Estudo da atividade antimelanoma de compostos 1,3,4- tiadiazóis mesoiônicos

RESUMO As características estruturais dos compostos mesoiônicos, os quais possuem regiões de carga positiva e negativa distintas associadas a um sistema poliheteroatômico, possibilitam que estes compostos atravessem membranas e interajam com biomoléculas. Diversas aplicações biológicas já foram descritas para os compostos mesoiônicos. Uma série de cloretos de 4-fenil-5-[2'-Y, 4'-X ou 4'-X-cinamoil]-1,3,4- tiadiazólio-2-fenilamina foi avaliada em células de melanoma murino B16-F10 in vitro e em tumores resultantes da implantação subcutánea das células B16-F10 em camundongos C57BL/6. Os compostos diferem apenas no substituinte do anel cinamoil (Ml-J, X = OH; MI-2,4diF, X = Y = F; MMF, X = F e Ml-D, X = N02). A exposição das células B16-F10 aos compostos Ml-D, MI-2,4diF and MI-4F, todos na mesma concentração micromolar (50 nmol.L"1) diminuiu a viabilidade celular para 8%, 50% e 22%, respectivamente, enquanto o Ml-J não apresentou nenhum efeito significante nas mesmas condições. Contudo, baixas doses de Ml-D, como 10nmol.L"1, foram suficientes para impedir o crescimento celular por 72 h, mas para o MI-2,4diF e o MI-4F o efeito na proliferação das células B16-F10 foi observado apenas na concentração de 25 nmol.L"1. Apesar de o MI-4F ter apresentado um efeito ligeiramente melhor que o Ml- 2,4diF in vitro, seus efeitos no crescimento tumoral in vivo não foram significativos. O Ml-D inibiu o crescimento tumoral em 77%. A maior efetividade do Ml-D comparado aos compostos Mi-2,4diF, MI-4F and Ml-J contra as células de melanoma B16-F10 é provavelmente devido ao seu grupo atrator de elétrons (N02), que intensifica a carga positiva do anel mesoiônico e permite uma extensiva conjugação da cadeia lateral com a porção exocíclica. Isto parece ser importante para a atividade antitumoral destes compostos contra o melanoma. O Ml-D foi comparado com os antineopiásicos fotemustina e dacarbazina, cujos efeitos em melanoma são reconhecidos. O Ml-D mostrou-se mais citotóxico para as células B16-F10 que a fotemustina nas mesmas condições experimentais in vitro. A atividade antitumoral in vivo das drogas foi avaliada em camundongos C57BL/6 portadores de melanoma subcutáneo (B16-F10). Os animais foram tratados por via intraperitoneal (i.p.) com Ml-D, fotemustina ou dacarbazina utilizando uma única dose de 57 |¿mol.kg"1, 24 h após a inoculação das células. No 17° dia, os tumores foram extraídos e seus pesos determinados. O Ml-D inibiu o crescimento tumoral em 85%. O crescimento das células B16-F10 in vivo respondeu à fotemustina, e uma inibição do crescimento tumoral de -50% foi observada. Para a dacarbazina, 57 ^mol.kg"1 causou uma reproduzível tendência à inibição tumoral de 27%. O Ml-D também foi eficiente em inibir em 64% o crescimento de um tumor já desenvolvido de 8 dias. Os efeitos do composto Ml-D foram avaliados em diferentes linhagens de melanoma humano. O Ml-D diminui in vitro a viabilidade e a proliferação das células MEL-85, SK-MEL, A2058 e MEWO, mostrando uma elevada atividade citotóxica. A adesão das células MEL-85 a diferentes componentes da matriz extracelular foi avaliada em presença do Ml-D. O composto reduziu a adesão à laminina, fibronectina e matrigel. A morfología e a organização dos filamentos de actina do citoesqueleto foram alteradas pelo tratamento com Ml-D. Estes resultados nas células de melanoma humano apontam o Ml-D como uma droga bastante promissora contra o melanoma, que é extremamente resistente à quimioterapia. Este é um estudo encorajador no que diz respeito à possibilidade do Ml-D se tornar uma nova ferramenta para o estudo e o tratamento do melanoma

A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization

Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. the venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (similar to 45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in similar to 45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a spreading factor. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao Araucaria-PR (FAP)Secretaria de Estado de Ciencia, Tecnologia e Ensino Superior do Parana (SETI)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Fed Parana, Dept Cell Biol, BR-80060000 Curitiba, Parana, BrazilUniv Fed Parana, Clin Hosp, Dept Clin Pathol, BR-80060000 Curitiba, Parana, BrazilUniv Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Ponta Grossa, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilWeb of Scienc

Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: Endothelial cell membrane phospholipids as targets for toxicity

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. the mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids. (C) 2010 Elsevier B.V. All rights reserved.Secretaria de Estado de CienciaTecnologia e Ensino Superior (SETI) do ParanaFundacao Araucaria-PRFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Fed Parana, Dept Cell Biol, BR-81531990 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniv Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Ponta Grossa, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilWeb of Scienc

