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2.
AAPS PharmSciTech ; 21(4): 124, 2020 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-32342227

RESUMO

To achieve improved drug delivery efficiency to hepatocellular carcinoma (HCC), biodegradable poly (ethylene glycol)-poly (lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (NP), surface-modified with SP94 peptide, were designed for the efficient delivery of cryptotanshinone to the tumor for the treatment of HCC. Cryptotanshinone NP and SP94-NP were prepared by using nanoprecipitation. The physicochemical and pharmaceutical properties of the NP and SP94-NP were characterized, and the release kinetics suggested that both NP and SP94-NP provided continuous, slow release of cryptotanshinone for 48 h. The in vitro cellular experiment demonstrated that SP94-NP significantly enhanced the cellular uptake of cryptotanshinone and induced high cytotoxicity and cellular apoptosis of hepatocellular carcinoma (HepG2) cells. The in vivo detecting results of targeting effect using the Cy5.5 probe evidenced that SP94-NP showed an accumulation in tumor more efficiently than that of unconjugated ones. Meanwhile, SP94-NP exhibited the smallest tumor size than other groups and showed no toxicity to body. The results of this study provide a promising nanoplatform for the targeting of HCC.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Fenantrenos/administração & dosagem , Poliésteres/administração & dosagem , Polietilenoglicóis/administração & dosagem , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/síntese química , Medicamentos de Ervas Chinesas/metabolismo , Feminino , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Nanopartículas/metabolismo , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Fenantrenos/síntese química , Fenantrenos/metabolismo , Poliésteres/síntese química , Poliésteres/metabolismo , Polietilenoglicóis/síntese química , Polietilenoglicóis/metabolismo
3.
Microvasc Res ; 129: 103986, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32017943

RESUMO

Wet age-related macular degeneration (AMD) and diabetic retinopathy are the leading causes of blindness through increased angiogenesis. Although VEGF-neutralizing proteins provide benefit, inconsistent responses indicate a need for new therapies. We previously identified the Fibulin-7 C-terminal fragment (Fbln7-C) as an angiogenesis inhibitor in vitro. Here we show that Fbln7-C inhibits neovascularization in vivo, in both a model of wet AMD involving choroidal neovascularization (CNV) and diabetic retinopathy involving oxygen-induced ischemic retinopathy. Furthermore, a short peptide sequence from Fbln7-C is responsible for the anti-angiogenic properties of Fbln7-C. Our work suggests Fbln7-C as a therapeutic candidate for wet AMD and ischemic retinopathy.


Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , Corioide/irrigação sanguínea , Neovascularização de Coroide/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Neovascularização Retiniana/prevenção & controle , Vasos Retinianos/efeitos dos fármacos , Degeneração Macular Exsudativa/prevenção & controle , Animais , Proteínas de Ligação ao Cálcio/síntese química , Proteínas de Ligação ao Cálcio/genética , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/síntese química , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Degeneração Macular Exsudativa/genética , Degeneração Macular Exsudativa/metabolismo , Degeneração Macular Exsudativa/patologia
4.
Amino Acids ; 52(3): 387-396, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31902008

RESUMO

Alzheimer's disease (AD), the most common form of dementia, is a growing problem worldwide, with 10 million incident cases registered every year. The complex etiology of AD has not been clarified yet and represents an active research topic. In this work, we studied the inhibitory properties of Hp-1935, a natural peptide extracted from the skin secretions of an Argentinian frog (Boana pulchella). It was initially isolated as an antimicrobial peptide by our group, but we later discovered its anti-AChE action. Since not many peptides with this activity have been reported, we focused on defining the basis of its inhibitory mechanism against acetylcholinesterase (AChE) and on finding the primary portion for the inhibitory activity in its sequence, through the combination of an experimental strategy of design and synthesis with molecular dynamics simulations. We also tested its cytotoxicity. We found that Hp-1935 is an interesting sequence for the development of new AChE inhibitors. This peptide is a peripheral anionic site inhibitor with an inhibitory activity that collocates it between the most potent natural amino acids peptides against AChE reported. We also demonstrate that its inhibitory activity is concentrated on the central part of the sequence.


