Hydrophobicity: The door to drug delivery.

The engineering of intracellular delivery systems with the goal of achieving personalized medicine has been encouraged by advances in nanomaterial science as well as a greater understanding of diseases and of the biochemical pathways implicated in many disorders. The development of vectors able to transport the drug to a target location and release it only on demand is undoubtedly the primary issue. From a molecular perspective, the topography of drug carrier surfaces is directly related to the design of an effective drug carrier because it provides a physical hint to modifying its interactions with biological systems. For instance, the initial ratio of hydrophilic to hydrophobic surfaces and the changes brought about by external factors enable the release or encapsulation of a therapeutic molecule and the ability of the nanosystem to cross biological barriers and reach its target without causing systemic toxicity. The first step in creating new materials with enhanced functionality is to comprehend and characterize the interplay between hydrophilic and hydrophobic molecules at the molecular level. Therefore, the focus of this review is on the function of hydrophobicity, which is essential for matching the complexity of biological environments with the intended functionality.

New biologic (Ab-IPL-IL-17) for IL-17-mediated diseases: identification of the bioactive sequence (nIL-17) for IL-17A/F function.

OBJECTIVES: Interleukin (IL) 17s cytokines are key drivers of inflammation that are functionally dysregulated in several human immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). Targeting these cytokines has some therapeutic benefits, but issues associated with low therapeutic efficacy and immunogenicity for subgroups of patients or IMIDs reduce their clinical use. Therefore, there is an urgent need to improve the coverage and efficacy of antibodies targeting IL-17A and/or IL-17F and IL-17A/F heterodimer. METHODS AND RESULTS: Here, we initially identified a bioactive 20 amino acid IL-17A/F-derived peptide (nIL-17) that mimics the pro-inflammatory actions of the full-length proteins. Subsequently, we generated a novel anti-IL-17 neutralising monoclonal antibody (Ab-IPL-IL-17) capable of effectively reversing the pro-inflammatory, pro-migratory actions of both nIL-17 and IL-17A/F. Importantly, we demonstrated that Ab-IPL-IL-17 has less off-target effects than the current gold-standard biologic, secukinumab. Finally, we compared the therapeutic efficacy of Ab-IPL-IL-17 with reference anti-IL-17 antibodies in preclinical murine models and samples from patients with RA and IBD. We found that Ab-IPL-IL-17 could effectively reduce clinical signs of arthritis and neutralise elevated IL-17 levels in IBD patient serum. CONCLUSIONS: Collectively, our preclinical and in vitro clinical evidence indicates high efficacy and therapeutic potency of Ab-IPL-IL-17, supporting the rationale for large-scale clinical evaluation of Ab-IPL-IL-17 in patients with IMIDs.

Unveiling the mechanism of action of acylated temporin L analogues against multidrug-resistant Candida albicans.

The increasing resistance of fungi to conventional antifungal drugs has prompted worldwide the search for new compounds. In this work, we investigated the antifungal properties of acylated Temporin L derivatives, Pent-1B and Dec-1B, against Candida albicans, including the multidrug-resistant strains. Acylated peptides resulted to be active both on reference and clinical strains with MIC values ranging from 6.5 to 26 µM, and they did not show cytotoxicity on human keratinocytes. In addition, we also observed a synergistic or additive effect with voriconazole for peptides Dec-1B and Pent-1B through the checkerboard assay on voriconazole-resistant Candida strains. Moreover, fluorescence-based assays, NMR spectroscopy, and confocal microscopy elucidated a potential membrane-active mechanism, consisting of an initial electrostatic interaction of acylated peptides with fungal membrane, followed by aggregation and insertion into the lipid bilayer and causing membrane perturbation probably through a carpeting effect.

Myxinidin-Derived Peptide against Biofilms Caused by Cystic Fibrosis Emerging Pathogens.

