemical shift nonequivalences of nonprotonated aromatic carbons in proteins first identified over 30 years ago and opens up the possibility of using aromatic carbon chemical shift information in structure determination.

Animals↗

PubMed2004

Cytochrome P450: an investigation of the Mössbauer spectra of a reaction intermediate and an Fe(IV)[double bond]O model system.

We have carried out a series of density functional theory (DFT) calculations to predict the 57Fe Mössbauer quadrupole splittings (DeltaEQ) and isomer shifts (deltaFe) for an Fe(IV)=O model compound ([Fe(O)(TMC)(NCCH3)](OTf)2, TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane, OTf = CF3SO3-) as well as a cytochrome P450 reaction intermediate (P450-RI). The DFT predictions on the model compound are in almost exact agreement with experiment. The same DFT methods did not enable the prediction of the experimental DeltaEQ results for P450-RI when using the experimental protein crystal structure and seven different spin/charge/protonation state combinations, but did permit good predictions of both DeltaEQ and deltaFe when using a geometry optimized structure having a porphyrin dianion, a protonated cysteine, and S = 1 (the same as that found from previous ESR studies).

Cytochrome P-450 Enzyme System↗

PubMed2004

Quantitative structure-activity relationships for gammadelta T cell activation by bisphosphonates.

gammadelta T cells are the first line of defense against many infectious organisms and are also involved in tumor cell surveillance and killing. They are stimulated by a broad range of small, phosphorus-containing antigens (phosphoantigens) as well as by the bisphosphonates commonly used in bone resorption therapy, such as pamidronate and risedronate. Here, we report the activation of gammadelta T cells by a broad range of bisphosphonates and develop a pharmacophore model for gammadelta T cell activation, in addition to using a comparative molecular similarity index analysis (CoMSIA) approach to make quantitative relationships between gammadelta T cell activation by bisphosphonates and their three-dimensional structures. The CoMSIA analyses yielded R(2) values of approximately 0.8-0.9 and q(2) values of approximately 0.5-0.6 for a training set of 45 compounds. Using an external test set, the activities (IC(50) values) of 16 compounds were predicted within a factor of 4.5, on average. The CoMSIA fields consisted of approximately 40% hydrophobic, approximately 40% electrostatic, and approximately 20% steric interactions. Since bisphosphonates are known to be potent, nanomolar inhibitors of the mevalonate/isoprene pathway enzyme farnesyl pyrophosphate synthase (FPPS), we also compared the pharmacophores for gammadelta T cell activation with those for FPPS inhibition, using the Catalyst program. The pharmacophores for gammadelta T cell activation and FPPS inhibition both consisted of two negative ionizable groups, a positive charge feature and an endocyclic carbon feature, all having very similar spatial dispositions. In addition, the CoMSIA fields were quite similar to those found for FPPS inhibition by bisphosphonates. The activities of the bisphosphonates in gammadelta T cell activation were highly correlated with their activities in FPPS inhibition: R = 0.88, p = 0.002, versus a human recombinant FPPS (N = 9 compounds); R = 0.82, p < 0.0001, for an expressed Leishmania major FPPS (N = 45 compounds). The bisphosphonate gammadelta T cell activation pharmacophore differs considerably, however, from that reported previously for gammadelta T cell activation by phosphoantigens (Gossman, W.; Oldfield, E. J. Med. Chem. 2002, 45, 4868-4874), suggesting different primary targets for the two classes of compounds. The ability to quite accurately predict the activity of bisphosphonates as gammadelta T cell activators by using 3D QSAR techniques can be expected to help facilitate the design of additional bisphosphonates for potential use in immunotherapy.

Alkyl and Aryl Transferases↗

PubMed2004

Effects of bisphosphonates on the growth of Entamoeba histolytica and Plasmodium species in vitro and in vivo.

The effects of a series of 102 bisphosphonates on the inhibition of growth of Entamoeba histolytica and Plasmodium falciparum in vitro have been determined, and selected compounds were further investigated for their in vivo activity. Forty-seven compounds tested were active (IC(50) < 200 microM) versus E. histolytica growth in vitro. The most active compounds (IC(50) approximately 4-9 microM) were nitrogen-containing bisphosphonates with relatively large aromatic side chains. Simple n-alkyl-1-hydroxy-1,1-bisphosphonates, known inhibitors of the enzyme farnesylpyrophosphate (FPP) synthase, were also active, with optimal activity being found with C9-C10 side chains. However, numerous other nitrogen-containing bisphosphonates known to be potent FPP synthase inhibitors, such as risedronate or pamidronate, had little or no activity. Several pyridine-derived bisphosphonates were quite active (IC(50) approximately 10-20 microM), and this activity was shown to correlate with the basicity of the aromatic group, with activity decreasing with increasing pK(a) values. The activities of all compounds were tested versus a human nasopharyngeal carcinoma (KB) cell line to enable an estimate of the therapeutic index (TI). Five bisphosphonates were selected and then screened for their ability to delay the development of amebic liver abscess formation in an E. histolytica infected hamster model. Two compounds were found to decrease liver abscess formation at 10 mg/kg ip with little or no effect on normal liver mass. With P. falciparum, 35 compounds had IC(50) values <200 microM in an in vitro assay. The most active compounds were also simple n-alkyl-1-hydroxy-1,1-bisphosphonates, having IC(50) values around 1 microM. Five compounds were again selected for in vivo investigation in a Plasmodium berghei ANKA BALB/c mouse suppressive test. The most active compound, a C9 n-alkyl side chain containing bisphosphonate, caused an 80% reduction in parasitemia with no overt toxicity. Taken together, these results show that bisphosphonates appear to be useful lead compounds for the development of novel antiamebic and antimalarial drugs.

Animals↗

PubMed2004

Antiparasitic activity of risedronate in a murine model of acute Chagas' disease.

We report the results of a study on the activity of the farnesyl-pyrophosphate synthase inhibitor risedronate (Ris) in a murine model of acute Chagas' disease. This compound displays rapid, cytocidal activity in vitro against Trypanosoma cruzi, but its in vivo activity had not been investigated previously. A murine model of acute Chagas' disease was used, in which experimental animals were infected with 10(3) trypomastigotes and intravenous treatment was started 24 h post-infection. In this model, Ris, at doses as low as 1 mg/kg per day given for 7 days, induced > 90% reductions in parasitaemia and increased very significantly (P = 0.001) the survival of treated animals. Higher doses (up to 10 mg/kg per day) led to further reductions in parasitaemia and mortality, with no deleterious effects on weight gain and general physical condition of the treated animals. There was no relapse of parasitaemia after discontinuation of treatment, suggesting trypanocidal, rather than trypanostatic, activity. This interpretation was confirmed by the almost complete disappearance of amastigote nests in the hearts of treated animals. However, no parasitological cures were observed in infected animals that received the bisphosphonate, probably due to the short treatment period. Taken together, these results indicate that Ris could be a useful lead compound for the development of new drugs effective against Chagas' disease.

Acute Disease↗