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins

Estudo da atividade antimelanoma de compostos 1,3,4- tiadiazóis mesoiônicos

RESUMO As características estruturais dos compostos mesoiônicos, os quais possuem regiões de carga positiva e negativa distintas associadas a um sistema poliheteroatômico, possibilitam que estes compostos atravessem membranas e interajam com biomoléculas. Diversas aplicações biológicas já foram descritas para os compostos mesoiônicos. Uma série de cloretos de 4-fenil-5-[2'-Y, 4'-X ou 4'-X-cinamoil]-1,3,4- tiadiazólio-2-fenilamina foi avaliada em células de melanoma murino B16-F10 in vitro e em tumores resultantes da implantação subcutánea das células B16-F10 em camundongos C57BL/6. Os compostos diferem apenas no substituinte do anel cinamoil (Ml-J, X = OH; MI-2,4diF, X = Y = F; MMF, X = F e Ml-D, X = N02). A exposição das células B16-F10 aos compostos Ml-D, MI-2,4diF and MI-4F, todos na mesma concentração micromolar (50 nmol.L"1) diminuiu a viabilidade celular para 8%, 50% e 22%, respectivamente, enquanto o Ml-J não apresentou nenhum efeito significante nas mesmas condições. Contudo, baixas doses de Ml-D, como 10nmol.L"1, foram suficientes para impedir o crescimento celular por 72 h, mas para o MI-2,4diF e o MI-4F o efeito na proliferação das células B16-F10 foi observado apenas na concentração de 25 nmol.L"1. Apesar de o MI-4F ter apresentado um efeito ligeiramente melhor que o Ml- 2,4diF in vitro, seus efeitos no crescimento tumoral in vivo não foram significativos. O Ml-D inibiu o crescimento tumoral em 77%. A maior efetividade do Ml-D comparado aos compostos Mi-2,4diF, MI-4F and Ml-J contra as células de melanoma B16-F10 é provavelmente devido ao seu grupo atrator de elétrons (N02), que intensifica a carga positiva do anel mesoiônico e permite uma extensiva conjugação da cadeia lateral com a porção exocíclica. Isto parece ser importante para a atividade antitumoral destes compostos contra o melanoma. O Ml-D foi comparado com os antineopiásicos fotemustina e dacarbazina, cujos efeitos em melanoma são reconhecidos. O Ml-D mostrou-se mais citotóxico para as células B16-F10 que a fotemustina nas mesmas condições experimentais in vitro. A atividade antitumoral in vivo das drogas foi avaliada em camundongos C57BL/6 portadores de melanoma subcutáneo (B16-F10). Os animais foram tratados por via intraperitoneal (i.p.) com Ml-D, fotemustina ou dacarbazina utilizando uma única dose de 57 |¿mol.kg"1, 24 h após a inoculação das células. No 17° dia, os tumores foram extraídos e seus pesos determinados. O Ml-D inibiu o crescimento tumoral em 85%. O crescimento das células B16-F10 in vivo respondeu à fotemustina, e uma inibição do crescimento tumoral de -50% foi observada. Para a dacarbazina, 57 ^mol.kg"1 causou uma reproduzível tendência à inibição tumoral de 27%. O Ml-D também foi eficiente em inibir em 64% o crescimento de um tumor já desenvolvido de 8 dias. Os efeitos do composto Ml-D foram avaliados em diferentes linhagens de melanoma humano. O Ml-D diminui in vitro a viabilidade e a proliferação das células MEL-85, SK-MEL, A2058 e MEWO, mostrando uma elevada atividade citotóxica. A adesão das células MEL-85 a diferentes componentes da matriz extracelular foi avaliada em presença do Ml-D. O composto reduziu a adesão à laminina, fibronectina e matrigel. A morfología e a organização dos filamentos de actina do citoesqueleto foram alteradas pelo tratamento com Ml-D. Estes resultados nas células de melanoma humano apontam o Ml-D como uma droga bastante promissora contra o melanoma, que é extremamente resistente à quimioterapia. Este é um estudo encorajador no que diz respeito à possibilidade do Ml-D se tornar uma nova ferramenta para o estudo e o tratamento do melanoma

Acetylation of translationally controlled tumor protein promotes its degradation through chaperone-mediated autophagy

International audienc

Highlights in the knowledge of brown spider toxins

Abstract Brown spiders are venomous arthropods that use their venom for predation and defense. In humans, bites of these animals provoke injuries including dermonecrosis with gravitational spread of lesions, hematological abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins (4–40 kDa). Characterization of the venom confirmed the presence of three highly expressed protein classes: phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels of expression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins), hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D family (also known as the dermonecrotic toxin) is the most studied class of brown spider toxins. This class of toxins single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure. The functional role of the hyaluronidase toxin as a spreading factor in loxoscelism has also been demonstrated. However, the biological characterization of other toxins remains unclear and the mechanism by which Loxosceles toxins exert their noxious effects is yet to be fully elucidated. The aim of this review is to provide an insight into brown spider venom toxins and toxicology, including a description of historical data already available in the literature. In this review article, the identification processes of novel Loxosceles toxins by molecular biology and proteomic approaches, their biological characterization and structural description based on x-ray crystallography and putative biotechnological uses are described along with the future perspectives in this field