Chronic lung infections in cystic fibrosis (CF) patients are triggered by multidrug-resistant bacteria such as Pseudomonas aeruginosa, Achromobacter xylosoxidans, and Stenotrophomonas maltophilia. The CF airways are considered ideal sites for the colonization and growth of bacteria and fungi that favor the formation of mixed biofilms that are difficult to treat. The inefficacy of traditional antibiotics reinforces the need to find novel molecules able to fight these chronic infections. Antimicrobial peptides (AMPs) represent a promising alternative for their antimicrobial, anti-inflammatory, and immunomodulatory activities. We developed a more serum-stable version of the peptide WMR (WMR-4) and investigated its ability to inhibit and eradicate C. albicans, S. maltophilia, and A. xylosoxidans biofilms in both in vitro and in vivo studies. Our results suggest that the peptide is able better to inhibit than to eradicate both mono and dual-species biofilms, which is further confirmed by the downregulation of some genes involved in biofilm formation or in quorum-sensing signaling. Biophysical data help to elucidate its mode of action, showing a strong interaction of WMR-4 with lipopolysaccharide (LPS) and its insertion in liposomes mimicking Gram-negative and Candida membranes. Our results support the promising therapeutic application of AMPs in the treatment of mono- and dual-species biofilms during chronic infections in CF patients.

Discovery of a new class of triazole based inhibitors of acetyl transferase KAT2A.

We have recently developed a new synthetic methodology that provided both N-aryl-5-hydroxytriazoles and N-pyridine-4-alkyl triazoles. A selection of these products was carried through virtual screening towards targets that are contemporary and validated for drug discovery and development. This study determined a number of potential structure target dyads of which N-pyridinium-4-carboxylic-5-alkyl triazole displayed the highest score specificity towards KAT2A. Binding affinity tests of abovementioned triazole and related analogs towards KAT2A confirmed the predictions of the in-silico assay. Finally, we have run in vitro inhibition assays of selected triazoles towards KAT2A; the ensemble of binding and inhibition assays delivered pyridyl-triazoles carboxylates as the prototype of a new class of inhibitors of KAT2A.

WMR Peptide as Antifungal and Antibiofilm against Albicans and Non-Albicans Candida Species: Shreds of Evidence on the Mechanism of Action.

Candida species are the most common fungal pathogens infecting humans and can cause severe illnesses in immunocompromised individuals. The increased resistance of Candida to traditional antifungal drugs represents a great challenge in clinical settings. Therefore, novel approaches to overcome antifungal resistance are desired. Here, we investigated the use of an antimicrobial peptide WMR against Candida albicans and non-albicans Candida species in vitro and in vivo. Results showed a WMR antifungal activity on all Candida planktonic cells at concentrations between 25 µM to >50 µM and exhibited activity at sub-MIC concentrations to inhibit biofilm formation and eradicate mature biofilm. Furthermore, in vitro antifungal effects of WMR were confirmed in vivo as demonstrated by a prolonged survival rate of larvae infected by Candida species when the peptide was administered before or after infection. Additional experiments to unravel the antifungal mechanism were performed on C. albicans and C. parapsilosis. The time-killing curves showed their antifungal activity, which was further confirmed by the induced intracellular and mitochondrial reactive oxygen species accumulation; WMR significantly suppressed drug efflux, down-regulating the drug transporter encoding genes CDR1. Moreover, the ability of WMR to penetrate within the cells was demonstrated by confocal laser scanning microscopy. These findings provide novel insights for the antifungal mechanism of WMR against Candida albicans and non-albicans, providing fascinating scenarios for the identification of new potential antifungal targets.

Broad-Spectrum Antiviral Activity of the Amphibian Antimicrobial Peptide Temporin L and Its Analogs.

The COVID-19 pandemic has evidenced the urgent need for the discovery of broad-spectrum antiviral therapies that could be deployed in the case of future emergence of novel viral threats, as well as to back up current therapeutic options in the case of drug resistance development. Most current antivirals are directed to inhibit specific viruses since these therapeutic molecules are designed to act on a specific viral target with the objective of interfering with a precise step in the replication cycle. Therefore, antimicrobial peptides (AMPs) have been identified as promising antiviral agents that could help to overcome this limitation and provide compounds able to act on more than a single viral family. We evaluated the antiviral activity of an amphibian peptide known for its strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, namely Temporin L (TL). Previous studies have revealed that TL is endowed with widespread antimicrobial activity and possesses marked haemolytic activity. Therefore, we analyzed TL and a previously identified TL derivative (Pro3, DLeu9 TL, where glutamine at position 3 is replaced with proline, and the D-Leucine enantiomer is present at position 9) as well as its analogs, for their activity against a wide panel of viruses comprising enveloped, naked, DNA and RNA viruses. We report significant inhibition activity against herpesviruses, paramyxoviruses, influenza virus and coronaviruses, including SARS-CoV-2. Moreover, we further modified our best candidate by lipidation and demonstrated a highly reduced cytotoxicity with improved antiviral effect. Our results show a potent and selective antiviral activity of TL peptides, indicating that the novel lipidated temporin-based antiviral agents could prove to be useful additions to current drugs in combatting rising drug resistance and epidemic/pandemic emergencies.