Assuntos
Acetilcolinesterase/metabolismo , Doença de Alzheimer/enzimologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Inibidores da Colinesterase/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Anuros , Células CHO , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Cricetulus , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Relação Estrutura-Atividade
5.
Nat Commun ; 11(1): 467, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980626

RESUMO

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived ß-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.


Assuntos
Corantes Fluorescentes , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Linhagem Celular , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Receptor do Peptídeo Semelhante ao Glucagon 1/deficiência , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Células HEK293 , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Transdução de Sinais , Distribuição Tecidual
6.
Clin Cancer Res ; 26(1): 232-241, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31624103

RESUMO

PURPOSE: Advances in immunotherapy have revolutionized care for some patients with cancer. However, current checkpoint inhibitors are associated with significant toxicity and yield poor responses for patients with central nervous system tumors, calling into question whether cancer immunotherapy can be applied to glioblastoma multiforme. We determined that targeting the CD200 activation receptors (CD200AR) of the CD200 checkpoint with a peptide inhibitor (CD200AR-L) overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the CD200AR-L in a trial in companion dogs with spontaneous high-grade glioma. Addition of the peptide to autologous tumor lysate vaccines significantly increased the median overall survival to 12.7 months relative to tumor lysate vaccines alone, 6.36 months. EXPERIMENTAL DESIGN: This study was developed to elucidate the mechanism of the CD200ARs and develop a humanized peptide inhibitor. We developed macrophage cell lines with each of four CD200ARs knocked out to determine their binding specificity and functional response. Using proteomics, we developed humanized CD200AR-L to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in human CD14+ cells. GMP-grade peptide was further validated for activity. RESULTS: We demonstrated that the CD200AR-L specifically targets a CD200AR complex. Moreover, we developed and validated a humanized CD200AR-L for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhanced an antigen-specific response, and determined that the use of the CD200AR-L downregulated the expression of CD200 inhibitory and PD-1 receptors. CONCLUSIONS: These results support consideration of a CD200AR-L as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.


Assuntos
Antígenos CD/metabolismo , Células Dendríticas/imunologia , Glioblastoma/tratamento farmacológico , Imunoterapia/métodos , Receptores de Orexina/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Antígenos CD/química , Células Cultivadas , Glioblastoma/imunologia , Glioblastoma/metabolismo , Humanos , Tolerância Imunológica , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Receptores de Orexina/química , Fragmentos de Peptídeos/síntese química , Receptor de Morte Celular Programada 1/imunologia
7.
Amino Acids ; 52(3): 361-369, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31875260

RESUMO

Breast cancer susceptibility gene 2 (BRCA2) is an important tumor suppressor, which is participated in repair of damaged DNA by its highly conserved BRC repeat motifs regulating RAD51 protein homologous recombination and thereby preventing cell carcinogenesis. In this study, the BRCA2(1524-1548)-RAD51(241-260) complex structure was obtained based on PDB bank data 1N0W, which provided the basis for site-specific mutation of BRCA2(1524-1548). The BRC4 and BRC4 analogous peptides were synthesized, and the interaction between BRC peptide and RAD51(241-260) was studied by fluorescence spectroscopy, circular dichroism spectroscopy and microscale thermophoresis (MST). The results of circular dichroism showed that the changes in secondary structures of RAD51(241-260) occurred after adding BRC4 analogous peptides, and the α-helix content increased significantly. Fluorescence spectral data demonstrated that the model of BRC peptide binding to RAD51(241-260) was static quenching, and the binding constants of BRC4, P1, P2, P4 with RAD51(241-260) were 1.647 × 10-4 L mol-1, 2.532 × 10-4 L mol-1, 3.161 × 10-4 L mol-1, 1.705 × 10-4 L mol-1, respectively. The results of MST indicated that P2 and RAD51(241-260) have better affinity for dissociation constant 44.286 µM. The strongest affinity between P2 and RAD51(241-260) indicated that the mutation of amino acid residue constituting BRC α-helix affects the structure and interaction of BRC peptide and RAD51(241-260).


