Cancer-Related Mutations in the Sam Domains of EphA2 Receptor and Ship2 Lipid Phosphatase: A Computational Study.

The lipid phosphatase Ship2 interacts with the EphA2 receptor by forming a heterotypic Sam (sterile alpha motif)-Sam complex. Ship2 works as a negative regulator of receptor endocytosis and consequent degradation, and anti-oncogenic effects in cancer cells should be induced by hindering its association with EphA2. Herein, a computational approach is presented to investigate the relationship between Ship2-Sam/EphA2-Sam interaction and cancer onset and further progression. A search was first conducted through the COSMIC (Catalogue of Somatic Mutations in Cancer) database to identify cancer-related missense mutations positioned inside or close to the EphA2-Sam and Ship2-Sam reciprocal binding interfaces. Next, potential differences in the chemical-physical properties of mutant and wild-type Sam domains were evaluated by bioinformatics tools based on analyses of primary sequences. Three-dimensional (3D) structural models of mutated EphA2-Sam and Ship2-Sam domains were built as well and deeply analysed with diverse computational instruments, including molecular dynamics, to classify potentially stabilizing and destabilizing mutations. In the end, the influence of mutations on the EphA2-Sam/Ship2-Sam interaction was studied through docking techniques. This in silico approach contributes to understanding, at the molecular level, the mutation/cancer relationship by predicting if amino acid substitutions could modulate EphA2 receptor endocytosis.

Virtual Screening of Peptide Libraries: The Search for Peptide-Based Therapeutics Using Computational Tools.

Over the last few decades, we have witnessed growing interest from both academic and industrial laboratories in peptides as possible therapeutics. Bioactive peptides have a high potential to treat various diseases with specificity and biological safety. Compared to small molecules, peptides represent better candidates as inhibitors (or general modulators) of key protein-protein interactions. In fact, undruggable proteins containing large and smooth surfaces can be more easily targeted with the conformational plasticity of peptides. The discovery of bioactive peptides, working against disease-relevant protein targets, generally requires the high-throughput screening of large libraries, and in silico approaches are highly exploited for their low-cost incidence and efficiency. The present review reports on the potential challenges linked to the employment of peptides as therapeutics and describes computational approaches, mainly structure-based virtual screening (SBVS), to support the identification of novel peptides for therapeutic implementations. Cutting-edge SBVS strategies are reviewed along with examples of applications focused on diverse classes of bioactive peptides (i.e., anticancer, antimicrobial/antiviral peptides, peptides blocking amyloid fiber formation).

EPHA2 Receptor as a Possible Therapeutic Target in Viral Infections.

BACKGROUND: The receptor tyrosine kinase EphA2 plays a role in many diseases, like cancer, cataracts, and osteoporosis. Interestingly, it has also been linked to viral infections. OBJECTIVE: Herein, current literature has been reviewed to clarify EphA2 functions in viral infections and explore its potential role as a target in antiviral drug discovery strategies. METHODS: Research and review articles and preprints connecting EphA2 to different viruses have been searched through PubMed and the web. Structures of complexes between EphA2 domains and viral proteins have been retrieved from the PDB database. RESULTS: EphA2 assumes a key role in Kaposi's sarcoma-associated herpes virus (KSHV) and Epstein Barr virus (EBV) infections by directly binding, through its ligand binding domain, viral glycoproteins. For human cytomegalovirus (HCMV), the role of EphA2 in maintaining virus latency state, through cooperation with specific viral proteins, has also been speculated. In certain cells, with high EphA2 expression levels, following ligand stimulation, receptor activation might contribute to severe symptoms accompanying a few viral infections, including lung injuries often related to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). CONCLUSION: Since EphA2 works as a host receptor for certain viruses, it might be worth more deeply investigating known compounds targeting its extracellular ligand binding domain as antiviral therapeutics. Due to EphA2's function in inflammation, its possible correlation with SARS-CoV-2 cannot be excluded, but more experimental studies are needed in this case to undoubtedly attribute the role of this receptor in viral infections.

Tryptophan-PNA gc Conjugates Self-Assemble to Form Fibers.

