In vitro and in vivo trypanocidal activity of the ethyl esters of N-allyl and N-propyl oxamates using different Trypanosoma cruzi strains

The trypanocidal activity of N-allyl (NAOx) and N-propyl (NPOx) oxamates and that of the ethyl esters ofN-allyl (Et-NAOx) and N-propyl (Et-NPOx) oxamates were tested on cultured epimastigotes (in vitro) and murine trypanosomiasis (in vivo) using five different T. cruzi strains. NAOx and NPOx did not penetrate intact epimastigotes and therefore we were not able to detect any trypanocidal effect with these oxamates. Whereas the ethyl esters (Et-NAOx and Et-NPOx), acting as prodrugs, exhibited in vitro and in vivo trypanocidal activity on the five tested T. cruzi strains. On the contrary, when Nifurtimox and Benznidazole used as reference drugs were tested, we found that only three of the five tested T cruzi strains were affected, whereas the other two strains, Miguz and Compostela, were resistant to the in vitro and in vivo trypanocidal activity of these compounds.     

In vitro and in vivo trypanocidal activity of the ethyl esters of N-allyl and N-propyl oxamates using different Trypanosoma cruzi strains

The trypanocidal activity of N-allyl (NAOx) and N-propyl (NPOx) oxamates and that of the ethyl esters of N-allyl (Et-NAOx) and N-propyl (Et-NPOx) oxamates were tested on cultured epimastigotes (in vitro) and murine trypanosomiasis (in vivo) using five different T. cruzi strains. NAOx and NPOx did not penetrate intact epimastigotes and therefore we were not able to detect any trypanocidal effect with these oxamates. Whereas the ethyl esters (Et-NAOx and Et-NPOx), acting as prodrugs, exhibited in vitro and in vivo trypanocidal activity on the five tested T. cruzi strains. On the contrary, when Nifurtimox and Benznidazole used as reference drugs were tested, we found that only three of the five tested T. cruzi strains were affected, whereas the other two strains, Miguz and Compostela, were resistant to the in vitro and in vivo trypanocidal activity of these compounds.     

Inhibition of Trypanosoma cruzi alpha-hydroxyacid dehydrogenase-isozyme II by N-isopropyl oxamate and its effect on intact epimastigotes

The effect of N-isopropyl oxamate on the activity of alpha-hydroxyacid dehydrogenase-isozyme II (HADH-isozyme II) from Trypanosoma cruzi was investigated. The kinetic studies showed that this substance was a competitive inhibitor of this isozyme. The attachment of the nonpolar isopropylic branched chain to the nitrogen of oxamate increased 12-fold the affinity of N-isopropyl oxamate for the active site of T. cruzi HADH-isozyme II. N-isopropyl oxamate was a selective inhibitor of HADH-isozyme II, since other T. cruzi dehydrogenases were not inhibited by this substance. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with inhibitors of this isozyme. However, although it was not possible to detect any trypanocidal activity with N-isopropyl oxamate when the ethyl ester was tested as a possible trypanocidal prodrug, the expected trypanocidal effect was obtained, comparable to that obtained with nifurtimox and benznidazole.     

Inhibition of Trypanosoma cruzi α-Hydroxyacid Dehydrogenase-isozyme II by N-Isopropyl Oxamate and its Effect on Intact Epimastigotes

The effect of N-isopropyl oxamate on the activity of α-hydroxyacid dehydrogenase-isozyme II (HADH-isozyme II) from Trypanosoma cruzi was investigated. The kinetic studies showed that this substance was a competitive inhibitor of this isozyme. The attachment of the nonpolar isopropylic branched chain to the nitrogen of oxamate increased 12-fold the affinity of N-isopropyl oxamate for the active site of T. cruzi HADH-isozyme II. N-isopropyl oxamate was a selective inhibitor of HADH-isozyme II, since other T. cruzi dehydrogenases were not inhibited by this substance. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with inhibitors of this isozyme. However, although it was not possible to detect any trypanocidal activity with N-isopropyl oxamate when the ethyl ester was tested as a possible trypanocidal prodrug, the expected trypanocidal effect was obtained, comparable to that obtained with nifurtimox and benznidazole.     

