Jimena Fernández‐Carneado

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.

Differentiation Restricted Endocytosis of Cell Penetrating Peptides in MDCK Cells Corresponds with Activities of Rho-GTPases

PurposeCellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a γ-zein related sequence.MethodsInternalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis.ResultsWe observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement.ConclusionOur findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery.

D-SAP: a new, noncytotoxic, and fully protease resistant cell-penetrating peptide.

Protease resistant cell‐penetrating peptides (CPPs) are promising carriers for drugs unable to cross the cell membrane. As these CPPs are stable in vivo for much longer periods of time compared to other classes of therapeutic peptides, noncytotoxicity is a property sine qua non for their pharmacological development. Described herein is a fully protease resistant CPP that is noncytotoxic at concentrations up to 1 mM. Proteolytic stability was obtained by chiral inversion of the residues of a known self‐assembling CPP—from all L‐amino acids to all D‐amino acids—and then assessed against trypsin and human serum. Circular dichroism studies confirmed the enantiomeric structure of the analogue, and transmission electron microscopy (TEM) studies indicated that the new inverso analogue retains the ability of the original peptide to self‐assemble. The results of uptake experiments indicate that the protease‐stable (that is, D‐amino acid) analogue of the peptide is internalised by cells to the same extent as the protease‐susceptible (that is, L‐amino acid) parent peptide. Also reported herein are the results of studies on the cellular internalisation mechanism of the all‐D analogue, which reveal the steps followed by the peptide upon its entry into the cell.

Synthetic ligands able to interact with the P53 tetramerization domain. Towards understanding a protein surface recognition event

The applied interaction of synthetic molecules with defined regions of protein surfaces is an emerging strategy for the modulation of protein activity and/or stability. In spite of recent advances, the design of these molecules is not trivial. Among the most challenging aspects in designing these compounds is that they must compete with water molecules for interaction with polar patches of protein surfaces. Herein is reported the preparation of an arginine‐rich peptide that interacts in aqueous solution with a very hydrophilic patch at the surface of the tetramerization domain of the tumor suppressor protein p53. The interaction has been studied by several complementary techniques. By using this peptide as a template, a library of peptides has been prepared and evaluated in order to examine the different factors that contribute to the recognition event. The conclusions extracted from this work could be useful for the design of ligands directed at highly hydrophilic protein surface patches.

A Template‐Assembled Synthetic Protein Surface Mimetic of the von Willebrand Factor A1 domain Inhibits Botrocetin‐Induced Platelet Aggregation

Platelet adhesion, the initial step of platelet activation, is mediated by the interaction of von Willebrand factor (VWF) with its platelet receptor, the GPIb–IX complex. The binding of VWF to GPIb–IX is induced either by increased shear stress or by exogenous modulators, such as botrocetin. At a molecular level, this interaction takes place between the A1 domain of VWF and the GPIbα chain of the GPIb–IX complex. We report here the design and functional characteristics of a VWF template‐assembled synthetic protein (TASP), a chimeric four‐helix‐bundle TASP scaffold mimicking the surface of the A1 domain. Twelve residues located on helices α3 and α4 in the native A1 domain were grafted onto a surface formed by two neighboring helices of the TASP. VWF TASP was found to inhibit specifically botrocetin‐induced platelet aggregation and to bind both botrocetin and GPIbα. However, in contrast to the native A1 domain, VWF TASP did not bind simultaneously to both ligands. Modeling studies revealed that the relative orientation of the α helices in VWF TASP led to a clash of bound botrocetin and GPIbα. These results demonstrate that a chimeric four‐helix‐bundle TASP as a scaffold offers a suitable surface for presenting crucial residues of the VWF A1 domain; the potential of the TASP approach for de novo protein design and mimicry is thereby illustrated.

A tetradecapeptide somatostatin dicarba-analog: Synthesis, structural impact and biological activity

We described here the first tetradecapeptide somatostatin-analogue where the disulfide bridge has been replaced by a carbon-carbon double bond. This analogue was prepared using microwave assisted ring closing metathesis (RCM) using the 2nd generation Grubbs as catalyst. Under our optimized conditions the cyclization between allylGly 3 and 14 proceeded in moderate yield, excellent cyclic/linear ratio and very high Z-double bond selectivity. NMR studies also demonstrated that the conformational flexibility of this peptide is increased in comparison to that of the natural hormone. Remarkably, this alkene-bridged somatostatin analog is highly selective against somatostatin receptors 1 and 5, suggesting that conformational rigidity is not required for the efficient interaction of somatostatin analogues with these two receptors.

SSTR1- and SSTR3-selective somatostatin analogues.

We prepared the two enantiomers of 3‐(3′‐quinolyl)‐alanine (Qla, 1) in multigram scale by asymmetric hydrogenation. These amino acids, protected as Fmoc derivatives, were then used in the solid‐phase synthesis of two new somatostatin 14 (SRIF‐14) analogues 8 a and 8 b, tetradecapeptides in which the tryptophan residue (Trp8) is replaced by one of the two enantiomers of 3‐(3′‐quinolyl)‐alanine (Qla8) and therefore lack the NH bond in residue 8. The selectivity of these new analogues for the somatostatin receptors, SSTR1–5, was measured. Substitution with L‐Qla8 yielded peptide 8 a, which was highly selective for SSTR1 and SSTR3, with an affinity similar to that of SRIF‐14. Substitution by D‐Qla gave the relatively selective analogue 8 b, which showed high affinity for SSTR3 and significant affinity for SSTR1, SSTR2 and SSTR5. The biological results demonstrate that bulky and electronically poor aromatic amino acids at position 8 are compatible with strong activity with SSTR1 and SSTR3. Remarkably, these high affinity levels were achieved with peptides in which the conformational mobility was increased with respect to that of SRIF‐14. This observation suggests that conformational rigidity is not required, and might be detrimental to the interaction with receptors SSTR1 and SSTR3. The absence of an indole N proton in Qla8 might also contribute to the increased flexibility observed in these analogues.

Targeting molecular recognition: exploring the dual role of functional pseudoprolines in the design of SH3 ligands

Pseudoprolines (ΨPro) have been developed as tools for inducing bioactive conformations that allow for optimal spatial complementation in protein-protein interactions. This dual function of ΨPro was explored for tuning proline-rich peptides as potent ligands for SH3 domains.

Insights into Structure-Activity Relationships of Somatostatin Analogs Containing Mesitylalanine

The non-natural amino acid mesitylalanine (2,4,6-trimethyl-L-phenylalanine; Msa) has an electron-richer and a more conformationally restricted side-chain than that of its natural phenylalanine counterpart. Taking these properties into account, we have synthesized ten somatostatin analogs containing Msa residues in different key positions to modify the intrinsic conformational flexibility of the natural hormone. We have measured the binding affinity of these analogs and correlated it with the main conformations they populate in solution. NMR and computational analysis revealed that analogs containing one Msa residue were conformationally more restricted than somatostatin under similar experimental conditions. Furthermore, we were able to characterize the presence of a hairpin at the pharmacophore region and a non-covalent interaction between aromatic residues 6 and 11. In all cases, the inclusion of a D-Trp in the eighth position further stabilized the main conformation. Some of these peptides bound selectively to one or two somatostatin receptors with similar or even higher affinity than the natural hormone. However, we also found that multiple incorporations of Msa residues increased the life span of the peptides in serum but with a loss of conformational rigidity and binding affinity.

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 aromatic ring in the network of aromatic 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 aqueous solution 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, respectively. 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.