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1.
Bioorg Med Chem Lett ; 22(19): 6256-60, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22926067

RESUMO

Cryptolepine derivatives containing alkyldiamine side-chains, 2, with potent inhibitory activity against Trypanosoma brucei brucei are reported. Compounds 2 showed improved activity and selectivity to T. b. brucei when compared to the lead compound. The most selective compound, 2k, presents a selectivity index value of 6200 and an IC(50) of 10nM against the parasite. These derivatives are also potent inhibitors of the trypanosome papain-like cysteine proteases cruzain, which could, at least in part, explain their antitrypanosomal activity. Overall, these compounds with good antitrypanosomal activity and selectivity provide an encouraging starting point for the rational design of new and effective antitrypanosomal agents.


Assuntos
Antiprotozoários/farmacologia , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Diaminas/farmacologia , Alcaloides Indólicos/farmacologia , Quinolinas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Antiprotozoários/síntese química , Antiprotozoários/química , Domínio Catalítico/efeitos dos fármacos , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Diaminas/síntese química , Diaminas/química , Relação Dose-Resposta a Droga , Alcaloides Indólicos/síntese química , Alcaloides Indólicos/química , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-Atividade
2.
PLoS Negl Trop Dis ; 5(9): e1337, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21980548

RESUMO

BACKGROUND: Skin invasion is the initial step in infection of the human host by schistosome blood flukes. Schistosome larvae have the remarkable ability to overcome the physical and biochemical barriers present in skin in the absence of any mechanical trauma. While a serine peptidase with activity against insoluble elastin appears to be essential for this process in one species of schistosomes, Schistosoma mansoni, it is unknown whether other schistosome species use the same peptidase to facilitate entry into their hosts. METHODS: Recent genome sequencing projects, together with a number of biochemical studies, identified alternative peptidases that Schistosoma japonicum or Trichobilharzia regenti could use to facilitate migration through skin. In this study, we used comparative proteomic analysis of human skin treated with purified cercarial elastase, the known invasive peptidase of S. mansoni, or S. mansoni cathespin B2, a close homolog of the putative invasive peptidase of S. japonicum, to identify substrates of either peptidase. Select skin proteins were then confirmed as substrates by in vitro digestion assays. CONCLUSIONS: This study demonstrates that an S. mansoni ortholog of the candidate invasive peptidase of S. japonicum and T. regenti, cathepsin B2, is capable of efficiently cleaving many of the same host skin substrates as the invasive serine peptidase of S. mansoni, cercarial elastase. At the same time, identification of unique substrates and the broader species specificity of cathepsin B2 suggest that the cercarial elastase gene family amplified as an adaptation of schistosomes to human hosts.


Assuntos
Peptídeo Hidrolases/metabolismo , Proteoma/análise , Schistosoma mansoni/enzimologia , Pele/química , Pele/parasitologia , Animais , Humanos , Larva/enzimologia , Larva/patogenicidade , Proteólise , Schistosoma japonicum/enzimologia , Schistosoma japonicum/patogenicidade , Schistosoma mansoni/patogenicidade
3.
Antiviral Res ; 92(2): 187-94, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21820471

RESUMO

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/normas , Ensaios de Triagem em Larga Escala/normas , Humanos , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Cultura de Vírus/métodos , Cultura de Vírus/normas
4.
PLoS Negl Trop Dis ; 5(5): e1023, 2011 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-21572521

RESUMO

The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas' disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique ∼ 2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts.


Assuntos
Antiparasitários/isolamento & purificação , Antiparasitários/metabolismo , Cisteína Proteases/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/metabolismo , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos
5.
PLoS Negl Trop Dis ; 4(9)2010 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-20856868

RESUMO

BACKGROUND: Cruzain, the major cysteine protease of Trypanosoma cruzi, is an essential enzyme for the parasite life cycle and has been validated as a viable target to treat Chagas' disease. As a proof-of-concept, K11777, a potent inhibitor of cruzain, was found to effectively eliminate T. cruzi infection and is currently a clinical candidate for treatment of Chagas' disease. METHODOLOGY/PRINCIPAL FINDINGS: WRR-483, an analog of K11777, was synthesized and evaluated as an inhibitor of cruzain and against T. cruzi proliferation in cell culture. This compound demonstrates good potency against cruzain with sensitivity to pH conditions and high efficacy in the cell culture assay. Furthermore, WRR-483 also eradicates parasite infection in a mouse model of acute Chagas' disease. To determine the atomic-level details of the inhibitor interacting with cruzain, a 1.5 A crystal structure of the protease in complex with WRR-483 was solved. The structure illustrates that WRR-483 binds covalently to the active site cysteine of the protease in a similar manner as other vinyl sulfone-based inhibitors. Details of the critical interactions within the specificity binding pocket are also reported. CONCLUSIONS: We demonstrate that WRR-483 is an effective cysteine protease inhibitor with trypanocidal activity in cell culture and animal model with comparable efficacy to K11777. Crystallographic evidence confirms that the mode of action is by targeting the active site of cruzain. Taken together, these results suggest that WRR-483 has potential to be developed as a treatment for Chagas' disease.


