A Diruthenium Metallodrug as a Potent Inhibitor of Amyloid-ß Aggregation: Synergism of Mechanisms of Action.

The physical and chemical properties of paddlewheel diruthenium compounds are highly dependent on the nature of the ligands surrounding the bimetallic core. Herein, we compare the ability of two diruthenium compounds, [Ru2Cl(D-p-FPhF)(O2CCH3)3]·H2O (1) (D-p-FPhF- = N,N'-bis(4-fluorophenyl)formamidinate) and K3[Ru2(O2CO)4]·3H2O (2), to act as inhibitors of amyloid aggregation of the Aß1-42 peptide and its peculiar fragments, Aß1-16 and Aß21-40. A wide range of biophysical techniques has been used to determine the inhibition capacity against aggregation and the possible mechanism of action of these compounds (Thioflavin T fluorescence and autofluorescence assays, UV-vis absorption spectroscopy, circular dichroism, nuclear magnetic resonance, mass spectrometry, and electron scanning microscopy). Data show that the most effective inhibitory effect is shown for compound 1. This compound inhibits fiber formation and completely abolishes the cytotoxicity of Aß1-42. The antiaggregatory capacity of this complex can be explained by a binding mechanism of the dimetallic units to the peptide chain along with π-π interactions between the formamidinate ligand and the aromatic side chains. The results suggest the potential use of paddlewheel diruthenium complexes as neurodrugs and confirm the importance of the steric and charge effects on the properties of diruthenium compounds.

Comparative Analysis of the Inhibitory Mechanism of Aß1-42 Aggregation by Diruthenium Complexes.

There is a growing interest in the search for metal-based therapeutics for protein misfolding disorders such as Alzheimer's disease (AD). A novel and largely unexplored class of metallodrugs is constituted by paddlewheel diruthenium complexes, which exhibit unusual water solubility and stability and unique coordination modes to proteins. Here, we investigate the ability of the complexes [Ru2Cl(DPhF)(O2CCH3)3]·H2O (1), [Ru2Cl(DPhF)2(O2CCH3)2]·H2O (2), and K2[Ru2(DPhF)(CO3)3]·3H2O (3) (DPhF- = N,N'-diphenylformamidinate) to interfere with the amyloid aggregation of the Aß1-42 peptide. These compounds differ in charge and steric hindrance due to the coordination of a different number of bulky ligands. The mechanisms of action of the three complexes were studied by employing a plethora of physicochemical and biophysical techniques as well as cellular assays. All these studies converge on different mechanisms of inhibition of amyloid fibrillation: complexes 1 and 2 show a clear inhibitory effect due to an exchange ligand process in the Ru2 unit aided by aromatic interactions. Complex 3 shows no inhibition of aggregation, probably due to its negative charge in solution. This study demonstrates that slight variations in the ligands surrounding the bimetallic core can modulate the amyloid aggregation inhibition and supports the use of paddlewheel diruthenium complexes as promising therapeutics for Alzheimer's disease.

The effects of histidine substitution of aromatic residues on the amyloidogenic properties of the fragment 264-277 of nucleophosmin 1.

Histidine (His) plays a key role in mediating protein interactions and its unique side chain determines pH responsive self-assembling processes and thus in the formation of nanostructures. In this study, To identify novel self-assembling bioinspired sequences, we analyzed a series of peptide sequences obtained through the point mutation of aromatic residues of 264-277 fragment of nucleophosmin 1 (NPM1) with single and double histidines. Through several orthogonal biophysical techniques and under different pH and ionic strength conditions we evaluated the effects of these substitutions in the amyloidogenic features of derived peptides. The results clearly indicate that both the type of aromatic mutated residue and its position can have different effect on amyloid-like behaviors. They corroborate the crucial role exerted by Tyr271 in the self-assembling process of CTD of NPM1 in AML mutated form and add novel insights in the accurate investigation of how side chain orientations can determine successful design of innovative bioinspired materials.

Modulation of hydrogel networks by metal ions.

Self-assembling hydrogels are receiving great attention for both biomedical and technological applications. Self-assembly of protein/peptides as well as organic molecules is commonly induced in response to external triggers such as changes of temperature, concentration, or pH. An interesting strategy to modulate the morphology and mechanical properties of the gels implies the use of metal ions, where coordination bonds regulate the dynamic cross-linking in the construction of hydrogels, and coordination geometries, catalytic, and redox properties of metal ions play crucial roles. This review aims to discuss recent insights into the supramolecular assembly of hydrogels involving metal ions, with a focus on self-assembling peptides, as well as applications of metallogels in biomedical fields including tissue engineering, sensing, wound healing, and drug delivery.