Trypanocidal structure-activity relationship for cis- and trans-methylpluviatolide

The trypanocidal activity of racemic mixtures of cis- and trans-methylpluviatolides was evaluated in vitro against trypomastigote forms of two strains of Trypanosoma cruzi, and in the enzymatic assay of T. cruzi gGAPDH. The cytotoxicity of the compounds was assessed by the MTT method using LLC-MK2 cells. The effect of the compounds on peroxide and NO production were also investigated. The mixture of the trans stereoisomers displayed trypanocidal activity (IC50 approximately 89.3 microM). Therefore, it was separated by chiral HPLC, furnishing the (+) and (-)-enantiomers. Only the (-)-enantiomer was active against the parasite (IC50 approximately 18.7 microM). Despite being inactive, the (+)-enantiomer acted as an antagonistic competitor. Trans-methylpluviatolide displayed low toxicity for LLC-MK2 cells, with an IC50 of 6.53 mM. Furthermore, methylpluviatolide neither inhibited gGAPDH activity nor hindered peroxide and NO production at the evaluated concentrations.     

Polyamines with N-(3-phenylpropyl) substituents are effective competitive inhibitors of trypanothione reductase and trypanocidal agents

Several N-(3-phenylpropyl)-substituted spermidine and spermine derivatives were prepared and found to be potent competitive inhibitors of Trypanosoma cruzi trypanothione reductase (seven compounds with Ki values < 5 microM are described). The most effective inhibitor studied was compound 12 with a Ki value of 0.151 microM. Six of the compounds described are also effective trypanocides with IC50 values < 1 microM.     

Trypanothione reductase from Trypanosoma cruzi. Catalytic properties of the enzyme and inhibition studies with trypanocidal compounds

Trypanothione reductase of Trypanosoma cruzi is a key enzyme in the antioxidant metabolism of the parasite. Here we report on the enzymic and pharmacological properties of trypanothione reductase using glutathionylspermidine disulfide as a substrate. 1. Both pH optimum (7.5) and the ionic strength optimum (at 30 mM) are unusually narrow for this enzyme. 40 mM Hepes, 1 mM EDTA, pH 7.5 was chosen as a standard assay buffer because in this system the kcat/Km ratio had the highest values for both natural substrates, glutathionylspermidine disulfide (2.65 x 10(6) M-1 s-1) and trypanothione disulfide (4.63 x 10(6) M-1 s-1). 2. Using the standardized assay, trypanothione reductase and the phylogenetically related host enzyme, human glutathione reductase, were studied as targets of inhibitors. Both enzymes, in their NADPH-reduced forms, were irreversibly modified by the cytostatic agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Nifurtimox, the drug used in the treatment of Chagas' disease, is a stronger inhibitor of glutathione reductase (Ki = 40 microM) than of trypanothione reductase (IC50 = 200 microM). 3. Of the newly synthesized trypanocidal compounds [Henderson, G. B., Ulrich, P., Fairlamb, A. H., Rosenberg, I., Pereira, M., Sela, M. & Cerami, A. (1988) Proc. Natl Acad. Sci., 85, 5374-5378] a nitrofuran derivative, 2-(5-nitro-2-furanylmethylidene)-N,N'-[1,4-piperazinediylbis (1,3-propanediyl)]bishydrazinecarboximidamide tetrahydrobromide, was found to be a better inhibitor for trypanothione reductase (Ki = 0.5 microM) than for glutathione reductase (IC50 = 10 microM). A naphthoquinone derivative, 2,3-bis[3-(2-amidinohydrazono)-butyl]-1,4-naphthoquinone dihydrochloride, turned out to be both an inhibitor (IC50 = 1 microM) and an NADPH-oxidation-inducing substrate (Km = 14 microM). This effect was not observed with human glutathione reductase. Such compounds which lead to oxidative stress by more than one mechanism in the parasite are promising starting points for drug design based on the three-dimensional structures of glutathione and trypanothione reductases.     

Kinetics of gossypol inhibition of bovine lactate dehydrogenase X

Gossypol, a polyphenolic binaphthalene dialdehyde isolated from cotton meal is a potent inhibitor of lactate dehydrogenase-X purified from bovine testis. For the conversion of pyruvate to lactate the IC50 for gossypol is 200 microM for the reverse reaction the IC50 is 12 microM. Gossypol is a competitive inhibitor of NADH, Ki = 30 microM (Km = 17 microM), and NAD+, Ki = 6 microM (Km = 130 microM), and noncompetitive for pyruvate, Ki = 220 microM (Km = 224 microM), and lactate, Ki = 52 microM (Km = 5.6 mM).     