Assuntos
Proteína BRCA2 , Fragmentos de Peptídeos , Rad51 Recombinase/química , Substituição de Aminoácidos , Proteína BRCA2/síntese química , Proteína BRCA2/química , Proteína BRCA2/genética , Dicroísmo Circular , Humanos , Mutação , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Conformação Proteica em alfa-Hélice/genética , Espectrometria de Fluorescência
8.
J Pept Sci ; 25(12): e3218, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31755207

RESUMO

Human laminin-511 (α5ß1γ1) and its truncated protein, laminin-511 E8 fragment, bind to integrin α6ß1 and have been widely used for embryonic stem cell and induced pluripotent stem cell culture under feeder-free conditions. In this study, we focused on human laminin α5 chain G domain, which is thought to be critical for the biological functions of laminin-511, and screened its biologically active sequences using a synthetic peptide library. We synthesized 115 peptides (hA5G1-hA5G115) covering the entire laminin α5 chain G domain and evaluated cell attachment activity using both the peptide-coated plate and peptide-chitosan matrix (peptide-ChtM) assays. Seventeen peptides demonstrated cell attachment activity in the assays. Both hA5G18 and hA5G26-coated plates and hA5G74-ChtMs promoted integrin ß1-mediated cell attachment. These findings are useful for the study of molecular mechanisms of laminin-511, and the active peptides have a potential for use as a molecular probe for cell adhesion receptors.


Assuntos
Fibroblastos/efeitos dos fármacos , Laminina/química , Laminina/metabolismo , Fragmentos de Peptídeos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/síntese química , Domínios Proteicos , Relação Estrutura-Atividade
9.
Molecules ; 24(22)2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31731725

RESUMO

N-Alkylation and N-acylation of the prostaglandin-F2α allosteric modulator l-PDC31 were performed to install various alkyl, PEG and isoprenoid groups onto the l-enantiomer of the peptide. Among the different bio-conjugates studied, the N-dodecyl analog reduced prostaglandin-F2α-induced mouse myometrium contractions ex vivo. Furthermore, N-dodecyl-l-PDC31 exhibited improved stability in a mouse serum assay, likely due to protection from protease degradation by the lipid chain.


Assuntos
Proteína Básica da Mielina , Miométrio/metabolismo , Fragmentos de Peptídeos , Contração Uterina/efeitos dos fármacos , Animais , Dinoprosta/química , Feminino , Camundongos , Proteína Básica da Mielina/síntese química , Proteína Básica da Mielina/química , Proteína Básica da Mielina/farmacologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia
10.
Amino Acids ; 51(10-12): 1527-1545, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31576456

RESUMO

A new series of N-modified analogues of the VV-hemorphin-5 with aminophosphonate moiety have been synthesized, characterized and investigated for anticonvulsant activity. The novel peptide analogues were prepared by solid-phase peptide synthesis-Fmoc-strategy and were evaluated in the timed intravenous pentylenetetrazole infusion test (ivPTZ) and 6-Hz psychomotor seizure test in mice. The acute neurological toxicity was determined using the rotarod test. The redox potentials at glass carbonic electrode (GC) and the acid/base properties as pKa values of these peptide analogues were compared with the electrochemical behaviour of tyrosine- and tryptophan-containing peptides using different voltamperometric modes. Among the five tested peptide analogues, V3p was the most active against the ivPTZ test with effect comparable to that of the VV-hemorphin-5 (V1) used as a positive control. Dose-dependent elevation of the seizure threshold for myoclonic twitch and generalized clonic seizures was observed after i.c.v. administration of V2p, V4p and V5p as well as for forelimbs tonus in V4p peptides. The peptide analogues V2p-V5p were able to suppress dose-dependent psychomotor seizures in the 6-Hz test. In contrast, the V6p peptide showed either a pro-convulsant effect in the iv PTZ test or was inactive in the 6-Hz test. No changes in motor coordination were noted with the novel peptides. Docking study results suggest that kappa opioid receptor binding could be the mechanism of action of peptide derivatives with anticonvulsant activity. The results suggest that incorporation of aminophosphonate moiety at position 1 of the VV-hemorphin-5 scaffold deserve further evaluation in models of epilepsy and derivatization.


