Modulation du récepteur de la neurotensine de type 1 par des pepducines mimant sa première boucle intracellulaire

Abstract: The neurotensin receptor type I (NTS1) belongs to the G protein coupled receptor (GPCR) superfamily, which induces after ligand-receptor interaction different physiological effects, such as analgesia, hypothermia, and hypotension. The diversity in the physiological response reveals that several pathways are being activated and that several cytosolic effectors are also involved. Previous studies conducted with lipopeptide conjugates whose peptide portion mimics the first intracellular loop (ICL1) of NTS1 (also known as pepducins) have shown that some pathways are favored or inhibited, demonstrating the influence of these ICLs on the interaction with the cytosolic effectors and in the modulation of signaling pathways. The evaluation of this pepducin (PP-ICL1) in acute, tonic, and chronic pain models has revealed that the activation of the Gα13 and Gαq proteins signaling pathway could be associated with an antinociceptive action. In this study, we have evaluated the effect of enhancing the hydrophobic properties in punctual positions of PP-ICL1 sequence on its biological potential. Accordingly, we synthetized a series of pepducins based on the sequence of PP-ICL1 in which each amino acid was substituted by tryptophan. The whole cellular response induced by our pepducins was monitored using electrical cell impedance sensing and used to evaluate the effect of the tryptophan introduction. The signaling profile of the pepducins was determined using BRET biosensors, which revealed a biased modulation. Indeed, the substitution of certain residues seemed to favor the engagement of the Gα13 and Gαq signaling pathways without causing any effect on the recruitment of β-arrestins 1 and 2. Using a radiolabeled probe in a competitive radioligand binding assay on the hNTS1 receptor, we demonstrated that our pepducins could displace the bounded orthosteric ligand by probably acting in an allosteric site. Finally, the most potent compounds of this series tested in vitro were screened in a model of an acute pain model (tail-flick test). Intrathecal administration of pepducins PP-W11 at 275 nmol/kg induced a potent antinociceptive response which outperformed all the analgesic responses reported by any pepducin synthesized so far. Taken together, these results confirm that enhancing selectively the hydrophobic potential of PP-ICL1 could constitute a valid approach in the refinement of these pepducin properties as well as in the discovery of new drugs candidates.Le récepteur de la neurotensine de type 1 (NTS1) appartient à la superfamille des récepteurs couplés aux protéines G (RCPG), qui après son interaction avec son ligand peut exercer différents effets physiologiques tels qu’analgésie, hypothermie et hypotension. La diversité des réponses physiologiques possibles indique que plusieurs voies de signalisation sont activées et que plusieurs effecteurs cytosoliques sont impliqués. Des études précédentes menées avec des conjugués lipopeptidiques, dont la partie peptidique imite la première boucle intracellulaire (ICL1) de NTS1 (également appelées pepducines), ont montré que certaines voies de signalisation sont favorisées ou inhibées, démontrant l'influence de ces conjugués sur l'interaction avec les effecteurs cytosoliques et sur la modulation des voies de signalisation. De plus, l'évaluation in vivo de cette pepducine dans des modèles de douleur aiguë, douleur tonique et douleur chronique a révélé un effet antinociceptif ainsi qu'une forte capacité à diminuer la pression artérielle. Dans cette étude, nous avons évalué l'effet de l'augmentation de l’hydrophobicité des pepducines sur leurs activités biologiques. En conséquence, nous avons généré une série de pepducines basées sur la séquence de ICL1 de NTS1 dans laquelle chaque acide aminé a été remplacé par un tryptophane. L'impact de l’introduction du tryptophane dans la séquence lipopeptidique de nos pepducines a été évalué en mesurant la réponse cellulaire globale par un essai d’impédance électrique. Le profil de signalisation de nos pepducines a aussi été déterminé à l'aide de biosenseurs spécifique BRET (Gα13, Gαq et β-arrestines 1 et 2), qui ont révélé une probable modulation allostérique biaisée. En effet, la substitution de certains résidus semble favoriser l'activation des voies de signalisation Gα13 et Gαq sans recrutement des β-arrestines 1 et 2. De façon intéressante, en utilisant la neurotensine radiomarquée dans un test de liaison compétitif sur le récepteur hNTS1, nous avons également démontré que nos pepducines pouvaient induire le déplacement d’un ligand orthostérique via un site allostérique probablement placé à la surface intracellulaire du récepteur. Enfin, les composés les plus puissants des tests in vitro ont été évalués dans un modèle de douleur aiguë (test de retrait de la queue). L'administration intrathécale des pepducines PP-W11 à 275 nmol/kg a induit une réponse antinociceptive puissante qui a surpassé toutes les réponses analgésiques rapportées par toutes pepducines synthétisées jusqu'à présent. Ces résultats ont confirmé que l'augmentation du potentiel hydrophobe de PP-ICL1 pourrait constituer une approche intéressante dans le raffinement des propriétés des pepducines de NTS1 ainsi que dans la découverte de nouveaux candidats médicaments

