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1.
Biopolymers ; 106(3): 245-59, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26916937

RESUMO

Deamination of vasopressin (AVP) enhances its antidiuretic activity. Moreover, introduction of D-Arg8 instead of its L enantiomer in deamino-vasopressin (dAVP) results in an extremely potent and selective antidiuretic agonist - desmopressin (dDAVP). In this study we describe the synthesis, pharmacological properties and structures of these two potent antidiuretic agonists, and their inverso analogs. The structures of the peptides are studied in micellar and liposomic models of cell membrane using CD spectroscopy. Additionally, three-dimensional structures in mixed anionic-zwitterionic micelles are obtained using NMR spectroscopy supported by molecular dynamics simulations. Our conformational studies have shown that desmopressin in a membrane mimicking environment adopts one of the characteristic for vasopressin-like peptides ß-turn - in position 3,4. Furthermore, dDAVP shows the tendency to create a ß-turn in the Cys6-Gly9 C-tail, considered to be important for the antidiuretic activity, and also some tendency to adopt a 5,6 ß-turn. In desmopressin, in contrast to the native vasopressin, deamino-vasopressin and [D-Arg8]-vasopressin (DAVP), the Arg8 side chain, crucial for the pressor and antidiuretic activities, is very well exposed for interaction with the receptor, whereas Gly9, crucial for the pressor and uterotonic activities, is situated together with the C-terminal amide group very close to the tocin ring. The arrangements of the Gln4 and Asn5 side chains, being crucial for OT activity, also differ in desmopressin as compared to those of AVP, dAVP and DAVP. These differences in arrangement of the important for activities side chains are likely to explain extremely potent and selective antidiuretic activities of desmopressin. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 245-259, 2016.


Assuntos
Antidiuréticos/síntese química , Desamino Arginina Vasopressina/síntese química , Lipossomos/química , Ocitócicos/síntese química , 1,2-Dipalmitoilfosfatidilcolina/química , Animais , Antidiuréticos/farmacologia , Ciclização , Desamino Arginina Vasopressina/farmacologia , Feminino , Fluorenos/química , Ligação de Hidrogênio , Micelas , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Ocitócicos/farmacologia , Fosfatidilgliceróis/química , Estrutura Secundária de Proteína , Ratos Wistar , Técnicas de Síntese em Fase Sólida/métodos , Útero/efeitos dos fármacos , Útero/fisiologia
2.
J Nat Prod ; 79(4): 1073-83, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-26998557

RESUMO

Venoms of hymenopteran insects have attracted considerable interest as a source of cationic antimicrobial peptides (AMPs). In the venom of the solitary bee Hylaeus signatus (Hymenoptera: Colletidae), we identified a new hexadecapeptide of sequence Gly-Ile-Met-Ser-Ser-Leu-Met-Lys-Lys-Leu-Ala-Ala-His-Ile-Ala-Lys-NH2. Named HYL, it belongs to the category of α-helical amphipathic AMPs. HYL exhibited weak antimicrobial activity against several strains of pathogenic bacteria and moderate activity against Candida albicans, but its hemolytic activity against human red blood cells was low. We prepared a set of HYL analogues to evaluate the effects of structural modifications on its biological activity and to increase its potency against pathogenic bacteria. This produced several analogues exhibiting significantly greater activity compared to HYL against strains of both Staphylococcus aureus and Pseudomonas aeruginosa even as their hemolytic activity remained low. Studying synergism of HYL peptides and conventional antibiotics showed the peptides act synergistically and preferentially in combination with rifampicin. Fluorescent dye propidium iodide uptake showed the tested peptides were able to facilitate entrance of antibiotics into the cytoplasm by permeabilization of the outer and inner bacterial cell membrane of P. aeruginosa. Transmission electron microscopy revealed that treatment of P. aeruginosa with one of the HYL analogues caused total disintegration of bacterial cells. NMR spectroscopy was used to elucidate the structure-activity relationship for the effect of amino acid residue substitution in HYL.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Venenos de Abelha/farmacologia , Abelhas/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade
3.
Eur Biophys J ; 44(8): 727-43, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26290060

