The Characterization of Multifaceted PREP1 Peptides Provides Insights into Correlations between Spectroscopic and Structural Properties of Amyloid-like Assemblies.

The widespread ability of proteins and peptides to self-assemble by forming cross-ß structure is one of the most significant discoveries in structural biology. Intriguingly, the cross-ß association of proteins/peptides may generate intricate supramolecular architectures with uncommon spectroscopic properties. We have recently characterized self-assembled peptides extracted from the PREP1 protein that are endowed with interesting structural/spectroscopic properties. We here demonstrate that the green fluorescence emission of the peptide PREP1[117-132] (λem ~520 nm), can be induced by excitation with UV radiation. The associated unusually large Stokes shift (Δλ ~150 nm) represents, to the best of our knowledge, the first evidence of an internal resonance energy transfer in amyloid-like structures, where the blue emission of some assemblies becomes the excitation radiation for others. Moreover, the characterization of PREP1[117-132] variants provides insights into the sequence/structure and structure/spectroscopic properties relationships. Our data suggests that the green fluorescence is plausibly associated with antiparallel ß-sheet states of the peptide whereas parallel ß-sheet assemblies are only endowed with blue fluorescence. Notably, the different PREP1[117-132] variants also form assemblies characterized by distinct morphologies. Indeed, the parent peptide and single mutants form compact but structured aggregates whereas most of the double mutants exhibit elongated and highly extended fibers.

Oreoch-1: A broad-spectrum virus and host-targeting peptide against animal infections.

In recent decades, the global rise of viral emerging infectious diseases has posed a substantial threat to both human and animal health worldwide. The rapid spread and accumulation of mutations into viruses, and the limited availability of antiviral drugs and vaccines, stress the urgent need for alternative therapeutic strategies. Antimicrobial peptides (AMPs) derived from natural sources present a promising avenue due to their specificity and effectiveness against a broad spectrum of pathogens. The present study focuses on investigating the antiviral potential of oreochromicin-1 (oreoch-1), a fish-derived AMP obtained from Nile tilapia, against a wide panel of animal viruses including canine distemper virus (CDV), Schmallenberg virus (SBV), caprine herpesvirus 1 (CpHV-1), and bovine herpesvirus 1 (BoHV-1). Oreoch-1 exhibited a strong antiviral effect, demonstrating an inhibition of infection at concentrations in the micromolar range. The mechanism of action involves the interference with viral entry into host cells and a direct interaction between oreoch-1 and the viral envelope. In addition, we observed that the peptide could also interact with the cell during the CDV infection. These findings not only highlight the efficacy of oreoch-1 in inhibiting viral infection but also emphasize the potential of fish-derived peptides, specifically oreoch-1, as effective antiviral agents against viral infections affecting animals, whose potential to spill into humans is high. This research contributes valuable insights to the ongoing quest for novel antiviral drugs with the potential to mitigate the impact of infectious diseases on a global scale.

Synthetic Peptides and Peptidomimetics: From Basic Science to Biomedical Applications-Second Edition.

Peptides are increasingly emerging as a drug class for a wide range of human diseases due to their intrinsic properties, such as excellent recognition abilities and biocompatibility [...].

SARS-CoV-2 Fusion Peptide Conjugated to a Tetravalent Dendrimer Selectively Inhibits Viral Infection.

Fusion is a key event for enveloped viruses, through which viral and cell membranes come into close contact. This event is mediated by viral fusion proteins, which are divided into three structural and functional classes. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein belongs to class I fusion proteins, characterized by a trimer of helical hairpins and an internal fusion peptide (FP), which is exposed once fusion occurs. Many efforts have been directed at finding antivirals capable of interfering with the fusion mechanism, mainly by designing peptides on the two heptad-repeat regions present in class I viral fusion proteins. Here, we aimed to evaluate the anti-SARS-CoV-2 activity of the FP sequence conjugated to a tetravalent dendrimer through a classical organic nucleophilic substitution reaction (SN2) using a synthetic bromoacetylated peptide mimicking the FP and a branched scaffold of poly-L-Lysine functionalized with cysteine residues. We found that the FP peptide conjugated to the dendrimer, unlike the monomeric FP sequence, has virucidal activity by impairing the attachment of SARS-CoV-2 to cells. Furthermore, we found that the peptide dendrimer does not have the same effects on other coronaviruses, demonstrating that it is selective against SARS-CoV-2.