Assuntos
Antiprotozoários/administração & dosagem , Antiprotozoários/farmacologia , Doença de Chagas/tratamento farmacológico , Inibidores de Cisteína Proteinase/administração & dosagem , Inibidores de Cisteína Proteinase/farmacologia , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Sulfonas/administração & dosagem , Sulfonas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antiprotozoários/síntese química , Antiprotozoários/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/metabolismo , Dipeptídeos/administração & dosagem , Dipeptídeos/síntese química , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C3H , Modelos Moleculares , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Testes de Sensibilidade Parasitária , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sulfonas/síntese química , Sulfonas/metabolismo , Resultado do Tratamento , Compostos de Vinila/administração & dosagem , Compostos de Vinila/síntese química , Compostos de Vinila/metabolismo , Compostos de Vinila/farmacologia
6.
J Biol Chem ; 285(24): 18516-27, 2010 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-20378535

RESUMO

Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.


Assuntos
Amebíase/parasitologia , Cisteína Proteases/química , Cisteína Proteases/fisiologia , Entamoeba histolytica/metabolismo , Animais , Linhagem Celular Tumoral , Desenho de Fármacos , Regulação Enzimológica da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Massas/métodos , Camundongos , Camundongos Endogâmicos C3H , Peptídeo Hidrolases/química , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/química , Tiorredoxinas/química
7.
J Med Chem ; 52(22): 7192-210, 2009 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-19848405

RESUMO

Aza-peptide Michael acceptors and epoxides with the general structure of YCO-Ala-Ala-AAsn-trans-CH horizontal lineCHCOR and YCO-Ala-Ala-AAsn-EP-COR, respectively, are shown to be potent inhibitors of asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE), and the hard tick, Ixodes ricinus (IrAE). Structure-activity relationships (SARs) were determined for a set of 41 aza-peptide Michael acceptors and eight aza-peptide epoxides. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position, and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues at P2. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors, and for some of these compounds, second-order inhibiton rate constants are the fastest yet discovered. Given the central functions of these enzymes in both parasites, the data presented here may facilitate the eventual design of selective antiparasitic drugs.


Assuntos
Compostos Aza/química , Cisteína Endopeptidases/metabolismo , Compostos de Epóxi/química , Ixodes/enzimologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Schistosoma mansoni/enzimologia , Sequência de Aminoácidos , Animais , Disponibilidade Biológica , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Cinética , Oligopeptídeos/síntese química , Oligopeptídeos/farmacocinética
8.
J Biol Chem ; 284(38): 25697-703, 2009 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-19620707

RESUMO

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.


Assuntos
Antiparasitários/química , Cisteína Endopeptidases/química , Plasmodium falciparum/enzimologia , Inibidores de Proteases/química , Proteínas de Protozoários/química , Sulfonas/química , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/enzimologia , Animais , Antiparasitários/uso terapêutico , Doença de Chagas/tratamento farmacológico , Doença de Chagas/enzimologia , Cristalografia por Raios X , Desenho de Fármacos , Cinética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/enzimologia , Inibidores de Proteases/uso terapêutico , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Sulfonas/uso terapêutico , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/enzimologia
9.
PLoS Negl Trop Dis ; 3(6): e449, 2009 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-19488406

