RESUMO
A series of novel water-soluble short peptide-bioconjugates containing a ferrocenoyl (Fc) or ruthenocenoyl (Rc) unit was synthesized and characterized to combine the unique activity of ferrocene and the isoelectronic ruthenocene with precisely designed peptide structures. We aim at evaluating these bioconjugates as a new class of OrganoMetallic Short AntiMicrobial Peptides (OM-SAMPs). The series of OM-SAMPs was designed with a set of linear and "head-to-tail" cyclic metallocene-based hexapeptides derived from the homo-sequence H-KKKKKK-NH2 by substitution of lysine (K) by tryptophan (W) and by orthogonal derivatization of the ε-N-amine group of lysine by a metallocene moiety. Peptide conjugates were characterized by RP-HPLC, mass spectrometry (ESI and MALDI-TOF) and circular dichroism (CD) spectroscopy. Gram-positive and Gram-negative antibacterial activity testings were carried out to explore the role of insertion of the metallocene fragment into the peptide, and the effect of the modification of the cationic charge and aromatic residues on the physiochemical properties of these OM-SAMPs. These results show that the insertion of two tryptophan residues and ferrocenoyl/ruthenocenoyl moieties into a linear homo-sequence peptides increase significantly their antibacterial activity with minimum inhibitory concentration values as low as 5 µM for the most active compounds. However, "head-to-tail" cyclic metallocene-based hexapeptides were not active against Gram-negative bacteria up to concentrations of 50 µM. These studies provide a better understanding of the role of structural modifications to enhance antibacterial peptide activity, which is promising for their therapeutic application.
Assuntos
Antibacterianos/farmacologia , Compostos Ferrosos/farmacologia , Metalocenos/farmacologia , Oligopeptídeos/farmacologia , Compostos Organometálicos/farmacologia , Técnicas de Síntese em Fase Sólida , Antibacterianos/síntese química , Antibacterianos/química , Compostos Ferrosos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Metalocenos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Compostos Organometálicos/química , Solubilidade , Água/químicaRESUMO
Big defensins is a large family of antimicrobial peptides found in restricted groups of invertebrates, in particular mollusks where they have highly diversified. Big defensins are composed of a highly hydrophobic N-terminal region and a C-terminal region containing six cysteine residues whose arrangement is identical to that of vertebrate ß-defensins. They have been shown to be active against both Gram-positive and Gram-negative bacteria and fungi. Antimicrobial aggregates called nanonets entrapping and killing bacteria have been recently described for the hydrophobic N-terminal region of the Cg-BigDef1 from the oyster Crassostrea gigas. To determine whether nanonets formation is a conserved trait of mollusk big defensins, we assessed the potential entrapping of bacteria through nanonets of the big defensin from the scallop Argopecten purpuratus, ApBD1. Recombinant ApBD1 was produced with a thrombin-cleavable N-terminal His6 tag, followed by the mature peptide carrying a mutation of the last cysteine residue of the C-terminal region by and arginine, named rApBD1(C87R). This mutation did not apparently affect the three-dimensional structure and the biological properties of rApBD1(C87R), as evidenced by in silico modeling and in vitro antimicrobial assays. Strong immune staining of rApBD1(C87R) in numerous areas surrounding bacteria was observed by confocal microscopy, suggesting that rApBD1(C87R) entraps bacteria in peptide aggregates similar to those reported to the oyster big defensin. This study suggests the conservation of bactericidal activity and nanonet formation across big defensins from bivalve mollusks.