Peptide nucleic acids and their conjugates to peptides can self-assemble and generate complex architectures. In this work, we explored the self-assembly of PNA dimers conjugated to the dipeptide WW. Our studies suggest that the indole ring of tryptophan promotes aggregation of the conjugates. The onset of fluorescence is observed upon self-assembly. The structure of self-assembled WWgc is concentration-dependent, being spherical at low concentrations and fibrous at high concentrations. As suggested by molecular modeling studies, fibers are stabilized by stacking interactions between tryptophans and Watson-Crick hydrogen bonds between nucleobases.

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.

Looking for SARS-CoV-2 Therapeutics Through Computational Approaches.

BACKGROUND: In the last few years, in silico tools, including drug repurposing coupled with structure-based virtual screening, have been extensively employed to look for anti-COVID-19 agents. OBJECTIVE: The present review aims to provide readers with a portrayal of computational approaches that could be conducted more quickly and cheaply to novel anti-viral agents. Particular attention is given to docking-based virtual screening. METHODS: The World Health Organization website was consulted to gain the latest information on SARS-CoV-2, its novel variants and their interplay with COVID-19 severity and treatment options. The Protein Data Bank was explored to look for 3D coordinates of SARS-CoV-2 proteins in their free and bound states, in the wild-types and mutated forms. Recent literature related to in silico studies focused on SARS-CoV-2 proteins was searched through PubMed. RESULTS: A large amount of work has been devoted thus far to computationally targeting viral entry and searching for inhibitors of the S-protein/ACE2 receptor complex. Another large area of investigation is linked to in silico identification of molecules able to block viral proteases -including Mpro- thus avoiding maturation of proteins crucial for virus life cycle. Such computational studies have explored the inhibitory potential of the most diverse molecule databases (including plant extracts, dietary compounds, FDA approved drugs). CONCLUSION: More efforts need to be dedicated in the close future to experimentally validate the therapeutic power of in silico identified compounds in order to catch, among the wide ensemble of computational hits, novel therapeutics to prevent and/or treat COVID- 19.

Hunting for Novel Routes in Anticancer Drug Discovery: Peptides against Sam-Sam Interactions.

Among the diverse protein binding modules, Sam (Sterile alpha motif) domains attract attention due to their versatility. They are present in different organisms and play many functions in physiological and pathological processes by binding multiple partners. The EphA2 receptor contains a Sam domain at the C-terminus (EphA2-Sam) that is able to engage protein regulators of receptor stability (including the lipid phosphatase Ship2 and the adaptor Odin). Ship2 and Odin are recruited by EphA2-Sam through heterotypic Sam-Sam interactions. Ship2 decreases EphA2 endocytosis and consequent degradation, producing chiefly pro-oncogenic outcomes in a cellular milieu. Odin, through its Sam domains, contributes to receptor stability by possibly interfering with ubiquitination. As EphA2 is upregulated in many types of tumors, peptide inhibitors of Sam-Sam interactions by hindering receptor stability could function as anticancer therapeutics. This review describes EphA2-Sam and its interactome from a structural and functional perspective. The diverse design strategies that have thus far been employed to obtain peptides targeting EphA2-mediated Sam-Sam interactions are summarized as well. The generated peptides represent good initial lead compounds, but surely many efforts need to be devoted in the close future to improve interaction affinities towards Sam domains and consequently validate their anticancer properties.

Structure-Activity Relationship Investigations of Novel Constrained Chimeric Peptidomimetics of SOCS3 Protein Targeting JAK2.

SOCS3 (suppressor of cytokine signaling 3) protein suppresses cytokine-induced inflammation and its deletion in neurons or immune cells increases the pathological growth of blood vessels. Recently, we designed several SOCS3 peptidomimetics by assuming as template structures the interfacing regions of the ternary complex constituted by SOCS3, JAK2 (Janus Kinase 2) and gp130 (glycoprotein 130) proteins. A chimeric peptide named KIRCONG chim, including non-contiguous regions demonstrated able to bind to JAK2 and anti-inflammatory and antioxidant properties in VSMCs (vascular smooth muscle cells). With the aim to improve drug-like features of KIRCONG, herein we reported novel cyclic analogues bearing different linkages. In detail, in two of them hydrocarbon cycles of different lengths were inserted at positions i/i+5 and i/i+7 to improve helical conformations of mimetics. Structural features of cyclic compounds were investigated by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopies while their ability to bind to catalytic domain of JAK2 was assessed through MST (MicroScale Thermophoresis) assay as well as their stability in biological serum. Overall data indicate a crucial role exerted by the length and the position of the cycle within the chimeric structure and could pave the way to the miniaturization of SOCS3 protein for therapeutic aims.

