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1.
Antibiotics (Basel) ; 10(10)2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34680817

RESUMO

The emergence and spread of multiple drug-resistant bacteria strains caused the development of new antibiotics to be one of the most important challenges of medicinal chemistry. Despite many efforts, the commercial availability of peptide-based antimicrobials is still limited. The presented study aims to explain that immobilized artificial membrane chromatography can support the characterization of antimicrobial peptides. Consequently, the chromatographic experiments of three groups of related peptide substances: (i) short cationic lipopeptides, (ii) citropin analogs, and (iii) conjugates of ciprofloxacin and levofloxacin, with a cell-penetrating peptide were discussed. In light of the discussion of the mechanisms of action of these compounds, the obtained results were interpreted.

2.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066491

RESUMO

Poor efficiency of chemotherapeutics in the eradication of Cancer Stem Cells (CSCs) has been driving the search for more active and specific compounds. In this work, we show how cell density-dependent stage culture profiles can be used in drug development workflows to achieve more robust drug activity (IC50 and EC50) results. Using flow cytometry and light microscopy, we characterized the cytological stage profiles of the HL-60-, A-549-, and HEK-293-derived sublines with a focus on their primitive cell content. We then used a range of cytotoxic substances-C-123, bortezomib, idarubicin, C-1305, doxorubicin, DMSO, and ethanol-to highlight typical density-related issues accompanying drug activity determination. We also showed that drug EC50 and selectivity indices normalized to primitive cell content are more accurate activity measurements. We tested our approach by calculating the corrected selectivity index of a novel chemotherapeutic candidate, C-123. Overall, our study highlights the usefulness of accounting for primitive cell fractions in the assessment of drug efficiency.


Assuntos
Antineoplásicos/farmacologia , Contagem de Células , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Humanos , Concentração Inibidora 50 , Estadiamento de Neoplasias , Espécies Reativas de Oxigênio/metabolismo
3.
Metallomics ; 12(12): 2199, 2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33320153

RESUMO

Correction for 'Stability of Cu(ii) complexes with FomA protein fragments containing two His residues in the peptide chain' by Monika Katarzyna Lesiów et al., Metallomics, 2019, 11, 1518-1531, DOI: 10.1039/C9MT00131J.

4.
J Inorg Biochem ; 212: 111250, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32920436

RESUMO

Mono- and dinuclear Cu(II) complexes with Ac-PTVHNEYH-NH2 (L1) and Ac-NHHTLND-NH2 (L2) peptides from FomA protein of Fusobacterium nucleatum were studied by potentiometry, spectroscopic methods (UV-Vis, CD, EPR) and MS technique. The dominant mononuclear complexes for L1 ligand are: CuHL (pH range 5.0-6.0) with 2N {2Nim}, CuH-2L (pH range 8.0-8.5) and CuH-3L species (above pH 9.0) with 4N {Nim, 3N-} coordination modes. The complexes: CuH-1L with 3N {2Nim, N-}, CuH-2L with 3N {Nim, 2N-} and CuH-3L with 4N {Nim, 3N-} binding sites are proposed for the L2 ligand. Probably in the CuH-2L complex for CuL2 system the second His residue in His-His sequence is bound to Cu(II) ion, while the first His residue may stabilize this complex by His-His and/or His-Cu(II) interactions. The dominant dinuclear Cu2L1 complexes in the pH range 6.5-10.5 are: the Cu2H-4L and Cu2H-6L species with 3N{Nim, 2N-}4N{Nim, 3N-} and 4N{Nim, 3N-}4N{Nim, 3N-} binding sites, respectively. In the case of the Cu2L2 complex in the pH range 7.2-10.5, the Cu2H-4L and Cu2H-7L species dominate with 2N{Nim, N-}4N{Nim, 3N-} and (Cu(OH)42-4N{Nim, 3N-}) coordination modes, respectively. The ability to generate reactive oxygen species (ROS) by uncomplexed Cu(II) ions, ligands and their complexes at pH 7.4 in the presence of hydrogen peroxide or ascorbic acid was studied. UV-Vis, luminescence, EPR spin trapping and gel electrophoresis methods were used. Both complexes produce higher level of ROS compared to those of their ligands. ROS produced by Cu(II) complexes are hydroxyl radical and singlet oxygen, which contribute to oxidative DNA cleavage.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Complexos de Coordenação/metabolismo , Cobre/metabolismo , DNA/metabolismo , Histidina/metabolismo , Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Motivos de Aminoácidos , Fusobacterium nucleatum/metabolismo , Histidina/química , Potenciometria , Análise Espectral/métodos
5.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32630159