Assuntos
Anti-Infecciosos , Pectinidae , Animais , Antibacterianos , Peptídeos Antimicrobianos , Cisteína , Defensinas/genética , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Pectinidae/genéticaRESUMO
Previous work demonstrated that Lys homopeptides with an odd number of residues (9, 11 and 13) were capable of inhibiting the growth of Gram-positive bacteria in a broader spectrum and more efficiently than those with an even number of Lys residues or Arg homopeptides of the same size. Indeed, all Gram-positive bacteria tested were totally inhibited by 11-residue Lys homopeptides. In the present work, a wide variety of Gram-negative bacteria were used to evaluate the inhibitory activity of chemically synthesized homopeptides of L-Lys and L-Arg ranging from 7 to 14 residues. Gram-negative bacteria were comparatively more resistant than Gram-positive bacteria to Lys homopeptides with an odd number of residues, but exhibited a similar inhibition pattern than on Gram-positive bacteria. CD spectra for the odd-numbered Lys homopeptides in anionic lipid dimyristoylphosphatidylglycerol, and Escherichia coli membrane extract increased polyproline II content, as compared to those measured in phosphate buffer solution. Lys and Arg homopeptides were covalently linked to rhodamine to visualize the peptide interactions with E. coli cells using confocal laser scanning microscopy. Analysis of Z-stack images showed that Arg homopeptides indeed appear to be localized intracellularly, while the Lys homopeptide is localized exclusively on the plasma membrane. Moreover, these Lys homopeptides induced membrane disruption since the Sytox fluorophore was able to bind to the DNA in E. coli cultures.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Bothrops , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Venenos de Crotalídeos/química , Escherichia coli/crescimento & desenvolvimento , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacologiaRESUMO
Endogenous peptides are valuable targets in the analysis of biological processes. The tear film contains proteins and peptides released by the tear duct mucosal cells, including antimicrobial peptides involved in the protection against exogenous pathogens; however, the peptide content of the tear liquid remains poorly characterized. We analyzed naturally occurring peptides isolated from human basal tears. Mass spectrometry analysis of endogenous peptides presents a number of drawbacks, including size heterogeneity and nonpredictable fragmentation patterns, among others. Therefore, CID, ETD, and HCD methods were used for the characterization of the tear peptide content. The contribution of DMSO as an additive of the chromatographic solvents was also evaluated. We identified 157, 131, and 122 peptides using CID-, ETD-, and HCD-based methods, respectively. Altogether, 234 different peptides were identified, leading to the generation of the biggest data set of endogenous tear peptides to date. The antimicrobial activity prediction analysis performed in silico revealed different putative antimicrobial peptides. Two of the extracellular glycoprotein lacritin peptides were de novo synthesized, and their antimicrobial activity was confirmed in vitro. Our findings demonstrate the benefits of using different fragmentation methods for the analysis of endogenous peptides and provide a useful approach for the discovery of peptides with antimicrobial activity.
Assuntos
Antibacterianos/química , Peptídeos/química , Proteoma , Lágrimas/química , Sequência de Aminoácidos , Antibacterianos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/farmacologiaRESUMO
In the selection or design of antimicrobial peptides, the key role played by cationic amino acids and chain length on the inhibitory potency and specificity is not clear. A fundamental study was conducted using chemically synthesized homopeptides of L-Lys and L-Arg ranging from 7 to 14 residues. Their effect on growth inhibition was evaluated over a wide range of Gram-positive bacteria at different levels of concentration. Interestingly, at lower concentrations (10 µM), Lys homopeptides with odd number of residues, especially with 11 residues, showed a broader inhibitory activity than those with even number of residues. At higher peptide concentrations (>20 µM), the inhibitory activity of Lys homopeptides was directly related to the number of residues in the chain. In contrast, Arg homopeptides, at lower concentrations, did not exhibit a defined pattern of bacterial inhibition related to the number of residues; however, at higher concentrations (>20 µM), the inhibitory effects were more pronounced. Lys homopeptides at concentrations up to 300 µM showed a remarkably lower toxicity against CHSE-214 cells. Arg homopeptides exhibited negligible cytotoxicity up to chain length of 11 residues at concentrations lower than 100 µM, but an abrupt increase in toxicity resulted when the peptide chain length reached 12 amino acid residues and higher concentrations. All synthesized homopeptides displayed characteristic polyproline II helix conformation in both buffer and liposomes, as shown by CD spectroscopy. This result suggests that short Lys homopeptides with an odd number of residues (9 and 11) have a broad spectrum of activity against Gram-positive bacterial cells compared with Arg homopeptides, which in turn showed a considerably higher selectivity toward those cells. By investigating the differences between Lys and Arg homopeptides, this study contributes to the understanding of their mechanism of growth inhibition and selectivity. Thus, it provides further guidelines for a rational design of short antimicrobial peptides.
Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Peptídeos/farmacologia , Polilisina/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/toxicidade , Cátions , Linhagem Celular , Lipossomos/química , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/toxicidade , Polilisina/química , Polilisina/toxicidade , Estrutura Secundária de Proteína , Salmão , SoluçõesRESUMO
We report on the synthesis of two series of 1,4-naphthohydroquinone derivatives conjugated with amino acids (Gly, Ala, Phe, and Glu) and with substituted purines linked by an aliphatic chain. The compounds were obtained through Diels-Alder cycloaddition between myrcene and 1,4-benzoquinone and evaluated in vitro for their cytotoxicity (GI50 ) against cultured human cancer cells of A-549 lung carcinoma, HT-29 colon adenocarcinoma, and MCF-7 breast carcinoma. The GI50 values found for some hydroquinone-amino acid and hydroquinone-purine hybrids against MCF-7 are in an activity range comparable to that of the reference drug doxorubicin.