Structural Insights on Tiny Peptide Nucleic Acid (PNA) Analogues of miRNA-34a: An in silico and Experimental Integrated Approach.

In the present work, structural features of the interaction between peptide nucleic acid (PNA)-based analogs of the tumor-suppressor microRNA-34a with both its binding sites on MYCN mRNA were investigated. In particular, the region from base 1 to 8 ("seed" region) of miR-34a was reproduced in the form of an 8-mer PNA fragment (tiny PNA), and binding to target 3'UTR MYCN mRNA, was studied by a seldom reported and detailed NMR characterization, providing evidence for the formation of anti-parallel duplexes with a well-organized structural core. The formation of PNA-3'UTR duplexes was also confirmed by Circular Dichroism, and their melting curves were measured by UV spectroscopy. Nevertheless, this study offered a valuable comparison between molecular dynamics predictions and experimental evidence, which showed great correlation. Preliminary uptake assays were carried out in Neuroblastoma Kelly cells, using short peptide conjugates as carriers and FITC fluorescent tag for subcellular localization. Moderate internalization was observed without the use of transfecting agents. The reported results corroborate the interest toward the design and development of chimeric PNA/RNA sequences as effective RNA-targeting agents.

NMR Spectroscopy in the Conformational Analysis of Peptides: An Overview.

BACKGROUND: NMR spectroscopy is one of the most powerful tools to study the structure and interaction properties of peptides and proteins from a dynamic perspective. Knowing the bioactive conformations of peptides is crucial in the drug discovery field to design more efficient analogue ligands and inhibitors of protein-protein interactions targeting therapeutically relevant systems. OBJECTIVE: This review provides a toolkit to investigate peptide conformational properties by NMR. METHODS: Articles cited herein, related to NMR studies of peptides and proteins were mainly searched through PubMed and the web. More recent and old books on NMR spectroscopy written by eminent scientists in the field were consulted as well. RESULTS: The review is mainly focused on NMR tools to gain the 3D structure of small unlabeled peptides. It is more application-oriented as it is beyond its goal to deliver a profound theoretical background. However, the basic principles of 2D homonuclear and heteronuclear experiments are briefly described. Protocols to obtain isotopically labeled peptides and principal triple resonance experiments needed to study them, are discussed as well. CONCLUSION: NMR is a leading technique in the study of conformational preferences of small flexible peptides whose structure can be often only described by an ensemble of conformations. Although NMR studies of peptides can be easily and fast performed by canonical protocols established a few decades ago, more recently we have assisted to tremendous improvements of NMR spectroscopy to investigate instead large systems and overcome its molecular weight limit.

Chimeric Peptidomimetics of SOCS 3 Able to Interact with JAK2 as Anti-inflammatory Compounds.

The immunomodulatory effects of Suppressor of Cytokine Signaling (SOCS) proteins, that control the JAK/STAT pathway, indicate them as attractive candidates for immunotherapies. Recombinant SOCS3 protein suppresses the effects of inflammation, and its deletion in neurons or in immune cells increases pathological blood vessels growth. Recently, on the basis of the structure of the ternary complex among SOCS3, JAK2, and gp130, we focused on SOCS3 interfacing regions and designed several interfering peptides (IPs) that were able to mimic SOCS3 biological role in triple negative breast cancer (TNBC) models. Herein, to explore other protein regions involved in JAK2 recognition, several new chimeric peptides connecting noncontiguous SOCS3 regions and including a strongly aromatic fragment were investigated. Their ability to recognize the catalytic domain of JAK2 was evaluated through MST (microscale thermophoresis), and the most promising compound, named KIRCONG chim, exhibited a low micromolar value for dissociation constant. The conformational features of chimeric peptides were analyzed through circular dichroism and NMR spectroscopies, and their anti-inflammatory effects were assessed in cell cultures. Overall data suggest the importance of aromatic contribution in the recognition of JAK2 and that SOCS3 peptidomimetics could be endowed with a therapeutic potential in diseases with activated inflammatory cytokines.