RESUMO

BACKGROUND: Blood flukes of the genus Schistosoma are platyhelminth parasites that infect 200 million people worldwide. Digestion of nutrients from the host bloodstream is essential for parasite development and reproduction. A network of proteolytic enzymes (proteases) facilitates hydrolysis of host hemoglobin and serum proteins. METHODOLOGY/PRINCIPAL FINDINGS: We identified a new cathepsin L termed SmCL3 using PCR strategies based on S. mansoni EST sequence data. An ortholog is present in Schistosoma japonicum. SmCL3 was heterologously expressed as an active enzyme in the yeast, Pichia pastoris. Recombinant SmCL3 has a broad pH activity range against peptidyl substrates and is inhibited by Clan CA protease inhibitors. Consistent with a function in degrading host proteins, SmCL3 hydrolyzes serum albumin and hemoglobin, is localized to the adult gastrodermis, and is expressed mainly in those life stages infecting the mammalian host. The predominant form of SmCL3 in the parasite exists as a zymogen, which is unusual for proteases. This zymogen includes an unusually long prodomain with alpha helical secondary structure motifs. The striking specificity of SmCL3 for amino acids with large aromatic side chains (Trp and Tyr) at the P2 substrate position, as determined with positional scanning-synthetic combinatorial library, is consistent with a molecular model that shows a large and deep S2 pocket. A sequence similarity network (SSN) view clusters SmCL3 and other cathepsins L in accordance with previous large-scale phylogenetic analyses that identify six super kingdoms. CONCLUSIONS/SIGNIFICANCE: SmCL3 is a gut-associated cathepsin L that may contribute to the network of proteases involved in degrading host blood proteins as nutrients. Furthermore, this enzyme exhibits some unusual sequence and biophysical features that may result in additional functions. The visualization of network inter-relationships among cathepsins L suggests that these enzymes are suitable 'marker sequences' for inclusion in future phylogenetic analyses.


Assuntos
Catepsina L/isolamento & purificação , Catepsina L/metabolismo , Schistosoma mansoni/enzimologia , Sequência de Aminoácidos , Animais , Catepsina L/genética , Cricetinae , DNA de Protozoário/genética , Estabilidade Enzimática , Etiquetas de Sequências Expressas , Trato Gastrointestinal/química , Expressão Gênica , Hemoglobinas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Pichia/genética , Reação em Cadeia da Polimerase/métodos , Inibidores de Proteases/farmacologia , Schistosoma mansoni/genética , Homologia de Sequência de Aminoácidos , Albumina Sérica/metabolismo , Caramujos , Especificidade por Substrato
10.
Mol Biochem Parasitol ; 164(1): 86-94, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19111576

RESUMO

Toxoplasma gondii is an obligate intracellular parasite of all vertebrates, including man. Successful invasion and replication requires the synchronized release of parasite proteins, many of which require proteolytic processing. Unlike most parasites, T. gondii has a limited number of Clan CA, family C1 cysteine proteinases with one cathepsin B (TgCPB), one cathepsin L (TgCPL) and three cathepsin Cs (TgCPC1, 2, 3). Previously, we characterized toxopain, the only cathepsin B enzyme, which localizes to the rhoptry organelle. Two cathepsin Cs are trafficked through dense granules to the parasitophorous vacuole where they degrade peptides. We now report the cloning, expression, and modeling of the sole cathepsin L gene and the identification of two new endogenous inhibitors. TgCPL differs from human cathepsin L with a pH optimum of 6.5 and its substrate preference for leucine (vs. phenylalanine) in the P2 position. This distinct preference is explained by homology modeling, which reveals a non-canonical aspartic acid (Asp 216) at the base of the predicted active site S2 pocket, which limits substrate access. To further our understanding of the regulation of cathepsins in T. gondii, we identified two genes encoding endogenous cysteine proteinase inhibitors (ICPs or toxostatins), which are active against both TgCPB and TgCPL in the nanomolar range. Over expression of toxostatin-1 significantly decreased overall cysteine proteinase activity in parasite lysates, but had no detectable effect on invasion or intracellular multiplication. These findings provide important insights into the proteolytic cascades of T. gondii and their endogenous control.


Assuntos
Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Catepsina L , Catepsinas/química , Catepsinas/genética , Clonagem Molecular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Toxoplasma/genética
11.
J Inorg Biochem ; 102(10): 1839-45, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18684510