Antiviral Potential of Naphthoquinones Derivatives Encapsulated within Liposomes.

HSV infections, both type 1 and type 2, are among the most widespread viral diseases affecting people of all ages. Their symptoms could be mild, with cold sores up to 10 days of infection, blindness and encephalitis caused by HSV-1 affecting immunocompetent and immunosuppressed individuals. The severe effects derive from co-evolution with the host, resulting in immune evasion mechanisms, including latency and growing resistance to acyclovir and derivatives. An efficient alternative to controlling the spreading of HSV mutations is the exploitation of new drugs, and the possibility of enhancing their delivery through the encapsulation of drugs into nanoparticles, such as liposomes. In this work, liposomes were loaded with a series of 2-aminomethyl- 3-hydroxy-1,4-naphthoquinones derivatives with n-butyl (compound 1), benzyl (compound 2) and nitrobenzene (compound 3) substituents in the primary amine of naphthoquinone. They were previously identified to have significant inhibitory activity against HSV-1. All of the aminomethylnaphthoquinones derivatives encapsulated in the phosphatidylcholine liposomes were able to control the early and late phases of HSV-1 replication, especially those substituted with the benzyl (compound 2) and nitrobenzene (compound 3), which yields selective index values that are almost nine times more efficient than acyclovir. The growing interest of the industry in topical administration against HSV supports our choice of liposome as a drug carrier of aminomethylnaphthoquinones derivatives for formulations of in vivo pre-clinical assays.

Bicyclic ß-Sheet Mimetics that Target the Transcriptional Coactivator ß-Catenin and Inhibit Wnt Signaling.

Protein complexes are defined by the three-dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been applied for example, as inhibitors of protein-protein interactions (PPIs). Even though ß-sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure-based design of ß-sheet mimetics targeting the intracellular protein ß-catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of ß-catenin, a macrocyclic peptide was designed and its crystal structure in complex with ß-catenin obtained. Using this structure, we designed a library of bicyclic ß-sheet mimetics employing a late-stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to ß-catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other ß-sheet-mediated PPIs.

Novel temporin L antimicrobial peptides: promoting self-assembling by lipidic tags to tackle superbugs.

The rapid development of antimicrobial resistance is pushing the search in the discovering of novel antimicrobial molecules to prevent and treat bacterial infections. Self-assembling antimicrobial peptides, as the lipidated peptides, are a novel and promising class of molecules capable of meeting this need. Based on previous work on Temporin L analogs, several new molecules lipidated at the N- or and the C-terminus were synthesised. Our goal is to improve membrane interactions through finely tuning self-assembly to reduce oligomerisation in aqueous solution and enhance self-assembly in bacterial membranes while reducing toxicity against human cells. The results here reported show that the length of the aliphatic moiety is a key factor to control target cell specificity and the oligomeric state of peptides either in aqueous solution or in a membrane-mimicking environment. The results of this study pave the way for the design of novel molecules with enhanced activities.

Exploring Fe(III) coordination and membrane interaction of a siderophore-peptide conjugate: Enhancing synergistically the antimicrobial activity.

