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- ItemSomente MetadadadosAvaliação de peptídeos sintéticos selecionados por phage display para o sorodiagnóstico da Paracoccidioidomicose(Universidade Federal de São Paulo, 2017-06-29) Portes, Leticia da Silva [UNIFESP]; Xander, Patricia [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Paraccocidioidomycosis (PCM) is a systemic granulomatous disease caused by the thermo-dimorphic fungus from complex Paraccocidioides. The disease diagnosis uses both direct detection of the fungus in biological samples and serological tests, which have also important role to follow disease progression and response to treatment. Currently, the use of synthetic peptides in the serological diagnosis of infectious diseases may represents a valuable strategy since, in comparison with natural or recombinant antigens, assays using these molecules are cheaper, simple, reproducible, and in some cases more specific and sensitive. Thus, the aim of this study was to screen and to identify peptides reactive against sera from patients with PCM by using Phage Display approach. First, a selection using immunoglobulins purified from sera of individuals without PCM (normal) were performed to eliminate phages clones not specific to the fungus. Then, positive selection was carried out against immunoglobulins purified from pool of sera from patients with PCM. The binding-phages were sequenced and tested in a binding assay to evaluate the interaction between the selected phages and the sera of normal individuals and patients with PCM. LP2 and LP15 samples showed higher affinity with sera of patients with PCM, compared to normal sera. The synthetic peptides were tested by ELISA and the LP15 peptide was recognized by sera of patients with PCM. The detection was higher when we used a mixture of LP15 and P2 peptide (mimetope of gp75 of P. brasiliensis). Thus, the Phage Display technology were useful to identify peptides with potential applications in PCM serodiagnosis.
- ItemAcesso aberto (Open Access)Caracterização de marcadores de virulência em Paracoccidioides brasiliensis(Universidade Federal de São Paulo (UNIFESP), 2009-06-24) Kioshima, Érika Seki [UNIFESP]; Lopes, Jose Daniel [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Paracoccidioidomycosis (PCM) is a human systemic granulomatous disease, prevalent in South America, caused by a thermodimorphic fungus, Paracoccidioides brasiliensis. This fungus presents complex antigenic structure and some of these components have been related with its pathogenicity, of which little is known. The virulence recovery of isolates by passage in vivo was performed in our laboratory. Attenuated and virulent Pb18 isolates were analyzed from various angles to confirm this change. The results of the survival curve, the number of CFUs and histology, showed clear differences in pathogenicity pattern of these isolates. Other features were also evaluated as morphology, growth curve and cell ultrastructure. Analysis of differential gene expression profile showed positive regulation genes related to metabolisms of proteins, lipids and amino acids. Some molecules, previously described as putative virulence factors, were positive regulated, among which calmodulin, kex-like protein and Hsp70. However, the number of defined virulence factors for dimorphic fungal pathogens, up to now, is relatively small. Several techniques have unsuccessfully been employed to characterize these elusive antigenic structures. Using phage display methodology, three peptide-displaying phages that bound preferentially to virulent isolates of P. brasiliensis were selected. By binding assay, p04 phage distinguished predefined degrees of virulence of isolates. Using confocal microscopy, the homologue synthetic peptide (pep04), labeled with 6-FAM, was internalized by only virulent isolate yeast cells. Sequential optical section imaging indicated that the labeling was within the intracellular milieu and frequently close or overlapping DAPI staining. These results showed that both, phage p04 and pep04, can be considered as biomarkers of virulence in PCM since both bound to virulent P. brasiliensis. To evaluate the consequences of interactions between the biomarkers and fungal cells, in vitro and in vivo experiments were performed. The latter demonstrated that p04 phage was sufficient to prevent the implantation of the fungus in the lungs and its migration to spleen and liver. In addition, this phage reduced colony-forming units in the lungs of mice infected with P. brasiliensis as compared to controls. In vitro experiments showed that pep04 exhibited fungicidal activity only against virulent P. brasiliensis, leaving unaltered the growth of the attenuated isolate. Therefore, these biomarkers may be useful tools for prognosis in PCM and may be possibly used in the routine clinical practice as therapeutic drug adjuvants.