RESUMO

The cysteine proteases of the trypanosomatid parasitic protozoa have been validated as targets for chemotherapy of Chagas' disease and leishmaniasis. Metal complexes of gold, platinum, iridium, palladium, rhodium and osmium have been reported to have activity against a variety of trypanosomatids, but the molecular target of these compounds has not been defined. The activity of gold(III) and palladium(II) cyclometallated complexes, and oxorhenium(V) complexes against mammalian and parasitic cysteine proteases was investigated. All gold(III) complexes (1-6) inhibited cathepsin B with IC(50) values in the range of 0.2-1.4 microM. Of the six palladium compounds, aceto[2,6-bis[(butylthio-kappa S)methyl]phenyl-kappa C]-, (SP-4-3)-palladium(II) (11) was the most potent inhibitor of cathepsin B with an IC(50) of 0.4 microM. A clear structure-activity relationship was observed with the oxorhenium(V) complexes with chloro[2,2'-(thio-kappa S)bis[ethanethiolato-kappa S)]] oxorhenium(V) (16) being the most potent inhibitor of cathepsin B with an IC(50) of 0.009 microM. Six complexes were further tested against the parasite cysteine proteases, cruzain from T. cruzi, and cpB from L. major; the most potent inhibitors were the two rhenium complexes (2(1H)-pyridinethionato-kappa S(2))[2,6-bis[(mercapto-kappa S)methyl]pyridine-kappa N(1)] oxorhenium(V) (15) and chloro[2,2'-(thio-kappa S)bis[ethanethiolato-kappa S)]] oxorhenium(V) (16). The compounds were also evaluated in assays for parasite growth. Two oxorhenium(V) compounds ((p-methoxyphenylthiolato-S)[2,6-bis[(mercapto-kappa S)methyl]pyridine-kappa N(1)] oxorhenium(V) (14) and (methanethiolato)[2,2'-(thio-kappa S)bis[ethanethiolato-kappa S)]] oxorhenium (V) (18)) and the palladium compound 11 inhibited T. cruzi intracellular growth, and compound 11 inhibited promastigote growth in three Leishmania species. In conclusion this preliminary data indicates that metal complexes targeted at parasite cysteine proteases show promise for the treatment of both Chagas' disease and leishmaniasis.


Assuntos
Catepsina B/metabolismo , Doença de Chagas/tratamento farmacológico , Inibidores de Cisteína Proteinase/farmacologia , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Metais/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma/efeitos dos fármacos , Animais , Catepsina B/antagonistas & inibidores , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/uso terapêutico , Humanos , Compostos Inorgânicos/química , Compostos Inorgânicos/farmacologia , Metais/uso terapêutico , Tripanossomicidas/uso terapêutico
12.
Bioorg Med Chem Lett ; 18(22): 5860-3, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18585034

RESUMO

The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.


Assuntos
Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Sulfonas/síntese química , Sulfonas/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Compostos de Vinila/síntese química , Compostos de Vinila/farmacologia , Animais , Cisteína Endopeptidases/efeitos dos fármacos , Inibidores de Cisteína Proteinase/química , Dipeptídeos/química , Desenho de Fármacos , Estrutura Molecular , Fenilalanina/análogos & derivados , Piperazinas , Sulfonas/química , Compostos de Tosil , Tripanossomicidas/química , Compostos de Vinila/química
13.
PLoS Negl Trop Dis ; 2(7): e262, 2008 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-18629379

RESUMO

BACKGROUND: During invasion of human skin by schistosome blood fluke larvae (cercariae), a multicellular organism breaches the epidermis, basement membrane, and dermal barriers of skin. To better understand the pathobiology of this initial event in schistosome infection, a proteome analysis of human skin was carried out following invasion by cercariae of Schistosoma mansoni. METHODOLOGY AND RESULTS: Human skin samples were exposed to cercariae for one-half hour to two hours. Controls were exposed to water used to collect cercariae in an identical manner, and punctured to simulate cercarial tunnels. Fluid from both control and experimental samples was analyzed by LC/MS/MS using a linear ion trap in "triple play" mode. The coexistence of proteins released by cercariae and host skin proteins from epidermis and basement membrane confirmed that cercarial tunnels in skin were sampled. Among the abundant proteins secreted by cercariae was the cercarial protease that has been implicated in degradation of host proteins, secreted proteins proposed to mediate immune invasion by larvae, and proteins implicated in protection of parasites against oxidative stress. Components of the schistosome surface tegument, previously identified with immune serum, were also released. Both lysis and apoptosis of epidermal cells took place during cercarial invasion of the epidermis. Components of lysed epidermal cells, including desmosome proteins which link cells in the stratum granulosum and stratum spinosum, were identified. While macrophage-derived proteins were present, no mast cell or lymphocyte cytokines were identified. There were, however, abundant immunoglobulins, complement factors, and serine protease inhibitors in skin. Control skin samples incubated with water for the same period as experimental samples ensured that invasion-related proteins and host protein fragments were not due to nonspecific degeneration of the skin samples. CONCLUSIONS: This analysis identified secreted proteins from invasive larvae that are released during invasion of human skin. Analysis of specific host proteins in skin invaded by cercariae served to highlight both the histolytic events facilitating cercarial invasion, and the host defenses that attempt to arrest or retard invasion. Proteins abundant in psoriatic skin or UV and heat-stressed skin were not abundant in skin invaded by cercariae, suggesting that results did not reflect general stress in the surgically removed skin specimen. Abundant immunoglobulins, complement factors, and serine protease inhibitors in skin form a biochemical barrier that complements the structural barrier of the epidermis, basement membrane, and dermis. The fragmentation of some of these host proteins suggests that breaching of host defenses by cercariae includes specific degradation of immunoglobulins and complement, and either degradation of, or overwhelming the host protease inhibitor repertoire.


