Pharmacological activation of insulin-degrading enzyme improves insulin secretion and glucose tolerance in diet-induced obese mice.

AIM: To investigate the use of synthetic preimplantation factor (sPIF) as a potential therapeutic tool for improving glucose-stimulated insulin secretion (GSIS), glucose tolerance and insulin sensitivity in the setting of diabetes. MATERIALS AND METHODS: We used a preclinical murine model of type 2 diabetes (T2D) induced by high-fat diet (HFD) feeding for 12 weeks. Saline or sPIF (1 mg/kg/day) was administered to mice by subcutaneously implanted osmotic mini-pumps for 25 days. Glucose tolerance, circulating insulin and C-peptide levels, and GSIS were assessed. In addition, ß-cells (Min-6) were used to test the effects of sPIF on GSIS and insulin-degrading enzyme (IDE) activity in vitro. The effect of sPIF on GSIS was also tested in human islets. RESULTS: GSIS was enhanced 2-fold by sPIF in human islets ex vivo. Furthermore, continuous administration of sPIF to HFD mice increased circulating levels of insulin and improved glucose tolerance, independently of hepatic insulin clearance. Of note, islets isolated from mice treated with sPIF exhibited restored ß-cell function. Finally, genetic (shRNA-IDE) or pharmacological (6bK) inactivation of IDE in Min-6 abolished sPIF-mediated effects on GSIS, showing that both the protein and its protease activity are required for its action. CONCLUSIONS: We conclude that sPIF is a promising secretagogue for the treatment of T2D.

Selectively Targeting Breast Cancer Stem Cells by 8-Quinolinol and Niclosamide.

Cancer maintenance, metastatic dissemination and drug resistance are sustained by cancer stem cells (CSCs). Triple negative breast cancer (TNBC) is the breast cancer subtype with the highest number of CSCs and the poorest prognosis. Here, we aimed to identify potential drugs targeting CSCs to be further employed in combination with standard chemotherapy in TNBC treatment. The anti-CSC efficacy of up to 17 small drugs was tested in TNBC cell lines using cell viability assays on differentiated cancer cells and CSCs. Then, the effect of 2 selected drugs (8-quinolinol -8Q- and niclosamide -NCS-) in the cancer stemness features were evaluated using mammosphere growth, cell invasion, migration and anchorage-independent growth assays. Changes in the expression of stemness genes after 8Q or NCS treatment were also evaluated. Moreover, the potential synergism of 8Q and NCS with PTX on CSC proliferation and stemness-related signaling pathways was evaluated using TNBC cell lines, CSC-reporter sublines, and CSC-enriched mammospheres. Finally, the efficacy of NCS in combination with PTX was analyzed in vivo using an orthotopic mouse model of MDA-MB-231 cells. Among all tested drug candidates, 8Q and NCS showed remarkable specific anti-CSC activity in terms of CSC viability, migration, invasion and anchorage independent growth reduction in vitro. Moreover, specific 8Q/PTX and NCS/PTX ratios at which both drugs displayed a synergistic effect in different TNBC cell lines were identified. The sole use of PTX increased the relative presence of CSCs in TNBC cells, whereas the combination of 8Q and NCS counteracted this pro-CSC activity of PTX while significantly reducing cell viability. In vivo, the combination of NCS with PTX reduced tumor growth and limited the dissemination of the disease by reducing circulating tumor cells and the incidence of lung metastasis. The combination of 8Q and NCS with PTX at established ratios inhibits both the proliferation of differentiated cancer cells and the viability of CSCs, paving the way for more efficacious TNBC treatments.

Hierarchical Quatsome-RGD Nanoarchitectonic Surfaces for Enhanced Integrin-Mediated Cell Adhesion.