Brown Spider (Loxosceles) Venom Toxins as Potential Biotools for the Development of Novel Therapeutics

Brown spider envenomation results in dermonecrosis with gravitational spreading characterized by a marked inflammatory reaction and with lower prevalence of systemic manifestations such as renal failure and hematological disturbances. Several toxins make up the venom of these species, and they are mainly peptides and proteins ranging from 5–40 kDa. The venoms have three major families of toxins: phospholipases-D, astacin-like metalloproteases, and the inhibitor cystine knot (ICK) peptides. Serine proteases, serpins, hyaluronidases, venom allergens, and a translationally controlled tumor protein (TCTP) are also present. Toxins hold essential biological properties that enable interactions with a range of distinct molecular targets. Therefore, the application of toxins as research tools and clinical products motivates repurposing their uses of interest. This review aims to discuss possibilities for brown spider venom toxins as putative models for designing molecules likely for therapeutics based on the status quo of brown spider venoms. Herein, we explore new possibilities for the venom components in the context of their biochemical and biological features, likewise their cellular targets, three-dimensional structures, and mechanisms of action

Biological and structural comparison of recombinant phospholipase D toxins from Loxosceles intermedia (brown spider) venom

The clinical features of brown spider bites are the appearance of necrotic skin lesions, which can also be accompanied by systemic involvement, including weakness, vomiting, fever, convulsions, disseminated intravascular coagulation, intravascular hemolysis and renal disturbances. Severe systemic loxoscelism is much less common than the cutaneous form, but it may be the cause of clinical complications and even death following envenomation. Here, by using three recombinant dermonecrotic toxins, LiRecDT1, LiRecDT2 and LiRecDT3 (the major toxins found in the venom), we report the biological, immunological and structural differences for these members of this toxin family. Purified toxins evoked similar inflammatory reactions following injections into rabbit skin. Recombinant toxin treatments of MDCK cells with LiRecDT1 and LiRecDT2 changed cell viability, as evaluated by neutral red uptake and assessment of cell morphology through inverted microscopy, whereas LiRecDT3 caused only residual activity. Differences in cell cytotoxicity triggered by recombinant toxins were confirmed through a human red blood lysis assay, during which LiRecDT1 and LiRecDT2 caused a high degree of hemolysis compared to LiRecDT3, which induced only a small hemolytic effect. Additionally, biological differences for recombinant toxins were corroborated through mice lethality experiments, which showed animal mortality after LiRecDT1 and LiRecDT2 treatments, but an absence of lethality following LiRecDT3 exposure. Moreover, in experiments for edema, both the LiRecDT1 and the LiRecDT2 toxins evoked similar results, causing edema following toxin exposure, whereas LiRecDT3 caused only residual effects. Characterization of antigenic cross-reactivity using sera against crude venom toxins by immunoWestern blotting and immunodot blotting with recombinant LiRecDT1, LiRecDT2 and LiRecDT3 compared among themselves pointed to a higher cross-reactivity for LiRecDT1 compared to LiRecDT2 and LiRecDT3, corroborating structural and antigenic differences for these three toxins. Finally, evidence for structural differences among the recombinant toxins was strengthened by circular dichroism spectra, which suggested that the toxins were folded, and not aggregated or denatured proteins. (c) 2007 Elsevier B.V. All rights reserved.Univ Fed Parana, Dept Cell Biol, BR-81531990 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniv Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Med, São Paulo, BrazilUniv Fed Parana, Dept Basic Pathol, BR-81531990 Curitiba, Parana, BrazilUniv Fed Minas Gerais, Dept Biochem & Immunol, Belo Horizonte, MG, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, São Paulo, BrazilWeb of Scienc

Brown Spider (Loxosceles) Venom Toxins as Potential Biotools for the Development of Novel Therapeutics

Brown spider envenomation results in dermonecrosis with gravitational spreading characterized by a marked inflammatory reaction and with lower prevalence of systemic manifestations such as renal failure and hematological disturbances. Several toxins make up the venom of these species, and they are mainly peptides and proteins ranging from 5-40 kDa. The venoms have three major families of toxins: phospholipases-D, astacin-like metalloproteases, and the inhibitor cystine knot (ICK) peptides. Serine proteases, serpins, hyaluronidases, venom allergens, and a translationally controlled tumor protein (TCTP) are also present. Toxins hold essential biological properties that enable interactions with a range of distinct molecular targets. Therefore, the application of toxins as research tools and clinical products motivates repurposing their uses of interest. This review aims to discuss possibilities for brown spider venom toxins as putative models for designing molecules likely for therapeutics based on the status quo of brown spider venoms. Herein, we explore new possibilities for the venom components in the context of their biochemical and biological features, likewise their cellular targets, three-dimensional structures, and mechanisms of action