Assuntos
Interações Hospedeiro-Parasita , Proteômica , Schistosoma mansoni/química , Esquistossomose mansoni/metabolismo , Pele/química , Animais , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Técnicas In Vitro , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/parasitologia , Pele/metabolismo , Pele/parasitologia , Caramujos
14.
J Med Chem ; 51(9): 2816-32, 2008 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-18416543

RESUMO

Aza-peptide Michael acceptors with the general structure of Cbz-Ala-Ala-AAsn- trans-CH=CHCOR are a new class of inhibitors specific for the asparaginyl endopeptidases (AE) (legumains). Structure-activity relationships (SARs) were characterized for a set of 31 aza-peptide Michael acceptors with AEs derived from three medically important parasites: the protist Trichomonas vaginalis, the hard tick Ixodes ricinus, and the flatworm Schistosoma mansoni. Despite arising from phylogenetically disparate organisms, all three AEs shared a remarkably similar SAR with lowest IC50 values extending into the picomolar range. The results suggest an evolutionary constraint on the topography of the prime side of the active site. SAR also revealed that esters in the P1' position are more potent than disubstituted amides and that monosubstituted amides and alkyl derivatives show little or no inhibition. The preferred P1' residues have aromatic substituents. Aza-asparaginyl Michael acceptors react with thiols, which provides insight into the mechanism of their inhibition of asparaginyl endopeptidases.


Assuntos
Compostos Aza/síntese química , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/síntese química , Ixodes/enzimologia , Oligopeptídeos/síntese química , Schistosoma mansoni/enzimologia , Trichomonas vaginalis/enzimologia , Animais , Compostos Aza/química , Biotina/química , Inibidores de Cisteína Proteinase/química , Ditiotreitol/química , Concentração Inibidora 50 , Oligopeptídeos/química , Relação Estrutura-Atividade , Compostos de Sulfidrila/química
15.
Biochimie ; 90(2): 345-58, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17936488

RESUMO

Schistosomes are parasitic platyhelminths (flatworms) of birds and mammals. As a parasitic disease of humans, schistosomiasis ranks second only to malaria in global importance. Schistosome larvae (cercariae) must invade and penetrate skin as an initial step to successful infection of the vertebrate host. Proteolytic enzymes secreted from the acetabular glands of cercariae contribute significantly to the invasion process. In this comparative study, we analyzed protease activities secreted by cercariae of Schistosoma mansoni, Schistosoma japonicum and Schistosomatium douthitti. Using protease-family specific, irreversible active-site probes, fluorogenic peptidyl substrates, immuno-histochemistry and high-resolution mass spectrometry, considerable species differences were noted in the quantity and character of proteases. Serine proteases, the most abundant enzymes secreted by S. mansoni cercariae, were not identified in S. japonicum. In contrast, the acetabular gland contents of S. japonicum cercariae had a 40-fold greater cathepsin B-like activity than those of S. mansoni. Based on the present data and previous reports, we propose that cysteine proteases represent an archetypal tool for tissue invasion among primitive metazoa and the use of serine proteases arose later in schistosome evolution. Computational analysis of serine protease phylogeny revealed an extraordinarily distant relationship between S. mansoni serine proteases and other members of the Clan PA family S1 proteases.