Protein Interaction Domains: Structural Features and Drug Discovery Applications (Part 2).

BACKGROUND: Proteins present a modular organization made up of several domains. Apart from the domains playing catalytic functions, many others are crucial to recruit interactors. The latter domains can be defined as "PIDs" (Protein Interaction Domains) and are responsible for pivotal outcomes in signal transduction and a certain array of normal physiological and disease-related pathways. Targeting such PIDs with small molecules and peptides able to modulate their interaction networks, may represent a valuable route to discover novel therapeutics. OBJECTIVE: This work represents a continuation of a very recent review describing PIDs able to recognize post-translationally modified peptide segments. On the contrary, the second part concerns with PIDs that interact with simple peptide sequences provided with standard amino acids. METHODS: Crucial structural information on different domain subfamilies and their interactomes was gained by a wide search in different online available databases (including the PDB (Protein Data Bank), the Pfam (Protein family), and the SMART (Simple Modular Architecture Research Tool)). Pubmed was also searched to explore the most recent literature related to the topic. RESULTS AND CONCLUSION: PIDs are multifaceted: they have all diverse structural features and can recognize several consensus sequences. PIDs can be linked to different diseases onset and progression, like cancer or viral infections and find applications in the personalized medicine field. Many efforts have been centered on peptide/peptidomimetic inhibitors of PIDs mediated interactions but much more work needs to be conducted to improve drug-likeness and interaction affinities of identified compounds.

Monoacylglycerides from the Diatom Skeletonema marinoi Induce Selective Cell Death in Cancer Cells.

Microalgae are an excellent source of valuable compounds for nutraceutical and cosmeceutical applications. These photosynthesizing microorganisms are amenable for large-scale production, thus overcoming the bottleneck of biomass supply for chemical and activity characterization of bioactive compounds. This characteristic has recently also prompted the screening of microalgae for potential pharmaceutical applications. Here, we show that monoacylglycerides (MAGs) purified from the marine diatom Skeletonema marinoi have selective cytotoxic activity against the haematological cancer cell line U-937 and colon cancer cell line HCT-116 compared to normal MePR-2B cells. LC-MS analysis of the raw extract revealed that in their natural form, MAGs occur as 2-monoacyl derivatives and include mainly C16 and C20 analogues, but they are converted into the corresponding 1-isomers during purification processes. Pure compounds along with the synthetic 1-monoarachidonoylglycerol tested on HCT-116 and U-937 tumor cell lines induced cell death via apoptosis. The mechanism of action was investigated, and we show that it involves the induction of apoptosis through caspase 3/7 activation. These findings pave the way for the possible use of these molecules as potential anticancer agents or as precursors for the generation of new and more potent and selective compounds against tumor cells.

Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications.

BACKGROUND: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as Protein Interaction Domains (PIDs). OBJECTIVE: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. METHOD: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns were retrieved through Pubmed and analyzed. RESULTS AND CONCLUSION: PIDs are rather versatile as concerned with their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

The Growth Differentiation Factor 11 is Involved in Skin Fibroblast Ageing and is Induced by a Preparation of Peptides and Sugars Derived from Plant Cell Cultures.