Oreoch-1: A Peptide from Oreochromis niloticus as a Potential Tool against Staphylococci.

Staphylococci, including Staphylococcus aureus and Staphylococcus epidermidis, are important human pathogens associated with potentially life-threatening infections. Their great biofilm-producing ability and the development of resistance mechanisms often account for therapeutic failure. Hence, the scientific community has devoted intensive efforts to the development of antimicrobial compounds active against both planktonic and sessile bacterial populations. Contextually, antimicrobial peptides (AMPs) are natural peptides produced by the innate immunity of every organism, representing a potential new therapeutic solution against human microbial pathogens. Our work focused on the in vitro activity of Oreoch-1, an AMP from the gills of Nile tilapia (Oreochromis niloticus), against standard and clinical S. aureus and S. epidermidis strains. Firstly, the cytotoxicity profile of Oreoch-1 was determined in human colon carcinoma cells. Secondly, its antibacterial spectrum was explored against staphylococcal strains to set up the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Our results highlighted an antibacterial activity in the range 6.25-25 µM, with a general bacteriostatic effect. Therefore, the biofilm-inhibitory property was assessed against S. aureus ATCC 25923 and S. epidermidis ATCC 35984, indicating a significant reduction in S. aureus biomass at sub-MIC concentrations. Overall, our study indicates Oreoch-1 as a promising new therapeutic weapon against staphylococcal infections.

Melittin-Related Peptides Interfere with Sandfly Fever Naples Virus Infection by Interacting with Heparan Sulphate.

Emerging viruses pose an important global public health challenge, and early action is needed to control their spread. The Bunyaviridae family contains a great number of arboviruses which are potentially pathogenic for humans. For example, phleboviruses affect a large range of hosts, including humans and animals. Some infections usually have an asymptomatic course, but others lead to severe complications, such as Toscana virus, which is able to cause meningitis and encephalitis. Unfortunately, to date, no vaccines or antiviral treatments have been found. In the present study, we evaluated the effect of melittin-related peptides, namely the frog-derived RV-23 and AR-23, on sandfly fever Naples virus infection in vitro. Both peptides exhibited a strong antiviral activity by targeting the viral particles and blocking the virus-cell interaction. Their action was directed to an early phase of SFNV infection, in particular at viral adsorption on host cells, by interfering with the binding of common glycosaminoglycan receptors. Given the better antimicrobial behavior of AR-23 and RV-23 compared to melittin in terms of selectivity, our studies expand our understanding of the potential of these peptides as antimicrobials and stimulate further investigations in the direction of novel antiviral strategies against phlebovirus infection.

Hylin-a1: A Pan-Inhibitor against Emerging and Re-Emerging Respiratory Viruses.

Pandemic and epidemic outbreaks of respiratory viruses are a challenge for public health and social care system worldwide, leading to high mortality and morbidity among the human populations. In light of the limited efficacy of current vaccines and antiviral drugs against respiratory viral infections and the emergence and re-emergence of new viruses, novel broad-spectrum antiviral drugs are needed for the prevention and treatment of these infections. Antimicrobial peptides with an antiviral effect, also known as AVPs, have already been reported as potent inhibitors of viral infections by affecting different stages of the virus lifecycle. In the present study, we analyzed the activity of the AVP Hylin-a1, secreted by the frog Hypsiboas albopunctatus, against a wide range of respiratory viruses, including the coronaviruses HCoV-229E and SARS-CoV-2, measles virus, human parainfluenza virus type 3, and influenza virus H1N1. We report a significant inhibitory effect on infectivity in all the enveloped viruses, whereas there was a lack of activity against the naked coxsackievirus B3. Considering the enormous therapeutic potential of Hylin-a1, further experiments are required to elucidate its mechanism of action and to increase its stability by modifying the native sequence.