RESUMO

We describe the synthesis, pharmacological properties, and structures of antidiuretic agonists, arginine vasopressin (AVP) and [D-Arg(8)]-vasopressin (DAVP), and their inverso analogues. The structures of the peptides are studied based on micellar and liposomic models of cell membranes using CD spectroscopy. Additionally, three-dimensional structures in mixed anionic-zwitterionic micelles are obtained using NMR spectroscopy and molecular dynamics simulations. NMR data have shown that AVP and DAVP tend to adopt typical of vasopressin-like peptides ß-turns: in the 2-5 and 3-6 fragments. The inverso-analogues also adopt ß-turns in the 3-6 fragments. For this reason, their inactivity seems to be due to the difference in side chains orientations of Tyr(2), Phe(3), and Arg(8), important for interactions with the receptors. Again, the potent antidiuretic activity of DAVP can be explained by CD data suggesting differences in mutual arrangement of the aromatic side chains of Tyr(2) and Phe(3) in this peptide in liposomes rather than of native AVP. In the presence of liposomes, the smallest conformational changes of the peptides are noticed with DPPC and the largest with DPPG liposomes. This suggests that electrostatic interactions are crucial for the peptide-membrane interactions. We obtained similar, probably active, conformations of the antidiuretic agonists in the mixed DPC/SDS micelles (5:1) and in the mixed DPPC/DPPG (7:3) liposomes. Thus it can be speculated that the anionic-zwitterionic liposomes as well as the anionic-zwitterionic micelles, mimicking the eukaryotic cell membrane environment, partially restrict conformational freedom of the peptides and probably induce conformations resembling those of biologically relevant ones.


Assuntos
Antidiuréticos/química , Arginina Vasopressina/análogos & derivados , Membrana Celular/química , Lipossomos/química , Micelas , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Animais , Antidiuréticos/síntese química , Antidiuréticos/farmacologia , Arginina Vasopressina/síntese química , Arginina Vasopressina/química , Arginina Vasopressina/farmacologia , Membrana Celular/efeitos dos fármacos , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Ratos Wistar
4.
J Pept Sci ; 20(11): 885-95, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25123582

RESUMO

The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan-containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detecting cytoplasmic ß-galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Venenos de Abelha/química , Escherichia coli/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antibacterianos/síntese química , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Venenos de Abelha/genética , Venenos de Abelha/isolamento & purificação , Venenos de Abelha/farmacologia , Abelhas/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Fluorescência , Hemólise/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/metabolismo , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Estrutura Secundária de Proteína , Triptofano/química
5.
J Pept Sci ; 20(9): 725-35, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24920043

RESUMO

The antimicrobial 40-amino-acid-peptide lucifensin was synthesized by native chemical ligation (NCL) using N-acylbenzimidazolinone (Nbz) as a linker group. NCL is a method in which a peptide bond between two discreet peptide chains is created. This method has been applied to the synthesis of long peptides and proteins when solid-phase synthesis is imcompatible. Two models of ligation were developed: [15+25] Ala-Cys and [19+21] His-Cys. The [19+21] His-Cys method gives lower yield because of the lower stability of 18-peptide-His-Nbz-CONH2 peptide, as suggested by density functional theory calculation. Acetamidomethyl-deprotection and subsequent oxidation of the ligated linear lucifensin gave a mixture of lucifensin isomers, which differed in the location of their disulfide bridges only. The dominant isomer showed unnatural pairing of cysteines [C1-6], [C3-5], and [C2-4], which limits its ability to form α-helical structure. The activity of isomeric lucifensin toward Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus was lower than that of the natural lucifensin. The desired product native lucifensin was prepared from this isomer using a one-pot reduction with dithiotreitol and subsequent air oxidation in slightly alkaline medium.


Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Defensinas , Bactérias Gram-Positivas/crescimento & desenvolvimento , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Defensinas/síntese química , Defensinas/química , Defensinas/farmacologia , Estrutura Secundária de Proteína
6.
J Pept Sci ; 20(6): 375-84, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24616110

RESUMO

A novel antimicrobial peptide, designated macropin (MAC-1) with sequence Gly-Phe-Gly-Met-Ala-Leu-Lys-Leu-Leu-Lys-Lys-Val-Leu-NH2 , was isolated from the venom of the solitary bee Macropis fulvipes. MAC-1 exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l- or d-lysine in selected positions. Furthermore, all-d analog and analogs with d-amino acid residues introduced at the N-terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α-helical secondary structure in the presence of trifluoroethanol or membrane-mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure-activity relationship for the effect of d-amino acid substitutions in MAC-1 using NMR spectroscopy.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Venenos de Abelha/química , Abelhas/química , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antifúngicos/síntese química , Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Bacillus subtilis/efeitos dos fármacos , Venenos de Abelha/isolamento & purificação , Venenos de Abelha/farmacologia , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Micrococcus luteus/efeitos dos fármacos , Modelos Moleculares , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade
7.
Amino Acids ; 45(1): 143-57, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23483218

RESUMO

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH2 (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8-Cys23 and Cys11-Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Venenos de Abelha/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Venenos de Abelha/química , Venenos de Abelha/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Himenópteros/metabolismo , Testes de Sensibilidade Microbiana , Análise de Sequência de Proteína , Relação Estrutura-Atividade , Tensoativos , Lipossomas Unilamelares/metabolismo
8.
Cell Mol Life Sci ; 69(17): 2951-66, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22527714

RESUMO

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.


Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/farmacocinética , Sistemas de Liberação de Medicamentos , Endocitose , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Astrocitoma/tratamento farmacológico , Astrocitoma/metabolismo , Astrocitoma/patologia , Encéfalo/metabolismo , Bovinos , Células Cultivadas , Quimiotaxia , Membrana Corioalantoide , AMP Cíclico/metabolismo , Derme/citologia , Derme/efeitos dos fármacos , Derme/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Hepacivirus , Hepatite C/tratamento farmacológico , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Transporte Proteico , Relação Quantitativa Estrutura-Atividade , Ratos , Ratos Wistar , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Contração Uterina/efeitos dos fármacos
9.
Amino Acids ; 42(5): 1715-25, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-21416381

RESUMO

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with D-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The D-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either D-aspartic acid or D-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the D-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the D-amino acid residues in the corresponding peptides. The introduction of the non-coded D: -amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.


Assuntos
Alanina/química , Ácido D-Aspártico/química , Ovário/citologia , Peptídeos/química , Alanina/metabolismo , Aminoácidos/biossíntese , Aminoácidos/química , Animais , Ácido D-Aspártico/metabolismo , Feminino , Espectroscopia de Ressonância Magnética , Ovário/crescimento & desenvolvimento , Proteólise , Sarcofagídeos/química , Sarcofagídeos/crescimento & desenvolvimento , Trítio/química
10.
Amino Acids ; 43(2): 617-27, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22038179

RESUMO

In this study we present the synthesis and some pharmacological properties of fourteen new analogues of neurohypophyseal hormones conformationally restricted in the N-terminal part of the molecule. All new peptides were substituted at position 2 with cis-1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc). Moreover, one of the new analogues: [cis-Apc(2), Val(4)]AVP was also prepared in N-acylated forms with various bulky acyl groups. All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of the selected compounds to human OT receptor. Our results showed that introduction of cis -Apc(2) in position 2 of either AVP or OT resulted in analogues with high antioxytocin potency. Two of the new compounds, [Mpa(1),cis-Apc(2)]AVP and [Mpa(1),cis-Apc(2),Val(4)]AVP, were exceptionally potent antiuterotonic agents (pA(2) = 8.46 and 8.40, respectively) and exhibited higher affinities for the human OT receptor than Atosiban (K (i) values 5.4 and 9.1 nM). Moreover, we have demonstrated for the first time that N -terminal acylation of AVP analogue can improve its selectivity. Using this approach, we obtained compound Aba[cis-Apc(2),Val(4)]AVP (XI) which turned out to be a moderately potent and exceptionally selective OT antagonist (pA(2) = 7.26).


Assuntos
Arginina Vasopressina/análogos & derivados , Arginina Vasopressina/farmacologia , Ocitocina/análogos & derivados , Ocitocina/farmacologia , Receptores de Ocitocina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antidiuréticos , Arginina Vasopressina/síntese química , Ácidos Carboxílicos/química , Cicloexanos/química , Desenho de Fármacos , Feminino , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Ocitocina/síntese química , Ligação Proteica , Estrutura Secundária de Proteína , Ratos , Ratos Wistar , Receptores de Ocitocina/metabolismo , Vasoconstritores
11.
Amino Acids ; 43(5): 2047-58, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22526241

RESUMO

The impact of inserting hydrocarbon staples into short α-helical antimicrobial peptides lasioglossin III and melectin (antimicrobial peptides of wild bee venom) on their biological and biophysical properties has been examined. The stapling was achieved by ring-closing olefin metathesis, either between two S-2-(4'-pentenyl) alanine residues (S (5)) incorporated at i and i + 4 positions or between R-2-(7'-octenyl) alanine (R (8)) and S (5) incorporated at the i and i + 7 positions, respectively. We prepared several lasioglossin III and melectin analogs with a single staple inserted into different positions within the peptide chains as well as analogs with double staples. The stapled peptides exhibited a remarkable increase in hemolytic activity, while their antimicrobial activities decreased. Some single stapled peptides showed a higher resistance against proteolytic degradation than native ones, while the double stapled analogs were substantially more resistant. The CD spectra of the singly stapled peptides measured in water showed only a slightly better propensity to form α-helical structure when compared to native peptides, whereas the doubly stapled analogs exhibited dramatically enhanced α-helicity.