Many microbes produce siderophores, which are extremely potent weapons capable of stealing iron ions from human tissues, fluids and cells and transferring them into bacteria through their appropriate porins. We have recently designed a multi-block molecule, each block having a dedicated role. The first component is an antimicrobial peptide, whose good effectiveness against some bacterial strains was gradually improved through interactive sequence modifications. Connected to this block is a flexible bio-band, also optimized in length, which terminates in a hydroxyamide unit, a strong metal binder. Thus, the whole molecule brings together two pieces that work synergistically to fight infection. To understand if the peptide unit, although modified with a long tail, preserves the structure and therefore the antimicrobial activity, and to characterize the mechanism of interaction with bio-membrane models mimicking Gram-negative membranes, we performed a set of fluorescence-based experiments and circular dichroism studies, which further supported our design of a combination of two different entities working synergistically. The chelating activity and iron(III) binding of the peptide was confirmed by iron(III) paramagnetic NMR analyses, and through a competitive assay with ethylenediamine-tetra acetic acid by ultraviolet-visible spectroscopy. The complexation parameters, the Michaelis constant K, and the number of sites n, evaluated with spectrophotometric techniques are confirmed by Fe(III) paramagnetic NMR analyses here reported. In conclusion, we showed that the coupling of antimicrobial capabilities with iron-trapping capabilities works well in the treatment of infectious diseases caused by Gram-negative pathogens.

Tuning Peptide-Based Nanofibers for Achieving Selective Doxorubicin Delivery in Triple-Negative Breast Cancer.

Introduction: The design of delivery tools that efficiently transport drugs into cells remains a major challenge in drug development for most pathological conditions. Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer with poor prognosis and limited effective therapeutic options. Purpose: In TNBC treatment, chemotherapy remains the milestone, and doxorubicin (Dox) represents the first-line systemic treatment; however, its non-selective distribution causes a cascade of side effects. To address these problems, we developed a delivery platform based on the self-assembly of amphiphilic peptides carrying several moieties on their surfaces, aimed at targeting, enhancing penetration, and therapy. Methods: Through a single-step self-assembly process, we used amphiphilic peptides to obtain nanofibers decorated on their surfaces with the selected moieties. The surface of the nanofiber was decorated with a cell-penetrating peptide (gH625), an EGFR-targeting peptide (P22), and Dox bound to the cleavage sequence selectively recognized and cleaved by MMP-9 to obtain on-demand drug release. Detailed physicochemical and cellular analyses were performed. Results: The obtained nanofiber (NF-Dox) had a length of 250 nm and a diameter of 10 nm, and it was stable under dilution, ionic strength, and different pH environments. The biological results showed that the presence of gH625 favored the complete internalization of NF-Dox after 1h in MDA-MB 231 cells, mainly through a translocation mechanism. Interestingly, we observed the absence of toxicity of the carrier (NF) on both healthy cells such as HaCaT and TNBC cancer lines, while a similar antiproliferative effect was observed on TNBC cells after the treatment with the free-Dox at 50 µM and NF-Dox carrying 7.5 µM of Dox. Discussion: We envision that this platform is extremely versatile and can be used to efficiently carry and deliver diverse moieties. The knowledge acquired from this study will provide important guidelines for applications in basic research and biomedicine.

Strategic Single-Residue Substitution in the Antimicrobial Peptide Esc(1-21) Confers Activity against Staphylococcus aureus, Including Drug-Resistant and Biofilm Phenotype.

Staphylococcus aureus, a bacterium resistant to multiple drugs, is a significant cause of illness and death worldwide. Antimicrobial peptides (AMPs) provide an excellent potential strategy to cope with this threat. Recently, we characterized a derivative of the frog-skin AMP esculentin-1a, Esc(1-21) (1) that is endowed with potent activity against Gram-negative bacteria but poor efficacy against Gram-positive strains. In this study, three analogues of peptide 1 were designed by replacing Gly8 with α-aminoisobutyric acid (Aib), Pro, and dPro (2-4, respectively). The single substitution Gly8 → Aib8 in peptide 2 makes it active against the planktonic form of Gram-positive bacterial strains, especially Staphylococcus aureus, including multidrug-resistant clinical isolates, with an improved biostability without resulting in cytotoxicity to mammalian cells. Moreover, peptide 2 showed a higher antibiofilm activity than peptide 1 against both reference and clinical isolates of S. aureus. Peptide 2 was also able to induce rapid bacterial killing, suggesting a membrane-perturbing mechanism of action. Structural analysis of the most active peptide 2 evidenced that the improved biological activity of peptide 2 is the consequence of a combination of higher biostability, higher α helical content, and ability to reduce membrane fluidity and to adopt a distorted helix, bent in correspondence of Aib8. Overall, this study has shown how a strategic single amino acid substitution is sufficient to enlarge the spectrum of activity of the original peptide 1, and improve its biological properties for therapeutic purposes, thus paving the way to optimize AMPs for the development of new broad-spectrum anti-infective agents.