Synthetic Frog-Derived-like Peptides: A New Weapon against Emerging and Potential Zoonotic Viruses.

Given the emergence of the coronavirus disease 2019 (COVID-19), zoonoses have raised in the spotlight of the scientific community. Animals have a pivotal role not only for this infection, but also for many other recent emerging and re-emerging viral diseases, where they may represent both intermediate hosts and/or vectors for zoonoses diffusion. Today, roughly two-thirds of human infections are derived from animal origins; therefore, the search for new broad-spectrum antiviral molecules is mandatory to prevent, control and eradicate future epidemic outbreaks. Host defense peptides, derived from skin secretions of amphibians, appear as the right alternative to common antimicrobial drugs. They are cationic peptides with an amphipathic nature widely described as antibacterial agents, but less is reported about their antiviral potential. In the present study, we evaluated the activity of five amphibian peptides, namely RV-23, AR-23, Hylin-a1, Deserticolin-1 and Hylaseptin-P1, against a wide panel of enveloped animal viruses. A strong virucidal effect was observed for RV-23, AR-23 and Hylin-a1 against bovine and caprine herpesviruses, canine distemper virus, bovine viral diarrhea virus, and Schmallenberg virus. Our results identified these three peptides as potential antiviral-led candidates with a putative therapeutic effect against several animal viruses.

Antiviral Peptides Delivered by Chitosan-Based Nanoparticles to Neutralize SARS-CoV-2 and HCoV-OC43.

The COVID-19 pandemic has made it clear that there is a crucial need for the design and development of antiviral agents that can efficiently reduce the fatality rate caused by infectious diseases. The fact that coronavirus mainly enters through the nasal epithelial cells and spreads through the nasal passage makes the nasal delivery of antiviral agents a promising strategy not only to reduce viral infection but also its transmission. Peptides are emerging as powerful candidates for antiviral treatments, showing not only a strong antiviral activity, but also improved safety, efficacy, and higher specificity against viral pathogens. Based on our previous experience on the use of chitosan-based nanoparticles to deliver peptides intra-nasally the current study aimed to explore the delivery of two-novel antiviral peptides making use of nanoparticles consisting of HA/CS and DS/CS. The antiviral peptides were chemically synthesized, and the optimal conditions for encapsulating them were selected through a combination of physical entrapment and chemical conjugation using HA/CS and DS/CS nanocomplexes. Finally, we evaluated the in vitro neutralization capacity against SARS-CoV-2 and HCoV-OC43 for potential use as prophylaxis or therapy.

Targeting Protein-Protein Interfaces with Peptides: The Contribution of Chemical Combinatorial Peptide Library Approaches.

Protein-protein interfaces play fundamental roles in the molecular mechanisms underlying pathophysiological pathways and are important targets for the design of compounds of therapeutic interest. However, the identification of binding sites on protein surfaces and the development of modulators of protein-protein interactions still represent a major challenge due to their highly dynamic and extensive interfacial areas. Over the years, multiple strategies including structural, computational, and combinatorial approaches have been developed to characterize PPI and to date, several successful examples of small molecules, antibodies, peptides, and aptamers able to modulate these interfaces have been determined. Notably, peptides are a particularly useful tool for inhibiting PPIs due to their exquisite potency, specificity, and selectivity. Here, after an overview of PPIs and of the commonly used approaches to identify and characterize them, we describe and evaluate the impact of chemical peptide libraries in medicinal chemistry with a special focus on the results achieved through recent applications of this methodology. Finally, we also discuss the role that this methodology can have in the framework of the opportunities, and challenges that the application of new predictive approaches based on artificial intelligence is generating in structural biology.

A Comprehensive Analysis of the Intrinsic Visible Fluorescence Emitted by Peptide/Protein Amyloid-like Assemblies.