Assuntos
Alcenos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Venenos de Abelha/química , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Abelhas/química , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Cromatografia de Fase Reversa , Dicroísmo Circular , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteólise
12.
Amino Acids ; 43(2): 751-61, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22038181

RESUMO

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8-Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17-Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Venenos de Abelha/química , Abelhas/química , Peptídeos Cíclicos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antifúngicos/síntese química , Antifúngicos/química , Venenos de Abelha/síntese química , Venenos de Abelha/farmacologia , Candida albicans/efeitos dos fármacos , Cistina/síntese química , Cistina/química , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/ultraestrutura , Hemólise , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Estrutura Secundária de Proteína , Análise de Sequência de Proteína
13.
Biomedicines ; 10(8)2022 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-36009516

RESUMO

This work aimed to test the effect of 7-day exposure of rats to multifunctional enkephalin analogs LYS739 and LYS744 at doses of 3 mg/kg and 10 mg/kg on the protein composition of rat spleen lymphocytes, brain cortex, and hippocampus. Alterations of proteome induced by LYS739 and LYS744 were compared with those elicited by morphine. The changes in rat proteome profiles were analyzed by label-free quantification (MaxLFQ). Proteomic analysis indicated that the treatment with 3 mg/kg of LYS744 caused significant alterations in protein expression levels in spleen lymphocytes (45), rat brain cortex (31), and hippocampus (42). The identified proteins were primarily involved in RNA processing and the regulation of cytoskeletal dynamics. In spleen lymphocytes, the administration of the higher 10 mg/kg dose of both enkephalin analogs caused major, extensive modifications in protein expression levels: LYS739 (119) and LYS744 (182). Among these changes, the number of proteins associated with immune responses and apoptotic processes was increased. LYS739 treatment resulted in the highest number of alterations in the rat brain cortex (152) and hippocampus (45). The altered proteins were functionally related to the regulation of transcription and cytoskeletal reorganization, which plays an essential role in neuronal plasticity. Administration with LYS744 did not increase the number of altered proteins in the brain cortex (26) and hippocampus (26). Our findings demonstrate that the effect of κ-OR full antagonism of LYS744 is opposite in the central nervous system and the peripheral region (spleen lymphocytes).

14.
Chembiochem ; 12(9): 1352-61, 2011 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-21560219

RESUMO

Recently, we identified a new insect defensin, named lucifensin that is secreted/excreted by the blowfly Lucilia sericata larvae into a wound as a disinfectant during the medicinal process known as maggot therapy. Here, we report the total chemical synthesis of this peptide of 40 amino acid residues and three intramolecular disulfide bridges by using three different protocols. Oxidative folding of linear peptide yielded a peptide with a pattern of disulfide bridges identical to that of native lucifensin. The synthetic lucifensin was active against Gram-positive bacteria and was not hemolytic. We synthesized three lucifensin analogues that are cyclized through one native disulfide bridge in different positions and having the remaining four cysteines substituted by alanine. Only the analogue cyclized through a Cys16-Cys36 disulfide bridge showed weak antimicrobial activity. Truncating lucifensin at the N-terminal by ten amino acid residues resulted in a drop in antimicrobial activity. Linear lucifensin having all six cysteine residues alkylated was inactive. Circular dichroism spectra measured in the presence of α-helix-promoting compounds showed different patterns for lucifensin and its analogues. Transmission electron microscopy revealed that Bacillus subtilis treatment with lucifensin induced significant changes in its envelope.