RESUMO

Seven conjugates composed of well-known fluoroquinolone antibacterial agents, ciprofloxacin (CIP) or levofloxacin (LVX), and a cell-penetrating peptide transportan 10 (TP10-NH2) were synthesised. The drugs were covalently bound to the peptide via an amide bond, methylenecarbonyl moiety, or a disulfide bridge. Conjugation of fluoroquinolones to TP10-NH2 resulted in congeners demonstrating antifungal in vitro activity against human pathogenic yeasts of the Candida genus (MICs in the 6.25 - 100 µM range), whereas the components were poorly active. The antibacterial in vitro activity of most of the conjugates was lower than the activity of CIP or LVX, but the antibacterial effect of CIP-S-S-TP10-NH2 was similar to the mother fluoroquinolone. Additionally, for two representative CIP and LVX conjugates, a rapid bactericidal effect was shown. Compared to fluoroquinolones, TP10-NH2 and the majority of its conjugates generated a relatively low level of reactive oxygen species (ROS) in human embryonic kidney cells (HEK293) and human myeloid leukemia cells (HL-60). The conjugates exhibited cytotoxicity against three cell lines, HEK293, HepG2 (human liver cancer cell line), and LLC-PK1 (old male pig kidney cells), with IC50 values in the 10 - 100 µM range and hemolytic activity. The mammalian toxicity was due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH2 since fluoroquinolones themselves were not cytotoxic. Nevertheless, the selectivity index values of the conjugates, both for the bacteria and human pathogenic yeasts, remained favourable.


Assuntos
Anti-Infecciosos/síntese química , Antineoplásicos/síntese química , Peptídeos Penetradores de Células , Ciprofloxacina , Levofloxacino , Proteínas Recombinantes de Fusão , Animais , Anti-Infecciosos/farmacologia , Candida/efeitos dos fármacos , Candida/metabolismo , Farmacorresistência Bacteriana , Células HEK293 , Células HL-60 , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Suínos
6.
Appl Microbiol Biotechnol ; 104(11): 4675-4703, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32274562

RESUMO

This article reviews mushrooms with anti-breast cancer activity. The mushrooms covered which are better known include the following: button mushroom Agaricus bisporus, Brazilian mushroom Agaricus blazei, Amauroderma rugosum, stout camphor fungus Antrodia camphorata, Jew's ear (black) fungus or black wood ear fungus Auricularia auricula-judae, reishi mushroom or Lingzhi Ganoderma lucidum, Ganoderma sinense, maitake mushroom or sheep's head mushroom Grifola frondosa, lion's mane mushroom or monkey head mushroom Hericium erinaceum, brown beech mushroom Hypsizigus marmoreus, sulfur polypore mushroom Laetiporus sulphureus, Lentinula edodes (shiitake mushroom), Phellinus linteus (Japanese "meshimakobu," Chinese "song gen," Korean "sanghwang," American "black hoof mushroom"), abalone mushroom Pleurotus abalonus, king oyster mushroom Pleurotus eryngii, oyster mushroom Pleurotus ostreatus, tuckahoe or Fu Ling Poria cocos, and split gill mushroom Schizophyllum commune. Antineoplastic effectiveness in human clinical trials and mechanism of anticancer action have been reported for Antrodia camphorata, Cordyceps sinensis, Coriolus versicolor, Ganoderma lucidum, Grifola frondosa, and Lentinula edodes.


Assuntos
Agaricales/química , Agaricales/classificação , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Misturas Complexas/química , Misturas Complexas/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Ratos
7.
Biochimie ; 171-172: 178-186, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32169666

RESUMO

A gradual truncation of the primary structure of frog skin-derived Huia versabilis Bowman-Birk peptidic inhibitor (HV-BBI) resulted in 18-times stronger inhibitor of matriptase-1 (peptide 6, Ki = 8 nm) in comparison to the full-length HV-BBI (Ki = 155 nm). Analogous increase in the inhibitory activity in correlation with the peptide length reduction was not observed in case of other serine proteases, bovine trypsin (Ki = 151 nm for peptide 6 and Ki = 120 nm for HV-BBI) and plasmin (Ki = 120 nm for peptide 6 and 82 nm for HV-BBI). Weaker binding affinity to these enzymes emphasized an inhibitory specificity of peptide 6. Molecular dynamic analysis revealed that the observed variations in the binding affinity of peptide 6 and HV-BBI with matriptase-1 are associated with the entropic differences of the unbound peptides. Moreover, several aspects explaining differences in the inhibition of matriptase-1 by peptide 6 (bearing the C-terminal amide group) and its two analogues, peptide 6∗ (having the C-terminal carboxyl group, Ki = 473 nm) and cyclic peptide 6∗∗ (Ki = 533 nm), both exhibiting more than 50-fold reduced inhibitory potency, were discovered. It was also shown that peptide 6 presented significantly higher resistance to proteolytic degradation in human serum than HV-BBI. Additional investigations revealed that, in contrast to some amphibian-derived inhibitors, HV-BBI and its truncated analogues do not possess bactericidal activity, thus they cannot be considered as bifunctional agents.