The synthesis and study of the tripeptide Arg-Gly-Asp (RGD), the binding site of different extracellular matrix proteins, e.g., fibronectin and vitronectin, has allowed the production of a wide range of cell adhesive surfaces. Although the surface density and spacing of the RGD peptide at the nanoscale have already shown a significant influence on cell adhesion, the impact of its hierarchical nanostructure is still rather unexplored. Accordingly, a versatile colloidal system named quatsomes, based on fluid nanovesicles formed by the self-assembling of cholesterol and surfactant molecules, has been devised as a novel template to achieve hierarchical nanostructures of the RGD peptide. To this end, RGD was anchored on the vesicle's fluid membrane of quatsomes, and the RGD-functionalized nanovesicles were covalently anchored to planar gold surfaces, forming a state of quasi-suspension, through a long poly(ethylene glycol) (PEG) chain with a thiol termination. An underlying self-assembled monolayer (SAM) of a shorter PEG was introduced for vesicle stabilization and to avoid unspecific cell adhesion. In comparison with substrates featuring a homogeneous distribution of RGD peptides, the resulting hierarchical nanoarchitectonic dramatically enhanced cell adhesion, despite lower overall RGD molecules on the surface. The new versatile platform was thoroughly characterized using a multitechnique approach, proving its enhanced performance. These findings open new methods for the hierarchical immobilization of biomolecules on surfaces using quatsomes as a robust and novel tissue engineering strategy.

Direct Quantitative Immunochemical Analysis of Autoinducer Peptide IV for Diagnosing and Stratifying Staphylococcus aureus Infections.

An immunochemical strategy to detect and quantify AIP-IV, the quorum sensing (QS) signaling molecule produced by Staphylococcus aureusagr type IV, is reported here for the first time. Theoretical calculations and molecular modeling studies have assisted on the design and synthesis of a suitable peptide hapten (AIPIVS), allowing to obtain high avidity and specific antibodies toward this peptide despite its low molecular weight. The ELISA developed achieves an IC50 value of 2.80 ± 0.17 and an LOD of 0.19 ± 0.06 nM in complex media such as 1/2 Tryptic Soy Broth. Recognition of other S. aureus AIPs (I-III) is negligible (cross-reactivity below 0.001%), regardless of the structural similarities. A pilot study with a set of clinical isolates from patients with airways infection or colonization demonstrates the potential of this ELISA to perform biomedical investigations related to the role of QS in pathogenesis and the association between dysfunctional agr or the agr type with unfavorable clinical outcomes. The AIP-IV levels could be quantified in the low nanomolar range in less than 1 h after inoculating agr IV-genotyped isolates in the culture broth, while those genotyped as I-III did not show any immunoreactivity after a 48 h growth, pointing to the possibility to use this technology for phenotyping S. aureus. The research strategy here reported can be extended to the rest of the AIP types of S. aureus, allowing the development of powerful multiplexed chips or point-of-care (PoC) diagnostic devices to unequivocally identify its presence and its agr type on samples from infected patients.

Heterobivalent Ligand for the Adenosine A2A-Dopamine D2 Receptor Heteromer.

A G protein-coupled receptor heteromer that fulfills the established criteria for its existence in vivo is the complex between adenosine A2A (A2AR) and dopamine D2 (D2R) receptors. Here, we have designed and synthesized heterobivalent ligands for the A2AR-D2R heteromer with various spacer lengths. The indispensable simultaneous binding of these ligands to the two different orthosteric sites of the heteromer has been evaluated by radioligand competition-binding assays in the absence and presence of specific peptides that disrupt the formation of the heteromer, label-free dynamic mass redistribution assays in living cells, and molecular dynamic simulations. This combination of techniques has permitted us to identify compound 26 [KDB1 (A2AR) = 2.1 nM, KDB1 (D2R) = 0.13 nM], with a spacer length of 43-atoms, as a true bivalent ligand that simultaneously binds to the two different orthosteric sites. Moreover, bioluminescence resonance energy transfer experiments indicate that 26 favors the stabilization of the A2AR-D2R heteromer.

Polyhydroxylated Cyclopentane ß-Amino Acids Derived from d-Mannose and d-Galactose: Synthesis and Protocol for Incorporation into Peptides.

A stereoselective synthesis of polyhydroxylated cyclopentane ß-amino acids from hexoses is reported. The reaction sequence comprises, as key steps, ring-closing metathesis of a polysubstituted diene intermediate followed by the stereoselective aza-Michael functionalization of the resulting cyclopent-1-ene-1-carboxylic acid ester. Examples of synthesis of polysubstituted 2-aminocyclopentanecarboxylic acid derivatives starting from protected d-mannose and d-galactose are presented. A general protocol for the incorporation of these highly functionalized alicyclic ß-amino acids into peptides is also reported.

