Phenotypic investigation of 4-nitrophenylacetyl- and 4-nitro-1H-imidazoyl-based compounds as antileishmanial agents.

Cutaneous leishmaniasis (CL) is a spectrum of clinical manifestations characterized by severe skin ulcerations that leads to social stigma. There are limited treatment options for CL, and the available drugs are becoming less efficacious due to drug resistance. More efficacious and safer antileishmanial drugs are needed. In this study, the biological effect of seven synthetically accessible nitroaromatic compounds was evaluated in vitro against amastigotes of Leishmania amazonensis, followed by in vivo evaluation using mouse models of CL. Two compounds (6 and 7) were active against amastigotes in vitro [half-maximal effective concentration (EC50): 4.57 ± 0.08 and 9.19 ± 0.68 µm, respectively], with selectivity indexes >50, and the other compounds were not selective. In vivo, compounds 6 and 7 (10 mg kg−1, twice a day for 14 days) failed to reduce skin lesion sizes and parasite loads determined by light microscopy of lesion imprints and quantitative polymerase chain reaction. Nevertheless, the in vitro leishmanicidal efficacy sustained their use as templates for nitroimidazole-based antileishmanial drug discovery programmes focusing on analogues with more suitable properties.

In Vitro and In Vivo Evaluation of an Adamantyl-Based Phenyl Sulfonyl Acetamide against Cutaneous Leishmaniasis Models of Leishmania amazonensis.

Phenotypic assay against Leishmania amazonensisin vitro and in vivo led to identification of an adamantyl-based phenyl sulfonyl acetamide (compound 1) as a promising antileishmanial agent. Compound 1 inhibited the growth of intracellular forms of L. amazonensis (50% inhibitory concentration [IC50] = 4 µM) and exhibited low toxicity to host cells, with a selectivity index (SI) of >125. However, in a cutaneous leishmaniasis (CL) mouse model, compound 1 did not reduce lesions and parasite load when administered as monotherapy or when given simultaneously with a suboptimal dose of miltefosine.

In Vitro, In Silico, and In Vivo Analyses of Novel Aromatic Amidines against Trypanosoma cruzi.

Five bis-arylimidamides were assayed as anti-Trypanosoma cruzi agents by in vitro, in silico, and in vivo approaches. None were considered to be pan-assay interference compounds. They had a favorable pharmacokinetic landscape and were active against trypomastigotes and intracellular forms, and in combination with benznidazole, they gave no interaction. The most selective agent (28SMB032) tested in vivo led to a 40% reduction in parasitemia (0.1 mg/kg of body weight/5 days intraperitoneally) but without mortality protection. In silico target fishing suggested DNA as the main target, but ultrastructural data did not match.

Gene expression profile in long-term non progressor HIV infected patients: in search of potential resistance factors.

Long-term non-progressors (LTNP) represent a minority (1-5%) of HIV-infected individuals characterized by documented infection for more than 7-10 years, a stable CD4+ T cell count over 500/mm(3) and low viremia in the absence of antiretroviral treatment. Protective factors described so far such as the CCR5delta32 deletion, protective HLA alleles, or defective viruses fail to fully explain the partial protection phenotype. The existence of additional host resistance mechanisms in LTNP patients was investigated here using a whole human genome microarray study comparing gene expression profiles of unstimulated peripheral blood mononuclear cells from LTNP patients, HIV-1 infected patients under antiretroviral therapy with CD4+ T cell levels above 500/mm(3) (ST), as well as healthy individuals. Genes that were up- or downregulated exclusively in LTNP, ST or in both groups in comparison to controls were identified and classified in functional categories using Ingenuity Pathway Analysis. ST and LTNP patient groups revealed distinct genetic profiles, regarding gene number in each category and up- or downregulation of specific genes, which could have a bearing on the outcome of each group. We selected some relevant genes to validate the differential expression using quantitative real-time qRT-PCR. Among others, we found several genes related to the canonical Wnt/beta-catenin signaling pathway. Our results identify new possible host genes and molecules that could be involved in the mechanisms leading to the slower progression to AIDS and sustained CD4+ T cell counts that is peculiar to LTNP patients.

Antinociceptive and anti-inflammatory effects of the monoterpene α,ß-epoxy-carvone in mice.

α,ß-Epoxy-carvone (EC) is a monoterpene found in the essential oils of many species of plants. It can also be obtained by organic synthesis. EC exerts a depressant effect on the central nervous system and is also known to have anticonvulsant, antimicrobial and antioxidant effects. The present study investigated the antinociceptive and anti-inflammatory effects of EC. Intraperitoneal administration of EC at doses of 100, 200 or 300 mg/kg promoted a significant antinociceptive effect, as shown in the acetic acid-induced abdominal writhing test. EC also provoked a reduction in formalin-induced nociception in the first (300 mg/kg) and second phases (200 and 300 mg/kg). In the hot-plate test, an increase in response latency was found at 30 min (at 100, 200 and 300 mg/kg), and at 60 and 120 min (at 300 mg/kg) following administration of EC, an effect that was reversed by naloxone. Intraperitoneal administration of EC (300 mg/kg) inhibited the increased vascular permeability provoked by acetic acid. These findings suggest that EC inhibited the acute inflammatory reaction, with a pronounced peripheral and central antinociceptive effect in mice that is probably associated with activation of the opioidergic system, which appears to play a role in the antinociceptive activity induced by EC.

