RESUMO
This study describes the interaction of human serum albumin (HSA) with the binol derivative (R)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (R-BrB), which has its optical activity based on the prohibitive energetic barrier for conversion into the enantiomer (S)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (S-BrB). The objective was to assess the ability of HSA to differentiate axial enantiomers based on their binding efficiency and their impact on the CD spectra. We discovered that both enantiomers were effective ligands, and the CD signal disappeared when equimolar amounts of R-BrB and S-BrB were simultaneously added, indicating no preference for either enantiomer. The complexation resulted in a significant signal increase at 250 nm and a bathochromic effect at 370 nm. Molecular docking simulations were performed, and the lower energy pose of R-BrB was selected for DFT calculations. The theoretical CD spectra of free and complexed R-BrB were obtained and showed alterations corroborating the experimental results. By comparing the difference spectrum (HSA:R-BrB minus HSA) with the spectrum of free RBrB in water or ethyl alcohol, we concluded that the CD signal intensification was due to the increased solubilization of R-BrB upon binding to HSA.
Assuntos
Dicroísmo Circular , Simulação de Acoplamento Molecular , Naftóis , Albumina Sérica Humana , Dicroísmo Circular/métodos , Naftóis/química , Albumina Sérica Humana/química , Estereoisomerismo , Humanos , Teoria da Densidade Funcional , Simulação por Computador , Ligação ProteicaRESUMO
In this study, we report the synthesis of eight novel indole-thiazole and indole-thiazolidinone derivatives, as well as their ability to interact with DNA, analysed through the UV-vis absorption, fluorescence, circular dichroism (CD), viscosity techniques and molecular docking. The ctDNA interaction analysis demonstrated different spectroscopic effects and the affinity constants (Kb) calculated by the UV-vis absorption method were between 2.08 × 105 and 6.99 × 106 M-1, whereas in the fluorescence suppression constants (Ksv) ranged between 0.38 and 0.77 × 104 M-1 and 0.60-7.59 × 104 M-1 using Ethidium Bromide (EB) and 4',6-Diamidino-2-phenylindole (DAPI) as fluorescent probes, respectively. Most derivatives did not alter significantly the secondary structure of the ctDNA according to the CD results. None of the compounds was able to change the relative viscosity of the ctDNA. These results prove that compounds interact with ctDNA via groove binding, which was confirmed by A-T rich oligonucleotide sequence assay with compound JF-252, suggesting the importance of both the phenyl ring coupled to C-4 thiazole ring and the bromo-unsubstituted indole nucleus.
Assuntos
DNA/química , Indóis/química , Tiazóis/química , Dicroísmo Circular/métodos , Etídio/química , Corantes Fluorescentes/química , Simulação de Acoplamento Molecular/métodos , Espectrometria de Fluorescência/métodos , TermodinâmicaRESUMO
The multi-copper Laccase enzyme corresponds to one of the most investigated oxidoreductases for potential uses in xenobiotic bioremediation. In this work, we have investigated the photo-degradation process of Laccase from Trametesversicolor induced by UVB light and the influence on its activity over selected substrates. Laccase undergoes photo-degradation when irradiated with UVB light, and the process depends on the presence of oxygen in the medium. With the kinetic data obtained from stationary and time resolved measurements, a photo-degradation mechanism of auto-sensitization was proposed for the enzyme. Laccase generates singlet oxygen, by UVB light absorption, and this reactive oxygen species can trigger the photo-oxidation of susceptible amino acids residues present in the protein structure. The catalytic activity of Laccase was evaluated before and after UVB photolysis over hydroxy-aromatic compounds and substituted phenols which represent potential pollutants. The dye bromothymol blue, the antibiotic rifampicin and the model compound syringaldazine, were selected as substrates. The values of the kinetic parameters determined in our experiments indicate that the photo-oxidative process of Laccase has a very negative impact on its overall catalytic function. Despite this, we have not found evidence of structural damage by SDS-PAGE and circular dichroism experiments, which indicate that the enzyme retained its secondary structure. We believe that, given the importance of Laccase in environmental bioremediation, the information found about the stability of this kind of biomolecule exposed to UV solar irradiation may be relevant in the technological design and/or optimization of decontamination strategies.