Expanding Structure-Activity Relationships of Human Urotensin II Peptide Analogues: A Proposed Key Role of the N-Terminal Region for Novel Urotensin II Receptor Modulators.

While the urotensinergic system plays a role in influencing various pathologies, its potential remains untapped because of the absence of therapeutically effective urotensin II receptor (UTR) modulators. Herein, we developed analogues of human urotensin II (hU-II) peptide in which, along with well-known antagonist-oriented modifications, the Glu1 residue was subjected to single-point mutations. The generated library was tested by a calcium mobilization assay and ex vivo experiments, also in competition with selected ligands. Interestingly, many derivatives showed noncompetitive modulation that was rationalized by the lateral allostery concept applied to a G protein-coupled receptor (GPCR) multimeric model. UPG-108 showed an unprecedented ability to double the efficacy of hU-II, while UPG-109 and UPG-111 turned out to be negative allosteric modulators of UTR. Overall, our investigation will serve to explore and highlight the expanding possibilities of modulating the UTR system through N-terminally modified hU-II analogues and, furthermore, will aim to elucidate the intricate nature of such a GPCR system.

Targetable domains for the design of peptide-dendrimer inhibitors of SARS-CoV-2.

We have recently witnessed that considerable progresses have been made in the rapid detection and appropriate treatments of COVID-19, but still this virus remains one of the main targets of world research. Based on the knowledge of the complex mechanism of viral infection we designed peptide-dendrimer inhibitors of SARS-CoV-2with the aim to block cell infection through interfering with the host-pathogen interactions. We used two different strategies: i) the first one aims at hindering the virus anchorage to the human cell; ii) the second -strategy points to interfere with the mechanism of virus-cell membrane fusion. We propose the use of different nanosized carriers, formed by several carbosilane dendritic wedges to deliver two different peptides designed to inhibit host interaction or virus entry. The antiviral activity of the peptide-dendrimers, as well as of free peptides and free dendrimers was evaluated through the use of SARS-CoV-2 pseudotyped lentivirus. The results obtained show that peptides designed to block host-pathogen interaction represent a valuable strategy for viral inhibition.

The Outstanding Chemodiversity of Marine-Derived Talaromyces.

Fungi in the genus Talaromyces occur in every environment in both terrestrial and marine contexts, where they have been quite frequently found in association with plants and animals. The relationships of symbiotic fungi with their hosts are often mediated by bioactive secondary metabolites, and Talaromyces species represent a prolific source of these compounds. This review highlights the biosynthetic potential of marine-derived Talaromyces strains, using accounts from the literature published since 2016. Over 500 secondary metabolites were extracted from axenic cultures of these isolates and about 45% of them were identified as new products, representing a various assortment of chemical classes such as alkaloids, meroterpenoids, isocoumarins, anthraquinones, xanthones, phenalenones, benzofurans, azaphilones, and other polyketides. This impressive chemodiversity and the broad range of biological properties that have been disclosed in preliminary assays qualify these fungi as a valuable source of products to be exploited for manifold biotechnological applications.

An Overview of Supramolecular Platforms Boosting Drug Delivery.

Numerous supramolecular platforms inspired by natural self-assembly are exploited as drug delivery systems. The spontaneous arrangement of single building blocks into inorganic and organic structures is determined and controlled by noncovalent forces such as electrostatic interactions, π-π interactions, hydrogen bonds, and van der Waals interactions. This review describes the main structures and characteristics of several building blocks used to obtain stable, self-assembling nanostructures tailored for numerous biological applications. Owing to their versatility, biocompatibility, and controllability, these nanostructures find application in diverse fields ranging from drug/gene delivery, theranostics, tissue engineering, and nanoelectronics. Herein, we described the different approaches used to design and functionalize these nanomaterials to obtain selective drug delivery in a specific disease. In particular, the review highlights the efficiency of these supramolecular structures in applications related to infectious diseases and cancer.