The trypanocidal effect of sesquiterpene lactones helenalin and mexicanin on cultured epimastigotes

Sesquiterpene lactones constitute a large group of biologically active compounds obtained from plants. The lactones, mexicanin (MXN) and helenalin (HLN), were reported recently as active against the infective form of Trypanosoma cruzi. In this work, we studied the effects of these compounds on the growth and viability of the noninfective epimastigote, to compare the sensitivity of the 2 stages and to characterize their actions. Both compounds were cytotoxic to the parasites, with HLN (inhibitory concentration 50% [IC50] 1.9 +/- 0.08 microM) more potent than MXN (IC50 3.8 +/- 0.19 microM) and the typanocidal drug, benznidazole (IC50 8.6 +/- 2.5 microM). The results showed that epimastigotes are less sensitive than trypomastigotes to the compounds. The trypanocidal effect of these lactones, irreversible after 12-hr exposure, was not reversed by the reducing agents dithiotreitol or beta-mercaptoethanol. Ultrastructurally, we observed cytoplasmic vacuolization and nuclear disorganization. Although concentrations between 0.5 and 1.5 microM of the drugs were not lethal to the parasites, epimastigotes became thinner and their nuclei became more pycnotic after exposure. We conclude that MXN and HLN are deleterious for T. cruzi epimastigotes and that their mechanism of action is different than that of the related lactone, dehydroleucodine.     

Synthesis and trypanocidal activity of 1,4-bis-(3,4,5-trimethoxy-phenyl)-1,4-butanediol and 1,4-bis-(3,4-dimethoxyphenyl)-1,4-butanediol

Chagas' disease is endemic in Central and South American countries. Specific chemotherapy with nifurtimox or benznidazole has been recommended for treatment of recent infection but they have limited efficacy. The natural products veraguensin (1) and grandisin (2) have shown potent in vitro activity against trypomastigote parasite (Y strain) with IC(50) 2.3 microM (1) and 3.7 microM (2). We report herein the synthesis and in vitro trypanocidal evaluation of symmetrical and unsymmetrical 1,4-diaryl-1,4-diol derivatives as potential trypanocidal analogs of natural compounds 1 and 2. Among the synthesized products, compounds 1,4-bis-(3,4,5-trimethoxyphenyl)-1,4-butanediol (6a) and 1,4-bis-(3,4-dimethoxyphenyl)-1,4-butanediol (6b) showed better activity against Trypanosoma cruzi trypomastigotes with IC(50) 100 and 105 microM (Y strain), respectively, and 110 microM (Bolivia strain) for both compounds. However, the most active compound of this series was 1,4-bis-(3,4-dimethoxyphenyl)butane-1,4-dione (7b) with IC(50) 10 and 200 microM against Y and Bolivia strains, respectively.     

Cysteine proteinase inhibitors kill cultured bloodstream forms of Trypanosoma brucei brucei

Trypanosoma brucei brucei is a causative agent of bovine trypanosomiasis (nagana), a disease of considerable economic significance in much of Africa. Here we report investigations on the effects of various irreversible cysteine proteinase inhibitors, including vinyl sulfones (VS), peptidyl chloromethylketones (CMK), diazomethylketones, and fluoromethyl ketones, on the major lysosomal cysteine proteinase (trypanopain-Tb) of T. b. brucei and on in vitro-cultured bloodstream forms of the parasite. Many of the tested inhibitors were trypanocidal at low micromolar concentrations. Methylpiperazine urea-Phe-homoPhe-VS was the most effective trypanocidal agent, killing 50% of test populations at a work ing concentration of 0.11 microM, while carbobenzoxy-Phe-Phe-CMK was the most trypanocidal of the methylketones with an IC50 of 3.6 microM. Labelling of live and lysed T. b. brucei with biotinylated inhibitor derivatives suggests that trypanopain-Tb is the likely intracellular target for these inhibitors. Kinetic analysis of the inhibition of purified trypanopain-Tb by the inhibitors showed that most had kass values in the 10(6) M-1 s-1 range. We conclude that cysteine proteinase inhibitors have potential as trypanocidal agents and that a major target of these compounds is the lysosomal enzyme trypanopain-Tb.     