Assuntos
Biodegradação Ambiental/efeitos da radiação , Poluentes Ambientais , Lacase/metabolismo , Lacase/efeitos da radiação , Absorção de Radiação , Dicroísmo Circular/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Fluorescência , Oxirredução , FotóliseRESUMO
Control of amylin agglomeration is of interest for both the study of pathophysiology and the design of amylin-based pharmaceutical products. Here we report the effects of a large set of common buffering agents, aminoacids and nucleoside phosphates over the amylin amyloid aggregation. Circular dichroism showed no apparent effects of the co-solutes over the secondary-structure of soluble amylin. Instead, we found a large dependence of the fibrillation process on the total amount of co-solute charged groups. The amyloid nature of the aggregates was confirmed by transmission electron microscopy, X-ray diffraction and infrared spectroscopy. While acidic pH and low-ionic co-solutes shows the largest size effect in hampering aggregation, no further effect was observed that could identify a single compound as a major direct heterotropic determinants of the amyloid process. These data suggest a more physico-chemical effect of co-solutes over the modulation of amylin instead of a chemical entity-related causal factor.
Assuntos
Amiloide/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Agregação Patológica de Proteínas , Soluções Tampão , Dicroísmo Circular/métodos , Diabetes Mellitus/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão/métodos , Estrutura Secundária de Proteína , Espectrofotometria Infravermelho/métodos , Difração de Raios X/métodosRESUMO
Micro Exon Gene (MEG) proteins are thought to play major roles in the infection and survival of parasitic Schistosoma mansoni worms in host organisms. Here, the physical chemical properties of two small MEG proteins found in the genome of S. mansoni, named MEG-24 and MEG-27, were examined by a combination of biophysical techniques such as differential scanning calorimetry, tensiometry, circular dichroism, fluorescence, and electron spin resonance spectroscopies. The proteins are surface active and structurally arranged as cationic amphipathic α-helices that can associate with lipid membranes and cause their disruption. Upon adsorption to lipid membranes, MEG-27 strongly affects the fluidity of erythrocyte ghost membranes, whereas MEG-24 forms pores in erythrocytes without modifying the ghost membrane fluidity. Whole-mount in situ hybridization experiments indicates that MEG-27 and MEG-24 transcripts are located in the parasite esophagus and subtegumental cells, respectively, suggesting a relevant role of these proteins in the host-parasite interface. Taken together, these characteristics lead us to propose that these MEG proteins may interact with host cell membranes and potentially modulate the immune process using a similar mechanism as that described for α-helical membrane-active peptides.
Assuntos
Éxons/genética , Membranas/química , Schistosoma mansoni/genética , Sequência de Aminoácidos , Animais , Varredura Diferencial de Calorimetria/métodos , Dicroísmo Circular/métodos , Peptídeos/química , Conformação Proteica em alfa-Hélice , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/genética , Esquistossomose mansoni/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Croton floribundus Spreng. (Euphorbiaceae), popularly known as Capixinguí, stands out due to its widespread use in traditional medicine to treat wounds, syphilis, hemorrhoids, eye diseases and as a purgative. AIM OF THE STUDY: To characterize clerodanes diterpenes from C. floribundus and to evaluate the effects of the fraction and diterpenes (1-5) on inhibition of nitrite production. MATERIALS AND METHODS: The hydroethanolic root extract of C. floribundus was fractionated on a solid phase extraction column to obtain the fraction named Fr80%. From this, five compounds were obtained and characterized. The absolute configuration of compound 1 was determined by a combination of electronic and vibrational circular dichroism spectroscopies. Additionally, compounds 1-5 were evaluated for their inhibitory effects on nitrite production induced by lipopolysaccharide (LPS) in RAW 264 macrophage cell. RESULTS: Five clerodane diterpenoids were characterized, and the absolute stereochemistry of 1 was established as 3R,4R,5R,8R,9R,10S,12S. The IC50 values obtained through inhibition of nitrite production were 28.52⯱â¯2.21⯵M (1), 40.26⯱â¯2.79⯵M (2), 25.47⯱â¯2.16⯵M (3), 35.78⯱â¯2.93⯵M (4) and 40.58⯱â¯4.78⯵M (5). In the tested concentrations, the samples presented low toxicity in macrophages. CONCLUSIONS: Four new diterpenes were characterized from C. floribundus, these being croflorins A-D (1-4) and a known halimane (5). These compounds exhibited inhibitory effect on nitrite production.