Hydrogelation tunability of bioinspired short peptides.

Supramolecular assemblies of short peptides are experiencing a stimulating flowering. Herein, we report a novel class of bioinspired pentapeptides, not bearing Phe, that form hydrogels with fibrillar structures. The inherent sequence comes from the fragment 269-273 of nucleophosmin 1 protein, that is normally involved in liquid-liquid phase separation processes into the nucleolus. By means of rheology, spectroscopy, and scanning microscopy the crucial roles of the extremities in the modulation of the mechanical properties of hydrogels were elucidated. Three of four peptide showed a typical shear-thinning profile and a self-assembly into hierarchical nanostructures fibers and two of them resulted biocompatible in MCF7 cells. The presence of an amide group at C-terminal extremity caused the fastest aggregation and the major content of structured intermediates during gelling process. The tunable mechanical and structural features of this class of hydrogels render derived supramolecular systems versatile and suitable for future biomedical applications.

Glucosyl Platinum(II) Complexes Inhibit Aggregation of the C-Terminal Region of the Aß Peptide.

Neurodegenerative diseases are often caused by uncontrolled amyloid aggregation. Hence, many drug discovery processes are oriented to evaluate new compounds that are able to modulate self-recognition mechanisms. Herein, two related glycoconjugate pentacoordinate Pt(II) complexes were analyzed in their capacity to affect the self-aggregation processes of two amyloidogenic fragments, Aß21-40 and Aß25-35, of the C-terminal region of the ß-amyloid (Aß) peptide, the major component of Alzheimer's disease (AD) neuronal plaques. The most water-soluble complex, 1Ptdep, is able to bind both fragments and to deeply influence the morphology of peptide aggregates. Thioflavin T (ThT) binding assays, electrospray ionization mass spectrometry (ESI-MS), and ultraviolet-visible (UV-vis) absorption spectroscopy indicated that 1Ptdep shows different kinetics and mechanisms of inhibition toward the two sequences and demonstrated that the peptide aggregation inhibition is associated with a direct coordinative bond of the compound metal center to the peptides. These data support the in vitro ability of pentacoordinate Pt(II) complexes to inhibit the formation of amyloid aggregates and pave the way for the application of this class of compounds as potential neurotherapeutics.

Targeting Ship2-Sam with peptide ligands: Novel insights from a multidisciplinary approach.

The lipid phosphatase Ship2 binds the EphA2 receptor through a heterotypic Sam-Sam (Sterile alpha motif) interaction. Inhibitors of the Ship2-Sam/EphA2-Sam complex hold a certain potential as novel anticancer agents. The previously reported "KRI3" peptide binds Ship2-Sam working as a weak antagonist of the EphA2-Sam/Ship2-Sam interaction. Herein, the design and functional evaluation of KRI3 analogues, both linear and cyclic, are described. A multidisciplinary study was conducted through computational docking techniques, and conformational analyses by CD and NMR spectroscopies. The ability of new peptides to bind Ship2-Sam was analysed by NMR, MST and SPR assays. Studies on linear KRI3 analogues pointed out that aromatic interactions through tyrosines are important for the association with Ship2-Sam whereas, an increase of the net positive charge of the sequence or peptide cyclization through a disulfide bridge can favour unspecific interactions without a substantial improvement of the binding affinity to Ship2-Sam. Interestingly, preliminary cell-based assays demonstrated KRI3 cellular uptake even without the conjugation to a cell penetrating sequence with a main cytosolic localization. This work highlights important features of the KRI3 peptide that can be further exploited to design analogues able to hamper Sam-Sam interactions driven by electrostatic contacts.

Insights into Network of Hot Spots of Aggregation in Nucleophosmin 1.

In a protein, point mutations associated with diseases can alter the native structure and provide loss or alteration of functional levels, and an internal structural network defines the connectivity among domains, as well as aggregate/soluble states' equilibria. Nucleophosmin (NPM)1 is an abundant nucleolar protein, which becomes mutated in acute myeloid leukemia (AML) patients. NPM1-dependent leukemogenesis, which leads to its aggregation in the cytoplasm (NPMc+), is still obscure, but the investigations have outlined a direct link between AML mutations and amyloid aggregation. Protein aggregation can be due to the cooperation among several hot spots located within the aggregation-prone regions (APR), often predictable with bioinformatic tools. In the present study, we investigated potential APRs in the entire NPM1 not yet investigated. On the basis of bioinformatic predictions and experimental structures, we designed several protein fragments and analyzed them through typical aggrsegation experiments, such as Thioflavin T (ThT), fluorescence and scanning electron microscopy (SEM) experiments, carried out at different times; in addition, their biocompatibility in SHSY5 cells was also evaluated. The presented data clearly demonstrate the existence of hot spots of aggregation located in different regions, mostly in the N-terminal domain (NTD) of the entire NPM1 protein, and provide a more comprehensive view of the molecular details potentially at the basis of NPMc+-dependent AML.

