Topical Administration of Somatostatin Prevents Retinal Neurodegeneration in Experimental Diabetes

Retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR). Somatostatin (SST) is an endogenous neuroprotective peptide that is downregulated in the diabetic eye. The aim of the study was to test the usefulness of topical administration of SST in preventing retinal neurodegeneration. For this purpose, rats with streptozotocin-induced diabetes mellitus (STZ-DM) were treated with either SST eye drops or vehicle for 15 days. Nondiabetic rats treated with vehicle served as a control group. Functional abnormalities were assessed by electroretinography (ERG), and neurodegeneration was assessed by measuring glial activation and the apoptotic rate. In addition, proapoptotic (FasL, Bid, and activation of caspase-8 and caspase-3) and survival signaling pathways (BclxL) were examined. Intraretinal concentrations of glutamate and its main transporter glutamate/aspartate transporter (GLAST) were also determined. Treatment with SST eye drops prevented ERG abnormalities, glial activation, apoptosis, and the misbalance between proapoptotic and survival signaling detected in STZ-DM rats. In addition, SST eye drops inhibited glutamate accumulation in the retina and GLAST downregulation induced by diabetes mellitus. We conclude that topical administration of SST has a potent effect in preventing retinal neurodegeneration induced by diabetes mellitus. In addition, our findings open up a new preventive pharmacological strategy targeted to early stages of DR

Lamellarin D bioconjugates II: synthesis and cellular internalization of dendrimer and nuclear location signal derivatives

The design and synthesis of Lamellarin D conjugates with a nuclear localization signal peptide and a poly(ethylene glycol)-based dendrimer are described. Conjugates 1-4 were obtained in 8-84% overall yields from the corresponding protected Lamellarin D. Conjugates 1 and 4 are 1.4 to 3.3-fold more cytotoxic than the parent compound against three human tumor cell lines(MDA-MB-231 breast, A-549 lung, and HT-29 colon). Besides, conjugates 3, 4 showed a decrease in activity potency in BJ skin fibroblasts, a normal cell culture. Cellular internalization was analyzed and nuclear distribution pattern was observed for 4, which contains a nuclear localization signalling sequence

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 analogues adopting selected native Cortistatin conformations in solution. One of them, A5, preserves the anti-inflammatory and immunomodulatory activities of Cortistatin in vitro and in mouse models of the disease. Additionally, 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 analogues and opens up new possibilities for the treatment of patients that fail to respond to other therapies.A.Rol was a recipient of a PhD fellowship from the Generalitat de Catalunya (FI) and A.E. and E.P. were recipients of PhD fellowships granted by the Severo Ochoa Program(FPI). T.T. was a postdoctoral fellow co-funded by the Marie Skłodowska-CurieCOFUND actions (IRB Barcelona Interdisciplinary Postdoc Programme). This work wassupported by the following grants: CTQ2014-56361-P and CTQ2017-87840-P (A.Riera)and RTI2018-100700-B-100 (M.D.) from the Spanish Ministry of Economy, Industryand Competitiveness (MINECO); and by AGAUR (SGR-50). We also acknowledge institutional funding from MINECO through the Centers of Excellence Severo OchoaAward given to IRB Barcelona, as well as from the CERCA Program of the Generalitat deCatalunya. M.J.M. is an ICREA Programme Investigator

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

Noves estructures basades en aminoàcids trifuncionals: foldàmers i quimioteques basades en derivats de prolina