Ageing is a complex and progressive phenomenon, during which the accumulation of morphological and chemical changes seriously compromises the capacity of the cells to proliferate and fulfil their biological tasks. The increase in the average age of the world population, associated with a higher occurrence of age-related diseases, is prompting scientific research to look for new strategies and molecular targets that may help in alleviating age-related phenotypes. Growth factors, responsible for modulating several aging markers in many tissues and organs, represent valuable targets to fight age-associated dysfunctions. The growth differentiation factor GDF11, a TGF-ß family member, has been associated with the maintenance of youth phenotypes in different human tissues and organs, and in the skin has been related to an inhibition of the inflammatory response. We investigated the role of GDF11 in skin dermal fibroblasts, and we observed that its expression and activity were reduced in fibroblasts deriving from adult donors compared to neonatal ones. The main effect of GDF11 was the induction of collagen I and III, in both neonatal and adult fibroblasts, by triggering Smad signalling in a TGF-ß-like fashion. Moreover, by analysing a number of plant extracts having GDF11 inducing activity, we found that a peptide/sugar preparation, obtained from Lotus japonicus somatic embryo cultures, was capable of restoring GDF11 expression in older fibroblasts and to activate the synthesis of collagen I, collagen III and periostin, an important protein involved in collagen assembly.

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.

Sam Domains in Multiple Diseases.

BACKGROUND: The sterile alpha motif (Sam) domain is a small helical protein module, able to undergo homo- and hetero-oligomerization, as well as polymerization, thus forming different types of protein architectures. A few Sam domains are involved in pathological processes and consequently, they represent valuable targets for the development of new potential therapeutic routes. This study intends to collect state-of-the-art knowledge on the different modes by which Sam domains can favor disease onset and progression. METHODS: This review was build up by searching throughout the literature, for: a) the structural properties of Sam domains, b) interactions mediated by a Sam module, c) presence of a Sam domain in proteins relevant for a specific disease. RESULTS: Sam domains appear crucial in many diseases including cancer, renal disorders, cataracts. Often pathologies are linked to mutations directly positioned in the Sam domains that alter their stability and/or affect interactions that are crucial for proper protein functions. In only a few diseases, the Sam motif plays a kind of "side role" and cooperates to the pathological event by enhancing the action of a different protein domain. CONCLUSION: Considering the many roles of the Sam domain into a significant variety of diseases, more efforts and novel drug discovery campaigns need to be engaged to find out small molecules and/or peptides targeting Sam domains. Such compounds may represent the pillars on which to build novel therapeutic strategies to cure different pathologies.

The antimicrobial peptides casocidins I and II: Solution structural studies in water and different membrane-mimetic environments.

Antimicrobial peptides (AMPs) represent crucial components of the natural immune defense machinery of different organisms. Generally, they are short and positively charged, and bind to and destabilize bacterial cytoplasmic membranes, ultimately leading to cell death. Natural proteolytic cleavage of αs2-casein in bovine milk generates the antimicrobial peptides casocidin I and II. In the current study, we report for the first time on a detailed structure characterization of casocidins in solution by means of Nuclear Magnetic Resonance spectroscopy (NMR). Structural studies were conducted in H2O and different membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE) and lipid anionic and zwitterionic vesicles. For both peptides, results indicate a mainly disordered conformation in H2O, with a few residues in a partial helical structure. No wide increase of order occurs upon interaction with lipid vesicles. Conversely, peptide conformation becomes highly ordered in presence of TFE, with both casocidins presenting a large helical content. Our data point out a preference of casocidins to interact with model anionic membranes. These results are compatible with possible mechanisms of action underlying the antimicrobial activity of casocidins that ultimately may affect membrane bilayer stability.

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.

Self-Assembling of Fmoc-GC Peptide Nucleic Acid Dimers into Highly Fluorescent Aggregates.

The study of molecules that self-assemble through noncovalent interactions is one of the most attractive topics in supramolecular chemistry. The use of short peptides or modified nucleotides as building blocks for the aggregates is particularly intriguing because these are very easy to synthesize; moreover, subtle changes in the chemical structure of such building blocks may drastically affect the properties of the aggregates. The ability of peptide nucleic acids (PNA) to aggregate has been very little explored, despite its practical applications. In this work we investigated the self-assembling properties of a PNA dimer, conjugated at the N-terminus to a fluorenylmethoxycarbonyl group. This PNA dimer forms nano-aggregates at low concentration in CHCl3 /CH3 OH mixtures. The aggregates retain very interesting fluorescent properties (high quantum yield in the visible region with lifetimes on the nanosecond scale), which make them promising materials for applications in optoelectronics.