Assuntos
Peptídeos , Serina Endopeptidases/metabolismo , Animais , Bovinos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Peptídeos/química , Peptídeos/farmacologia , Proteólise
8.
Appl Microbiol Biotechnol ; 104(10): 4211-4226, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32193575

RESUMO

Ribosome-inactivating proteins (RIPs) consist of three varieties. Type 1 RIPs are single-chained and approximately 30-kDa in molecular weight. Type 2 RIPs are double-chained and composed of a type 1 RIP chain and a lectin chain. Type III RIPs, such as maize b-32 barley and JIP60 which are produced as single-domain proenzymes, possess an N-terminal domain corresponding to the A domain of RIPs and fused to a C-terminal domain. In addition to the aforementioned three types of RIPs originating from flowering plants, there are recently discovered proteins and peptides with ribosome-inactivating and protein synthesis inhibitory activities but which are endowed with characteristics such as molecular weights distinctive from those of the regular RIPs. These new/unusual RIPs discussed in the present review encompass metazoan RIPs from Anopheles and Culex mosquitos, antimicrobial peptides derived from RIP of the pokeweed Phytolacca dioica, maize RIP (a type III RIP derived from a precursor form), RIPs from the garden pea and the kelp. In addition, RIPs with a molecular weight smaller than those of regular type 1 RIPs are produced by plants in the Cucurbitaceae family including the bitter gourd, bottle gourd, sponge gourd, ridge gourd, wax gourd, hairy gourd, pumpkin, and Chinese cucumber. A small type II RIP from camphor tree (Cinnamomum camphora) seeds and a snake gourd type II RIP with its catalytic chain cleaved into two have been reported. RIPs produced from mushrooms including the golden needle mushroom, king tuber mushroom, straw mushroom, and puffball mushroom are also discussed in addition to a type II RIP from the mushroom Polyporus umbellatus. Bacterial (Spiroplasma) RIPs associated with the fruitfly, Shiga toxin, and Streptomyces coelicolor RIP are also dealt with. The aforementioned proteins display a diversity of molecular weights, amino acid sequences, and mechanisms of action. Some of them are endowed with exploitable antipathogenic activities.


Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Inativadoras de Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Culicidae/química , Proteínas de Insetos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Inativadoras de Ribossomos/classificação , Proteínas Inativadoras de Ribossomos/farmacologia , Sementes/química
9.
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 , Ciprofloxacina/síntese química , Ciprofloxacina/farmacologia , Ciprofloxacina/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
10.
Peptides ; 117: 170079, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30959143

RESUMO

Eight new peptide conjugates composed of modified bovine lactoferricin truncated analogues (LFcinB) and one of the three antimicrobials - ciprofloxacin (CIP), levofloxacin (LVX), and fluconazole (FLC) - were synthesized. Four different linkers were applied to connect a peptide and an antimicrobial agent. The FLC-containing peptidic conjugates were synthesized using the "click chemistry" method. This novel approach is reported here for the first time. Unlike their components, CIP- and LVX-based conjugates exerted activity against Candida yeast. Similarly to the constituent peptides, synthesized conjugates showed activity against Gram-positive bacteria, especially S. epidermidis. The most active were the conjugates containing CIP linked to the peptide by the redox-sensitive disulfide bridge. Our results show a significant role of a linker between antimicrobial agent and a peptide. This was also confirmed by the lack of synergistic effects on the antimicrobial activity of the constituent compounds. Moreover, cytotoxicity assays revealed that the proposed conjugates cause a comparatively low cytotoxic effect in reference to antibiotics widely used in therapies. Therefore, they can be deliberated as attractive leading structures for the development of drugs.