- ItemEmbargoConstrução de bibliotecas de inibidores de serinoproteases em sistema phage display: seleção de inibidores para enzimas de flavivírus e do mosquito Aedes Aegypti(Universidade Federal de São Paulo, 2023-02-09) Manzato, Verônica de Moraes [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Torquato, Ricardo José Soares [UNIFESP]; http://lattes.cnpq.br/3995278540776284; http://lattes.cnpq.br/1168789309568199; http://lattes.cnpq.br/3139881734780671Introdução: No Brasil, ocorrem, de forma endêmica, arboviroses como Dengue, Zika, Chikungunya e Febre amarela, que possuem um vetor comum, o mosquito Aedes aegypti. As enzimas NS2B-NS3 dos flavivírus e as tripsinas nos Aedes aegypti desempenham funções essenciais no ciclo viral e na digestão de Aedes spp, respectivamente, o que justifica a busca por inibidores dessas enzimas como opções para romper esses ciclos. Nesse contexto, a técnica phage display tem sido uma aliada. Os inibidores Boophilina domínio 1 (D1) e Sunflower trypsin inhibitor (SFTI), são representantes das famílias de inibidores Kunitz-BPTI (I2) e sunflower cyclic trypsin inhibitor (família I12), respectivamente, e inibem serinoproteases na ordem de nano molar. Objetivo: Selecionar através da técnica de phage display, inibidores das bibliotecas BoophilinaD1 e SFTI, com a utilização de 3 alvos (proteases ZIKV, DENV2 e tripsina5607). Seguido pela caracterização cinética e in silico dos mutantes mais frequentes. Métodos: As proteases DENV2Pro e tripsina5607, foram expressas em bactéria E coli SHuffleT7 e purificadas por afinidade. As bibliotecas de inibidores foram sintetizadas utilizando técnicas moleculares de PCR, incluindo o plasmídeo pCANTAB5E e bactérias E. coli TG1. As seleções foram realizadas para enzimas do vírus ZIKVPro, DENV2Pro e do mosquito vetor tripsina5607. Os mutantes de BoophilinaD1 mais frequentes, selecionados para a enzima do ZIKVPro foram produzidos e utilizados nos ensaios cinéticos para determinação do perfil inibitório para as enzimas (ZIKVPro e DENV2Pro), utilizados também na modelagem, docking molecular e refinamento para as enzimas de flavivírus, via MODELLER, ClusPro e RosettaDock, respectivamente. Resultados: As enzimas alvo purificadas apresentaram bandas predominantes e ativas em torno de 28 e 26 kDa, para DENV2Pro e tripsina5607, respectivamente. A ZIKVPro usada nas seleções foi gentilmente cedida por Dr. Martin Wurtele. Os títulos das bibliotecas utilizadas nos processos de seleção foram de 2,9 x 106 CFU e 2,1 x 105 CFU para BoophilinaD1 e SFTI, respectivamente. Dentre os inibidores BophD1 mut selecionados, 76,5% apresentam aminoácido básico Arg ou Lys na posição P1 e 70,6% e 58,8% Ala na posição P1’e P4’, respectivamente. O inibidor BoophD1 WT e os BoophD1 mut12 e mut14 apresentam Ki entre 1 e 2 nM para tripsina bovina e aproximadamente 100 e 300 nM para as enzimas ZIKVPro e DENV2Pro, respectivamente. As análises in silico de docking molecular indicam diferenças de interação dos mutantes com as enzimas. Os mutantes da biblioteca SFTI selecionados para os três alvos são, em sua maioria, compartilhados e sem consenso. Subtraindo-se os ligantes inespecíficos, destaca-se as sequências (P1-P4’) ARQLL, KSQWD e LSRHP por serem representativas para ZIKVPro, DENV2Pro e tripsina5607, respectivamente. Esses SFTIs mutantes foram sintetizados e serão utilizados futuramente nos ensaios cinéticos para caracterização inibitória com diferentes alvos. Discussão: As enzimas proteolíticas desempenham papéis importantes em diversas funções, no caso das DENV2Pro e ZIKVPro, estão relacionadas com a clivagem da poliproteína e no caso da tripsina5607, relacionada a digestão de proteínas ingeridas pela larva de Ae. aegypti. Obtê-las de forma recombinante, solúvel e ativa é importante para aprofundar nossos conhecimentos sobre elas e fornecem informações para o desenvolvimento de inibidores com alta afinidade, podendo ser um inibidor específico ou pan-inibidores (para diferentes flavivírus). Conclusão: Este trabalho traz dados importantes sobre a busca de inibidores para as proteases de flavivírus e de mosquito utilizando bibliotecas de inibidores em sistema phage display. Inibidores para ZIKVPro foram selecionados por phage display e apresentaram constantes de inibição em 10-7 M assim como para a DENV2Pro, estes resultados sugerem que os inibidores selecionados são candidatos a pan-inibidores para proteases de flavivírus.
