Sensitization of melanoma cells to standard chemotherapy: G-quadruplex binders as synergistic agents.

The use of chemotherapeutics has achieved considerable success in cancer therapy; however, their toxicity can severely impact patients' health. In this study, aiming to reduce the doses and potential side effects of traditional chemotherapeutics, we systematically treated A375MM human melanoma cells with seven clinically approved antineoplastic drugs, in combination with three well-characterized G-quadruplex (G4) ligands, using either simultaneous or sequential dosing schedules. Interestingly, the G4 binders synergized with most of the investigated anticancer drugs, with the degree of synergism being strictly dependent on both the treatment schedule and the drug sequence employed. Notably, some of the synergistic combinations showed selective toxicity toward melanoma cells over nontumorigenic human keratinocytes. Furthermore, immunofluorescence experiments highlighted the potential implication of G4 structures in the molecular mechanisms driving the synergistic interaction between some chemotherapeutics and G4 binders. Overall, our systematic study supports the combination of G4-interacting molecules with standard antineoplastic drugs as a promising antitumor strategy.

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.

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.

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.

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.

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.

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.

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.

First-in-Class Cyclic Temporin L Analogue: Design, Synthesis, and Antimicrobial Assessment.

The pharmacodynamic and pharmacokinetic properties of bioactive peptides can be modulated by introducing conformational constraints such as intramolecular macrocyclizations, which can involve either the backbone and/or side chains. Herein, we aimed at increasing the α-helicity content of temporin L, an isoform of an intriguing class of linear antimicrobial peptides (AMPs), endowed with a wide antimicrobial spectrum, by the employment of diverse side-chain tethering strategies, including lactam, 1,4-substituted [1,2,3]-triazole, hydrocarbon, and disulfide linkers. Our approach resulted in a library of cyclic temporin L analogues that were biologically assessed for their antimicrobial, cytotoxic, and antibiofilm activities, leading to the development of the first-in-class cyclic peptide related to this AMP family. Our results allowed us to expand the knowledge regarding the relationship between the α-helical character of temporin derivatives and their biological activity, paving the way for the development of improved antibiotic cyclic AMP analogues.

Breast Tumor Cell Invasion and Pro-Invasive Activity of Cancer-Associated Fibroblasts Co-Targeted by Novel Urokinase-Derived Decapeptides.

Among peritumoral cells, cancer-associated fibroblasts (CAFs) are major facilitators of tumor progression. This study describes the effects of two urokinase-derived, novel decapeptides, denoted as Pep 1 and its cyclic derivative Pep 2. In a mouse model of tumor dissemination, using HT1080 fibrosarcoma cells, Pep 2 reduced the number and size of lung metastases. Specific binding of fluoresceinated Pep 2 to HT1080 and telomerase immortalised fibroblasts (TIF) cell surfaces was enhanced by αv overexpression or abolished by excess vitronectin, anti-αv antibodies or silencing of ITGAV αv gene, identifying αv-integrin as the Pep 2 molecular target. In 3D-organotypic assays, peptide-exposed TIFs and primary CAFs from breast carcinoma patients both exhibited a markedly reduced pro-invasive ability of either HT1080 fibrosarcoma or MDA-MB-231 mammary carcinoma cells, respectively. Furthermore, TIFs, either exposed to Pep 2, or silenced for αv integrin, were impaired in their ability to chemoattract cancer cells and to contract collagen matrices, exhibiting reduced α-smooth muscle actin (α-SMA) levels. Finally, peptide exposure of αv-expressing primary CAFs led to the downregulation of α-SMA protein and to a dramatic reduction of their pro-invasive capability. In conclusion, the ability of the novel decapeptides to interfere with tumor cell invasion directly and through the down-modulation of CAF phenotype suggests their use as lead compounds for co-targeting anti-cancer strategies.

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.

HOPPI-NMR: Hot-Peptide-Based Screening Assay for Inhibitors of Protein-Protein Interactions by NMR.