Anticonvulsant activity of thymoquinone and its structural analogues

It has been widely reported that the crude oil of Nigella sativa L., Ranunculaceae, seeds and its major chemical component thymoquinone present anticonvulsant activity. These facts led us to verify the pharmacological potential of five structurally related para-benzoquinones on the pentylenotetrazol-induced seizures model, and establish the structural characteristics that influence the anticonvulsant activity of thymoquinone. The unsubstituted para-benzoquinone was the compound that exhibited the highest potency, while 2-methyl-p-benzoquinone was inactive. It was found that the presence of alkyl groups attached to the ring influence the pharmacological activity of the para-benzoquinones. In addition, the number, position, and size of these groups change the anticonvulsant potency of the compounds.

Anticonvulsant activity of the linalool enantiomers and racemate: investigation of chiral influence.

The anticonvulsant activity of the racemate and enantiomers of linalool have been evaluated. Pretreatment of the mice with (S)-(+)-, (R)-(-)- and rac-linalool increased the latency of convulsions significantly in the PTZ model. Only rac-linalool had an effect at the dose of 200 mg/kg. The enantiomers and their racemic mixture were effective in inhibiting the convulsant effect of PTZ at the dose of 300 mg/kg. The linalools presented pharmacological activity close to that of diazepam. In the PIC seizure model, (R)-(-)-linalool and rac-linalool presented activity at the dose of 200 mg/kg, but the rac-linalool was more potent than (R)-(-)-linalool; (S)-(+)-linalool had no effect at this dose. On the other hand, at the dose of 300 mg/kg this enantiomer was effective, but less potent than (R)-(-)-linalool and rac-linalool. In the MES model, linalools decreased the convulsion time of the mice in the doses of 200 and 300 mg/kg. rac-Linalool presented maximum effect at 300 mg/kg. Surprisingly, it increased significantly the convulsion time at a dose of 100 mg/kg. Using the parameter of tonic hind convulsions, only (R)-(-)-linalool produced protection from tonic extension at the dose of 200 mg/kg. When the (+)- and (-)-enantiomers, and rac-linalool were administered at the dose of 300 mg/kg they were also effective in preventing tonic convulsions induced by transcorneal electroshock in the animals. The (+)- and (-)-forms were equipotent and the rac-linalool was more effective than phenytoin. We have demonstrated that the two enantiomers have similar qualitative anticonvulsant activity, but show different potencies.

The role of conserved residues of chagasin in the inhibition of cysteine peptidases.

We have evaluated the roles of key amino acids to the action of the natural inhibitor chagasin of papain-family cysteine peptidases. A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10-100-fold increases in the K(i) for cruzipain of Trypanosoma cruzi. A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases. These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.

Role of the Trypanosoma brucei natural cysteine peptidase inhibitor ICP in differentiation and virulence.

ICP is a chagasin-family natural tight binding inhibitor of Clan CA, family C1 cysteine peptidases (CPs). We investigated the role of ICP in Trypanosoma brucei by generating bloodstream form ICP-deficient mutants (Deltaicp). A threefold increase in CP activity was detected in lysates of Deltaicp, which was restored to the levels in wild type parasites by re-expression of the gene in the null mutant. Deltaicp displayed slower growth in culture and increased resistance to a trypanocidal synthetic CP inhibitor. More efficient exchange of the variant surface glycoprotein (VSG) to procyclin during differentiation from bloodstream to procyclic form was observed in Deltaicp, a phenotype that was reversed in the presence of synthetic CP inhibitors. Furthermore, we showed that degradation of anti-VSG IgG is abolished when parasites are pretreated with synthetic CP inhibitors, and that parasites lacking ICP degrade IgG more efficiently than wild type. In addition, Deltaicp reached higher parasitemia than wild type parasites in infected mice, suggesting that ICP modulates parasite infectivity. Taken together, these data suggest that CPs of T. brucei bloodstream form play a role in surface coat exchange during differentiation, in the degradation of internalized IgG and in parasite infectivity, and that their function is regulated by ICP.

Chagasin, the endogenous cysteine-protease inhibitor of Trypanosoma cruzi, modulates parasite differentiation and invasion of mammalian cells.

Chagasin is a Trypanosoma cruzi protein that was recently characterized as a tight-binding inhibitor of papain-like cysteine proteases (CPs). Considering that parasite virulence and morphogenesis depend on the endogenous activity of lysosomal CPs of the cruzipain family, we sought to determine whether chagasin and cruzipain interact in the living cell. Ultrastructural studies showed that chagasin and cruzipain both localize to the Golgi complex and reservosomes (lysosome-like organelles), whereas free chagasin was found in small intracellular vesicles, suggesting that chagasin trafficking pathways might intersect with those of cruzipain. Taking advantage of the fact that sodium dodecyl sulphate and beta-mercaptoethanol prevent binding between the isolated proteins but do not dismantle preformed cruzipain-chagasin complexes, we obtained direct evidence that chagasin-cruzipain complexes are indeed formed in epimastigotes. Chagasin transfectants (fourfold increase in CP inhibitory activity) displayed low rates of differentiation (metacyclogenesis) and exhibited increased resistance to a synthetic CP inhibitor. These phenotypic changes were accompanied by a drastic reduction of soluble cruzipain activity and by upregulated secretion of cruzipain-chagasin molecular complexes. Analysis of six T. cruzi strains revealed that expression levels of cruzipain and chagasin are variable, but the molar ratios are fairly stable ( approximately 50:1) in most strains, with the exception of the G strain (5:1), which is poorly infective. On the same vein, we found that trypomastigotes overexpressing chagasin are less infective than wild-type parasites in vitro. The deficiency of chagasin overexpressers is caused by lower activity of membrane-associated CPs, because membranes recovered from wild-type trypomastigotes restored infectivity and this effect was nullified by the CP inhibitor E-64. In summary, our studies suggest that chagasin regulates the endogenous activity of CP, thus indirectly modulating proteolytic functions that are essential for parasite differentiation and invasion of mammalian cells.