Assuntos
Defensinas/química , Defensinas/síntese química , Larva/química , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Defensinas/genética , Dissulfetos/química , Dobramento de Proteína , Estrutura Secundária de Proteína
15.
J Pept Sci ; 17(5): 366-72, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21412955

RESUMO

In the present work, achiral non-coded amino acids, N-(Bzl)-Gly, X(1) or X(2) , were substituted at position 7 of the model B(2) receptor antagonist [D-Arg(0) , Hyp(3) , Thi(5, 8) , D-Phe(7) ]-BK. The N-terminal amino group of the analogues was either free or acylated with 1-Aca or Aaa. Biological activity of the compounds was assessed in the in vitro rat uterus test and the in vivo rat blood pressure test. The X(1) (7) substitution resulted in a decrease in antagonistic potency of the new peptide in both assays. The X(2) (7) and N-(Bzl)-Gly(7) substituted analogues showed weak agonistic properties in the rat uterus test. Interestingly, the latter compound exhibited dual activity in the pressor test, i.e. intrinsic vasodepressor action and at the same time a weak antagonistic effect. Acylation of the N-terminus enhanced antagonistic properties of the resulting peptides in the rat blood pressure test in the case of compounds containing X(1) or X(2) modification. Our studies provide new information about structure-activity relationship of the BK antagonists which may be helpful for designing more potent B(2) receptor blockers.


Assuntos
Bradicinina/química , Bradicinina/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Bradicinina/análogos & derivados , Antagonistas de Receptor B2 da Bradicinina , Feminino , Técnicas In Vitro , Ratos , Relação Estrutura-Atividade , Útero/efeitos dos fármacos
16.
Cell Mol Life Sci ; 67(16): 2815-24, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20376529

RESUMO

Progesterone non-genomically attenuates the calcium signaling of the human oxytocin receptor and several other Galpha(q) protein-coupled receptors. High progesterone concentrations are found in the endometrium during pregnancy opposing the responsiveness of the underlying myometrium to labor-inducing hormones. Here, we demonstrate that within minutes, progesterone inhibits oxytocin- and bradykinin-induced contractions of rat uteri, calcium responses induced by platelet-activating factor in the human endometrial cell line MFE-280, and oxytocin-induced calcium signals in PHM1-31 immortalized pregnant human myometrial cells. Using human embryonic kidney (HEK293) cells as model system, we analyzed the molecular mechanisms underlying these effects. Our data indicate that progesterone rapidly depletes intracellular calcium stores. The resulting desensitization of the cells might contribute to the quiescence of the uterus during pregnancy.


Assuntos
Cálcio/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Animais , Linhagem Celular , Endométrio/efeitos dos fármacos , Endométrio/fisiologia , Estradiol/farmacologia , Estro , Etanol/farmacologia , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/fisiologia , Nifedipino/farmacologia , Ocitocina/farmacologia , Progesterona/metabolismo , Progesterona/farmacologia , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Útero/efeitos dos fármacos , Útero/fisiologia
17.
Naunyn Schmiedebergs Arch Pharmacol ; 394(5): 955-965, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33404687

RESUMO

Lithium (Li) represents a first choice mood stabilizer for bipolar disorder (BD). Despite extensive clinical use, questions regarding its mechanism of action and pathological mechanism of renal function impairment by Li remain open. The present study aimed to improve our knowledge in this area paying special attention to the relationship between the length of Li action, lipid peroxidation (LP), and Na+/K+-ATPase properties. The effects of therapeutic Li doses, administered daily to male Wistar rats for 1 (acute), 7 (short term) and 28 days (chronic), were studied. For this purpose, Na+/K+-ATPase activity measurements, [3H]ouabain binding and immunoblot analysis of α-Na+/K+-ATPase were performed. Li-induced LP was evaluated by determining the malondialdehyde concentration by HPLC. Sleep deprivation (SD) was used as an experimental approach to model the manic phase of BD. Results obtained from the kidney were compared to those obtained from erythrocytes and different brain regions in the same tested animals. Whereas treatment with therapeutic Li concentration did not bring any LP damage nor significant changes of Na+/K+-ATPase expression and [3H]ouabain binding in the kidney, it conferred strong protection against this type of damage in the forebrain cortex. Importantly, the observed changes in erythrocytes indicated changes in forebrain cortices. Thus, different resistance to SD-induced changes of LP and Na+/K+-ATPase was detected in the kidney, erythrocytes and the brain of Li-treated rats. Our study revealed the tissue-specific protective properties of Li against LP and Na+/K+-ATPase regulation.