Assuntos
Anticonvulsivantes/síntese química , Anticonvulsivantes/uso terapêutico , Hemoglobinas/síntese química , Hemoglobinas/uso terapêutico , Organofosfonatos/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/uso terapêutico , Animais , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hemoglobinas/química , Hemoglobinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores Opioides kappa/metabolismo , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Técnicas de Síntese em Fase Sólida , Relação Estrutura-Atividade
11.
Amino Acids ; 51(10-12): 1633-1648, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31654210

RESUMO

For medical use of proteins and peptide-based drugs, it is desirable to have small biologically active sequences because they improve stability, reduce side effects, and production costs. Several plant defensins have their biological activities imparted by a sequence named γ-core. Vu-Def, a Vigna unguiculata defensin, has activity against Leishmania amazonensis, which is one etiological agent of leishmaniasis and for which new drugs are needed. Our intention was to understand if the region comprising the Vu-Def γ-core is responsible for the biological activity against L. amazonensis and to unveil its mechanism of action. Different microbiological assays with L. amazonensis in the presence of the synthetic peptide A36,42,44γ32-46Vu-Def were done, as well as ultrastructural and fluorescent analyses. A36,42,44γ32-46Vu-Def showed biological activity similar to Vu-Def. A36,42,44γ32-46Vu-Def (74 µM) caused 97% inhibition of L. amazonensis culture and parasites were unable to regrow in fresh medium. The cells of the treated parasites showed morphological alterations by ultrastructural analysis and fluorescent labelings that corroborate with the data of the organelles alterations. The general significance of our work is based on the description of a small synthetic peptide, A36,42,44γ32-46Vu-Def, which has activity on L. amazonensis and that the interaction between A36,42,44γ32-46Vu-Def-L. amazonensis results in parasite inhibition by the activation of an apoptotic-like cell death pathway.


Assuntos
Apoptose/efeitos dos fármacos , Defensinas/química , Leishmania/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Vigna/química , Sequência de Aminoácidos , Defensinas/farmacologia , Leishmania/crescimento & desenvolvimento , Modelos Moleculares , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Sementes/química
12.
Biochemistry ; 58(44): 4398-4407, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31625391

RESUMO

γ-Secretase is a membrane-embedded aspartyl protease complex with presenilin as the catalytic component that cleaves within the transmembrane domain (TMD) of >90 known substrates, including the amyloid precursor protein (APP) of Alzheimer's disease. Processing by γ-secretase of the APP TMD produces the amyloid ß-peptide (Aß), including the 42-residue variant (Aß42) that pathologically deposits in the Alzheimer brain. Complex proteolysis of APP substrate by γ-secretase involves initial endoproteolysis and subsequent carboxypeptidase trimming, resulting in two pathways of Aß production: Aß49 → Aß46 → Aß43 → Aß40 and Aß48 → Aß45 → Aß42 → Aß38. Dominant mutations in APP and presenilin cause early onset familial Alzheimer's disease (FAD). Understanding how γ-secretase processing of APP is altered in FAD is essential for elucidating pathogenic mechanisms in FAD and developing effective therapeutics. To improve our understanding, we designed synthetic APP-based TMD substrates as convenient functional probes for γ-secretase. Installation of the helix-inducing residue α-aminoisobutyric acid provided full TMD helical substrates while also facilitating their synthesis and increasing the solubility of these highly hydrophobic peptides. Through mass spectrometric analysis of proteolytic products, synthetic substrates were identified that were processed in a manner that reproduced physiological processing of APP substrates. Validation of these substrates was accomplished through mutational variants, including the installation of two natural APP FAD mutations. These FAD mutations also resulted in increased levels of formation of Aß-like peptides corresponding to Aß45 and longer, raising the question of whether the levels of such long Aß peptides are indeed increased and might contribute to FAD pathogenesis.


