Unlocking the potential of protein-derived peptides to target G-quadruplex DNA: from recognition to anticancer activity.

Noncanonical nucleic acid structures, particularly G-quadruplexes, have garnered significant attention as potential therapeutic targets in cancer treatment. Here, the recognition of G-quadruplex DNA by peptides derived from the Rap1 protein is explored, with the aim of developing novel peptide-based G-quadruplex ligands with enhanced selectivity and anticancer activity. Biophysical techniques were employed to assess the interaction of a peptide derived from the G-quadruplex-binding domain of the protein with various biologically relevant G-quadruplex structures. Through alanine scanning mutagenesis, key amino acids crucial for G-quadruplex recognition were identified, leading to the discovery of two peptides with improved G-quadruplex-binding properties. However, despite their in vitro efficacy, these peptides showed limited cell penetration and anticancer activity. To overcome this challenge, cell-penetrating peptide (CPP)-conjugated derivatives were designed, some of which exhibited significant cytotoxic effects on cancer cells. Interestingly, selected CPP-conjugated peptides exerted potent anticancer activity across various tumour types via a G-quadruplex-dependent mechanism. These findings underscore the potential of peptide-based G-quadruplex ligands in cancer therapy and pave the way for the development of novel therapeutic strategies targeting these DNA structures.

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.

The urotensin-II receptor: A marker for staging and steroid outcome prediction in ulcerative colitis.

BACKGROUND: Urotensin-II receptor- (UTR) related pathway exerts a key-role in promoting inflammation. The aim was to assess the relationship between UTR expression and clinical, endoscopic and biochemical severity of ulcerative colitis (UC), exploring its predictivity of intravenous (iv) steroid administration therapeutic outcome. METHODS: One-hundred patients with first diagnosis of UC and 44 healthy subjects were enrolled. UTR expression was assessed by qPCR, Western Blot (WB) and immunohistochemistry (IHC). Clinical, endoscopic and histological activity of UC were evaluated by using Truelove and Witts (T&W) severity index, Mayo Endoscopic Score (MES), and Truelove and Richards Index (TRI). The partial and full Mayo scores (PMS and FMS) were assessed to stage the disease. RESULTS: The UTR expression, resulted higher in the lesioned mucosa of UC patients in comparison to healthy subjects (p < .0001 all). Direct relationship between UTR (mRNA and protein) expression and disease severity assessment (T&W, PMS, MES and TRI) was highlighted (p < .0001 all). UTR expression resulted also higher in the 72 patients requiring iv steroids administration compared to those who underwent alternative medications, (p < .0001). The 32 steroid-non-responders showed an increased UTR expression (WB, IHC and qPCR from lesioned mucosa), compared to 40 steroid-responders (p: .0002, .0001, p < .0001 respectively). The predictive role of UTR expression (p < .05) on the negative iv steroids administration therapeutic outcome was highlighted and ROC curves identified the thresholds expressing the better predictive performance. CONCLUSIONS: UTR represents a promising inflammatory marker related to clinical, endoscopic, and histological disease activity as well as a predictive marker of steroid administration therapeutic outcome in the UC context.

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.

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.

Disulfide Bond Replacement with 1,4- and 1,5-Disubstituted [1,2,3]-Triazole on C-X-C Chemokine Receptor Type 4 (CXCR4) Peptide Ligands: Small Changes that Make Big Differences.

Here we investigated the structural and biological effects ensuing from the disulfide bond replacement of a potent and selective C-X-C chemokine receptor type 4 (CXCR4) peptide antagonist, with 1,4- and 1,5- disubstituted 1,2,3-triazole moieties. Both strategies produced candidates that showed high affinity and selectivity against CXCR4. Notably, when assessed for their ability to modulate the CXCL12-mediated cell migration, the 1,4-triazole variant conserved the antagonistic effect in the low-mid nanomolar range, while the 1,5-triazole one displayed the ability to activate the migration, becoming the first in class low-molecular-weight CXCR4 peptide agonist. By combining NMR and computational studies, we provided a valuable model that highlighted differences in the interactions of the two peptidomimetics with the receptor that could account for their different functional profile. Finally, we envisage that our findings could be translated to different GPCR-interacting peptides for the pursuit of novel chemical probes that could assist in dissecting the complex puzzle of this fundamental class of transmembrane receptors.

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.

SIRT6 interacts with TRF2 and promotes its degradation in response to DNA damage.

Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.

From a Helix to a Small Cycle: Metadynamics-Inspired αvß6 Integrin Selective Ligands.