Discovery of potent and selective inhibitors of the Escherichia coli M1-aminopeptidase via multicomponent solid-phase synthesis of tetrazole-peptidomimetics

The Escherichia coli neutral M1-aminopeptidase (ePepN) is a novel target identified for the development of antimicrobials. Here we describe a solid-phase multicomponent approach which enabled the discovery of potent ePepN inhibitors. The on-resin protocol, developed in the frame of the Distributed Drug Discovery (D3) program, comprises the implementation of parallel Ugi-azide four-component reactions with resin-bound amino acids, thus leading to the rapid preparation of a focused library of tetrazole-peptidomimetics (TPMs) suitable for biological screening. By dose-response studies, three compounds were identified as potent and selective ePepN inhibitors, as little inhibitory effect was exhibited for the porcine ortholog aminopeptidase. The study allowed for the identification of the key structural features required for a high ePepN inhibitory activity. The most potent and selective inhibitor (TPM 11) showed a non-competitive inhibition profile of ePepN. We predicted that both diastereomers of compound TPM 11 bind to a site distinct from that occupied by the substrate. Theoretical models suggested that TPM 11 has an alternative inhibition mechanism that doesn't involve Zn coordination. On the other hand, the activity landscape analysis provided a rationale for our findings. Of note, compound TMP 2 showed in vitro antibacterial activity against Escherichia coli. Furthermore, none of the three identified inhibitors is a potent haemolytic agent, and only two compounds showed moderate cytotoxic activity toward the murine myeloma P3X63Ag cells. These results point to promising compounds for the future development of rationally designed TPMs as antibacterial agents

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Gestión del conocimiento. Perspectiva multidisciplinaria. Volumen 17

El libro “Gestión del Conocimiento. Perspectiva Multidisciplinaria”, Volumen 17 de la Colección Unión Global, es resultado de investigaciones. Los capítulos del libro, son resultados de investigaciones desarrolladas por sus autores. El libro es una publicación internacional, seriada, continua, arbitrada, de acceso abierto a todas las áreas del conocimiento, orientada a contribuir con procesos de gestión del conocimiento científico, tecnológico y humanístico. Con esta colección, se aspira contribuir con el cultivo, la comprensión, la recopilación y la apropiación social del conocimiento en cuanto a patrimonio intangible de la humanidad, con el propósito de hacer aportes con la transformación de las relaciones socioculturales que sustentan la construcción social de los saberes y su reconocimiento como bien público

Self-homodimerization of an actinoporin by disulfide bridging reveals implications for their structure and pore formation

Abstract The Trp111 to Cys mutant of sticholysin I, an actinoporin from Stichodactyla helianthus sea anemone, forms a homodimer via a disulfide bridge. The purified dimer is 193 times less hemolytic than the monomer. Ultracentrifugation, dynamic light scattering and size-exclusion chromatography demonstrate that monomers and dimers are the only independent oligomeric states encountered. Indeed, circular dichroism and fluorescence spectroscopies showed that Trp/Tyr residues participate in homodimerization and that the dimer is less thermostable than the monomer. A homodimer three-dimensional model was constructed and indicates that Trp147/Tyr137 are at the homodimer interface. Spectroscopy results validated the 3D-model and assigned 85° to the disulfide bridge dihedral angle responsible for dimerization. The homodimer model suggests that alterations in the membrane/carbohydrate-binding sites in one of the monomers, as result of dimerization, could explain the decrease in the homodimer ability to form pores

Contribución al mecanismo de formación de poros de sticholisina I, una proteína formadora de poros de la anémona Stichodactyla helianthus, mediante el empleo de mutantes de Cys en zonas funcionalmente relevantes de la proteína