Assuntos
Aminoácidos/síntese química , Aminoácidos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Hidroquinonas/síntese química , Hidroquinonas/farmacologia , Purinas/síntese química , Purinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Células HT29 , Humanos , Concentração Inibidora 50 , Células MCF-7 , Estrutura MolecularRESUMO
Several new 6-(3-pyrazolylpropyl) derivatives of 1,4-naphthohydroquinone-1,4-diacetate (NHQ-DA) have been prepared by chemical modifications of the Diels-Alder adduct of alpha-myrcene and 1,4-benzoquinone. All these new compounds and precursors have been evaluated in vitro for their cytotoxicity against cultured human cancer cells of MB-231 breast-adeno carcinoma, A-549 lung carcinoma, and HT-29 colon carcinoma. GI(50) values ranged in and below the micromolar concentration level.
Assuntos
Acetatos/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Hidroquinonas/farmacologia , Acetatos/síntese química , Antineoplásicos/síntese química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HT29 , Humanos , Hidroquinonas/síntese química , Concentração Inibidora 50 , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Several new prenyl-1,2-naphthohydroquinone derivatives have been prepared by chemical modifications of Diels-Alder products which were obtained from cycloaddition of alpha-myrcene to 1,2-benzoquinone and then evaluated in vitro for their cytotoxic activity against A-549 lung carcinoma, HT-29 colon carcinoma, and MB-231 breast adeno-carcinoma culture cells. Most of them exhibited GI50 values in the microM-concentration level.
Assuntos
Antineoplásicos/síntese química , Hidroquinonas/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Hidroquinonas/farmacologia , Relação Estrutura-AtividadeRESUMO
Several new prenylnaphthohydroquinone derivatives have been prepared through the Diels-Alder condensation between alpha-myrcene and 1,2-benzoquinone and evaluated for their cytotoxic activity against A-549, HT-29 and MB-231 cultured cell lines. All of them have shown GI50 values in the microM level.
Assuntos
Antineoplásicos/química , Antineoplásicos/toxicidade , Naftoquinonas/química , Naftoquinonas/toxicidade , Prenilamina/análogos & derivados , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Isomerismo , Estrutura Molecular , Naftoquinonas/síntese química , Prenilamina/síntese química , Prenilamina/química , Prenilamina/toxicidadeRESUMO
From the Diels-Alder adduct between alpha-myrcene and 2-chloro-1,4-benzoquinone, a family of chloro derivatives of prenylnaphthohydroquinone have been synthesised and evaluated for their cytotoxicity against 14 neoplastic cell lines.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Hidroquinonas/química , Hidroquinonas/farmacologia , Antineoplásicos/síntese química , Humanos , Hidroquinonas/síntese química , Células Jurkat , Espectroscopia de Ressonância Magnética , Espectrofotometria InfravermelhoRESUMO
El aneurisma paraanastomótico es el que ocurre luego de una reconstrucción quirúrgica previa, en este caso en la aorta, el cual debe de ser reparado en cuanto se diagnostica. Presentamos el caso de un paciente con antecedente de reparo quirúrgico de aneurisma de aorta abdominal infrarenal, un año siete meses atrás. El paciente ingresa con un cuadro urgente de rotura de aneurisma aórtico paraanastomótico en la aorta remanente infrarenal adyacente a la anastomosis aórtica proximal. El paciente fue tratado con intervención endovascular mediante la aplicación de dos stent-graft aórticos, tubulares, uno con sistema de fijación transrenal, y el segundo telescopado entre el primero y el injerto tubular antiguo. Se obtuvo completa exclusión angiográfica del aneurisma roto y mejoría clínica inmediata con desaparición del dolor. Fue dado de alta al cuarto día post intervención. En el seguimiento a los 45 días con control tomográfico se demuestra exclusión completa del aneurisma roto y reducción significativa del hematoma retroperitoneal con los stent grafts in situ y buena permeabilidad de las arterias renales. En los casos de aneurisma aórtico paraanastomótico electivo o roto el tratamiento endovascular con stent-graft es factible y exitoso; presenta menos morbimortalidad y debe considerarse como de primera elección.
The paraanastomotic aneurysm is the one that occurs after aortic reconstructive surgery and needs to be repaired at the time of diagnosis. We present the case of a patient with a previous infrarenal aortic aneurysm surgical repair 19 months back. The patient is admitted with an urgent case of ruptured paraanastomotic aortic aneurysm in the infrarenal remanent aorta next to the proximal aortic anastomosis. The patient underwent succesful endovascular intervention by placement of two aortic tube stent grafts, the first with a transrenal fixation system and the second one overlapped between the first stent graft and the previous Dacron tubular graft. Complete angiographic aortic aneurysm exclusion was obtained and we observed immediate clinical improvement with no pain. He was released from the hospital at the 4th day post endovascular repair. During the follow-up 45 days later the CT Scan showed complete exclusion of the aneurysm, reduction of the retroperitoneal haematoma, stent grafts in situ and bilateral renal artery patency. In elective or emergency paraanastomotic aortic aneurysms the endovascular stent-graft repair is feasible and successful; has lower perioperative morbidity and mortality and must be considered as the first line approach.