Electroanalytical Sensor Based on Gold-Nanoparticle-Decorated Paper for Sensitive Detection of Copper Ions in Sweat and Serum.

The growth of (bio)sensors in analytical chemistry is mainly attributable to the development of affordable, effective, portable, and user-friendly analytical tools. In the field of sensors, paper-based devices are gaining a relevant position for their outstanding features including foldability, ease of use, and instrument-free microfluidics. Herein, a multifarious use of filter paper to detect copper ions in bodily fluids is reported by exploiting this eco-friendly material to (i) synthesize AuNPs without the use of reductants and/or external stimuli, (ii) print the electrodes, (iii) load the reagents for the assay, (iv) filter the gross impurities, and (v) preconcentrate the target analyte. Copper ions were detected down to 3 ppb with a linearity up to 400 ppb in standard solutions. The applicability in biological matrices, namely, sweat and serum, was demonstrated by recovery studies and by analyzing these biofluids with the paper-based platform and the reference method (atomic absorption spectroscopy), demonstrating satisfactory accuracy of the novel eco-designed analytical tool.

Easy Surface Functionalization and Bioconjugation of Peptides as Capture Agents of a Microfluidic Biosensing Platform for Multiplex Assay in Serum.

The development of assays for protein biomarkers in complex matrices is a demanding task that still needs implementation of new approaches. Antibodies as capture agents have been largely used in bioassays but their low stability, low-efficiency production, and cross-reactivity in multiplex approaches impairs their larger applications. Instead, synthetic peptides, even with higher stability and easily adapted amino acid sequences, still remain largely unexplored in this field. Here, we provide a proof-of-concept of a microfluidic device for direct detection of biomarker overexpression. The multichannel microfluidic polydimethylsiloxane (PDMS) device was first derivatized with PAA (poly(acrylic acid)) solution. CRP-1, VEGF-114, and ΦG6 peptides were preliminarily tested to respectively bind the biomarkers, C-reactive protein (CRP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α). Each PDMS microchannel was then respectively bioconjugated with a specific peptide (CRP-1, VEGF-114, or ΦG6) to specifically capture CRP, VEGF, and TNF-α. With such microdevices, a fluorescence bioassay has been set up with sensitivity in the nanomolar range, both in buffered solution and in human serum. The proposed multiplex assay worked with a low amount of sample (25 µL) and detected biomarker overexpression (above nM concentration), representing a noninvasive and inexpensive screening platform.

Stimuli-responsive transdermal microneedle patches.

Microneedle (MN) patches consisting of miniature needles have emerged as a promising tool to perforate the stratum corneum and translocate biomolecules into the dermis in a minimally invasive manner. Stimuli-responsive MN patches represent emerging drug delivery systems that release cargos on-demand as a response to internal or external triggers. In this review, a variety of stimuli-responsive MN patches for controlled drug release are introduced, covering the mechanisms of action toward different indications. Future opportunities and challenges with respect to clinical translation are also discussed.

Conformational consequences of NPM1 rare mutations: An aggregation perspective in Acute Myeloid Leukemia.

Often proteins association is a physiological process used by cells to regulate their growth and to adapt to different stress conditions, including mutations. In the case of a subtype of Acute Myeloid Leukemia (AML), mutations of nucleophosmin 1 (NPM1) protein cause its aberrant cytoplasmatic mislocalization (NPMc+). We recently pointed out an amyloidogenic propensity of protein regions including the most common mutations of NPMc+ located in the C-terminal domain (CTD): they were able to form, in vitro, amyloid cytotoxic aggregates with fibrillar morphology. Herein, we analyzed the conformational characteristics of several peptides including rare AML mutations of NPMc+. By means of different spectroscopic, microscopic and cellular assays we evaluated the importance of amino acid composition, among rare AML mutations, to determine amyloidogenic propensity. This study could add a piece of knowledge to the structural consequences of mutations in cytoplasmatic NPM1c+.

Self-assembly of bio-inspired heterochiral peptides.

