RESUMO
The combination of a tumor-penetrating peptide (TPP) with a peptide able to interfere with a given protein-protein interaction (IP) is a promising strategy with potential clinical application. Little is known about the impact of fusing a TPP with an IP, both in terms of internalization and functional effect. Here, we analyze these aspects in the context of breast cancer, targeting PP2A/SET interaction, using both in silico and in vivo approaches. Our results support the fact that state-of-the-art deep learning approaches developed for protein-peptide interaction modeling can reliably identify good candidate poses for the IP-TPP in interaction with the Neuropilin-1 receptor. The association of the IP with the TPP does not seem to affect the ability of the TPP to bind to Neuropilin-1. Molecular simulation results suggest that peptide IP-GG-LinTT1 in a cleaved form interacts with Neuropilin-1 in a more stable manner and has a more helical secondary structure than the cleaved IP-GG-iRGD. Surprisingly, in silico investigations also suggest that the non-cleaved TPPs can bind the Neuropilin-1 in a stable manner. The in vivo results using xenografts models show that both bifunctional peptides resulting from the combination of the IP and either LinTT1 or iRGD are effective against tumoral growth. The peptide iRGD-IP shows the highest stability to serum proteases degradation while having the same antitumoral effect as Lin TT1-IP, which is more sensitive to proteases degradation. Our results support the development of the TPP-IP strategy as therapeutic peptides against cancer.
RESUMO
Gastroretentive drug delivery systems (GRDDS) are modified-release dosage forms designed to prolong their residence time in the upper gastrointestinal tract, where some drugs are preferentially absorbed, and increase the drug bioavailability. This work aimed the development of a novel GRDDS containing 60 mg of sildenafil citrate, and the evaluation of the feasibility of the proposed formulation for use in the treatment of pulmonary arterial hypertension (PAH), for once a day administration, by using in silico pharmacokinetic (PK) modeling and simulations using GastroPlusTM. The Model-Informed Drug Development (MIDD) approach was used in formulation design and pharmacokinetic exposure prospecting. A 22 factorial design with a central point was used for optimization of the formulation, which was produced by direct compression and characterized by some tests, including buoyancy test, assay, impurities, and in vitro dissolution. A compartmental PK model was built using the GatroPlusTM software for virtual bioequivalence of the proposed formulations in comparison with the defined target release profile provided by an immediate release (IR) tablet formulation containing 20 mg of sildenafil administered three times a day (TID). The results of the factorial design showed a direct correlation between the dissolution rate and the amount of hydroxypropyl methyl cellulose (HPMC) in the formulations. By comparing the PK parameters predicted by the virtual bioequivalence, the formulations F1, F2, F3 and F5 failed on bioequivalence. The F4 showed bioequivalence to the reference and was considered the viable formulation to substitute the IR. Thus, GRDDS could be a promising alternative for controlling the release of drugs with a pH-dependent solubility and narrow absorption window, specifically in the gastric environment, and an interesting way to reduce dose frequency and increase the drug bioavailability. The MIDD approach increases the level of information about the pharmaceutical product and guide the drug development for more assertive ways.
Assuntos
Sistemas de Liberação de Medicamentos , Desenvolvimento de Medicamentos , Citrato de Sildenafila , Preparações de Ação Retardada/farmacocinética , Disponibilidade Biológica , Solubilidade , Comprimidos/farmacocinéticaRESUMO
Infectious salmon anemia (ISA) can be devastating in farmed Atlantic salmon (Salmo salar). The disease can evolve into epidemics if it is not contained and controlled. ISA epidemics were seen in Norway in the early 1990s and Chile in 2007-2009. Consequently, there is an urgent need to develop a vaccine to prevent or treat the infection. In this study, an immunoinformatic approach was employed to predict 32 lineal B-cell epitopes based on antigenicity and surface accessibility prediction for ISAV fusion (F), hemagglutinin-esterase (HE), and matrix (M) proteins. On the other hand, twelve conformational B-cell epitopes were also predicted. We further identified six antigenic cytotoxic T lymphocyte (CTL) epitopes and investigated the binding interactions with five salmon MHC-I proteins after docking the peptides to the binding groove of the MHC-I proteins. Our results showed that all the predicted epitopes could bind to salmon MHC-I with high negative ΔG values with medium to high binding affinities. Hence, the predicted epitopes have a high potential of being recognized by Atlantic salmon MHC-I to elicit a CD8+ T cell response in salmon. The predicted and analyzed B and T cell antigenic epitopes in this work might present an initial set of peptides for future vaccine development against ISAV. The ability to model and predict these interactions will ultimately lead to the ability to predict potential binding for MHCs and epitopes that were not studied previously. As current knowledge of salmon MHC specificity is limited, studying and modeling interactions in the peptide/MHC complex is a key to resolving unknown epitope specificity.