- ItemAcesso aberto (Open Access)Efeito do peptídeo ligante de heparam sulfato na progressão tumoral(Universidade Federal de São Paulo (UNIFESP), 2018-02-22) Melo, Carina Mucciolo [UNIFESP]; Pinhal, Maria Aparecida Da Silva [UNIFESP]; http://lattes.cnpq.br/7511274763693292; http://lattes.cnpq.br/1814847465463146; Universidade Federal de São Paulo (UNIFESP)O heparam sulfato (HS) é amplamente distribuído e conservado evolutivamente, um fato que remonta à importância vital desta molécula no desenvolvimento celular e funções como proliferação, migração, adesão e diferenciação celular. Os proteoglicanos de heparam sulfato podem atuar como co-receptores, interagindo com numerosos fatores de crescimento, fatores angiogênicos e citocinas e modulam a patogênese de muitas doenças, incluindo a carcinogênese e inflamação. O objetivo deste estudo foi avaliar o peptídeo ligante de HS e verificar o efeitosobre a progressão tumoral. O peptídeo ligante de HS foi selecionado usando a técnica de Phage display. A ligação do peptídeo ao HS foi avaliado in vitro, utilizando a emissão de triptofano e a análise de FRET (transferência de energia ressonante por fluorescência), enquanto, o ensaio in vivo foi determinado por análise em microscopia confocal time-lapse. e imunomarcação dos embriões de Danio rerio. Os resultados mostraram que o peptídeo ligante de HS interage especificamente com HS/heparina. Os ensaios In vivo (modelo zebrafish), ex vivo (ensaio de aorta de camundongos) e in vitro, utilizando células endoteliais de veia umbilical humana (HUVEC), evidenciaram que o peptídeo ligante de HS é capaz de inibir a angiogênese. O teste de proliferação celular mostrou que FGF-2, mas não o VEGF-A, está envolvido com a resposta do peptídeo ligante de HS no processo angiogênico. A migração de células humanas tumorais de mama triplo negativas, MDA-MB-231, diminuiu na presença do peptídeo ligante de HS. No entanto, a linhagem MDA-MB-231-BR, que apresentam alta capacidade de desenvolver metástase cerebral, quando injetadas em embriões de galinha em presença do peptídeo ligante de HS, mostrou que tal peptídeo não foi capaz de inibir extravasão e colonização metastática. As células de câncer de mama triplo negativas, derivadas da ressecção cirúrgica de paciente com câncer de mama (pacient derived xenotransplant, PDX) foram plaqueadas em cultura 3D. A proliferação e a viabilidade de tais células de câncer de mama PDX não foram afetadas pelo peptídeo ligante de HS. As células PDX estavelmente transduzidas com proteína fluorescente vermelha (RFP), foram injetadas no saco de vitelínico de embriões de zebrafish e tratados com peptídeo ligante de HS. O peptídeo diminuiu a progressão do tumor in vivo e aumentou significativamente a sobrevida dos animais. Os resultados combinados permitem concluir que o peptídeo se liga ao HS e inibe a angiogênese, possivelmente por sinalização mediada pelo FGF-2. Ainda, a inibição da angiogênese parece ser suficiente para diminuir o desenvolvimento do tumor nos ensaios in vivo. O peptídeo ligante de HS pode ser útil como um potencial composto anti-neoplásico.
- ItemAcesso aberto (Open Access)Lepstospira interrogans shotgun phage display identified LigB as a heparin-binding protein(Elsevier B.V., 2012-11-02) Ching Ching, Ana Tung; Favaro, Regiane Degan; Lima, Swiany Silveira; Muniz Chaves, Agtha de Alencar; Lima, Marcelo Andrade de [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Estima Abreu, Patricia A.; Ho, Paulo Lee; Inst Butantan; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)LigB is an adhesin from pathogenic Leptospira that is able to bind to extracellular matrix and is considered a virulence factor. A shotgun phage display genomic library was constructed and used for panning against Heparan Sulfate Proteoglycan (HSPG). A phage clone encoding part of LigB protein was selected in panning experiments and showed specific binding to heparin. To validate the selected clone, fragments of LigB were produced as recombinant proteins and showed affinity to heparin and to mammalian cells. Heparin was also able