Assuntos
Secretases da Proteína Precursora do Amiloide/química , Precursor de Proteína beta-Amiloide/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/genética , Sequência de Aminoácidos , Ácidos Aminoisobutíricos/química , Precursor de Proteína beta-Amiloide/síntese química , Precursor de Proteína beta-Amiloide/genética , Espectrometria de Massas , Mutação , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/genética , Conformação Proteica em alfa-Hélice , Proteólise
13.
Eur J Med Chem ; 182: 111623, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31473417

RESUMO

Fowlicidin-1 (Fowl-1), a cathelicidin expressed in chicken intestine, is known to have both antimicrobial and anti-inflammatory properties. However, its pharmaceutical development has been ultimately compromised by its high host cytotoxicity. In this study, a series of N- and C-terminal-truncated 19-meric Fowl-1 peptides were synthesized. Among these truncated peptides, Fowl-1 (8-26) exhibited broad-spectrum antimicrobial activity without human erythrocyte cytotoxicity while reducing anti-inflammatory activity. Further, Fowl-1 (8-26)-WRK was designed via Thr5→Trp, Ile7→Arg, and Asn11→Lys substitutions in Fowl-1 (8-26) to exhibit more amphipathicity. The results revealed that it exhibited both antimicrobial and anti-inflammatory properties. This study also demonstrated that the inhibitory activity of Fowl-1 (8-26)-WRK against LPS-induced inflammation was mainly due to the binding of LPS to the peptide. Interestingly, compared with human cathelicidin LL-37 and melittin, Fowl-1 (8-26)-WRK showed more potent activity against drug-resistant bacteria. It was also resistant to physiological salts and human serum and acted synergistically in combination with conventional antibiotics, such as chloramphenicol, ciprofloxacin, and oxacillin, suggesting that combined with conventional antibiotics, it is a promising adjuvant. Furthermore, membrane depolarization, SYTOX Green uptake, and flow cytometry revealed that it kills bacteria by damaging their membrane integrity. Therefore, this study suggests that Fowl-1 (8-26)-WRK has considerable potential for future development as an antimicrobial and anti-inflammatory agent for treating antibiotic-resistant infections.


Assuntos
Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Catelicidinas/farmacologia , Desenho de Fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Catelicidinas/síntese química , Catelicidinas/química , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Células RAW 264.7 , Ovinos , Relação Estrutura-Atividade
14.
Sci Rep ; 9(1): 13575, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537869

RESUMO

Since metastatic melanoma is deadly, early diagnosis thereof is crucial for managing the disease. We recently developed α-melanocyte-stimulating hormone (αMSH) derivatives, [68Ga]Ga-CCZ01048 and [18F]CCZ01064, that target the melanocortin 1 receptor (MC1R) for mouse melanoma imaging. In this study, we aim to evaluate [18F]CCZ01064 as well as a novel dual-ammoniomethyl-trifluoroborate (AmBF3) derivative, [18F]CCZ01096, for targeting human melanoma xenograft using µPET imaging. The peptides were synthesized on solid phase using Fmoc chemistry. Radiolabeling was achieved in a one-step 18F-19F isotope-exchange reaction. µPET imaging and biodistribution studies were performed in NSG mice bearing SK-MEL-1 melanoma xenografts. The MC1R density on the SK-MEL-1 cell line was determined to be 972 ± 154 receptors/cell (n = 4) via saturation assays. Using [18F]CCZ01064, moderate tumor uptake (3.05 ± 0.47%ID/g) and image contrast were observed at 2 h post-injection. Molar activity was determined to play a key role. CCZ01096 with two AmBF3 motifs showed comparable sub-nanomolar binding affinity to MC1R and much higher molar activity. This resulted in improved tumor uptake (6.46 ± 1.42%ID/g) and image contrast (tumor-to-blood and tumor-to-muscle ratios were 30.6 ± 5.7 and 85.7 ± 11.3, respectively) at 2 h post-injection. [18F]CCZ01096 represents a promising αMSH-based µPET imaging agent for human melanoma and warrants further investigation for potential clinical translation.