Amide Formation: Choosing the Safer Carbodiimide in Combination with OxymaPure to Avoid HCN Release.

It has been reported that DIC can react with OxymaPure to render an oxadiazole compound with the concomitant formation of HCN. Here we demonstrate that this reaction is not a feature of all carbodiimides but rather depends on the alkyl structure that flanks the two N atoms of the carbodiimide. Furthermore, we have identified two carbodiimides, TBEC and EDC·HCl, whose reaction with OxymaPure is exempt from HCN formation.

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method.

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.

Hybrid Cyclobutane/Proline-Containing Peptidomimetics: The Conformational Constraint Influences Their Cell-Penetration Ability.

A new family of hybrid ß,γ-peptidomimetics consisting of a repetitive unit formed by a chiral cyclobutane-containing trans-ß-amino acid plus a Nα-functionalized trans-γ-amino-l-proline joined in alternation were synthesized and evaluated as cell penetrating peptides (CPP). They lack toxicity on the human tumoral cell line HeLa, with an almost negligible cell uptake. The dodecapeptide showed a substantial microbicidal activity on Leishmania parasites at 50 µM but with a modest intracellular accumulation. Their previously published γ,γ-homologues, with a cyclobutane γ-amino acid, showed a well-defined secondary structure with an average inter-guanidinium distance of 8-10 Å, a higher leishmanicidal activity as well as a significant intracellular accumulation. The presence of a very rigid cyclobutane ß-amino acid in the peptide backbone precludes the acquisition of a defined conformation suitable for their cell uptake ability. Our results unveiled the preorganized charge-display as a relevant parameter, additional to the separation among the charged groups as previously described. The data herein reinforce the relevance of these descriptors in the design of CPPs with improved properties.

Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment.

Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems-quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.

Chiral Cyclobutane-Containing Cell-Penetrating Peptides as Selective Vectors for Anti-Leishmania Drug Delivery Systems.

Two series of new hybrid γ/γ-peptides, γ-CC and γ-CT, formed by (1S,2R)-3-amino-2,2,dimethylcyclobutane-1-carboxylic acid joined in alternation to a Nα-functionalized cis- or trans-γ-amino-l-proline derivative, respectively, have been synthesized and evaluated as cell penetrating peptides (CPP) and as selective vectors for anti-Leishmania drug delivery systems (DDS). They lacked cytotoxicity on the tumoral human cell line HeLa with a moderate cell-uptake on these cells. In contrast, both γ-CC and γ-CT tetradecamers were microbicidal on the protozoan parasite Leishmania beyond 25 µM, with significant intracellular accumulation. They were conjugated to fluorescent doxorubicin (Dox) as a standard drug showing toxicity beyond 1 µM, while free Dox was not toxic. Intracellular accumulation was 2.5 higher than with Dox-TAT conjugate (TAT = transactivator of transcription, taken as a standard CPP). The conformational structure of the conjugates was approached both by circular dichroism spectroscopy and molecular dynamics simulations. Altogether, computational calculations predict that the drug-γ-peptide conjugates adopt conformations that bury the Dox moiety into a cavity of the folded peptide, while the positively charged guanidinium groups face the solvent. The favorable charge/hydrophobicity balance in these CPP improves the solubility of Dox in aqueous media, as well as translocation across cell membranes, making them promising candidates for DDS.

Radical Dendrimers Based on Biocompatible Oligoethylene Glycol Dendrimers as Contrast Agents for MRI.

Finding alternatives to gadolinium (Gd)-based contrast agents (CA) with the same or even better paramagnetic properties is crucial to overcome their established toxicity. Herein we describe the synthesis and characterization of entirely organic metal-free paramagnetic macromolecules based on biocompatible oligoethylene glycol dendrimers fully functionalized with 5 and 20 organic radicals (OEG Gn-PROXYL (n = 0, 1) radical dendrimers) with the aim to be used as magnetic resonance imaging (MRI) contrast agents. Conferring high water solubility on such systems is often a concern, especially in large generation dendrimers. Our approach to overcome such an issue in this study is by synthesizing dendrimers with highly water-soluble branches themselves. In this work, we show that the highly water-soluble OEG Gn-PROXYL (n = 0, 1) radical dendrimers obtained showed properties that convert them in good candidates to be studied as contrast agents for MRI applications like diagnosis and follow-up of infectious diseases, among others. Importantly, with the first generation radical dendrimer, a similar r1 relaxivity value (3.4 mM-1s-1) in comparison to gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) used in clinics (3.2 mM-1s-1, r.t. 7T) has been obtained, and it has been shown to not be cytotoxic, avoiding the toxicity risks associated with the unwanted accumulation of Gd in the body.