Assuntos
Doenças dos Peixes , Isavirus , Infecções por Orthomyxoviridae , Salmo salar , Animais , Epitopos de Linfócito B , Epitopos de Linfócito T , Esterases , Hemaglutininas , Antígenos de Histocompatibilidade Classe I , Complexo Principal de HistocompatibilidadeRESUMO
In mammals, the T lymphocyte receptor (TCR) is a multiprotein complex formed by the proteins TCRα, TCRß, CD3ε, CD3γ, CD3δ, and CD3ζ. It is responsible for recognizing antigens processed and presented by antigen-presenting cells (APC). The TCR is located at the cytoplasmic membrane of the T lymphocyte but is functional assembled in the rough endoplasmic reticulum (RER). Most of the available information on TCR constituents in salmonids comes from numerous nucleotide sequences available in different databases. In this work, by in silico homology modeling, we generated the TCRαß/CD3 complex of rainbow trout (Oncorhynchus mykiss) and characterized the structure of the different proteins and their potential interactions. The results show that the main structural features described in mammalian TCR/CD3 are present in the model predicted for trout. Furthermore, we highlighted several aminoacidic interactions between TCRα, TCRß, CD3γδ, and CD3ε. In silico structural analyses suggest that trout TCRαß complex would fit similarly to that described for mammals. Herein, we explore the implications of the modeled trout complex and the leukocyte phenotypes, mainly associated with different regulation mechanisms of trout TCRαß/CD3 subunits gene expression or may be due to differences in the assembly process of the complex in the RER. However, further studies will be needed to study deeper the mechanisms involved.
Assuntos
Oncorhynchus mykiss , Receptores de Antígenos de Linfócitos T alfa-beta , Animais , Complexo CD3 , Mamíferos , Complexo Receptor-CD3 de Antígeno de Linfócitos T , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
This study aimed to investigate the synthesis and potential vasodilator effect of a novel ruthenium complex, cis-[Ru(bpy)2(2-MIM)(NO2)]PF6 (bpy = 2,2'-bipyridine and 2-MIM = 2-methylimidazole) (FOR711A), containing an imidazole derivative via an in silico molecular docking model using ß1 H-NOX (Heme-nitric oxide/oxygen binding) domain proteins of reduced and oxidized soluble guanylate cyclase (sGC). In addition, pharmacokinetic properties in the human organism were predicted through computational simulations and the potential for acute irritation of FOR711A was also investigated in vitro using the hen's egg chorioallantoic membrane (HET-CAM). FOR711A interacted with sites of the ß1 H-NOX domain of reduced and oxidized sGC, demonstrating shorter bond distances to several residues and negative values of total energy. The predictive study revealed molar refractivity (RM): 127.65; Log Po/w = 1.29; topological polar surface area (TPSA): 86.26 Å2; molar mass (MM) = 541.55 g/mol; low solubility, high unsaturation index, high gastrointestinal absorption; toxicity class 4; failure to cross the blood-brain barrier and to react with cytochrome P450 (CYP) enzymes CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4. After the HET-CAM assay, the FOR711A complex was classified as non-irritant (N.I.) and its vasodilator effect was confirmed through greater evidence of blood vessels after the administration and ending of the observation period of 5 min. These results suggest that FOR711A presented a potential stimulator/activator effect of sGC via NO/sGC/cGMP. However, results indicate it needs a vehicle for oral administration.
Assuntos
Complexos de Coordenação/química , Óxido Nítrico/química , Rutênio/química , Vasodilatadores/química , Vasodilatadores/farmacologia , Animais , Galinhas , Membrana Corioalantoide/metabolismo , Heme/química , Humanos , Imidazóis/química , Simulação de Acoplamento Molecular/métodos , Óxido Nítrico/metabolismo , Oxigênio/química , Domínios Proteicos , Guanilil Ciclase Solúvel/química , Guanilil Ciclase Solúvel/metabolismoRESUMO
Good water quality for livestock is essential for animal health, supply of safe food and food production economy. Few countries have established water quality criteria for livestock for chemical contaminants. For those that have them, the values are quite variable among each other for the same substance due to differences in the approach for the acceptable daily intakes and algorithms for the calculation. In several countries, including Brazil, these standards are based on international or other countries' data, which differ among protected species, exposure scenarios, and levels of protection. The objective of this work was to discuss critical issues to establish chemical water quality criteria for livestock in Brazil. A discussion about the difficulties involved and the alternatives when sufficient data are not available is presented. Using the Canadian framework for reference, we provide recommendations on a revised framework and alternatives regarding the use of chronic studies as mandatory, defaulting to human health drinking water quality guidelines/criteria for livestock, the use of additional safety factors, and alternatives in the absence of toxicological data.