Peptide hydrogels, deriving from natural protein fragments, present unique advantages as compatibility and low cost of production that allow their wide application in different fields as wound healing, cell delivery and tissue regeneration. To engineer new biomaterials, the change of the chirality of single amino acids demonstrated a powerful approach to modulate the self-assembly mechanism. Recently we unveiled that a small stretch spanning residues 268-273 in the C-terminal domain (CTD) of Nucleophosmin 1 (NPM1) is an amyloid sequence. Herein, we performed a systematic D-scan of this sequence and analyzed the structural properties of obtained peptides. The conformational and kinetic features of self-aggregates and the morphologies of derived microstructures were investigated by means of different biophysical techniques, as well as the compatibility of hydrogels was evaluated in HeLa cells. All the investigated hexapeptides formed hydrogels even if they exhibited different conformational intermediates during aggregation, and they structural featured are finely tuned by introduced chiralities.

Self-Assembling Peptides: From Design to Biomedical Applications.

Self-assembling peptides could be considered a novel class of agents able to harvest an array of micro/nanostructures that are highly attractive in the biomedical field. By modifying their amino acid composition, it is possible to mime several biological functions; when assembled in micro/nanostructures, they can be used for a variety of purposes such as tissue regeneration and engineering or drug delivery to improve drug release and/or stability and to reduce side effects. Other significant advantages of self-assembled peptides involve their biocompatibility and their ability to efficiently target molecular recognition sites. Due to their intrinsic characteristics, self-assembled peptide micro/nanostructures are capable to load both hydrophobic and hydrophilic drugs, and they are suitable to achieve a triggered drug delivery at disease sites by inserting in their structure's stimuli-responsive moieties. The focus of this review was to summarize the most recent and significant studies on self-assembled peptides with an emphasis on their application in the biomedical field.

Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study.

Sterile alpha motif (SAM) domains are protein interaction modules with a helical fold. SAM-SAM interactions often adopt the mid-loop (ML)/end-helix (EH) model, in which the C-terminal helix and adjacent loops of one SAM unit (EH site) bind the central regions of another SAM domain (ML site). Herein, an original strategy to attack SAM-SAM associations is reported. It relies on the design of cyclic peptides that target a region of the SAM domain positioned at the bottom side of the EH interface, which is thought to be important for the formation of a SAM-SAM complex. This strategy has been preliminarily tested by using a model system of heterotypic SAM-SAM interactions involving the erythropoietin-producing hepatoma kinase A2 (EphA2) receptor and implementing a multidisciplinary plan made up of computational docking studies, experimental interaction assays (by NMR spectroscopy and surface plasmon resonance techniques) and conformational analysis (by NMR spectroscopy and circular dichroism). This work further highlights how only a specific balance between flexibility and rigidity may be needed to generate modulators of SAM-SAM interactions.

Engineered ß-hairpin scaffolds from human prion protein regions: Structural and functional investigations of aggregates.

The investigation of conformational features of regions of amyloidogenic proteins are of great interest to deepen the structural changes and consequent self-aggregation mechanisms at the basis of many neurodegenerative diseases. Here we explore the effect of ß-hairpin inducing motifs on regions of prion protein covering strands S1 and S2. In detail, we unveiled the structural and functional features of two model chimeric peptides in which natural sequences are covalently linked together by two dipeptides (l-Pro-Gly and d-Pro-Gly) that are known to differently enhance ß-hairpin conformations but both containing N- and the C-terminal aromatic cap motifs to further improve interactions between natural strands. Spectroscopic investigations at solution state indicate that primary assemblies of the monomers of both constructs follow different aggregativemechanisms during the self-assembly: these distinctions, evidenced by CD and ThT emission spectroscopies, reflect into great morphological differences of nanostructures and suggest that rigid ß-hairpin conformations greatly limit amyloid-like fibrillogenesis. Overall data confirm the important role exerted by the ß-structure of regions S1 and S2 during the aggregation process and lead to speculate to its persistence even in unfolding conditions.

Design and analysis of EphA2-SAM peptide ligands: A multi-disciplinary screening approach.