Assuntos
Anti-Infecciosos , Candida/crescimento & desenvolvimento , Lactoferrina , Peptídeos , Staphylococcus epidermidis/crescimento & desenvolvimento , Células A549 , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Células HL-60 , Humanos , Lactoferrina/química , Lactoferrina/farmacologia , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia
11.
Future Med Chem ; 2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30518272

RESUMO

Matriptase-2 (MT2) is a membrane-anchored proteolytic enzyme. It acts as the proteolytic key regulator in human iron homeostasis. A high expression level can lead to iron overload diseases, whereas mutations in the gene encoding MT2, TMPRSS6, may result in various forms of iron deficiency anemia. Recently, MT2 has been reported as a positive prognostic factor in breast and prostate cancers. However, the exact functions of MT2 in various pathophysiological conditions are still not fully understood. In this review, we describe the synthetic tools designed and synthesized to regulate or monitor MT2 proteolytic activity and present the latest knowledge about the role of MT2 in iron homeostasis and cancer.

12.
J Inorg Biochem ; 189: 69-80, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30243120

RESUMO

Fusobacterium nucleatum is an anaerobic, Gram-negative bacteria linked to colon cancer. It is interesting to determine how metal ions interact with bacterial adhesin proteins. To this end, the coordination of ATDAAS-NH2 and MKKFL-NH2 fragments of Fusobacterium adhesin A (FadA) to copper(II) ions was studied by potentiometry, spectroscopic techniques (UV-Vis, CD, EPR and NMR) and the density functional theory (DFT) methods. At pH 6.8 (colon physiological pH), the metal ion in the first peptide (ATDAAS-NH2) is coordinated by one oxygen and three nitrogen donors while in the second one (MKKFL-NH2) - by sulfur and three nitrogen atoms. Both complexes form two five- and one six-membered stable chelate rings. Moreover, reactivity studies confirmed the production of reactive oxygen species such as hydroxyl radical, superoxide anion radical and singlet oxygen. Generation of reactive oxygen species (ROS) was observed during gel electrophoresis and spectroscopic assays with reporting molecules like NDMA (N,N-dimethyl-p-nitrosoaniline) and NBT (Nitrotetrazolium Blue Chloride). All reactions were conducted in the presence of hydrogen peroxide as endogenous oxidant.


Assuntos
Adesinas Bacterianas/química , Cobre/química , Fusobacterium nucleatum/química , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Potenciometria , Espécies Reativas de Oxigênio/química , Superóxidos/química
13.
Bioconjug Chem ; 29(9): 3060-3071, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30048118

RESUMO

Three chimera peptides composed of bovine lactoferrampin and the analogue of truncated human neutrophil peptide 1 were synthesized by the solid-phase method. In two compounds peptide chains were connected via isopeptide bond, whereas in the third one disulfide bridge served as a linker. All three chimeras displayed significantly higher antimicrobial activity than the constituent peptides as well as their equimolar mixtures. The one with a disulfide bridge displayed selectivity toward Gram-positive bacteria and was able to penetrate bacterial cells. The chimeric peptides demonstrated low in vitro mammalian cytotoxicity, especially against benign cells. The significance of linker type was also reflected in the secondary structure and proteolytic stability of studied compounds. Presented results proved that such chimeras are good lead structures for designing antimicrobial drugs.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Lactoferrina/química , Fragmentos de Peptídeos/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , alfa-Defensinas/química , Animais , Candida/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , Dicroísmo Circular , Ensaios de Seleção de Medicamentos Antitumorais , Corantes Fluorescentes/química , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade
14.
Curr Med Chem ; 25(42): 5986-6001, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29768998

RESUMO

Lectins are a group of proteins or glycoproteins with various potentially exploitable bioactivities and have been capturing more interest recently. They have been isolated and reported from various tissues of a diversity of plant species. Tubers are modified and enlarged plant structures derived from stems or roots that are used for nutrient storage and asexual reproduction. A number of plants such as yam, taro and potato are grown for their edible tubers, and lectins are found to be one of the major storage proteins. These lectins exhibit potent bioactivities encompassing mitogenic, antitumor, antimicrobial, immunomodulatory, antioxidative, hypoglycemic, insecticidal and nematicidal activities. They are potential resources for development into functional or healthy foods and targets for food protein researchers.


Assuntos
Lectinas/metabolismo , Arisaema/metabolismo , Dioscorea/metabolismo , Lectinas/química , Tubérculos/metabolismo , Solanum tuberosum/metabolismo , Trichosanthes/metabolismo
15.
Biopolymers ; 108(6)2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28555756

RESUMO

Matriptase-2 plays a pivotal role in keeping iron concentrations within a narrow physiological range in humans. The opportunity to reduce matriptase-2 proteolytic activity may open a novel possibility to treat iron overload diseases, such as hereditary hemochromatosis and thalassemia. Here, we present 23 new analogues of trypsin inhibitor SFTI-1 designed to inhibit human matriptase-2. Influence of the modifications Gly1Lys, Ile10Arg, and Phe12His, as well as the introduction of Narg in P1 or P1 and P4 positions were examined. Selected peptides were further analyzed, together with previously reported peptides, for their inhibitory activity against related human proteases, that are, matriptase-1, plasmin, thrombin and trypsin. A highly potent inhibitor of matriptase-2, the bicycylic [Arg5 , Arg10 , His12 ]SFTI-1, with a Ki value of 15 nm was obtained.