Assuntos
Radioisótopos de Flúor/química , Melanoma/diagnóstico por imagem , Fragmentos de Peptídeos/administração & dosagem , alfa-MSH/análogos & derivados , Animais , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Melanoma/metabolismo , Camundongos , Estrutura Molecular , Transplante de Neoplasias , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacocinética , Tomografia por Emissão de Pósitrons , Receptor Tipo 1 de Melanocortina/metabolismo , alfa-MSH/química
15.
ACS Chem Biol ; 14(10): 2233-2242, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31513374

RESUMO

Recent studies have shown that modified human lactoferrin 20-31 fragment, named HLopt2, possesses antibacterial and antifungal activity. Thus, we decided to synthesize and evaluate the biological activity of a series of conjugates based on this peptide and one of the antimicrobials with proven antibacterial (ciprofloxacin, CIP, and levofloxacin, LVX) or antifungal (fluconazole, FLC) activity. The drugs were covalently connected to the peptide via amide, methylenecarbonyl moieties, or a disulfide bridge. The antibacterial and antifungal activities were evaluated under Clinical and Laboratory Standard Institute (CLSI) recommended conditions or in a low-salt brain-heart infusion diluted medium (BHI1/100). Results showed that conjugation of the peptide with the drug increased its antimicrobial activity up to 4-fold. Under CLSI-recommended conditions, all the compounds revealed rather low efficiency. Among conjugates, the highest antibacterial activity was recorded for the CIP-Cys-S-S-HLopt2-NH2 (III). In BHI1/100, which had lower differentiating properties, all of the conjugates revealed low MIC and MMC (minimum inhibitory and microbicidal concentrations) values. The disulfide bridge used as a linker in the most active conjugate (III) upon incubation with S. aureus cells is reduced, releasing constituent peptide and CIP-Cys. In addition, we showed that its fluorescently labeled analogue and constituent peptide are able to be internalized into both C. albicans and S. aureus cells. Moreover, the invaluable advantage of the presented conjugates was their low toxicity to mammalian cells and very low hemolytic activity. The current research can form a solid basis for further in vivo studies and drug development.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Imunoconjugados/farmacologia , Lactoferrina/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/toxicidade , Antifúngicos/síntese química , Antifúngicos/toxicidade , Candida albicans/efeitos dos fármacos , Ciprofloxacino/síntese química , Ciprofloxacino/farmacologia , Ciprofloxacino/toxicidade , Estabilidade de Medicamentos , Escherichia coli/efeitos dos fármacos , Fluconazol/síntese química , Fluconazol/farmacologia , Fluconazol/toxicidade , Células HEK293 , Células Hep G2 , Humanos , Imunoconjugados/toxicidade , Lactoferrina/síntese química , Lactoferrina/toxicidade , Levofloxacino/síntese química , Levofloxacino/farmacologia , Levofloxacino/toxicidade , Masculino , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/toxicidade , Staphylococcus aureus/efeitos dos fármacos , Suínos
16.
Biosci Rep ; 39(9)2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31477581

