RESUMO
Antimicrobial resistance (AMR) is one of the top ten threats to public health, as reported by the World Health Organization (WHO). One of the causes of the growing AMR problem is the lack of new therapies and/or treatment agents; consequently, many infectious diseases could become uncontrollable. The need to discover new antimicrobial agents that are alternatives to the existing ones and that allow mitigating this problem has increased, due to the rapid and global expansion of AMR. Within this context, both antimicrobial peptides (AMPs) and cyclic macromolecules, such as resorcinarenes, have been proposed as alternatives to combat AMR. Resorcinarenes present multiple copies of antibacterial compounds in their structure. These conjugate molecules have exhibited antifungal and antibacterial properties and have also been used in anti-inflammatory, antineoplastic, and cardiovascular therapies, as well as being useful in drug and gene delivery systems. In this study, it was proposed to obtain conjugates that contain four copies of AMP sequences over a resorcinarene core. Specifically, obtaining (peptide)4-resorcinarene conjugates derived from LfcinB (20-25): RRWQWR and BF (32-34): RLLR was explored. First, the synthesis routes that allowed obtaining: (a) alkynyl-resorcinarenes and (b) peptides functionalized with the azide group were established. These precursors were used to generate (c) (peptide)4-resorcinarene conjugates by azide-alkyne cycloaddition CuAAC, a kind of click chemistry. Finally, the conjugates' biological activity was evaluated: antimicrobial activity against reference strains and clinical isolates of bacteria and fungi, and the cytotoxic activity over erythrocytes, fibroblast, MCF-7, and HeLa cell lines. Our results allowed establishing a new synthetic route, based on click chemistry, for obtaining macromolecules derived from resorcinarenes functionalized with peptides. Moreover, it was possible to identify promising antimicrobial chimeric molecules that may lead to advances in the development of new therapeutic agents.
RESUMO
Click chemistry is currently one of the most used tools for the generation of complex organic molecules. The advantages of using click chemistry in organic synthesis are remarkable; in many cases, the reactions occur under mild conditions and are free of solvents, with high yields and short reaction times. This makes it an extraordinarily effective and viable alternative for obtaining complex/conjugated molecules. In this review, the use of click chemistry CuAAC is especially emphasized for polyhydroxylated platforms such as resorcinarenes or calixarenes, focusing mainly on aspects of synthesis, specifically conditions, reagents, and methodologies.
RESUMO
A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to study the conformer formation generated during the reaction for obtaining C-tetramethylcalix[4]resorcinarene. The chromatographic method was used to design a strategy for purifying the reaction products, using solid-phase extraction columns (RP-SPE) and gradient elution. The chromatographic profiles of the cyclocondensation reaction between resorcinol and acetaldehyde show the presence of three products under the different reaction and precipitation conditions studied. Using RP-SPE, it was possible to enrich the products, which were later characterized by means of RP-HPLC and 1H nuclear magnetic resonance (NMR). This investigation explored and established a new method for RP-HPLC analysis and RP-SPE separation of conformational isomers obtained in the formation reaction of C-tetramethylcalix[4]resorcinarene.
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Cryptococcosis is associated with high rates of morbidity and mortality. The limited number of antifungal agents, their toxicity, and the difficulty of these molecules in crossing the blood-brain barrier have made the exploration of new therapeutic candidates against Cryptococcus neoformans a priority task. To optimize the antimicrobial functionality and improve the physicochemical properties of AMPs, chemical strategies include combinations of peptide fragments into one. This study aimed to evaluate the binding of the minimum activity motif of bovine lactoferricin (LfcinB) and buforin II (BFII) against C. neoformans var. grubii. The antifungal activity against these chimeras was evaluated against (i) the reference strain H99, (ii) three Colombian clinical strains, and (iii) eleven mutant strains, with the aim of evaluating the possible antifungal target. We found high activity against these strains, with a MIC between 6.25 and 12.5 µg/mL. Studies were carried out to evaluate the effect of the combination of fluconazole treatments, finding a synergistic effect. Finally, when fibroblast cells were treated with 12.5 µg/mL of the chimeras, a viability of more than 65% was found. The results obtained in this study identify these chimeras as potential antifungal molecules for future therapeutic applications against cryptococcosis.
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Antimicrobial peptides (AMPs) are considered to be a valuable source for the identification and/or design of promising candidates for the development of antifungal treatments, since they have advantages such as lower tendency to induce resistance, ease of production, and high purity and safety. Bovine lactoferricin (LfcinB) and buforin II (BFII) are AMPs to which great antimicrobial potential has been attributed. The minimum motives with antimicrobial activity derived from LfcinB and BFII are RRWQWR and RLLR, respectively. Nine chimeras containing the minimum motives of both peptides were synthesized and their antifungal activity against fluconazole (FLC)-sensitive and resistant C. albicans, C. glabrata, and C. auris strains was evaluated. The results showed that peptides C9: (RRWQWR)2K-Ahx-RLLRRRLLR and C6: KKWQWK-Ahx-RLLRRLLR exhibited the greatest antifungal activity against two strains of C. albicans, a FLC-sensitive reference strain and a FLC-resistant clinical isolate; no medically significant results were observed with the other chimeras evaluated (MIC ~200 µg/mL). The chimera C6 was also active against sensitive and resistant strains of C. glabrata and C. auris. The combination of branched polyvalent chimeras together with FLC showed a synergistic effect against C. albicans. In addition to exhibiting antifungal activity against reference strains and clinical isolates of Candida spp., they also showed antibacterial activity against both Gram-positive and Gram-negative bacteria, suggesting that these chimeras exhibit a broad antimicrobial spectrum and can be considered to be promising molecules for therapeutic applications.