Competitive inhibitors of rabbit hepatic microsomal steroid 12 alpha-hydroxylase

The sterols 7 alpha-hydroxycholest-4-en-3-one (I) and 5 alpha-cholestane-3 alpha,7 alpha-diol (II) are competitive inhibitors for rabbit hepatic microsomal preparations of steroid 12 alpha-hydroxylase with apparent Ki values of 56 and 93 microM, respectively. To ascertain the optimum structure for a substrate with maximal enzymic activity, nine sterols or steroidal acids containing the 7 alpha-hydroxy-4-en-3-one or 3 alpha,7 alpha-dihydroxy-5 alpha configuration were prepared and studied as inhibitors with enzyme preparations in the presence of NADPH, oxygen and appropriate cofactors. Although each of these compounds exhibited competitive inhibition, the best inhibitor for sterol (I) was 7 alpha,25-dihydroxycholest-4-en-3-one (IV) (Ki 36 microM). Steroidal acids (3-oxo-7 alpha-hydroxychol-4-enoic acid and 3-oxo-7 alpha-hydroxy-4-cholene-24-carboxylic acid) were poor inhibitors (Ki 1080 and 654 microM, respectively). For sterol (II) the best inhibitors were sterol (IV) (Ki 35 microM) and 5 alpha-cholestane-3 alpha,7 alpha,25-triol (VIII) (Ki 45 microM). The 12 alpha-hydroxylated products of sterols (I) and (IV) were less tightly bound to the enzyme (Ki 88 and 98 microM, respectively) in the presence of sterol (II). Allochenodeoxycholic acid (Ki 495 microM) was not a good inhibitor for sterol (II). 12 alpha-Hydroxylated products of sterols (IV) and (VIII) were isolated from larger scale incubations, separated by HPLC and identified by mass spectrometry.     

Iron-containing metallocenes as active site directed inhibitors of the proteinase that cleaves the NH2-terminal propeptides from type I procollagen

Derivatives of ferrocene (dicyclopentadienyliron) (Fc) were examined as active site directed inhibitors of type I procollagen N-proteinase, the enzyme that cleaves the NH2-terminal propeptides from type I procollagen. The compounds were shown here to be reversible, competitive inhibitors of the enzyme. The effectiveness of the Fc inhibitors varied with modification of the cyclopentadienyl (cp) rings. The monocarboxylic acid (I) and the 1,1'-dicarboxylic acid (II) derivatives of Fc inhibited 50% of the enzymic activity (I50) at concentrations of 1.0 and 0.5 mM, respectively. The Ki values were 0.3 mM for both I and II. Derivatization of the carbonyl alpha to the cp ring of compound I (FcCOCH2CH2COOH, III) increased the inhibitory activity (I50 = 0.100 mM; Ki = 0.065 mM). Removal of the carbonyl alpha to the cp ring of III did not improve inhibitory activity: FcCH2CH2COOH, I50 = 2 mM; FcCH = CHCOOH, I50 = 1.5 mM. The active inhibitory species apparently contained iron in the 3+ valence state since two ferrocenium derivatives were very effective inhibitors: ferrocenium tetrachloroferrate, IV (I50 = 0.030 mM; Ki = 0.004 mM), and carboxyferrocenium hexafluorophosphate, V (I50 less than 0.1 mM; Ki less than 0.05 mM). In addition, reduction of III with ascorbic acid abolished its inhibitory activity. Compounds I and III stabilized the enzyme to heat denaturation in the absence of exogenous calcium; compound IV did not stabilize the enzyme. Further observations indicated that Fc derivatives were specific inhibitors of procollagen N-proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of inositol-1,4-bisphosphate 4-phosphohydrolase from rat brain