Amyloid aggregation is a widespread process that involves proteins and peptides with different molecular complexity and amino acid composition. The structural motif (cross-ß) underlying this supramolecular organization generates aggregates endowed with special mechanical and spectroscopic properties with huge implications in biomedical and technological fields, including emerging precision medicine. The puzzling ability of these assemblies to emit intrinsic and label-free fluorescence in regions of the electromagnetic spectrum, such as visible and even infrared, usually considered to be forbidden in the polypeptide chain, has attracted interest for its many implications in both basic and applied science. Despite the interest in this phenomenon, the physical basis of its origin is still poorly understood. To gain a global view of the available information on this phenomenon, we here provide an exhaustive survey of the current literature in which original data on this fluorescence have been reported. The emitting systems have been classified in terms of their molecular complexity, amino acid composition, and physical state. Information about the wavelength of the radiation used for the excitation as well as the emission range/peak has also been retrieved. The data collected here provide a picture of the complexity of this multifaceted phenomenon that could be helpful for future studies aimed at defining its structural and electronic basis and/or stimulating new applications.

Hylin-a1: A Host Defense Peptide with Antibacterial Potential against Staphylococcus aureus Multi-Resistant Strains.

In recent years, the resistance of pathogenic microorganisms to common antimicrobial agents has raised to a severe public health problem. The moderate and wise use of antimicrobials and the prevention of infections are the most effective strategies for decreasing the spread and development of resistance. Therefore, the World Health Organization (WHO) has intensified the search for new drugs to fight emerging pathogens. Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), play a crucial role in innate immunity, representing one of the first line of defense against microbial attacks. In this study, we evaluated the antibacterial activity of the AMP named Hylin-a1 (derived from the skin of the frog Heleioporus albopunctatus) against Staphylococcus aureus strains. S. aureus represents a commensal bacterium but also the principal causative agent of several human infections, including bacteremia, endocarditis, skin and device-related infections. Hylin-a1 toxicity was evaluated on human keratinocytes; once the non-cytotoxic concentration range was determined, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were analyzed, and time-killing assays were performed to verify the bacteriostatic and/or bactericidal activity of the peptide. We found that Hylin-a1 exerted a bacteriostatic action against most of the tested strains, with 90% inhibition at the concentration of 6.25 µM. Noteworthy, the peptide at a very low concentration (~3 µM) significantly blocked the growth of ß-lactam- and methicillin-resistant S. aureus. The levels of interleukin (IL)-1ß, IL-6 and IL-8 were quantified through a molecular assay, indicating that the peptide was able also to regulate the inflammatory response following bacterial infection. The effect of Hylin-a1 on S. aureus cell morphology was also evaluated. Altogether, these results indicate the high therapeutic potential of Hylin-a1 against a wide variety of clinical manifestations caused by S. aureus.

Expression, Purification, Structural and Functional Characterization of Recombinant Human Parvulin 17.

Parvulins, peptidyl-prolyl isomerase enzymes (PPIase), catalyze the cis-trans isomerization of prolyl bonds in polypeptides, contributing to folding and function regulation of many proteins. Among Parvulins, Par17, exclusively expressed in hominids, is the least examined in terms of structure, catalytic function and cellular activity. Setting the conditions for the preparation of recombinant active Par17 may therefore significantly foster future studies. Here, we comparatively evaluated the impact of several parameters, including host strains, culture media, isopropyl ß-D-1-thiogalactopyranoside concentration, post-induction incubation time and temperature, on the overexpression of Par17 in E. coli cells. A similar approach was also comparatively adopted for the preparation of the recombinant full-length Pin1 protein, the most representative Parvulin, and the catalytic domains of both enzymes. Proteins were efficiently expressed and purified to homogeneity and were subjected to a structural characterization by Size Exclusion Chromatography and Circular Dichroism. Moreover, a single-step homogeneous protease-based fluorimetric assay, potentially scalable in HTS format, has been developed for determining the peptidyl-prolyl cis-trans isomerase activity of recombinant Parvulins. Results obtained show that proteins are folded and active. These new data mark an important milestone for progressing the investigation of Parvulins.

Design of Three Residues Peptides against SARS-CoV-2 Infection.