Competitive ELISA for N-terminal pro-brain natriuretic peptide (NT-proBNP) determination in human plasma.

Brain natriuretic peptides (BNPs) are well-established cardiovascular disease (CVD) biomarkers that are released from the heart after ventricular wall stress. Particularly, the N-terminal proBNP (NT-proBNP) is a 76 aa long peptide and is recognized as an indicator for the diagnosis of heart failure (HF) and is being used in routine tests in emergency rooms when levels are above 0.4 ng mL-1. Herein, we report a new competitive ELISA for NT-proBNP, which is able to detect this biomarker directly in undiluted human plasma samples. The ELISA has been the result of a rational design of an immunizing peptide hapten and the investigation of different immunochemical conditions, including heterologous competitors and distinct physico-chemical conditions. The developed ELISA is able to detect NT-proBNP with a LOD of 0.40 ± 0.15 ng mL-1 in human plasma samples and quantify this biomarker in the range between 0.97 ± 0.38 and 23.10 ± 9.46 ng mL-1 with good accuracy. The ELISA can simultaneously measure many samples in 1.5 h and has been found to be robust, reproducible and shows great promise in diagnosis of heart failures.

Solid-Phase Synthesis of Head to Side-Chain Tyr-Cyclodepsipeptides Through a Cyclative Cleavage From Fmoc-MeDbz/MeNbz-resins.

Cyclic depsipeptides constitute a fascinating class of natural products. Most of them are characterized by an ester formed between the ß-hydroxy function of Ser/Thr -and related amino acids- and the carboxylic group of the C-terminal amino acid. Less frequent are those where the thiol of Cys is involved rendering a thioester (cyclo thiodepsipeptides) and even less common are the cyclo depsipeptides with a phenyl ester coming from the side-chain of Tyr. Herein, the preparation of the later through a cyclative cleavage using the Fmoc-MeDbz/MeNbz-resin is described. This resin has previously reported for the synthesis of cyclo thiodepsipeptides and homodetic peptides. The use of that resin for the preparation of all these peptides is also summarized.

Synthesis of Stable Cholesteryl-Polyethylene Glycol-Peptide Conjugates with Non-Disperse Polyethylene Glycol Lengths.

A method for conjugating cholesterol to peptide ligands through non-disperse polyethylene glycol (ND-PEG) through a non-hydrolysable linkage is described. The iterative addition of tetraethylene glycol macrocyclic sulfate to cholesterol (Chol) renders a family of highly pure well-defined Chol-PEG compounds with different PEG lengths from 4 up to 20 ethylene oxide units, stably linked through an ether bond. The conjugation of these Chol-PEG compounds to the cyclic (RGDfK) peptide though Lys5 side chains generates different lengths of Chol-PEG-RGD conjugates that retain the oligomer purity of the precursors, as analysis by HRMS and NMR has shown. Other derivatives were synthesized with similar results, such as Chol-PEG-OCH3 and Chol-PEG conjugated to glutathione and Tf1 peptides through maleimide-thiol chemoselective ligation. This method allows the systematic synthesis of highly pure uniform stable Chol-PEGs, circumventing the use of activation groups on each elongation step and thus reducing the number of synthesis steps.

Engineering a Nanostructured Nucleolin-Binding Peptide for Intracellular Drug Delivery in Triple-Negative Breast Cancer Stem Cells.