EphA2 receptor plays a critical and debatable function in cancer and is considered a target in drug discovery. Lately, there has been a growing interest in its cytosolic C-terminal SAM domain (EphA2-SAM) as it engages protein modulators of receptor endocytosis and stability. Interestingly, EphA2-SAM binds the SAM domain from the lipid phosphatase Ship2 (Ship2-SAM) mainly producing pro-oncogenic outcomes. In an attempt to discover novel inhibitors of the EphA2-SAM/Ship2-SAM complex with possible anticancer properties, we focused on the central region of Ship2-SAM (known as Mid-Loop interface) responsible for its binding to EphA2-SAM. Starting from the amino acid sequence of the Mid-Loop interface virtual peptide libraries were built through ad hoc inserted mutations with either l- or d- amino acids and screened against EphA2-SAM by docking techniques. A few virtual hits were synthesized and experimentally tested by a variety of direct and competition-type interaction assays relying on NMR (Nuclear Magnetic Resonance), SPR (Surface Plasmon Resonance), MST (Microscale Thermophoresis) techniques. These studies guided the discovery of an original EphA2-SAM ligand antagonist of its interaction with Ship2-SAM.

Mimetics of suppressor of cytokine signaling 3: Novel potential therapeutics in triple breast cancer.

Suppressor of cytokine signaling (SOCS) family of proteins plays critical role in the regulation of immune responses controlling JAK/STAT mediated inflammatory cytokines. Among the members, SOCS1 and SOCS3 contain a kinase inhibitory region (KIR) and SOCS3 binds to JAK/STAT/gp130 complex by inhibiting the downstream signaling and suppressing inflammatory cytokines. Loss or reduced levels of SOCS3 have been linked to cancer-associated inflammation and suppressive immunity leading to enhanced tumor growth and metastasis. In line with these reports, we previously demonstrated that proteolytic degradation of SOCS3 in triple negative breast cancer (TNBC) subtype drives the expression of inflammatory cytokines. Therefore, we postulated that SOCS3 mimetics might suppress the inflammatory cytokine production in TNBC subtype and inhibit tumor growth and metastasis. Here we designed and characterized five linear peptides derived from the N-terminal region of SOCS3 encompassing regions that interface with the JAK2/gp130 complex using the Circular Dichroism and Surface Plasmon Resonance spectroscopies. The KIRESS peptide resulted the sequence containing the most part of the hot-spots required for binding to JAK2 and was further investigated in vivo in mouse xenografts of MDA-MB-231-luci tumors as models of human TNBC subtype. Expectedly, this peptide showed a significant inhibition of primary tumor growth and pulmonary metastasis. Our studies suggest that SOCS3 peptidomimetics may possess a therapeutic potential in aggressive cancers, such as TNBC subtype, with activated inflammatory cytokines.

Sam domain-based stapled peptides: Structural analysis and interaction studies with the Sam domains from the EphA2 receptor and the lipid phosphatase Ship2.

Sam (Sterile alpha motif) domains represent small helical protein-protein interaction modules which play versatile functions in different cellular processes. The Sam domain from the EphA2 receptor binds the Sam domain of the lipid phosphatase Ship2 and this interaction modulates receptor endocytosis and degradation primarily generating pro-oncogenic effects in cell. To identify molecule antagonists of the EphA2-Sam/Ship2-Sam complex with anti-cancer activity, we focused on hydrocarbon helical stapled peptides. EphA2-Sam and one of its interactors (i.e., the first Sam domain of the adaptor protein Odin) were used as model systems for peptide design. Increase in helicity in the stapled peptides, with respect to the corresponding linear/native-like regions, was proved by structural studies conducted through CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance). Interestingly, interaction assays by means of NMR, SPR (Surface Plasmon Resonance) and MST (MicroScale Thermophoresis) techniques led to the discovery of a novel ligand of Ship2-Sam.

Peptides as Therapeutic Agents for Inflammatory-Related Diseases.

Inflammation is a physiological mechanism used by organisms to defend themselves against infection, restoring homeostasis in damaged tissues. It represents the starting point of several chronic diseases such as asthma, skin disorders, cancer, cardiovascular syndrome, arthritis, and neurological diseases. An increasing number of studies highlight the over-expression of inflammatory molecules such as oxidants, cytokines, chemokines, matrix metalloproteinases, and transcription factors into damaged tissues. The treatment of inflammatory disorders is usually linked to the use of unspecific small molecule drugs that can cause undesired side effects. Recently, many efforts are directed to develop alternative and more selective anti-inflammatory therapies, several of them imply the use of peptides. Indeed, peptides demonstrated as elected lead compounds toward several targets for their high specificity as well as recent and innovative synthetic strategies. Several endogenous peptides identified during inflammatory responses showed anti-inflammatory activities by inhibiting, reducing, and/or modulating the expression and activity of mediators. This review aims to discuss the potentialities and therapeutic use of peptides as anti-inflammatory agents in the treatment of different inflammation-related diseases and to explore the importance of peptide-based therapies.