Structure-based design of a Cortistatin analogue with immunomodulatory activity in models of inflammatory bowel disease

Ulcerative colitis and Crohn's disease are forms of inflammatory bowel disease whose incidence and prevalence are increasing worldwide. These diseases lead to chronic inflammation of the gastrointestinal tract as a result of an abnormal response of the immune system. Recent studies positioned Cortistatin, which shows low stability in plasma, as a candidate for IBD treatment. Here, using NMR structural information, we design five Cortistatin analogs adopting selected native Cortistatin conformations in soln. One of them, A5, preserves the anti-inflammatory and immunomodulatory activities of Cortistatin in vitro and in mouse models of the disease. Addnl., A5 displays an increased half-life in serum and a unique receptor binding profile, thereby overcoming the limitations of the native Cortistatin as a therapeutic agent. This study provides an efficient approach to the rational design of Cortistatin analogs and opens up new possibilities for the treatment of patients that fail to respond to other therapies

Peptide aromatic interactions modulated by fluorinated residues: Synthesis, structure and biological activity of Somatostatin analogs containing 3-(3',5'difluorophenyl)-alanine

Somatostatin is a 14-residue peptide hormone that regulates the endocrine system by binding to five G-protein-coupled receptors (SSTR1-5). We have designed six new Somatostatin analogs with L-3-(3',5'-difluorophenyl)-alanine (Dfp) as a substitute of Phe and studied the effect of an electron-poor arom. ring in the network of arom. interactions present in Somatostatin. Replacement of each of the Phe residues (positions 6, 7 and 11) by Dfp and use of a D-Trp8 yielded peptides whose main conformations could be characterized in aq. soln. by NMR. Receptor binding studies revealed that the analog with Dfp at position 7 displayed a remarkable affinity to SSTR2 and SSTR3. Analogs with Dfp at positions 6 or 11 displayed a π-π interaction with the Phe present at 11 or 6, resp. Interestingly, these analogs, particularly [D-Trp8,L-Dfp11]-SRIF, showed high selectivity towards SSTR2, with a higher value than that of Octreotide and a similar one to that of native Somatostatin

Análogos de somatostatina y cortistatina. Efecto de las interacciones aromáticas en sus estructuras y en la actividad biológica