The RGD-recognizing αvß6 integrin has only recently emerged as a major target for cancer diagnosis and therapy. Thus, the development of selective, low-molecular-weight ligands of this receptor is still in great demand. Here, a metadynamics-driven design strategy allowed us to successfully convert a helical nonapeptide into a cyclic pentapeptide (6) showing remarkable potency and αvß6 specificity. NMR and docking studies elucidated the reasons for the high affinity and selectivity of this compound, setting the ground for the rational design of new αvß6-specific small peptides or even peptidomimetics. In vivo PET imaging studies demonstrated the potential use of 6 for medical applications.

An investigation into the origin of the biased agonism associated with the urotensin II receptor activation.

The urotensin II receptor (UTR) has long been studied mainly for its involvement in the cardiovascular homeostasis both in health and disease state. Two endogenous ligands activate UTR, i.e. urotensin II (U-II) and urotensin II-related peptide (URP). Extensive expression of the two ligands uncovers the diversified pathophysiological effects mediated by the urotensinergic system such as cardiovascular disorders, smooth muscle cell proliferation, renal disease, diabetes, and tumour growth. As newly reported, U-II and URP have distinct effects on transcriptional activity, cell proliferation, and myocardial contractile activities supporting the idea that U-II and URP interact with UTR in a distinct manner (biased agonism). To shed light on the origin of the divergent activities of the two endogenous ligands, we performed a conformational study on URP by solution NMR in sodium dodecyl sulfate micelle solution and compared the obtained NMR structure of URP with that of hU-II previously determined. Finally, we undertook docking studies between URP, hU-II, and an UT receptor model.

Development and Nanoparticle-Mediated Delivery of Novel MDM2/MDM4 Heterodimer Peptide Inhibitors to Enhance 5-Fluorouracil Nucleolar Stress in Colorectal Cancer Cells.

Colorectal cancer (CRC) often involves wild-type p53 inactivation by MDM2 and MDM4 overexpression, promoting tumor progression and resistance to 5-fluoruracil (5-FU). Disrupting the MDM2/4 heterodimer can proficiently reactivate p53, sensitizing cancer cells to 5-FU. Herein, we developed 16 peptides based on Pep3 (1), the only known peptide acting through this mechanism. The new peptides, notably 3 and 9, showed lower IC50 values than 1. When incorporated into tumor-targeted biodegradable nanoparticles, these exhibited cytotoxicity against three different CRC cell lines. Notably, NPs/9 caused a significant increase in p53 levels associated with a strong increment of its main downstream target p21 inducing apoptosis. Also, the combined treatment of 9 with 5-FU caused the activation of nucleolar stress and a synergic apoptotic effect. Hence, the co-delivery of MDM2/4 heterodimer disruptors with 5-FU through nanoparticles might be a promising strategy to overcome drug resistance in CRC.

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.

Comprehensive structural investigation of a potent and selective CXCR4 antagonist via crosslink modification.

Macrocyclization presents a valuable strategy for enhancing the pharmacokinetic and pharmacodynamic profiles of short bioactive peptides. The exploration of various macrocyclic characteristics, such as crosslinking tethers, ring size, and orientation, is generally conducted during the early stages of development. Herein, starting from a potent and selective C-X-C chemokine receptor 4 (CXCR4) cyclic heptapeptide antagonist mimicking the N-terminal region of CXCL12, we demonstrated that the disulfide bridge could be successfully replaced with a side-chain to side-chain lactam bond, which is commonly not enlisted among the conventional disulfide mimetics. An extensive investigation was carried out to explore the chemical space of the resulting peptides, including macrocyclization width, stereochemical configuration, and lactam orientation, all of which were correlated with biochemical activity. We identified a novel heptapeptide that fully replicates the pharmacological profile of the parent peptide on CXCR4, including its potency, selectivity, and antagonistic activity, while demonstrating enhanced stability in a reductive environment. At this stage, computational studies were instructed to shed light on how the lactam cyclization features influenced the overall structure of 21 and, in turn, its ability to interact with the receptor. We envisage that these findings can give new momentum to the use of lactam cyclization as a disulfide bond mimetic and contribute to the enhancement of the repertoire for peptide-based drug development, thereby paving the way for novel avenues in therapeutic innovation.

Disulfide bond replacement with non-reducible side chain to tail macrolactamization for the development of potent and selective CXCR4 peptide antagonists endowed with flanking binding sites.