Inositol-1,4-bisphosphate 4-phosphohydrolase (inositol-1,4-bisphosphatase) was highly purified from a soluble fraction of rat brain. On SDS-polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band and its molecular weight was estimated to be 42000. The isoelectric point of the enzyme was 4.3. The enzyme specifically hydrolyzed the 4-phosphomonoester linkage of inositol 1,4-bisphosphate. The Km value for inositol 1,4-bisphosphate was 30 microM, and it required Mg2+ for activity. Ca2+ was a competitive inhibitor with a Ki value of 60 microM as regards the Mg2+ binding. Li+, which is known to be a strong inhibitor of inositol 1-phosphatase (EC 3.1.3.25), inhibited the enzyme activity and caused 50% inhibition at a concentration of 1 mM (IC50 = 1 mM). Li+ was an uncompetitive inhibitor of substrate binding with a Ki value of 0.6 mM. These inhibitory parameters of Li+ were quite similar to those for inositol 1-phosphatase (IC50 = 1 mM, Ki = 0.3 mM). Thus, the effect of Li+ on decreasing the free inositol level with a subsequent decrease in agonist-sensitive phosphoinositides, is caused by its inhibition of multiple enzymes involved in conversion of inositol 1,4-bisphosphate to inositol.     

Tyrosinase inhibitory effects and inhibition mechanisms of nobiletin and hesperidin from citrus peel crude extracts

The inhibitory effects of nobiletin and hesperidin from citrus peel crude extracts on tyrosinase diphenolase activity are evaluated. IC50 of nobiletin and hesperidin is 1.49 mM and 16.08mM, respectively and their inhibition mechanism is competitive type with Ki = 2.82 mM and noncompetitive with Ki = 9.16 mM, respectively. Crude extracts from citrus peel (C. unshiu Marc.) were extracted with 95% ethanol and fractionated by petroleum ether (PCPE). The ethanol phase (ECPE) was further desorbed from macroporous adsorption resin (FGRE). Their IC50 values were 8.09 mg/mL, 7.53 mg/mL and 4.80 mg/mL, respectively. Their inhibition on melanogenesis in B16 mouse melanoma cells was also evaluated. FGRE showed a significant inhibition (42.5% at 31.25 microg/mL, p < 0.01) while hesperidin showed almost no inhibition. Nobiletin and PCPE give efficacious antiproliferation effects on B16 mouse melanoma cell with IC50 values 88.6 microM and 62.96 microg/mL, respectively, by the MTT test. Hesperidin and other crude extracts showed very low cytotoxity to the B16 cell.     

Kinetic characterization of the pyruvate and oxoglutarate dehydrogenase complexes from human heart

The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruvate, CoA and NAD, respectively. Acetyl-CoA produces a competitive inhibition of PDC (Ki = 35 microM) with respect to CoA, whereas NADH produces the same type of inhibition with respect to NAD (Ki = 36 microM). The oxoglutarate dehydrogenase complex (OGDC) from human heart has active sites with two different affinities for 2-oxoglutarate ([S]0.5 of 30 and 120 microM). ADP (1 mM) decreases the [S]0.5 values by a half. The inhibition of OGDC (Ki = 81 microM) by succinyl-CoA is of a competitive type with respect to CoA (Km = 2.5 microM), whereas that of NADH (Ki = 25 microM) is of a mixed type with respect to NAD (Km = 170 microM).     

Inhibition of soybean lipoxygenase and mouse skin tumor promotion by onion and garlic components

Onion and garlic essential oils were previously shown to inhibit mouse skin tumor promotion, as were the enzymes, lipoxygenase, and cyclooxygenase. In the present study, the inhibition of soybean lipoxygenase (EC 1.13.11.12) by onion and garlic components and related compounds was investigated. The IC50 values as well as the kinetic inhibition constants were determined for the most active compounds. Di-(1-propenyl) sulfide, an analog of the substrate moiety required for oxygenase action, was the only irreversible inhibitor observed with Ki = 59 microM and k3 = 0.53/min. Inhibition in the presence of substrate was uncompetitive at 88 and 132 microM linoleic acid with Ki = 129 microM. At 173 microM linoleic acid, however, inhibition was competitive with Ki = 66 microM. Dially trisulfide, allyl methyl trisulfide, and diallyl disulfide were competitive inhibitors, while 1-propenylpropyl sulfide and (E, Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide (ajoene) were mixed inhibitors. Nordihydroguaiaretic acid (NDGA), the most potent lipoxygenase inhibitor, was a competitive inhibitor with Ki = 0.29 microM. The results indicate a relative potency of inhibition for structural features in the following order: di(1-propenyl) sulfide greater than an alkenyl trisulfide greater than an alkenyl disulfide. Di(n-propyl) disulfide, a major onion oil component, inhibited neither lipoxygenase nor promotion. Di(1-propenyl) sulfide and ajoene inhibited both. This suggests that the inhibition of lipoxygenase may be involved in antipromotion.     