Assuntos
Catepsina B/metabolismo , Schistosoma japonicum/enzimologia , Schistosoma mansoni/enzimologia , Serina Endopeptidases/metabolismo , Animais , Catepsina B/química , Quimotripsina/metabolismo , Transferência Genética Horizontal , Larva/enzimologia , Larva/patogenicidade , Espectrometria de Massas , Filogenia , Proteômica , Schistosoma japonicum/crescimento & desenvolvimento , Schistosoma japonicum/patogenicidade , Schistosomatidae/enzimologia , Serina Endopeptidases/classificação , Serina Endopeptidases/genética , Especificidade da Espécie
16.
Bioorg Med Chem Lett ; 18(2): 624-8, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-18055204

RESUMO

A systematic study of P2 and P3 substitution in a series of vinyl sulfone cysteine protease inhibitors is described. The introduction of a methyl substituent in the P2 phenylalanine aryl ring had a favorable effect on protease inhibition and conferred modest selectivity for rhodesain over cruzain. Rhodesain selectivity could be enhanced further by combining these P2 modifications with certain P3 amide substituents.


Assuntos
Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Etilenos/química , Etilenos/farmacologia , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma/efeitos dos fármacos , Animais , Relação Estrutura-Atividade , Trypanosoma/enzimologia
18.
FEBS Lett ; 581(22): 4260-4, 2007 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-17707374

RESUMO

The physiological significance of the squamous cell carcinoma antigens 1 (SCCA1) and SCCA2, members of the ovalbumin serpin family, remains unresolved. In this study, we examined whether SCCA1 or SCCA2 inhibits protozoa- or helminth-derived cysteine proteases. SCCA1, but not SCCA2, potently inhibited the cysteine protease activities of CPB2.8 from Leishmania mexicana, cruzain from Trypanosoma cruzi, rhodesain from Trypanosoma brucei rhodesience, and cathepsin L2 from Fasciola hepatica. The inhibitory activities of SCCA1 were due to its resistance to cleavage by the cysteine proteases. The findings indicate that induction of cysteine protease inhibitors might be a novel defense mechanism against parasite development.


Assuntos
Antígenos de Neoplasias/metabolismo , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Parasitos/enzimologia , Serpinas/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/química , Catálise , Linhagem Celular , Humanos , Queratinócitos/metabolismo , Dados de Sequência Molecular , Serpinas/química , Relação Estrutura-Atividade
19.
Chem Biol Drug Des ; 67(5): 355-63, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16784460

RESUMO

Chemotherapy against human African trypanosomiasis relies on four drugs that cause frequent and occasionally severe side-effects. Because human African trypanosomiasis is a disease of poor people in Africa, the traditional market-driven pathways to drug development are not available. One potentially rapid and cost-effective approach to identifying and developing new trypanocidal drugs would be high throughput-screening of existing drugs already approved for other uses, as well as clinical candidates in late development. We have developed an ATP-bioluminescence assay that could be used to rapidly and efficiently screen compound libraries against trypanosomes in a high throughput-screening format to validate this notion. We screened a collection of 2160 FDA-approved drugs, bioactive compounds and natural products to identify hits that were cytotoxic to cultured Trypanosoma brucei at a concentration of 1 mum or less. This meant that any hit identified would be effective at a concentration readily achievable by standard drug dosing in humans. From the screen, 35 hits from seven different drug categories were identified. These included the two approved trypanocidal drugs, suramin and pentamidine, several other drugs suspected but never validated as trypanocidal, and 17 novel trypanocidal drugs.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Sensibilidade Parasitária/métodos , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Diazometano/análogos & derivados , Humanos , Medições Luminescentes/métodos , Estrutura Molecular , Tripanossomicidas/química , Tripanossomicidas/economia , Trypanosoma brucei brucei/metabolismo
20.
Bioorg Med Chem Lett ; 16(10): 2753-7, 2006 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-16516467

RESUMO

The protozoan parasite Trypanosoma brucei causes Human African trypanosomiasis, which is fatal if left untreated. Due to the toxicity of currently used drugs and emerging drug resistance, there is an urgent need for novel therapies. The major trypanosome papain-like cysteine protease expressed by the parasite (e.g., rhodesain in T. b. rhodesiense) is considered an important target for the development of new trypanocidal drugs. Series of aziridine-2,3-dicarboxylate-based cysteine protease inhibitors have been tested, most of them inhibiting rhodesain in the low micromolar range. Among these, only dibenzyl aziridine-2,3-dicarboxylates display trypanocidal activity being equipotent to the drug eflornithine. The Leu-Pro-containing aziridinyl tripeptides 13a-f are the most promising as they are not cytotoxic to macrophages up to concentrations of 125microM.


Assuntos
Antiprotozoários/farmacologia , Ácidos Carboxílicos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Antiprotozoários/química , Ácidos Carboxílicos/química , Inibidores de Cisteína Proteinase/química , Trypanosoma brucei brucei/enzimologia
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