Glycosylation and Lipidation Strategies: Approaches for Improving Antimicrobial Peptide Efficacy.

Antimicrobial peptides (AMPs) have recently gained attention as a viable solution for combatting antibiotic resistance due to their numerous advantages, including their broad-spectrum activity, low propensity for inducing resistance, and low cytotoxicity. Unfortunately, their clinical application is limited due to their short half-life and susceptibility to proteolytic cleavage by serum proteases. Indeed, several chemical strategies, such as peptide cyclization, N-methylation, PEGylation, glycosylation, and lipidation, are widely used for overcoming these issues. This review describes how lipidation and glycosylation are commonly used to increase AMPs' efficacy and engineer novel AMP-based delivery systems. The glycosylation of AMPs, which involves the conjugation of sugar moieties such as glucose and N-acetyl galactosamine, modulates their pharmacokinetic and pharmacodynamic properties, improves their antimicrobial activity, and reduces their interaction with mammalian cells, thereby increasing selectivity toward bacterial membranes. In the same way, lipidation of AMPs, which involves the covalent addition of fatty acids, has a significant impact on their therapeutic index by influencing their physicochemical properties and interaction with bacterial and mammalian membranes. This review highlights the possibility of using glycosylation and lipidation strategies to increase the efficacy and activity of conventional AMPs.

Heavy Metal Pollution and Male Fertility: An Overview on Adverse Biological Effects and Socio-Economic Implications.

Trace metals can be divided into two subgroups considering their pathophysiological effects: the first consists of microelements essential for life (arsenic, cobalt, chromium, copper, fluorine, iron, iodine, manganese, molybdenum, nickel, selenium, silicon, tin, vanadium and zinc), implicated in important metabolic processes; the second includes toxic microelements, such as cadmium (Cd), mercury (Hg), chromium (Cr), and lead (Pb) for living organisms, even at low concentrations. These metals contribute to serious consequences for human health, including male infertility. Studies performed in several in vitro and in vivo models revealed that environmental exposure to toxic pollutants, as heavy metals, negatively affects human male fertility. Stem cells, due to their ability to self-renew and differentiate in several cell types, have been proposed as a useful tool in assisted reproductive technology, permitting the spermatogenesis recovery in patients with irreversible infertility. Considering the effects of heavy metals on male fertility and, from a demographic point of view, the decreased fertility ratio, further strategies are required to maintain a sustainable turn-over of 2 children for woman. We discuss here the findings on the biological effects of heavy metal pollution in the male fertility and underline the related socioeconomic impact on population demography.

Synthesis of temporin L hydroxamate-based peptides and evaluation of their coordination properties with iron(III ).

Ferric iron is an essential nutrient for bacterial growth. Pathogenic bacteria synthesize iron-chelating entities known as siderophores to sequestrate ferric iron from host organisms in order to colonize and replicate. The development of antimicrobial peptides (AMPs) conjugated to iron chelators represents a promising strategy for reducing the iron availability, inducing bacterial death, and enhancing simultaneously the efficacy of AMPs. Here we designed, synthesized, and characterized three hydroxamate-based peptides Pep-cyc1, Pep-cyc2, and Pep-cyc3, derived from a cyclic temporin L peptide (Pep-cyc) developed previously by some of us. The Fe3+ complex formation of each ligand was characterized by UV-visible spectroscopy, mass spectrometry, and IR and NMR spectroscopies. In addition, the effect of Fe3+ on the stabilization of the α-helix conformation of hydroxamate-based peptides and the cotton effect were examined by CD spectroscopy. Moreover, the antimicrobial results obtained in vitro on some Gram-negative strains (K. pneumoniae and E. coli) showed the ability of each peptide to chelate efficaciously Fe3+ obtaining a reduction of MIC values in comparison to their parent peptide Pep-cyc. Our results demonstrated that siderophore conjugation could increase the efficacy and selectivity of AMPs used for the treatment of infectious diseases caused by Gram-negative pathogens.