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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.
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.

3.
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
4.
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
5.
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
6.
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
7.
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.

8.
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
9.
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
10.
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
11.
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
12.
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
13.
J Pept Sci ; 21(11): 819-25, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26415697

RESUMO

N-substituted glycines constitute mimics of natural amino acids that are of great interest in the peptide-based drug development. Peptoids-oligo(N-substituted glycines) have been recently demonstrated to be highly active peptidomimetics in biological systems, resistant to proteolytic degradation. We developed a method of the deuterium labeling of peptidomimetics containing N-substituted glycine residues via H/D exchange of their α-carbon hydrogen atoms. The labeling was shown to be easy, inexpensive, and without the use of derivatization reagents or the need for a further purification. The deuterons introduced at the α-carbon atoms do not undergo a back exchange under acidic conditions during liquid chromatography mass spectrometry (LC-MS) analysis. The LC-MS analysis of a mixture of isotopologues revealed a co-elution of deuterated and nondeuterated forms of the peptidomimetics, which may be useful in the quantitative isotope dilution analysis of peptoids and other derivatives of N-substituted glycines.


Assuntos
Glicina/análogos & derivados , Marcação por Isótopo , Peptídeos/química , Peptoides/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Cistina , Deutério , Medição da Troca de Deutério , Glicina/química , Técnicas de Diluição do Indicador , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Peptoides/síntese química , Peptoides/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria , Espectrometria de Massas em Tandem
14.
Chembiochem ; 16(14): 2036-45, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26212347

RESUMO

Serine-proteinase-catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI-1: both single peptides and two-peptide chains (C- and N-terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl-enzyme intermediate was preceded by hydrolysis of the substrate Lys-Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two-peptide-chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl-enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl-enzyme were not observed. The peptide splicing was sequence- not structure-specific.


Assuntos
Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Peptídeos/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Helianthus/química , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos Cíclicos/síntese química , Serina Proteases/síntese química , Serina Proteases/química , Serina Proteases/farmacologia , Tripsina/química , Inibidores da Tripsina/síntese química
15.
Chembiochem ; 16(11): 1601-7, 2015 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-25999208

RESUMO

A series of 17 new analogues of trypsin inhibitor SFTI-1 were designed and synthesized to obtain matriptase-2 inhibitors. A number of the modified bicyclic peptides displayed much higher affinity towards matriptase-2 than towards the highly homologous matriptase-1. Replacement of Lys5 by Arg in the wild-type SFTI-1 led to an 11-fold increase in the matriptase-2 inhibitory activity. Replacement of Arg2 by its enantiomer (D-arginine) slightly lowered the inhibition of matriptase-2, but almost completely abolished the affinity towards matriptase-1, thus yielding the most selective matriptase-2 inhibitor. This is the first report describing inhibitors of the recently discovered matriptase-2 based on the SFTI-1 structure. The results showed that SFTI-1 is a promising scaffold for the design of potent and selective inhibitors of this enzyme.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Inibidores da Tripsina/farmacologia , Sequência de Aminoácidos , Células HEK293 , Humanos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Serina Endopeptidases , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/química
16.
Biopolymers ; 104(3): 206-12, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25904562

RESUMO

Recently, we described a process of trypsin-assisted peptide splicing of analogs of trypsin inhibitor SFTI-1, that seems to be very similar to proteasome-catalyzed peptide splicing. Here, we show, for the first time, that a peptide-peptoid hybrid (peptomer) can also be spliced by trypsin. Incubation of a double sequence SFTI-1 analog, containing two peptoid monomers, with equimolar amount of trypsin leads to formation of monocyclic peptomer as the main product. We proved that the peptide bond formed by a peptoid monomer is not only digested by trypsin but also participates in the enzyme-assisted splicing process.