Five peptide ligands of four different cell surface receptors (nucleolin, CXCR1, CMKLR1, and CD44v6) have been evaluated as targeting moieties for triple-negative human breast cancers. Among them, the peptide F3, derived from phage display, promotes the fast and efficient internalization of a genetically fused green fluorescent protein (GFP) inside MDA-MB-231 cancer stem cells in a specific receptor-dependent fashion. The further engineering of this protein into the modular construct F3-RK-GFP-H6 and the subsequent construct F3-RK-PE24-H6 resulted in self-assembling polypeptides that organize as discrete and regular nanoparticles. These materials, 15-20 nm in size, show enhanced nucleolin-dependent cell penetrability. We show that the F3-RK-PE24-H6, based on the Pseudomonas aeruginosa exotoxin A (PE24) as a core functional domain, is highly cytotoxic over target cells. The combination of F3, the cationic peptide (RK)n, and the toxin domain PE24 in such unusual presentation appears as a promising approach to cell-targeted drug carriers in breast cancers and addresses selective drug delivery in otherwise difficult-to-treat triple-negative breast cancers.

Optimized Stepwise Synthesis of the API Liraglutide Using BAL Resin and Pseudoprolines.

The number of peptide-based active pharmaceutical ingredients (APIs) has increased enormously in recent years. Furthermore, the emerging new peptide drug candidates are more complex and larger. For the industrial solid-phase synthesis of C-carboxylic acid peptides, the two main resins available, Wang and chlorotrityl chloride (CTC), have a number of drawbacks. In this context, resins that form an amide bond with the first amino acid are more robust than Wang and CTC resins. Here, we address the use of the backbone (BAL) resin for the synthesis of the peptide liraglutide. The BAL resin, in conjunction with the use of pseudoprolines to avoid aggregation, allows the stepwise solid-phase synthesis of this API in excellent purity and yield.

Carbosilane Dendron-Peptide Nanoconjugates as Antimicrobial Agents.

Over the last decades, multidrug-resistant bacteria have emerged and spread, increasing the number of bacteria, against which commonly used antibiotics are no longer effective. It has become a serious public health problem whose solution requires medical research in order to explore novel effective antimicrobial molecules. On the one hand, antimicrobial peptides (AMPs) are regarded as good alternatives because of their generally broad-spectrum activities, but sometimes they can be easily degraded by the organism or be toxic to animal cells. On the other hand, cationic carbosilane dendrons, whose focal point can be functionalized in many different ways, have also shown good antimicrobial activity. In this work, we synthetized first- and second-generation cationic carbosilane dendrons with a maleimide molecule on their focal point, enabling their functionalization with three different AMPs. After different microbiology studies, we found an additive effect between first-generation dendron and AMP3 whose study reveals three interesting effects: (i) bacteria aggregation due to AMP3, which could facilitate bacteria detection or even contribute to antibacterial activity by preventing host cell attack, (ii) bacteria disaggregation capability of second-generation cationic dendrons, and (iii) a higher AMP3 aggregation ability when dendrons were added previously to peptide treatment. These compounds and their different effects observed over bacteria constitute an interesting system for further mechanism studies.

Pseudo-Wang Handle for the Preparation of Fully Protected Peptides. Synthesis of Liraglutide by Fragment Condensation.

A handle for the protection of the C-terminus of peptides after cleaving with low concentration of trifluoroacetic acid (2-4%) is reported. The handle prevents polymerization reactions in the convergent condensation of peptidic fragments. Moreover, it is traceless, being removed during the final deprotection step of the peptide synthesis. This cheap and convenient handle is easily attached to the solid support, causing no disturbance to peptide elongation and thus proving to be useful in the convergent synthesis of long peptides.

Design of a True Bivalent Ligand with Picomolar Binding Affinity for a G Protein-Coupled Receptor Homodimer.

Bivalent ligands have emerged as chemical tools to study G protein-coupled receptor dimers. Using a combination of computational, chemical, and biochemical tools, here we describe the design of bivalent ligand 13 with high affinity ( KDB1 = 21 pM) for the dopamine D2 receptor (D2R) homodimer. Bivalent ligand 13 enhances the binding affinity relative to monovalent compound 15 by 37-fold, indicating simultaneous binding at both protomers. Using synthetic peptides with amino acid sequences of transmembrane (TM) domains of D2R, we provide evidence that TM6 forms the interface of the homodimer. Notably, the disturber peptide TAT-TM6 decreased the binding of bivalent ligand 13 by 52-fold and had no effect on monovalent compound 15, confirming the D2R homodimer through TM6 ex vivo. In conclusion, by using a versatile multivalent chemical platform, we have developed a precise strategy to generate a true bivalent ligand that simultaneously targets both orthosteric sites of the D2R homodimer.