Sticholisina I (St I) es una citolisina producida por la anémona marina Stichodactyla helianthus, que se caracteriza por formar poros oligoméricos en membranas naturales y artificiales. En este trabajo se describe la obtención por vía recombinante en Escherichia coli (E. coli) de una variante recombinante de St I (St Ir), así como su caracterización conformacional y funcional. Estos trabajos permitieron reducir el impacto ecológico que provoca la obtención de St I a partir de las anémonas como fuente natural y además realizar mutagénesis sitio-específica para su caracterización funcional. La ausencia de Cys en St I facilitó la introducción de este residuo aminoacídico, por mutagénesis dirigida, en zonas funcionalmente importantes de la toxina. En el trabajo se sustituyeron por Cys en St Ir de forma independiente, los aminoácidos Glu2 y Phe15 (localizados en el segmento de los primeros treinta aminoácidos que participan en la formación del canal) y la Arg52, Pro80 y Trp111 (en la región de interacción con las membranas). Así, se obtuvieron y purificaron de E. coli cinco proteínas mutadas: StI E2C, StI F15C, StI R52C, StI P80C y StI W111C. Los estudios de caracterización espectroscópicos permitieron establecer que las sustituciones aminoacídicas no provocaron cambios conformacionales en los mutantes con respecto a St Ir. Los monómeros de StI E2C, StI R52C y StI P80C mostraron capacidades similares de unión a las membranas en comparación con la variante recombinante. StI F15C mostró un ligero incremento en su capacidad de interacción con las membranas mientras que StI W111C resultó la de menor capacidad de unión. La capacidad formadora de poros de los monómeros, medida en vesículas liposomales y membrana eritrocitaria, resultó similar entre StI E2C, StI F15C y St Ir. Sin embargo, la actividad lítica de StI R52C, StI P80C y StI W111C disminuyó con respecto a St Ir. Los agregados diméricos por enlace disulfuro en StI R52C y StI W111C disminuyeron la actividad biológica de las proteínas. Una de las novedades científicas más importante del presente trabajo radica en que se demuestra que la presencia de agregados diméricos estabilizados por enlace disulfuro, en StI E2C, incrementa la actividad biológica formadora de poros tanto en vesículas liposomales como en eritrocitos. Estos resultados demuestran, por primera vez, que la unión covalente de los extremos aminos de St I potencia la formación de poros funcionales por un mecanismo hasta ahora desconocido. En la investigación se aportan nuevos elementos sobre la importancia de los residuos Glu2, Phe15, Arg52, Pro80 y Trp111 en las etapas de unión inicial a las membranas y de formación de poros durante el mecanismo lítico. Por último, la obtención de los mutantes de Cys abre las posibilidades para el marcaje de St I con sondas de espín o sondas fluorescentes para estudiar el mecanismo de formación de poros mediante las espectroscopias de resonancia paramagnética electrónica (EPR) y de fluorescencia, y sustentan otras aplicaciones nanobiotecnológicas actualmente en desarrollo en nuestro grupo de trabajo.Sticholisin I (St I) is a cytolysin produced by the sea anemone Stichodactyla helianthus, which is characterized by forming oligomeric pores in natural and artificial membranes. This work describes the recombinant production in Escherichia coli (E. coli) of a recombinant variant of St I (St Ir), as well as its conformational and functional characterization. These works allowed us to reduce the ecological impact caused by obtaining St I from anemones as a natural source and also perform site-specific mutagenesis for its functional characterization. The absence of Cys in St I facilitated the introduction of this amino acid residue, by directed mutagenesis, into functionally important areas of the toxin. In the work, the amino acids Glu2 and Phe15 (located in the segment of the first thirty amino acids that participate in the formation of the channel) and Arg52, Pro80 and Trp111 (in the interaction region) were independently replaced by Cys in St Ir. with the membranes). Thus, five mutated proteins were obtained and purified from E. coli: StI E2C, StI F15C, StI R52C, StI P80C and StI W111C. Spectroscopic characterization studies allowed us to establish that amino acid substitutions did not cause conformational changes in the mutants with respect to St Ir. The monomers of StI E2C, StI R52C and StI P80C showed similar binding capacities to membranes compared to the recombinant variant. StI F15C showed a slight increase in its interaction capacity with membranes while StI W111C had the lowest binding capacity. The pore-forming capacity of the monomers, measured in liposomal vesicles and erythrocyte membrane, was similar between StI E2C, StI F15C and St Ir. However, the lytic activity of StI R52C, StI P80C and StI W111C decreased with respect to St Ir The dimeric aggregates by disulfide bond in StI R52C and StI W111C decreased the biological activity of the proteins. One of the most important scientific novelties of the present work is that it is demonstrated that the presence of dimeric aggregates stabilized by disulfide bond, in StI E2C, increases the pore-forming biological activity in both liposomal vesicles and erythrocytes. These results demonstrate, for the first time, that covalent attachment of the amino termini of St I enhances the formation of functional pores by a previously unknown mechanism. The research provides new elements on the importance of residues Glu2, Phe15, Arg52, Pro80 and Trp111 in the stages of initial binding to membranes and pore formation during the lytic mechanism. Finally, obtaining the Cys mutants opens the possibilities for labeling St I with spin probes or fluorescent probes to study the mechanism of pore formation through electron paramagnetic resonance (EPR) and fluorescence spectroscopies, and support other nanobiotechnological applications currently under development in our working group.Universidad Nacional, Costa RicaAcademia de Ciencias de Cuba, CubaEscuela de Ciencias Biológica