[cat] Les molècules ramificades poden jugar un paper clau en la síntesi en fase sòlida de compostos amb una elevada complexitat estructural. En aquest sentit i concretament en la síntesi de pèptids i peptidomimètics, els aminoàcids trifuncionals hi juguen un paper fonamental. En aquesta tesi s'han utilitzat aquest tipus de molècules per tal d'obtenir estructures complexes com són els pèptids cíclics, els foldàmers i noves quimioteques. Com a plataformes ramificadores s'han utilitzat derivats trifuncionals de prolina, com la cis-gamma-amino-L-prolina i la trans-4-hidroxi-L-prolina. Així, la present tesi doctoral descriu tres projectes diferents els quals comparteixen l'ús de la síntesi en fase sòlida com a metodologia sintètica, així com la utilització d'aminoàcids trifuncionals. Els diferents projectes es basen en: (i) estudi de grups protectors ortogonals en la síntesi de pèptids cíclics en fase sòlida; (ii) síntesi, estudis estructurals i aplicacions de fòldamers gamma-peptídics derivats de la gamma-amino-L-prolina; i (iii) síntesi en fase sòlida d'una llibreria d'inhibidors de la lisil-tRNA sintetasa (LysRS) de Plasmodium falciparum, paràsit causant de la malària. CAPÍTOL 1: En la síntesi en fase sòlida és del tot imprescindible disposar de grups protectors ortogonals per tal d'obtenir uns resultats satisfactoris al final d'una síntesi complexa. Tot i això, no sempre s'arriba als resultats esperats. El primer capítol d'aquesta tesi descriu els problemes derivats de treballar amb els grups protectors d'amines Fmoc i Alloc quan estan continguts en el mateix aminoàcid trifuncional. El problema que es descriu és l'eliminació prematura del grup Fmoc en les condicions d'eliminació dels grup Alloc. Així, s'estableix l'origen del problema i s'estableixen metodologies alternatives d'acoblament per tal d'evitar subproductes no desitjats. Aquest mateix capítol descriu la síntesi en fase sòlida de tetra-beta-pèptids cíclics utilitzant un sistema de protecció tetraortogonal. CAPÍTOL 2: Els gamma-pèptids són els foldàmers més estudiats fins al moment, ja que se n'ha descrit propietats força interessants com ara l'antimicrobiana o la seva capacitat per travessar la membrana de cèl·lules eucariotes, fet que els converteix en bons candidats a transportadors de fàrmacs. La rellevància que estan adoptant aquests peptidomimètics es deu bàsicament al fet que aquestes molècules no naturals poden millorar alguns inconvenients dels pèptids naturals, com pot ser la baixa estabilitat en front a proteasses. El segon capítol d'aquesta tesi doctoral descriu la síntesi en fase sòlida de gamma-pèptids derivats de la cis-gamma-amino-L-prolina. A més a més, estudis estructurals mitjançant l'ús de tècniques de dicroïsme circular i ressonància magnètica nuclear estableixen que aquest tipus de compostos formen llaços C9, donant com a resultat en una estructura regular una sèrie de girs concatenats d'aquest tipus, estructura que també es pot descriure com a hèlix 9. Aquest mateix capítol descriu algunes propietats d'aquest compostos, com ara la seva capacitat de travessar membranes cel·lulars, la baixa toxicitat, l'elevada estabilitat en front a proteasses o l'activitat antimicrobiana. L'entrada cel·lular de cada pèptid es quantifica mitjançant tècniques de fluorimetria de plaques i de citometria de flux i es visualitza al microscopi confocal. CAPÍTOL 3: El tercer capítol descriu la síntesi d'una llibreria de compostos potencials inhibidors de la lisil-tRNA sintetassa (LysRS) del Plasmodium falciparum, paràsit causant de la malària. S'ha vist que les aminoacil-tRNA sintetasses poden ser dianes terapèutiques força vàlides, ja que tot i ser enzims essencials en totes les espècies, aquestes es poden inhibir d'una forma espècie-específica. En la ruta biològica de la traducció en la que participen les tRNA sintetasses es forma un complex intermedi, l'aminoacil adenilat, en el qual es va basar el disseny dels inhibidors. Així, aquest capítol descriu la síntesi de sulfamats en fase sòlida i la seva posterior utilització en la síntesi d'una llibreria de 90 inhibidors.[eng] Branched molecules, such as trifunctional amino acids, can play important roles in the creation of complex structures using solid-phase synthesis. This thesis describes the use of proline derived trifunctional amino acids such as cis-gamma-aminoproline and trans-4-hidroxyproline for the synthesis of cyclic peptides, foldamers and libraries. Thus, this thesis has been divided into three different chapters, which share both the use of these trifunctional molecules and the use of solid-phase peptide synthesis as a common methodology. The first chapter describes several problems derived from the use of orthogonal protecting groups in the same molecule. For instance, it describes that, in certain cases, the Fmoc protecting group can be removed when eliminating the Alloc one, not for its removing conditions but for the free amine that are kept free once the Alloc group has been removed. The use of several protecting groups at the same time was also tested in the synthesis of cyclic tetra-beta-peptides using a tetra-orthogonal protecting scheme. The second chapter describes the solid-phase synthesis of abiotic foldamers derived from proline derivatives. This chapter also describes that these compounds can fold forming a series of C9 turns, which, in an idealised structure, can form an helix 9. The labelling of these gamma-peptides with a fluorescent probe followed by their confocal microscopy study reveal that these gamma-peptides are able to cross the cell membrane of eukaryotic cells, a property that makes them good candidates as drug carriers for drug delivery. Actually, the conjugation of one of them with a PNA reveals that this gamma-peptide can transport this big non-membrane permeable molecule inside the cell, demonstrating its value for such application. In the third chapter, the solid phase synthesis of sulfamates is described, as well as their application in the synthesis of inhibitors of the lysil-tRNA synthetase (LysRS) of "Plasmodium falciparum

Acridine and quindoline oligomers linked through a 4-aminoproline backbone prefer G-quadruplex structures

[Background] DNA-intercalating drugs are planar molecules with several fused aromatic rings that form stacks between DNA base pairs, reducing the opening and unwinding of the double helix. Recently, interest on intercalating agents has moved in the search for new ligands to G-quadruplex structures.[Methods] The DNA binding properties of 4-aminoproline oligomers functionalized with one, two or three units of acridine and/or quindoline have been analyzed by competitive dialysis. A NMR/molecular dynamics study was performed on G-quadruplex telomeric sequence and the 4-aminoproline dimer carrying two quindolines. A model of the complex with the telomeric DNA quadruplex is described.[Results and conclusions] A selectivity of quindoline 4-aminoproline oligomers for G-quadruplex and triplex structures was observed, especially for those quadruplex sequences found in telomeres and in the promoter regions of c-myc and bcl-2 oncogenes. In this model the quindoline dimer is stabilized by π–π stacking interactions between the aromatic rings of the ligand and the nucleobases of the telomeric sequence that are located above and below the molecule.[General significance] The results of this work can be used for the design of new molecules with high affinity to telomeres which may have anticancer properties.This work was partially supported by grants from Spanish Ministerio de Ciencia e Innovación MICINN (CTQ2005-00315/BQU, CTQ2008-00177, BFU2007-63287, CTQ2009-20541), Generalitat de Catalunya, (2009/SGR/208), CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Institute for Research in Biomedicine, and the Barcelona Science Park. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Peer reviewe