RESUMO

The trimeric heptad repeat domains HR1 and HR2 of the human immunodeficiency virus 1 (HIV-1) gp41 play a key role in HIV-1-entry by membrane fusion. To develop efficient inhibitors against this step, the corresponding trimeric-N36 and C34 peptides were designed and synthesized. Analysis by circular dichroism of monomeric and trimeric N36 and C34 peptides showed their capacities to adopt α-helical structures and to establish physical interactions. At the virological level, while trimeric-C34 conserves the same high anti-fusion activity as monomeric-C34, trimerization of N36-peptide induced a significant increase, reaching 500-times higher in anti-fusion activity, against R5-tropic virus-mediated fusion. This result was associated with increased stability of the N36 trimer peptide with respect to the monomeric form, as demonstrated by the comparative kinetics of their antiviral activities during 6-day incubation in a physiological medium. Collectively, our findings demonstrate that while the trimerization of C34 peptide had no beneficial effect on its stability and antiviral activity, the trimerization of N36 peptide strengthened both stability and antiviral activity. This approach, promotes trimers as new promising HIV-1 inhibitors and point to future development aimed toward innovative peptide fusion inhibitors, microbicides or as immunogens.


Assuntos
Proteína gp41 do Envelope de HIV/química , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Fragmentos de Peptídeos/química , Sequência de Aminoácidos/genética , Dicroísmo Circular , Desenho de Fármacos , Proteína gp41 do Envelope de HIV/síntese química , Proteína gp41 do Envelope de HIV/farmacologia , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Fusão de Membrana/efeitos dos fármacos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Conformação Proteica em alfa-Hélice
17.
ACS Chem Biol ; 14(10): 2176-2184, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31498587

RESUMO

Mucin-1 (MUC1) is a highly attractive antigenic target for anticancer vaccines. Naturally existing MUC1 can contain multiple types of O-linked glycans, including the Thomsen-Friedenreich (Tf) antigen and the Sialyl Thomsen-nouveau (STn) antigen. In order to target these antigens as potential anticancer vaccines, MUC1 glycopeptides SAPDT*RPAP (T* is the glycosylation site) bearing the Tf and the STn antigen, respectively, have been synthesized. The bacteriophage Qß carrier is a powerful carrier for antigen delivery. The conjugates of MUC1-Tf and -STn glycopeptides with Qß were utilized to immunize immune-tolerant human MUC1 transgenic (MUC1.Tg) mice, which elicited superior levels of anti-MUC1 IgG antibodies with titers reaching over 2 million units. The IgG antibodies recognized a wide range of MUC1 glycopeptides bearing diverse glycans. Antibodies induced by Qß-MUC1-Tf showed strongest binding, with MUC1-expressing melanoma B16-MUC1 cells, and effectively killed these cells in vitro. Vaccination with Qß-MUC1-Tf first followed by tumor challenge in a lung metastasis model showed significant reductions of the number of tumor foci in the lungs of immunized mice as compared to those in control mice. This was the first time that a MUC1-Tf-based vaccine has shown in vivo efficacy in a tumor model. As such, Qß-MUC1 glycopeptide conjugates have great potential as anticancer vaccines.


Assuntos
Vacinas Anticâncer/uso terapêutico , Glicopeptídeos/uso terapêutico , Imunoconjugados/uso terapêutico , Mucina-1/imunologia , Fragmentos de Peptídeos/uso terapêutico , Proteínas Virais/uso terapêutico , Allolevivirus/química , Sequência de Aminoácidos , Animais , Antígenos Glicosídicos Associados a Tumores/imunologia , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/imunologia , Humanos , Imunoconjugados/imunologia , Imunoglobulina G/imunologia , Neoplasias Pulmonares/terapia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Proteínas Virais/síntese química , Proteínas Virais/imunologia
18.
Biomolecules ; 9(8)2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31412593