RESUMO
Chimeric peptides containing short sequences derived from bovine Lactoferricin (LfcinB) and Buforin II (BFII) were synthetized using solid-phase peptide synthesis (SPPS) and characterized via reversed-phase liquid chromatography and mass spectrometry. The chimeras were obtained with high purity, demonstrating their synthetic viability. The chimeras' antibacterial activity against Gram-positive and Gram-negative strains was evaluated. Our results showed that all the chimeras exhibited greater antibacterial activity against the evaluated strains than the individual sequences, suggesting that chemical binding of short sequences derived from AMPs significantly increased the antibacterial activity. For each strain, the chimera with the best antibacterial activity exerted a bacteriostatic and/or bactericidal effect, which was dependent on the concentration. It was found that: (i) the antibacterial activity of a chimera is mainly influenced by the linked sequences, the palindromic motif RLLRRLLR being the most relevant one; (ii) the inclusion of a spacer between the short sequences did not significantly affect the chimera's synthesis process; however, it enhanced its antibacterial activity against Gram-negative and Gram-positive strains; on the other hand, (iii) the replacement of Arg with Lys in the LfcinB or BFII sequences improved the chimeras' synthesis process without significantly affecting their antibacterial activity. These results illustrate the great importance of the synthesis of chimeric peptides for the generation of promising antibacterial peptides.
Assuntos
Antibacterianos/química , Lactoferrina/química , Fragmentos de Peptídeos/química , Proteínas/química , Animais , Antibacterianos/síntese química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Bovinos , Humanos , Lactoferrina/síntese química , Lactoferrina/farmacologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Proteínas/síntese química , Proteínas/farmacologia , Técnicas de Síntese em Fase SólidaRESUMO
A methodology was implemented for purifying peptides in one chromatographic run via solid-phase extraction (SPE), reverse phase mode (RP), and gradient elution, obtaining high-purity products with good yields. Crude peptides were analyzed by reverse phase high performance liquid chromatography and a new mathematical model based on its retention time was developed in order to predict the percentage of organic modifier in which the peptide will elute in RP-SPE. This information was used for designing the elution program of each molecule. It was possible to purify peptides with different physicochemical properties, showing that this method is versatile and requires low solvent consumption, making it the least polluting one. Reverse phase-SPE can easily be routinely implemented. It is an alternative to enrich and purified synthetic or natural molecules.
Assuntos
Peptídeos/isolamento & purificação , Extração em Fase Sólida/economia , Extração em Fase Sólida/métodos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Peptídeos/químicaRESUMO
BACKGROUND: Glycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of Escherichia coli physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes. RESULTS: Transcriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroG(fbr)tktA, when growing on glycerol, as compared to glucose. CONCLUSIONS: The overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.
Assuntos
Acetatos/metabolismo , Carbono/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Glicerol/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , ProteômicaRESUMO
Antecedentes: Una relación inversa entre estatura y riesgo de mortalidad ha sido establecida en estudios prospectivos de países desarrollados. Sin embargo, dicha asociación prácticamente permanece inexplorada en sociedades latinoamericanas en rápida transición económica y epidemiológica. Diseño: De un universo de 11.600 adultos viviendo en el centro urbano de Mostazal, Chile, se realizó un estudio de cohorte prospectivo de una muestra aleatoria ponderada de 795 sujetos seguidos durante 8 años. Método: Desde 1997 a 1999 se evaluó la estatura (percentiles 50 y 75 por sexo), hipertensión, diabetes, dislipidemia, obesidad, tabaquismo, consumo de alcohol y antecedentes hereditarios de enfermedad cardiovascular. El riesgo relativo de mortalidad por cualquier causa fue estimado a través de modelos de regresión de Cox ajustando por edad, sexo, factores de riesgo, educación e ingreso. Resultados: Se observó una relación inversa entre la estatura adulta y los factores de riesgo cardiovascular. En el modelo completamente ajustado el riesgo de mortalidad por cualquier causa asociado a la estatura fue 0,75 (IC 95 por ciento 0,66 - 0,85; p tendencia <0,001). Los factores de riesgo cardiovascular tradicionales solo explicaron 22 por ciento de la asociación inversa establecida entre estatura y mortalidad. Conclusión: En sociedades en transición económica como Chile, la estatura aparece como un factor de riesgo independiente de mortalidad. En esta cohorte de adultos chilenos, los factores de riesgo cardiovascular tradicionales solo explicaron parcialmente la relación inversa entre estatura y mortalidad.
Background: The inverse relationship between height and mortality risk has been well established in developed countries, but remains practically unexplored in Latin American societies. Setting: Chile, 11,600 adults living in the urban centre of Mostazal. Design: Prospective cohort study of a weighted random sample of 795 subjects followed during 8 years. Methods: Height (percentiles 50 and 75 by sex) along hypertension, diabetes, dyslipidemia, obesity, smoking, alcohol consumption and hereditary cardiovascular disease were assessed at baseline. Relative risks of all-cause mortality with 95% confidence intervals were computed in Cox regression models adjusting for age, gender, cardiovascular risk factors and joint-effects of other socioeconomic measures. Results: A significant inverse relationship between cardiovascular risk factors and height was observed. The risk of all cause mortality was 0.75 (95 percent CI 0.66 - 0.85; p-for trend <0.001) after full adjustments. Traditional cardiovascular risk factors explained only 22 percent of the association for height with mortality risk. Conclusions: In this adult cohort, traditional cardiovascular risk factors only explained partially the inverse relationship between height and all-cause mortality. Similar to developed countries, in economic transitioning societies such as Chile, height is an independent risk factor, likely reflecting different early exposure patterns that influence the health status during the life course.