to reduce the binding of rLB-Ct to mammalian cells. Our data suggests that the glycosaminoglycan moiety of the HSPG is responsible for its binding and could mediate the attachment of the recombinant protein rLB-Ct. Thus, heparin may act as a receptor for Leptospira to colonize and to invade the host tissue. (C) 2012 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosA novel melanoma-targeting peptide screened by phage display exhibits antitumor activity(Springer, 2010-12-01) Matsuo, Alisson L. [UNIFESP]; Tanaka, Aparecida S. [UNIFESP]; Juliano, Maria A. [UNIFESP]; Rodrigues, Elaine G. [UNIFESP]; Travassos, Luiz R. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Peptide display on the phage surface has been widely used to identify specific peptides targeting several in vivo and in vitro tumor cells and the tumor vasculature, playing a role in the discovery of bioactive antitumor agents. Bioactive peptides have been selected to target important tumor receptors or apoptosis-associated molecules such as p53. Presently, we attempted to identify potentially antitumor bioactive molecules using the whole cell surface as the recognizable static matrix. Such methodology could be advantageous in cancer therapy because it does not require previous characterization of target molecules. Using a C7C phage display library, we screened for peptides binding to the B16F10-Nex2 melanoma cell surface after pre-absorption on melan-A lineage. After a few rounds of enrichment, 50 phages were randomly selected, amplified, and tested for inhibition of tumor cell proliferation. Seven were active, and the corresponding peptide of each phage was chemically synthesized in the cyclic form and tested in vitro. Three peptides were able to preferentially inhibit the melanoma lineage. A unique peptide, [-CSSRTMHHC-], exhibited in vivo antitumor inhibitory activity against a subcutaneous melanoma challenge, rendering 60% of mice without tumor growth. Further, this peptide also markedly inhibited in vitro and in vivo the tumor cell invasion and cell-to-cell adhesiveness in vitro. This is the first report on a bioactive peptide derived from a C7C library active against whole melanoma cells in vitro and in vivo.
- ItemSomente MetadadadosProkineticin receptor identified by phage display is an entry receptor for Trypanosoma cruzi into mammalian cells(Springer, 2015-01-01) Khusal, Ketna Guilhermino; Tonelli, Renata Rosito [UNIFESP]; Mattos, Eliciane Cevolani; Soares, Chrislaine Oliveira; Di Genova, Bruno Martorelli [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Urias, Úrsula; Colli, Walter; Alves, Maria Julia Manso; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Trypanosoma cruzi trypomastigotes invade a great variety of mammalian cells, with several molecules being implicated in this complex event. Herein, the sequence GGIALAG present in prokineticin-2 receptor (PKR2), selected by phage display technology, is described as a new T. cruzi receptor for the Tc85 group of glycoproteins belonging to the gp85/TS superfamily and involved in cellular invasion of mammalian hosts. This finding is confirmed by the inhibitory activity of MCF10-A (human mammary) cell invasion by T. cruzi either by anti-PKR2 antibodies (77 %) or GGIALAG-synthetic peptide (42 %). Furthermore, interference RNA (iRNA) inhibition of PKR2 expression in MCF10-A cells reduces T. cruzi invasion by 50 %. the binding site of Tc85 to PKR2 was localized at the C-terminal end of the molecule, upstream of the conserved FLY sequence, previously implicated in parasite cell invasion. PKR2, a receptor formed by seven membrane-spanning alpha-helical segments, is mainly present in the central nervous system, peripheral organs, and mature blood cells. Due to its wide distribution, PKR2 could be a suitable receptor for T. cruzi natural infection, contributing to the parasite dissemination throughout the mammalian organism. These findings augment the number and diversity of possible in vivo receptors for T. cruzi and reassure the multiplicity of Tc85 binding sites to mammalian hosts.