Protein-protein interactions (PPIs) contribute to the onset and/or progression of several diseases, especially cancer, and this discovery has paved the way for considering disruption of the PPIs as an attractive anti-tumor strategy. In this regard, simple and efficient biophysical methods for detecting the interaction of the inhibitors with the protein counterpart are still in high demand. Herein, we describe a convenient NMR method for the screening of putative PPI inhibitors based on the use of "hot peptides" (HOPPI-NMR). As a case study, HOPPI-NMR was successful applied to the well-known p53/MDM2 system. Our outcomes highlight the main advantages of the method, including the use of a small amount of unlabeled proteins, the minimization of the risk of protein aggregation, and the ability to identify weak binders. The last leaves open the possibility for application of HOPPI-NMR in tandem with fragment-based drug discovery as a valid strategy for the identification of novel chemotypes acting as PPI inhibitors.

Urotensin II receptor expression in patients with ulcerative colitis: a pilot study.

BACKGROUND: Urotensin II (U-II) is a vasoactive peptide that interacts with a specific receptor named UTR. Recently, our group has demonstrated increased UTR expression in both human colon adenocarcinoma cell lines and adenomatous polyps, as well as in colon carcinoma samples if compared to healthy colon samples of the same patients. We also showed that an UTR agonist induced an increase in colon adenocarcinoma cell growth in vitro, whereas the UTR block with a specific antagonist caused an inhibition of their growth and an inhibition of about 50% of both motility and cell invasion. Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) associated with an increased baseline risk for colon cancer compared with the general population, and this risk is mostly attributed to chronic inflammation and immune dysregulation. This risk increases along with the duration of the disease, as demonstrated by many studies. There are no UTR expression data related to UC, and we therefore evaluated UTR expression in ill colon biopsies and in healthy colon ones of patients with UC and colon biopsies of healthy patients. METHODS: We enrolled, prior to informed consent, 11 patients (5 males and 6 females, age range 29-75 years, median age 52 years) with first UC diagnosis compared to 11 healthy controls (6 males and 5 females, age range 30-78 years, median age 55 years). We have therefore sampled inflammatory and healthy tissue in UC patients. We have also taken colic tissue samples in healthy subjects. Evaluation of receptor expression was performed by reverse transcription-polymerase chain reaction (RT-PCR), Western Blot analysis. The ANOVA Test (P<0.05) was used for statistical analysis. RESULTS: We found: 1) increased expression of UTR in 11/11 UC patients with ill mucosa biopsies compared to healthy controls in RT-PCR and in Western Blot analysis; 2) increased UTR expression in 11/11 UC patients with ill colon biopsies compared to the results obtained from healthy colon biopsies of the same patients both in RT-PCR and in Western Blot analysis; 3) increased UTR expression in 9/11 UC patients healthy colon biopsy specimens compared to healthy controls. CONCLUSIONS: UTR could be considered as an inflammatory UC disease marker because its expression is greater in the mucosa of ill colon than in the healthy colon of the same patients and compared to healthy controls.

Intestinal Anti-Inflammatory Effect of a Peptide Derived from Gastrointestinal Digestion of Buffalo (Bubalus bubalis) Mozzarella Cheese.

Under physiological conditions, the small intestine represents a barrier against harmful antigens and pathogens. Maintaining of the intestinal barrier depends largely on cell⁻cell interactions (adherent-junctions) and cell⁻matrix interactions (tight-junctions). Inflammatory bowel disease is characterized by chronic inflammation, which induces a destructuring of the architecture junctional epithelial proteins with consequent rupture of the intestinal barrier. Recently, a peptide identified by Bubalus bubalis milk-derived products (MBCP) has been able to reduce oxidative stress in intestinal epithelial cells and erythrocytes. Our aim was to evaluate the therapeutic potential of MBCP in inflammatory bowel disease (IBD). We studied the effect of MBCP on (i) inflamed human intestinal Caco2 cells and (ii) dinitrobenzene sulfonic acid (DNBS) mice model of colitis. We have shown that MBCP, at non-cytotoxic concentrations, both in vitro and in vivo induced the adherent epithelial junctions organization, modulated the nuclear factor (NF)-κB pathway and reduced the intestinal permeability. Furthermore, the MBCP reverted the atropine and tubocurarine injury effects on adherent-junctions. The data obtained showed that MBCP possesses anti-inflammatory effects both in vitro and in vivo. These results could have an important impact on the therapeutic potential of MBCP in helping to restore the intestinal epithelium integrity damaged by inflammation.