Aminothiol multidentate chelators against Chagas disease

Three compounds of an aminothiol family of iron chelators were examined for activity against trypomastigote (human) and epimastigote (vector) forms of Trypanosoma cruzi: tetraethyl and tetramethyl derivatives of ethane-1,2-bis (N-1-amino-3-ethyl butyl-3-thiol) (BAT-TE and BAT-TM) and N',N',N'-tris-(2-methyl-2-mercaptopriopyl)- 1,4,7-triazacyclonane (TAT). BAT-TE at 270 microM completely arrested the growth of trypomastigote forms in mouse blood stored at 4 degrees C for 24 h (IC(50) 67.7+/-7 microM), while BAT-TM arrested growth at 630 microM (IC(50) 158+/-17 microM) and TAT at concentrations >800 microM (IC(50) 415+/-55 microM). In T. cruzi-infected mice, BAT-TE and BAT-TM had no anti-trypanosomal activity in doses up to 200 mg/kg, whether the route of administration was intraperitoneal or oral, and TAT was not tested due to insufficient quantity. TAT had an IC(50) of 52+/-7 microM against the epimastigote forms while BAT-TM and BAT-TE were inhibitory only at concentrations >250 microM. The trypanocidal activity of BAT derivatives in blood stored at 4 degrees C makes these compounds potential candidates for the purpose of clearing donated blood of trypomastigotes.     

Effects of acyclovir and its metabolites on purine nucleoside phosphorylase

Acyclovir (9-(2-hydroxyethoxymethyl)guanine), the clinically useful antiherpetic agent, is an "acyclic" analogue of 2'-deoxyguanosine. Purine nucleoside phosphorylase partially purified from human erythrocytes did not catalyze detectable phosphorolysis of this drug or any of its metabolites (less than 0.07% of the rate with Guo). However, these compounds were competitive inhibitors of this enzyme with Ino as the variable substrate. Acyclovir per se was a relatively weak inhibitor. Its Ki value (91 microM) was much greater than that for its 8-hydroxy metabolite (Ki = 4.7 microM) but less than that for its carboxylic acid metabolite (9-carboxymethoxy-methylguanine) (K'i = 960 microM). The phosphorylated metabolites of acyclovir were more potent inhibitors than were their guanine nucleotide counterparts. At a phosphate concentration of 50 mM, the apparent Ki values for the mono- (120 microM), di- (0.51 microM), and tri (43 microM)-phosphate esters of acyclovir were 1/2, 1/1200, and 1/26 those for dGMP, dGDP, and dGTP, respectively. The concentration of phosphate did not markedly affect the Ki value of acyclovir but dramatically affected those of its phosphorylated metabolites and their nucleotide counterparts. Decreasing phosphate to a physiological concentration (1 mM) decreased the apparent Ki values for the mono-, di-, and triphosphate esters of acyclovir to 6.6, 0.0087, and 0.31 microM, respectively. Inhibition of the enzyme by acyclovir diphosphate was also influenced by pH. This metabolite of acyclovir is the most potent inhibitor of purine nucleoside phosphorylase reported to date. It has some features of a "multisubstrate" analogue inhibitor.     

In vitro anti-parasitic activity of Cyclosporin A analogs on Trypanosoma cruzi

Cyclosporin A (CsA) nonimmunosuppressive analogs were evaluated against Trypanosoma cruzi and on TcCyP19, a cyclophilin of 19 kDa. Two out of eight CsA analogs, H-7-94 and F-7-62 showed the best anti-parasitic effects on all in vitro assays. Their IC(50) values were 0.82 and 3.41 microM, respectively, compared to CsA IC(50) value 5.39 microM on epimastigote proliferation; and on trypomastigote lysis their IC(50) values were 0.97 and 2.66 microM compared to CsA IC(50) value 7.19 microM. H-7-94 and F-7-62 were also more effective than CsA in inhibiting trypomastigote infection. The enzymatic activity of TcCyP19 was inhibited by all CsA derivatives, suggesting this target is involved in the trypanocidal effects observed.