- ItemSomente MetadadadosSelective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display(Elsevier B.V., 2013-01-01) Soares, Tatiane Sanches [UNIFESP]; Soares Torquato, Ricardo Jose [UNIFESP]; Alves Lemos, Francisco Jose; Tanaka, Aparecida Sadae [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Estadual Norte FluminenseDengue is a serious disease transmitted by the mosquito Aedes aegypti during blood meal feeding. It is estimated that the dengue virus is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies have been based on controlling the vector, Ae. aegypti, using insecticide, but the emergence of resistance poses new challenges. the aim of this study was the identification of specific protease inhibitors of the digestive enzymes from Ae. aegypti larvae, which may serve as a prospective alternative biocontrol method. High affinity protein inhibitors were selected by all of the digestive serine proteases of the 4th instar larval midgut, and the specificity of these inhibitors was characterized. These inhibitors were obtained from a phage library displaying variants of HiTI, a trypsin inhibitor from Haematobia irritans, that are mutated in the reactive loop (P1 P4'). Based on the selected amino acid sequence pattern, seven HiTI inhibitor variants were cloned, expressed and purified. the results indicate that the HiTI variants named T6 (RGGAV) and T128 (WNEGL) were selected by larval trypsin-like (IC50 of 1.1 nM) and chymotrypsin-like enzymes (IC50 of 11.6 nM), respectively. the variants 123 (LLGGL) and 1149 (GGVWR) inhibited both larval chymotrypsin-like (IC50 of 4.2 nM and 29.0 nM, respectively) and elastase-like enzymes (IC50 of 1.2 nM for both). Specific inhibitors were successfully obtained for the digestive enzymes of Ae. aegypti larvae by phage display. Our data also strongly suggest the presence of elastase-like enzymes in Ae. aegypti larvae. the Hill variants T6 and T23 are good candidates for the development as a larvicide to control the vector. (C) 2012 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosSubtractive phage display selection for screening and identification of peptide sequences with potential use in serodiagnosis of paracoccidioidomycosis caused by Paracoccidioides brasiliensis(Wiley, 2017) Portes, Leticia da Silva [UNIFESP]; Kioshima, Érika Seki; Camargo, Zoilo Pires de [UNIFESP]; Batista, Wagner Luiz [UNIFESP]; Xander, Patricia [UNIFESP]Paracoccidioidomycosis (PCM) is a systemic granulomatous disease endemic in Latin America whose aetiologic agents are the thermodimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. Despite technological advances, some problems have been reported for the fungal antigens used for serological diagnosis, and inconsistencies among laboratories have been reported. The use of synthetic peptides in the serological diagnosis of infectious diseases has proved to be a valuable strategy because in some cases, the reactions are more specific and sensitive. In this study, we used a subtractive selection with a phage display library against purified polyclonal antibodies for negative and positive PCM sera caused by P. brasiliensis. The binding phages were sequenced and tested in a binding assay to evaluate its interaction with sera from normal individuals and PCM patients. Synthetic peptides derived from these phage clones were tested in a serological assay, and we observed a significant recognition of LP15 by sera from PCM patients infected with P. brasiliensis. Our results demonstrated that subtractive phage display selection may be useful for identifying new epitopes that can be applied to the serodiagnosis of PCM caused by P. brasiliensis.
- ItemAcesso aberto (Open Access)Synthetic Peptides Mimic gp75 from Paracoccidioides brasiliensis in the Diagnosis of Paracoccidioidomycosis(Springer, 2012-07-01) Caldini, Camila Pistelli [UNIFESP]; Xander, Patricia [UNIFESP]; Kioshima, Érika Seki [UNIFESP]; Bachi, Andre Luis Lacerda [UNIFESP]; Camargo, Zoilo Pires de [UNIFESP]; Mariano, Mario [UNIFESP]; Lopes, Jose Daniel [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. in this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. the protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients' sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. the test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.
- ItemSomente MetadadadosValidation of a Phage Display Method for Protease Inhibitor Selection Using SFTI and HiTI Synthetic Hybrid Peptides(Bentham Science Publ Ltd, 2010-11-01) Marco, Renato de [UNIFESP]; Azzolini, Simone Sant'Anna [UNIFESP]; Lovato, Diogo Ventura [UNIFESP]; Torquato, Ricardo Jose Soares [UNIFESP]; Amino, Rogerio [UNIFESP]; Miranda, Antonio de [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)A recombinant Haematobia irritans irritans trypsin inhibitor (HiTI - Mw 7030 kDa) phagemid library was constructed and displayed functionally on the tip of the filamentous M13 phage. A combinatorial library of 7.2 x 10(6) mutants was created with HiTI mutations restricted to the P1' - P3' and P5' positions of the reactive site. This combinatorial library was selected for trypsin-like Pr2 proteases of Metarhizium anisopliae fungus, and 11 HiTI mutants containing the following substitutions: K17G, S18R, D19G, S21A, among 60 sequenced clones, were obtained. In order to confirm the inhibitory activity of the selected sequences, we transferred the selected sequence to the shortest protease inhibitor, the sunflower trypsin inhibitor (SFTI - Mw 1533 Da), for inhibitory activity analysis. The hybrid peptide containing the mutated sequence (SFTI-Mut, GRCTRGRGLACFPD-NH(2); Ki = 14 mu M) presented an apparent inhibition constant (Ki(app)) for Pr2 proteases similar to 20-fold lower than the control peptide containing the original HiTI sequence (SFTI-HiTI, GRCTRKSDLSCFPD-NH(2); Ki = 259 mu M). In conclusion, the present work enabled the selection of a specific HiTI mutant for Pr2 proteases of M. anisopliae fungus using a HiTI combinatorial library on M13 phage surface. Selection of strong binders by phage display and their validation as inhibitors using synthetic hybrid peptides proved to be a powerful technique to generate specific serine protease inhibitors suitable for studies of drug design and enzyme-inhibitor interaction.