Assuntos
Água Potável/química , Água Potável/normas , Poluentes Químicos da Água/análise , Qualidade da Água , Animais , Brasil , Canadá , Água Potável/efeitos adversos , Humanos , Gado , Medição de Risco , Poluentes Químicos da Água/efeitos adversosRESUMO
Chemotherapy is limited in the treatment of leishmaniasis due to the toxic effects of drugs, low efficacy of alternative treatments, and resistance of the parasite. This work assesses the in vitro activity of flavopereirine on promastigote cultures of Leishmania amazonensis. In addition, an in silico evaluation of the physicochemical characteristics of this alkaloid is performed. The extract and fractions were characterized by thin-layer chromatography and HPLC-DAD, yielding an alkaloid identified by NMR. The antileishmanial activity and cytotoxicity were assayed by cell viability test (MTT). The theoretical molecular properties were calculated on the Molinspiration website. The fractionation made it possible to isolate a beta-carboline alkaloid (flavopereirine) in the alkaloid fraction. Moreover, it led to obtaining a fraction with greater antileishmanial activity, since flavopereirine is very active. Regarding the exposure time, a greater inhibitory effect of flavopereirine was observed at 24 h and 72 h (IC50 of 0.23 and 0.15 µg/mL, respectively). The extract, fractions, and flavopereirine presented low toxicity, with high selectivity for the alkaloid. Furthermore, flavopereirine showed no violation of Lipinski's rule of five, showing even better results than the known inhibitor of oligopeptidase B, antipain, with three violations. Flavopereirine also interacted with residue Tyr-499 of oligopeptidase B during the molecular dynamics simulations, giving a few insights of a possible favorable mechanism of interaction and a possible inhibitory pathway. Flavopereirine proved to be a promising molecule for its antileishmanial activity.
Assuntos
Antiprotozoários/farmacologia , Apocynaceae/química , Carbolinas/farmacologia , Alcaloides Indólicos/isolamento & purificação , Leishmania mexicana/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Serina Endopeptidases/química , Antipaína/química , Antipaína/farmacologia , Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Carbolinas/química , Carbolinas/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Humanos , Alcaloides Indólicos/química , Alcaloides Indólicos/classificação , Concentração Inibidora 50 , Leishmania mexicana/crescimento & desenvolvimento , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/fisiologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Casca de Planta/química , Extratos Vegetais/química , Proteínas de Protozoários/química , Células THP-1RESUMO
BACKGROUND: The Cry toxins, or δ-endotoxins, are a diverse group of proteins produced by Bacillus thuringiensis. While DNA secondary structures are biologically relevant, it is unknown if such structures are formed in regions encoding conserved domains of Cry toxins under shuffling conditions. We analyzed 5 holotypes that encode Cry toxins and that grouped into 4 clusters according to their phylogenetic closeness. The mean number of DNA secondary structures that formed and the mean Gibbs free energy [Formula: see text] were determined by an in silico analysis using different experimental DNA shuffling scenarios. In terms of spontaneity, shuffling efficiency was directly proportional to the formation of secondary structures but inversely proportional to ∆G. RESULTS: The results showed a shared thermodynamic pattern for each cluster and relationships among sequences that are phylogenetically close at the protein level. The regions of the cry11Aa, Ba and Bb genes that encode domain I showed more spontaneity and thus a greater tendency to form secondary structures (<∆G). In the region of domain III; this tendency was lower (>∆G) in the cry11Ba and Bb genes. Proteins that are phylogenetically closer to Cry11Ba and Cry11Bb, such as Cry2Aa and Cry18Aa, maintained the same thermodynamic pattern. More distant proteins, such as Cry1Aa, Cry1Ab, Cry30Aa and Cry30Ca, featured different thermodynamic patterns in their DNA. CONCLUSION: These results suggest the presence of thermodynamic variations associated to the formation of secondary structures and an evolutionary relationship with regions that encode highly conserved domains in Cry proteins. The findings of this study may have a role in the in silico design of cry gene assembly by DNA shuffling techniques.