The continuous and rapid spread of the COVID-19 pandemic has emphasized the need to seek new therapeutic and prophylactic treatments. Peptide inhibitors are a valid alternative approach for the treatment of emerging viral infections, mainly due to their low toxicity and high efficiency. Recently, two small nucleotide signatures were identified in the genome of some members of the Coronaviridae family and many other human pathogens. In this study, we investigated whether the corresponding amino acid sequences of such nucleotide sequences could have effects on the viral infection of two representative human coronaviruses: HCoV-OC43 and SARS-CoV-2. Our results showed that the synthetic peptides analyzed inhibit the infection of both coronaviruses in a dose-dependent manner by binding the RBD of the Spike protein, as suggested by molecular docking and validated by biochemical studies. The peptides tested do not provide toxicity on cultured cells or human erythrocytes and are resistant to human serum proteases, indicating that they may be very promising antiviral peptides.

Design and Synthesis of Oligopeptidic Parvulin Inhibitors.

Pin1 catalyzes the cis-trans isomerization of pThr-Pro or pSer-Pro amide bonds of various proteins involved in several physio/pathological processes. In this framework, recent research activity is directed toward the identification of new selective Pin1 inhibitors. Here, we developed a set of peptide-based Pin1 inhibitors. Direct-binding experiments allowed the identification of the peptide-based inhibitor 5 k (methylacetyl-l-alanyl-l-histidyl-l-prolyl-l-phenylalaninate) as a potent ligand of Pin1. Notably, 5 k binds Pin1 with higher affinity than Pin4. The comparative analysis of molecular models of Pin1 and Pin4 with the selected compound gave a rational explanation of the biochemical activity and pinpointed the chemical elements that, if opportunely modified, may further improve inhibitory potency, pharmacological properties, and selectivity of future peptide-based parvulin inhibitors. Since 5 k showed limited cell penetration and no antiproliferative activity, it was conjugated to a polyarginine stretch (R8), known to promote cell penetration of peptides, to obtain the R8-5 k derivative, which displayed antiproliferative effects on cancer cell lines over non-tumor cells. The effect of R8 on cell proliferation was also investigated. This work warrants caution about applying the R8 strategy in the development of cell-penetrating antiproliferative peptides, as it is not inert.

The Broad-Spectrum Antiviral Potential of the Amphibian Peptide AR-23.

Viral infections represent a serious threat to the world population and are becoming more frequent. The search and identification of broad-spectrum antiviral molecules is necessary to ensure new therapeutic options, since there is a limited availability of effective antiviral drugs able to eradicate viral infections, and consequently due to the increase of strains that are resistant to the most used drugs. Recently, several studies on antimicrobial peptides identified them as promising antiviral agents. In detail, amphibian skin secretions serve as a rich source of natural antimicrobial peptides. Their antibacterial and antifungal activities have been widely reported, but their exploitation as potential antiviral agents have yet to be fully investigated. In the present study, the antiviral activity of the peptide derived from the secretion of Rana tagoi, named AR-23, was evaluated against both DNA and RNA viruses, with or without envelope. Different assays were performed to identify in which step of the infectious cycle the peptide could act. AR-23 exhibited a greater inhibitory activity in the early stages of infection against both DNA (HSV-1) and RNA (MeV, HPIV-2, HCoV-229E, and SARS-CoV-2) enveloped viruses and, on the contrary, it was inactive against naked viruses (PV-1). Altogether, the results indicated AR-23 as a peptide with potential therapeutic effects against a wide variety of human viruses.

Synthetic Peptides That Antagonize the Angiotensin-Converting Enzyme-2 (ACE-2) Interaction with SARS-CoV-2 Receptor Binding Spike Protein.

The SARS-CoV-2 viral spike protein S receptor-binding domain (S-RBD) binds ACE2 on host cells to initiate molecular events, resulting in intracellular release of the viral genome. Therefore, antagonists of this interaction could allow a modality for therapeutic intervention. Peptides can inhibit the S-RBD:ACE2 interaction by interacting with the protein-protein interface. In this study, protein contact atlas data and molecular dynamics simulations were used to locate interaction hotspots on the secondary structure elements α1, α2, α3, ß3, and ß4 of ACE2. We designed a library of discontinuous peptides based upon a combination of the hotspot interactions, which were synthesized and screened in a bioluminescence-based assay. The peptides demonstrated high efficacy in antagonizing the SARS-CoV-2 S-RBD:ACE2 interaction and were validated by microscale thermophoresis which demonstrated strong binding affinity (∼10 nM) of these peptides to S-RBD. We anticipate that such discontinuous peptides may hold the potential for an efficient therapeutic treatment for COVID-19.