[spa] La somatostatina (SST), es un péptido natural de 14 residuos descubierto en 1973, con cinco subreceptores identificados, SSTR1-5. En la práctica clínica, y debido a su carácter inhibidor de secreción de otras hormonas endógenas, se utiliza para tratar los trastornos de la secreción de la hormona del crecimiento y tumores endocrinos. Además, la cortistatina (CST), un péptido relacionado con la somatostatina, ha mostrado un perfil farmacológico interesante contra enfermedades inflamatorias, como la enfermedad de Crohn, que la somatostatina no presenta. En esta tesis doctoral, se han diseñado tres familias de análogos de somatostatina de catorce residuos, sustituyendo a las L-β-fenilalanina en posiciones 6, 7 y 11 por los aminoácidos aromáticos no naturales L-β-3’,5’-difluorofenilalanina, L-β-3’-piridilalanina y L-β-4’piridilalanina. Al utilizar estos aminoácidos (de menor densidad electrónica aromática que L-β-fenilalanina), la potencia de las interacciones aromáticas intramoleculares aumenta, lo que ha permitido obtener la estructura de la conformación mayoritaria para cada uno de los derivados estudiados. Además, se incluyeron los aminoácidos L-β-mesitilalanina y L-β-3’,4’,5’-trimetilfenilalanina (de mayor densidad electrónica) en determinados péptidos, incrementando aún más la fuerza de las interacciones aromáticas. Los estudios conformacionales (realizados mediante RMN) demuestran que la inclusión de un aminoácido aromático pobre en electrones, en posición 7, provoca un acercamiento espacial de tipo clúster de los tres residuos aromáticos 6, 7 y 11. Este ordenamiento favorece la interacción con SSTR3. Cuando estos aminoácidos fueron incluidos en posiciones 6 u 11, se produjo una interacción entre 6-11, quedando el aminoácido 7 por la otra cara del péptido. En estos casos, al sustituirse la fenilalanina en posición 7 por L-β-mesitilalanina, se aumenta la selectividad por SSTR2. Concretamente, [L-4’Pya6_L-Msa7_D-Trp8]-SST presenta un perfil de actividad completamente selectivo para SSTR2, alcanzando además los niveles de afinidad de la SST. Por último, se sintetizaron diversos análogos de cortistatina, determinando de nuevo su conformación tridimensional mayoritaria, además de determinar su respuesta in vivo e in vitro frente a enfermedad de Crohn y perfil farmacológico en SSTR1-5. Sólo uno de ellos, con estructura tipo clúster, obtuvo un perfil de respuesta similar a CST frente a diferentes parámetros de enfermedad inflamatoria intestinal.[eng] Somatostatin (SST), also known as somatotropin release-inhibiting factor (SRIF), is a 14-amino-acid peptide discovered in 1973. It is a natural hormone whose biological activity is linked to five identified receptors: SSTR1-5. In clinical practice, somatostatin is currently used as a gastric anti-secretory drug as well as to treat growth hormone secretion disorders and endocrine tumors. Cortistatin (CST) is another natural hormone, structurally a related to somatostatin. It has shown an interesting inflammatory activity that somatostatin do not display. Therefore, it has potential applications against inflammatory diseases such as Crohn's disease. In the present thesis, three families of fourteen-residue somatostatin analogs have been designed and synthetized replacing L-β-phenylalanine in positions 6, 7 and 11 by the non-natural aromatic amino acids L-β-3’,5'-difluorophenylalanine, L-β-3'-pyridylalanine and L-β-4'-piridilalanine. The intramolecular aromatic interactions of these peptides (with amino acids whose aromatic electron-density is lower than Phe) increased substantially allowing us to obtain the major tridimensional conformation for each derivative by NMR. Furthermore, L-β-mesitylalanine and L-β-3’,5'-trimethylphenylalanine (with high aromatic electron density) were also included in certain peptides, to increase even further the strength of the aromatic interactions. Our NMR conformational studies demonstrated that the inclusion of an electron-poor aromatic amino acid in position 7 causes a three-dimensional cluster-type arrangement of the three aromatic residues in positions 6th, 7th and 11th. We have observed that peptides with this kind of conformation showed a good binding with SSTR3. On the other hand, when these amino acids were included in positions 6 or 11, only a strong 6-11 interaction was observed, being residue 7 placed on the other side of the peptide. In these cases, the additional replacement of the phenylalanine at position 7 by L-β-mesitylalanine provoked a further increase of the selectivity for SSTR2. Specifically, [L-4'Pya6_L-Msa7_D-Trp8] -SST showed a completely selective activity profile for SSTR2, with an affinity for this receptor at the same level of SST. Finally, various cortistatin analogs were synthesized, their major 3D conformation were determined by NMR and their binding profile with SSTR1-5 were measured. Some of these peptides showed a remarkable response against Crohn’s disease. One of them, with a cluster-type structure, showed a response similar to CST against different parameters of inflammatory bowel disease

Peptide aromatic interactions modulated by fluorinated residues: Synthesis, structure and biological activity of Somatostatin analogs containing 3-(3',5'difluorophenyl)-alanine

Somatostatin is a 14-residue peptide hormone that regulates the endocrine system by binding to five G-protein-coupled receptors (SSTR1-5). We have designed six new Somatostatin analogs with L-3-(3',5'-difluorophenyl)-alanine (Dfp) as a substitute of Phe and studied the effect of an electron-poor arom. ring in the network of arom. interactions present in Somatostatin. Replacement of each of the Phe residues (positions 6, 7 and 11) by Dfp and use of a D-Trp8 yielded peptides whose main conformations could be characterized in aq. soln. by NMR. Receptor binding studies revealed that the analog with Dfp at position 7 displayed a remarkable affinity to SSTR2 and SSTR3. Analogs with Dfp at positions 6 or 11 displayed a π-π interaction with the Phe present at 11 or 6, resp. Interestingly, these analogs, particularly [D-Trp8,L-Dfp11]-SRIF, showed high selectivity towards SSTR2, with a higher value than that of Octreotide and a similar one to that of native Somatostatin