Assuntos
Peptídeos Cíclicos/química , Processamento de Proteína , Inibidores da Tripsina/química , Tripsina/química , Animais , Bovinos
17.
Proteins ; 83(3): 582-9, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25546528

RESUMO

Protease inhibitors of the Bowman-Birk (BBI) family are commonly found in plants and animals where they play a protective role against invading pathogens. Here, we report an atomic resolution (1Å) crystal structure of a peptide inhibitor isolated from a skin secretion of a Chinese bamboo odorous frog Huia versabilis (HV-BBI) in complex with trypsin. HV-BBI shares significant similarities in sequence with a previously described inhibitor from a diskless-fingered odorous frog Odorrana graham (ORB). However, the latter is characterized by more than a 16,000 fold higher Ki against trypsin than HV-BBI. Comparative analysis of trypsin cocrystal structures of HV-BBI and ORB and additionally that of Sunflower Trypsin Inhibitor (SFTI-1) together with accessory information on the affinities of inhibitor variants allowed us to pinpoint the inhibitor moiety responsible for the observed large difference in activity and also to define the extent of modifications permissible within the common protease-binding loop scaffold of BBI inhibitors. We suggest that modifications outside of the inhibitory loop permit the evolution of specificity toward different enzymes characterized by trypsin-like specificity.


Assuntos
Peptídeos/química , Tripsina/química , Sequência de Aminoácidos , Animais , Anuros , Bovinos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Pele/química , Tripsina/metabolismo
18.
PLoS One ; 9(2): e89465, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24586798

RESUMO

Starting from the primary structure of sunflower trypsin inhibitor SFTI-1, we designed novel non-covalent inhibitors of human and yeast 20S proteasomes. Peptides with Arg residue in P1 position and two basic amino acid residues (Lys or/and Arg) in P2' and P3' positions strongly inhibited chymotrypsin-like and caspase-like activities, while trypsin-like activity was poorly modified. We found that some SFTI-1 analogues up-regulated exclusively the chymotrypsin-like activity of latent yeast 20S proteasome.


Assuntos
Peptídeos Cíclicos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores da Tripsina/metabolismo , Leveduras/metabolismo , Caspases/metabolismo , Quimotripsina/metabolismo , Humanos , Peptídeos/metabolismo , Tripsina/metabolismo
19.
Biopolymers ; 102(1): 124-35, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24272319

RESUMO

This article describes the synthesis and enzymatic study of newly synthesized analogs of trypsin inhibitors SFTI-1 that were fluorescent labeled on their N-terminal amino groups. Two fluorescent derivatives of benzoxazole (3-[2-(4-diphenylaminophenyl)benzoxazol-5-yl]-L-alanine-[(4NPh2 )Ph]Box-Ala and 3-[2-(2',4',5'-trimethoxyphenyl)benzoxazol-5-yl]-L-alanine-[2,4,5-(OMe)3Ph]Box-Ala) were used as efficient fluorescent labels. The compounds obtained preserved their inhibitory activity and were efficient inhibitors of bovine trypsin or chymotrypsin. Nevertheless, their association inhibition constants were one or two orders of magnitude lower than those determined for unlabeled monocyclic SFTI-1 or [Phe(5)]SFTI-1, respectively. The conjugates obtained were found to be proteolytically stable in the presence of cognate enzymes. Applying such fluorescent peptides, we were able to investigate enzyme-inhibitor complex formation using fluorescent techniques. We found that such compounds were rapidly internalized by the fibroblast or cancer cells with no cytotoxic effects.


Assuntos
Helianthus/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/isolamento & purificação , Sementes/química , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/isolamento & purificação , Sequência de Aminoácidos , Animais , Benzoxazóis/química , Bovinos , Linhagem Celular , Permeabilidade da Membrana Celular , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Citometria de Fluxo , Fluorescência , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Peptídeos Cíclicos/química , Fatores de Tempo , Inibidores da Tripsina/química
20.
FEBS J ; 280(23): 6213-22, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24112465

RESUMO

This study examines peptide splicing catalyzed by serine proteinases. A series of two-peptide-chain analogs of trypsin inhibitor SFTI-1 were designed and synthesized via the solid-phase method. All consisted of two peptide chains (also called N- and C-terminal fragments) joined together by one disulfide bridge. The analogs were incubated with bovine ß-trypsin or bovine α-chymotrypsin. Analysis of MS data analysis showed that, after enzyme-catalyzed degradation of the single peptide bond between the Lys and Ser residues located at the C-terminus of the C-terminal peptide chain, a new peptide bond was formed. This bond brought together the separated peptide chains, and, as a result, monocyclic SFTI-1 was recovered. This proteolytic route of peptide rearrangement appears to be similar to peptide splicing catalyzed by proteasomes. However, the proteasome is much more complex than 'classical' serine proteinases.


Assuntos
Quimotripsina/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Inibidores da Tripsina/farmacologia , Tripsina/química , Animais , Bovinos , Quimotripsina/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeos Cíclicos/síntese química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismo
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