Interleukin 6 reduces vascular smooth muscle cell apoptosis via Prep1 and is associated with aging.

Aging exacerbates neointimal formation by reducing apoptosis of vascular smooth muscle cells (VSMCs) and induces inflammation within vascular wall. Prep1 is a homeodomain transcription factor which stimulates the expression of proinflammatory cytokines in aortic endothelial cell models and plays a primary role in the regulation of apoptosis. In this study, we have investigated the role of Prep1 in aorta of Prep1 hypomorphic heterozygous mice (Prep1i/+ ) and in VSMCs, and its correlation with aging. Histological analysis from Prep1i/+ aortas revealed a 25% reduction in medial smooth muscle cell density compared to WT animals. This result paralleled higher apoptosis, caspase 3, caspase 9 and p53 levels in Prep1i/+ mice and lower Bcl-xL. Prep1 overexpression in VSMCs decreased apoptosis by 25% and caspase 3 and caspase 9 expression by 40% and 37%. In parallel, Bcl-xL inhibition by BH3I-1 and p53 induction by etoposide reverted the antiapoptotic effect of Prep1. Experiments performed in aorta from 18 months old WT mice showed a significant increase in Prep1, p16INK4 , p21Waf1 and interleukin 6 (IL-6) compared to youngest animals. Similar results have been observed in H2 O2 -induced senescent VSMCs. Interestingly, the synthetic Prep1 inhibitory peptide Prep1 (54-72) reduced the antiapoptotic effects mediated by IL-6, particularly in senescent VSMCs. These results indicate that IL-6-Prep1 signaling reduces apoptosis, by modulating Bcl-xL and p53 both in murine aorta and in VSMCs. In addition, age-dependent increase in IL-6 and Prep1 in senescent VSMCs and in old mice may be involved in the aging-related vascular dysfunction.

Design, Optimization, and Structural Characterization of an Apoptosis-Inducing Factor Peptide Targeting Human Cyclophilin A to Inhibit Apoptosis Inducing Factor-Mediated Cell Death.

Blocking the interaction between the apoptosis-inducing factor (AIF) and cyclophilin A (CypA) by the AIF fragment AIF(370-394) is protective against glutamate-induced neuronal cell death and brain injury in mice. Starting from AIF(370-394), we report the generation of the disulfide-bridged and shorter variant AIF(381-389) and its structural characterization by nuclear magnetic resonance (NMR) in the free and CypA-bound state. AIF(381-389) in both the free and bound states assumes a ß-hairpin conformation similar to that of the fragment in the AIF protein and shows a highly reduced conformational flexibility. This peptide displays a similar in vitro affinity for CypA, an improved antiapoptotic activity in cells and an enhanced proteolytic stability compared to the parent peptide. The NMR-based 3D model of the AIF(381-389)/CypA complex provides a better understanding of the binding hot spots on both the peptide and the protein and can be exploited to design AIF/CypA inhibitors with improved pharmacokinetic and pharmacodynamics features.

Recent Applications of Retro-Inverso Peptides.

Natural and de novo designed peptides are gaining an ever-growing interest as drugs against several diseases. Their use is however limited by the intrinsic low bioavailability and poor stability. To overcome these issues retro-inverso analogues have been investigated for decades as more stable surrogates of peptides composed of natural amino acids. Retro-inverso peptides possess reversed sequences and chirality compared to the parent molecules maintaining at the same time an identical array of side chains and in some cases similar structure. The inverted chirality renders them less prone to degradation by endogenous proteases conferring enhanced half-lives and an increased potential as new drugs. However, given their general incapability to adopt the 3D structure of the parent peptides their application should be careful evaluated and investigated case by case. Here, we review the application of retro-inverso peptides in anticancer therapies, in immunology, in neurodegenerative diseases, and as antimicrobials, analyzing pros and cons of this interesting subclass of molecules.