The present study describes a small library of peptides derived from a potent and selective CXCR4 antagonist (3), wherein the native disulfide bond is replaced using a side-chain to tail macrolactamization technique to vary ring size and amino acid composition. The peptides were preliminary assessed for their ability to interfere with the interaction between the receptor and anti-CXCR4 PE-conjugated antibody clone 12G5. Two promising candidates (13 and 17) were identified and further evaluated in a125I-CXCL12 competition binding assay, exhibiting IC50 in the low-nanomolar range. Furthermore, both candidates displayed high selectivity towards CXCR4 with respect to the cognate receptor CXCR7, ability to block CXCL12-dependent cancer cell migration, and receptor internalization, albeit at a higher concentration compared to 3. Molecular modeling studies on 13 and 17 produced a theoretical model that may serve as a guide for future modifications, aiding in the development of analogs with improved affinity. Finally, the study provides valuable insights into developing therapeutic agents targeting CXCR4-mediated processes, demonstrating the adaptability of our lead peptide 3 to alternative cyclization approaches and offering prospects for comprehensive investigations into the receptor region's interaction with its C-terminal region.

New insight into the binding mode of peptides at urotensin-II receptor by Trp-constrained analogues of P5U and urantide.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G-protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of human U-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-D-Trp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized four analogues of P5U and urantide in which the Trp(7) residue was replaced by the highly constrained L-Tpi and D-Tpi residues. The replacement of the Trp(7) by Tpi led to active analogues. Solution NMR analysis allowed improving the knowledge on conformation-activity relationships previously reported on UT receptor ligands.

Unveiling the interaction between DNA G-quadruplexes and RG-rich peptides.

G-quadruplexes are non-canonical DNA secondary structures formed within guanine-rich strands that play important roles in various biological processes, including gene regulation, telomere maintenance and DNA replication. The biological functions and formation of these DNA structures are strictly controlled by several proteins that bind and stabilize or resolve them. Many G-quadruplex-binding proteins feature an arginine and glycine-rich motif known as the RGG or RG-rich motif. Although this motif plays a crucial role in the recognition of such non-canonical structures, their interaction is still poorly understood. Here, we employed a combination of several biophysical techniques to provide valuable insights into the interaction between a peptide containing an RGG motif shared by numerous human G-quadruplex-binding proteins (NIQI) and various biologically relevant G-quadruplex DNA structures with different topologies. We also shed light on the key amino acids involved in the binding process. Our findings contribute to lay the basis for the development of a new class of peptide-based G-quadruplex ligands as an alternative to small molecules. These ligands may serve as valid tools for interfering in DNA-protein interactions, with potential therapeutic applications.

αV-Integrin-Dependent Inhibition of Glioblastoma Cell Migration, Invasion and Vasculogenic Mimicry by the uPAcyclin Decapeptide.

Among the deadliest human cancers is glioblastoma (GBM) for which new treatment approaches are urgently needed. Here, the effects of the cyclic decapeptide, uPAcyclin, are investigated using the U87-MG, U251-MG, and U138-MG human GBM and C6 rat cell models. All GBM cells express the αV-integrin subunit, the target of uPAcyclin, and bind specifically to nanomolar concentrations of the decapeptide. Although peptide exposure affects neither viability nor cell proliferation rate, nanomolar concentrations of uPAcyclin markedly inhibit the directional migration and matrix invasion of all GBM cells, in a concentration- and αV-dependent manner. Moreover, wound healing rate closure of U87-MG and C6 rat glioma cells is reduced by 50% and time-lapse videomicroscopy studies show that the formation of vascular-like structures by U87-MG in three-dimensional matrix cultures is markedly inhibited by uPAcyclin. A strong reduction in the branching point numbers of the U87-MG, C6, and U251-MG cell lines undergoing vasculogenic mimicry, in the presence of nanomolar peptide concentrations, was observed. Lysates from matrix-recovered uPAcyclin-exposed cells exhibit a reduced expression of VE-cadherin, a prominent factor in the acquisition of vascular-like structures. In conclusion, these results indicate that uPAcyclin is a promising candidate to counteract the formation of new vessels in novel targeted anti-GBM therapies.

Ultrasound-assisted Peptide Nucleic Acids synthesis (US-PNAS).

Herein, we developed an innovative and easily accessible solid-phase synthetic protocol for Peptide Nucleic Acid (PNA) oligomers by systematically investigating the ultrasonication effects in all steps of the PNA synthesis (US-PNAS). When compared with standard protocols, the application of the so-obtained US-PNAS approach succeeded in improving the crude product purities and the isolated yields of different PNA, including small or medium-sized oligomers (5-mer and 9-mer), complex purine-rich sequences (like a 5-mer Guanine homoligomer and the telomeric sequence TEL-13) and longer oligomers (such as the 18-mer anti-IVS2-654 PNA and the 23-mer anti-mRNA 155 PNA). Noteworthy, our ultrasound-assisted strategy is compatible with the commercially available PNA monomers and well-established coupling reagents and only requires the use of an ultrasonic bath, which is a simple equipment generally available in most synthetic laboratories.