Assuntos
Desenho de Fármacos , Helianthus/química , Proteínas de Membrana/antagonistas & inibidores , Peptídeos Cíclicos/química , Inibidores de Serino Proteinase/síntese química , Inibidores da Tripsina/química , Sequência de Aminoácidos , Helianthus/metabolismo , Humanos , Cinética , Proteínas de Membrana/metabolismo , Peptídeos Cíclicos/sangue , Estabilidade Proteica , Sementes/química , Sementes/metabolismo , Alinhamento de Sequência , Serina Endopeptidases/metabolismo , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/metabolismo , Trombina/antagonistas & inibidores , Trombina/metabolismo , Tripsina/química , Tripsina/metabolismo
16.
Biopolymers ; 108(2)2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27627696

RESUMO

A series of analogues of trypsin inhibitor SFTI-1 were designed and synthesized to monitor peptide splicing. In the middle part of the SFTI-1 analogues, which is released upon incubation with proteinase, the RGD sequence or an acceptor of fluorescence for FRET was introduced. The results of studies with trypsin confirmed that the designed analogues underwent peptide splicing. Furthermore, we showed that a FRET displaying SFTI-1 analogue was internalized into the HaCaT keratinocytes, where it was degraded. Therefore, both proteolysis and the reduction of the disulfide bridge of the peptide took place. As a result, such analogues are a convenient tool to trace the proteolytic activity inside the cell. However, the cytotoxicity of SFTI-1 analogues grafted with the RGD sequence did not correlate with their susceptibility to peptide splicing. Nevertheless, these peptides were slightly more active than the reference peptide (GRGDNP). Interestingly, one of the analogues assigned as [desSer6 ]VI, under experimental conditions, appeared significantly more cytotoxic towards cancer cells U87-MG in contrast to the reference peptide.


Assuntos
Queratinócitos/metabolismo , Peptídeos/metabolismo , Inibidores da Tripsina/metabolismo , Tripsina/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Humanos , Queratinócitos/citologia , Espectrometria de Massas , Microscopia de Fluorescência , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Proteólise , Tripsina/química , Inibidores da Tripsina/química
18.
Biopolymers ; 106(5): 685-96, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27258473

RESUMO

Sunflower trypsin inhibitor (SFTI-1) is recognized as an attractive scaffold to designed potent inhibitors of various proteases. We have recently found that its analogues inhibit noncovalently both human and yeast 20S proteasomes. Here, a set of novel and more potent in vitro inhibitors is presented. The inhibitory potency of the peptides was assessed with human 20S proteasome in the presence or absence of sodium dodecyl sulfate and with human 26 proteasome. Their antiproliferative action against tumor (human melanoma cells A375) and normal cells (46 BR.1N human fibroblasts and HaCaT keratinocytes) was determined. The selected fluoresceine-labeled inhibitors were able to internalize into A375 cells and were sometimes present as foci in the cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 685-696, 2016.


Assuntos
Peptídeos Cíclicos , Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma , Inibidores da Tripsina , Linhagem Celular Tumoral , Humanos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologia
19.
FEBS J ; 283(15): 2929-48, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27326540

RESUMO

The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2-aminobenzoic acid (ABZ)-Val-Val-Ser-Tyr-Ala-Met-Gly-Tyr(3-NO2 )-NH2 , is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 10(5)  m(-1) ·s(-1) ) and a high kcat (8 s(-1) ). Using this peptide, we identified chymotrypsin-like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods.


Assuntos
Aminobenzoatos/metabolismo , Oligopeptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/urina , Neoplasias da Bexiga Urinária/diagnóstico , Aminobenzoatos/química , Feminino , Corantes Fluorescentes , Humanos , Masculino , Oligopeptídeos/química , Complexo de Endopeptidases do Proteassoma/química , Neoplasias da Bexiga Urinária/enzimologia , ortoaminobenzoatos
20.
Sci Rep ; 6: 22856, 2016 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-26955973

RESUMO

Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface.


Assuntos
Mieloblastina/metabolismo , Peptidomiméticos/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Peptidomiméticos/química , Análise de Sequência de DNA , Especificidade por Substrato
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