Assuntos
Antimaníacos/farmacologia , Transtorno Bipolar/tratamento farmacológico , Peroxidação de Lipídeos/efeitos dos fármacos , Carbonato de Lítio/farmacologia , Animais , Antimaníacos/administração & dosagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Carbonato de Lítio/administração & dosagem , Masculino , Ratos , Ratos Wistar , Privação do Sono/psicologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Tempo
18.
Amino Acids ; 38(5): 1549-59, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19885720

RESUMO

We report the solid phase synthesis and some pharmacological properties of 24 oxytocin (OT) analogues. Basic modifications at position 9 (introduction of L- or D-beta-(2-thienyl)-alanine [L- or D-Thi], or L- or D-3-Pyridylalanine [L- or D-3-Pal]) were combined with D-tyrosine(OEthyl) [D-Tyr(Et)] or D-1-naphthylalanine [D-1-Nal] in position 2 and beta-mercaptopropionic acid (Mpa) in position 1 modifications in altogether 14 analogues. Additionally, 8 analogues having alpha-aminoisobutyric acid [Aib] or D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (D-Tic) or diethylglycine (Deg) in position 9 and D-Tyr(Et) or D-1-Nal or D-Tic in position 2 and Mpa or Pen (beta beta-dimethylcysteine) in position 1 were prepared. Two of these analogues have one more modification in position 6, i.e. Pen. Furthermore, two analogues having Mpa in position 1 and D-Tyr(Et) or D-1-Nal in position 2 were prepared for comparison purposes. The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OT receptor. The analogue having the highest anti-oxytocic activity was [Mpa(1), D-Tyr(Et)(2), Deg(9)]OT (pA(2) = 8.68 +/- 0.26); this analogue was also selective.


Assuntos
Aminoácidos/química , Ocitocina/análogos & derivados , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Ocitocina/síntese química , Ocitocina/farmacologia , Ratos , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade
19.
Amino Acids ; 39(2): 539-48, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20108008

RESUMO

Incorporation of L- or D-Tic into position 7 of oxytocin (OT) and its deamino analogue ([Mpa(1)]OT) resulted in four analogues, [L-Tic(7)]OT (1), [D-Tic(7)]OT (2), [Mpa(1),L-Tic(7)]OT (3) and [Mpa(1),D-Tic(7)]OT (4). Their biological properties were described by Fragiadaki et al. (Eur J Med Chem 42:799-806, 2007). Their NMR study (NOESY, TOCSY, (1)H-(13)C HSQC spectra) is presented here. Analogues 1, 3 and 4 showed partial agonistic activity, analogue 2 was pure antagonist, suggesting that a cis conformation between residues 6 and 7 of the molecule does not result in antagonistic activity. However, the reduction in agonistic activity of analogues 1, 3 and 4 in comparison to oxytocin is consistent with the reduction of the trans conformation form. Binding affinity for the human oxytocin receptor with IC(50) value of 130, 730, 103, and 380 nM for peptides 1, 2, 3, and 4, respectively, showed lower affinity in the case of D analogues. Deamination slightly increased the affinity. The existence of both cis and trans configurations of the Cys(6)-D-Tic(7) bond is supported by observation of two sets of cross-peaks for (1)H and (13)C nuclei for most of the residues of the peptide not only in NOESY and TOCSY but also in (1)H-(13)C HSQC spectra. The MS and HPLC indicate the presence of a single molecule/peptide, and NMR data thus suggest that this second set of peaks is due to the cis conformation.


Assuntos
Ocitocina/análogos & derivados , Tetra-Hidroisoquinolinas/química , Sequência de Aminoácidos , Modelos Moleculares , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular , Ocitocina/síntese química
20.
Amino Acids ; 39(5): 1553-61, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20499256

RESUMO

The recently described antimicrobial peptide melectin (MEP, GFLSILKKVLPKVMAHMK-NH2) exhibits high antimicrobial activity against Gram-positive and Gram-negative bacteria. Here we describe the synthesis and biological activities of 23 new analogues of MEP. We studied the influence of dimerization and tetramerization (MAP-constructs of MEP) on the antimicrobial and hemolytic activities, as well as the role of Met in positions 14 and 17 of the peptide chain. Oxidation of the Met to Met(O) and Met(O2) decreases antimicrobial activity of all tested bacteria if the peptide is in the monomeric form, however, only to Staphylococcus aureus if in the form of dimer or tetramer. Dimerization and tetramerization increase the undesirable hemolytic activity of the peptides. Interestingly, substitution of Leu for Val in position 6 leads to the decrease of hemolytic activity. Introduction of the isosteric amino acid Nle into positions 14 or 17 or both leads to slight increase of hemolytic activity under preservation of high antimicrobial activities. Unfortunately, dimerization again leads to an increase of hemolytic activity.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Aminoácidos/química , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Cromatografia Líquida de Alta Pressão , Dimerização , Eletroforese Capilar , Testes de Sensibilidade Microbiana
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