Assuntos
Croton/química , Diterpenos/química , Diterpenos/farmacologia , Nitritos/metabolismo , Animais , Linhagem Celular , Dicroísmo Circular/métodos , Diterpenos Clerodânicos/química , Diterpenos Clerodânicos/farmacologia , Euphorbiaceae/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Medicina Tradicional/métodos , Camundongos , Células RAW 264.7RESUMO
The search for plasma proteins precipitation methods has been increasing due to the plasma protein therapeutic needs in world-wide. Thus, this work evaluates the tannic acid (TA) ability to precipitate proteins from human plasma. In this study, TA-plasma protein complexes were studied at different pH conditions, tannin/plasma ratio and reaction mixing time. The complexes formed from combinations of TA and plasma proteins were analyzed by gel electrophoresis, protein quantification, particle size, charge, mass spectrometry, microscopic image, and circular dichroism. It was possible to verify the precipitate formation in all tested pH values, with high precipitation at pHâ¯5. The native PAGE analysis showed three mainly bands corresponding independent of the pHs used. It was possible to observe a gradual growing of precipitate protein in the first precipitation process (P1) when increased the TA/plasma ratio. 15â¯min of incubation was enough to precipitate 72.3% of proteins. Spectroscopic analyzes showed albumin signals and the electron microscopy analysis of IgG-TA confirmed the compact form of a precipitate. According to CD, formation of the IgG-TA complexes does not cause a major structural change of the protein. From the results obtained, it was possible to establish some parameters for plasma proteins precipitation using TA.
Assuntos
Plasma/química , Albumina Sérica Humana/química , Soroglobulinas/química , Taninos/química , Dicroísmo Circular/métodos , Humanos , Espectrometria de Massas/métodos , Tamanho da PartículaRESUMO
Antimicrobial peptides (AMPs) are biologically active molecules with a broad-spectrum activity against a myriad of microorganisms. Aside from their antimicrobial functions, AMPs present physicochemical and structural properties that allow them to exert activity against other kind of cells, such as cancer cells. VmCT1 is a potent cationic amphipathic AMP from the venom of the scorpion Vaejovis mexicanus. In this study, we designed lysine-substituted VmCT1 analogs for verifying the influence of changes in the net positive charge on biological activities. The increase in the net positive charge caused by lysine substitutions in the hydrophilic portion, led to higher antimicrobial activity values (0.1-6.3⯵molâ¯L-1) than VmCT1 (0.8-50⯵molâ¯L-1) and higher activity against mammary cancer cells MCF-7 (6.3-12.5⯵molâ¯L-1) than VmCT1 (12.5⯵molâ¯L-1). Contrarily, when lysine-substitutions were made at the hydrophobic portion of the helical projection, the activity values decreased. However, the lysine-substitution at the center of the hydrophobic face led to the generation of an analog with antiplasmodial activity at the same concentration presented by VmCT1 (0.8⯵molâ¯L-1). In this study, we demonstrated that it is possible to modulate biological activities and cytotoxicity of VmCT1 peptides by increasing their net positive charge using lysine residues, thus creating alternatives for standard-of-care therapeutics against different types of microorganisms and MCF-7 human breast cancer cells.
Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Lisina/química , Venenos de Escorpião/química , Escorpiões/química , Animais , Linhagem Celular Tumoral , Dicroísmo Circular/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células MCF-7 , Relação Estrutura-AtividadeRESUMO
BACKGROUND: RTXM83 is a rituximab biosimilar with proven clinical safety and efficacy. It is the first rituximab biosimilar developed and approved in South America and is currently marketed in several Latin American, Middle Eastern and African countries. OBJECTIVE: The aim of this study was to present the physicochemical and biological characterization studies utilized to demonstrate the similarity between RTXM83 and its reference product. METHODS: Primary and higher order protein structures were analysed using peptide mapping with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), fluorescence spectroscopy and circular dichroism, and micro-differential scanning calorimetry, among other techniques. Charge variants were determined by cation-exchange chromatography (CEX) and capillary isoelectric focusing (cIEF). Glycosylation and glycoforms distribution were analysed using MS, normal phase high-performance liquid chromatography (NP-HPLC) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Size variants were evaluated by size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (SV-AUC), dynamic light scattering (DLS), and capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Biological characterization included binding assays for complement C1q, CD20, and several Fc receptors (FcRs), as well as potency determination for in vitro apoptosis induction, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). RESULTS: RTXM83 and the reference product showed identical primary sequences and disulfide bridge patterns, and similarity at higher order protein structures, post-translational modification profiles (amino acid modifications, charge variants, and glycosylation) and levels of purity and process-related impurities. Functional studies demonstrated that RTXM83 is similar to the reference product regarding the three known mechanisms of action of rituximab: CDC, ADCC, and apoptosis induction. Binding affinities to CD20, complement component C1q, and different FcRs were also equivalent. CONCLUSION: RTXM83 is similar to its reference product in all critical quality attributes.
Assuntos
Medicamentos Biossimilares/química , Medicamentos Biossimilares/uso terapêutico , Rituximab/química , Rituximab/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Antígenos CD20/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Dicroísmo Circular/métodos , Complemento C1q/metabolismo , Difusão Dinâmica da Luz/métodos , Eletroforese Capilar/métodos , Glicosilação , Humanos , Mapeamento de Peptídeos/métodos , Receptores Fc/metabolismo , Espectrometria de Massas em Tandem/métodos , Ultracentrifugação/métodosRESUMO
A methodology to determine the enantiomeric excess and the absolute configuration (AC) of natural epoxythymols was developed and tested using five constituents of Ageratina glabrata. The methodology is based on enantiomeric purity determination employing 1,1'-bi-2-naphthol (BINOL) as a chiral solvating agent combined with vibrational circular dichroism (VCD) measurements and calculations. The conformational searching included an extensive Monte Carlo protocol that considered the rotational barriers to cover the whole conformational spaces. (+)-(8S)-10-Benzoyloxy-6-hydroxy-8,9-epoxythymol isobutyrate (1), (+)-(8S)-10-acetoxy-6-methoxy-8,9-epoxythymol isobutyrate (4), and (+)-(8S)-10-benzoyloxy-6-methoxy-8,9-epoxythymol isobutyrate (5) were isolated as enantiomerically pure constituents, while 10-isobutyryloxy-8,9-epoxythymol isobutyrate (2) was obtained as a 75:25 (8S)/(8R) scalemic mixture. In the case of 10-benzoyloxy-8,9-epoxythymol isobutyrate (3), the BINOL methodology revealed a 56:44 scalemic mixture and the VCD measurement was beyond the limit of sensitivity since the enantiomeric excess is only 12%. The racemization process of epoxythymol derivatives was studied using compound 1 and allowed the clarification of some stereochemical aspects of epoxythymol derivatives since their ACs have been scarcely analyzed and a particular behavior in their specific rotations was detected. In more than 30 oxygenated thymol derivatives, including some epoxythymols, the reported specific rotation values fluctuate from -1.6 to +1.4 passing through zero, suggesting the presence of scalemic and close to racemic mixtures, since enantiomerically pure natural constituents showed positive or negative specific rotations greater than 10 units.