RESUMO

Lung cancer is the leading cause of cancer-related deaths worldwide; hence novel treatments for this malignancy are eagerly needed. Since natural-based compounds represent a rich source of novel chemical entities in drug discovery, we have focused our attention on tambjamines, natural compounds isolated from marine invertebrates that have shown diverse pharmacological activities. Based on these structures, we have recently identified the novel indole-based tambjamine analog 21 (T21) as a promising antitumor agent, which modulates the expression of apoptotic proteins such as survivin. This antiapoptotic protein plays an important role in carcinogenesis and chemoresistance. In this work, we have elucidated the molecular mechanism by which the anticancer compound T21 exerts survivin inhibition and have validated this protein as a therapeutic target in different lung cancer models. T21 was able to reduce survivin protein levels in vitro by repressing its gene expression through the blockade of Janus kinase/Signal Transducer and Activator of Transcription-3 (JAK/STAT3)/survivin signaling pathway. Interestingly, this occurred even when the pathway was overstimulated with its ligand interleukin 6 (IL-6), which is frequently overexpressed in lung cancer patients who show poor clinical outcomes. Altogether, these results show T21 as a potent anticancer compound that effectively decreases survivin levels through STAT3 inhibition in lung cancer, appearing as a promising therapeutic drug for cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteína gp41 do Envelope de HIV/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Survivina/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Proteína gp41 do Envelope de HIV/síntese química , Proteína gp41 do Envelope de HIV/química , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Estrutura Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade , Survivina/metabolismo
19.
Colloids Surf B Biointerfaces ; 183: 110401, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31382050

RESUMO

Various techniques have been developed to determine protein's structure to understand how proteins work.  Compared with X-ray crystallography requiring proteins to form single crystal structure and NMR which usually needs long time measurement, surface FT-IR techniques are able to quickly determine the tilt angle (the key information to determine whether the α-helix is transmembrane) of peptides/proteins in a monolayer at the interface (e.g. membranes). Specifically, for α-helical peptides/proteins in membrane, the tilt angle of the axis is one of the key information. In this paper, Multiple Angle Incidence Resolution Spectroscopy (MAIRS), a recently developed surface FTIR technique, was applied for the first time to quantitatively determine the tilt angle of the axis of α-helical model peptide related to α-synuclein (α-syn). α-Syn is a 140-amino-acid presynaptic protein whose aggregation is the hallmark of Parkinson's disease (PD). It is difficult for α-syn to form a single crystal structure and the primary structure of α-syn constitutes three domains: the N-terminus containing residues 1-60; the nonamyloid component (NAC) which spans residues 61-95 and is highly prone to aggregation; and C-terminus with residues 96-140. Here, the NAC part (i.e., α-syn(61-95)) responsible for the aggregation was found to change its unstructured conformation in aqueous solution to α-helix at the air-water interface by circular dichroism and MAIRS. In addition, the instinct power of MAIRS to quantitatively measure the tilt angle of the axis of α-helical α-syn(61-95) in monolayer was fully exhibited. Therefore, MAIRS is a potential supplemental technique to X-ray crystallography and NMR to determine the structure of membrane peptides/proteins.


Assuntos
Ar/análise , Fragmentos de Peptídeos/química , Água/química , alfa-Sinucleína/química , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Fragmentos de Peptídeos/síntese química , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier/instrumentação , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Propriedades de Superfície , Termodinâmica , alfa-Sinucleína/síntese química
20.
Chem Commun (Camb) ; 55(65): 9649-9652, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31339160

RESUMO

Intracellular delivery of bioactive polyphenols is currently evaluated as a protective strategy for cells under pharmaceutical stress. To this end, the 20mer R5 peptide from the marine diatom C. fusiformis was N-terminally modified with a quercetin derivative. This polyphenol-peptide conjugate was used to generate homogeneous silica particles under biomimetic conditions that are efficiently taken up by eukaryotic cells without being cytotoxic. However, not only was accumulation in the cytoplasm of living cells observed via electron and fluorescence microscopy but also translocation into the nucleus. The latter was only seen when the quercetin-peptide conjugate was present within the silica particles and provides a novel targeting option for silica particles to nuclei.


Assuntos
Núcleo Celular/metabolismo , Corantes Fluorescentes/farmacocinética , Fragmentos de Peptídeos/farmacocinética , Quercetina/análogos & derivados , Quercetina/farmacocinética , Dióxido de Silício/farmacocinética , Transporte Ativo do Núcleo Celular , Biomimética , Diatomáceas/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/toxicidade , Células HT29 , Humanos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Quercetina/síntese química , Quercetina/toxicidade , Dióxido de Silício/química , Dióxido de Silício/toxicidade
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