Functional Selectivity Revealed by N-Methylation Scanning of Human Urotensin II and Related Peptides.

In accordance with their common but also divergent physiological actions, human urotensin II (1) and urotensin II-related peptide (2) could stabilize specific urotensin II receptor (UTR) conformations, thereby activating different signaling pathways, a feature referred to as biased agonism or functional selectivity. Sequential N-methylation of the amides in the conserved core sequence of 1, 2, and fragment U-II4-11 (3) shed light on structural requirements involved in their functional selectivity. Thus, 18 N-methylated UTR ligands were synthesized and their biological profiles evaluated using in vitro competition binding assays, ex vivo rat aortic ring bioassays and BRET-based biosensor experiments. Biological activity diverged from that of the parent structures contingent on the location of amide methylation, indicating relevant hydrogen-bond interactions for the function of the endogenous peptides. Conformational analysis of selected N-methyl analogs indicated the importance of specific amide residues of 2 for the distinct pharmacology relative to 1 and 3.

Urotensin-II-Targeted Liposomes as a New Drug Delivery System towards Prostate and Colon Cancer Cells.

Urotensin-II (UT-II) and its receptor (UTR) are involved in the occurrence of different epithelial cancers. In particular, UTR was found overexpressed on colon, bladder, and prostate cancer cells. The conjugation of ligands, able to specifically bind receptors that are overexpressed on cancer cells, to liposome surface represents an efficient active targeting strategy to enhance selectivity and efficiency of drug delivery systems. The aim of this study was to develop liposomes conjugated with UT-II (LipoUT) for efficient targeting of cancer cells that overexpress UTR. The liposomes had a mean diameter between 150 nm and 160 nm with a narrow size distribution (PI ≤ 0.1) and a doxo encapsulation efficiency of 96%. Moreover, the conjugation of UT-II to liposomes weakly reduced the zeta potential. We evaluated UTR expression on prostate (DU145, PC3, and LNCaP) and colon (WIDR and LoVo) cancer cells by FACS and western blotting analysis. UTR protein was expressed in all the tested cell lines; the level of expression was higher in WIDR, PC3, and LNCaP cells compared with LoVo and DU145. MTT cell viability assay showed that LipoUT-doxo was more active than Lipo-doxo on the growth inhibition of cells that overexpressed UTR (PC3, LNCaP, and WIDR) while in LoVo and DU145 cell lines, the activity was similar to or lower than that one of Lipo-doxo, respectively. Moreover, we found that cell uptake of Bodipy-labeled liposomes in PC3 and DU145 was higher for LipoUT than the not-armed counterparts but at higher extent in UTR overexpressing PC3 cells (about 2-fold higher), as evaluated by both confocal and FACS. In conclusion, the encapsulation of doxo in UT-II-targeted liposomes potentiated its delivery in UTR-overexpressing cells and could represent a new tool for the targeting of prostate and colon cancer.

Simultaneous Targeting of RGD-Integrins and Dual Murine Double Minute Proteins in Glioblastoma Multiforme.

In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5ß1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4. Herein, we present the discovery of compound 7, which inhibits both MDM2/4 and α5ß1/αvß3 integrins. A lead optimization campaign was carried out on 7 with the aim to preserve the activities on integrins while improving those on MDM proteins. Compound 9 turned out to be a potent MDM2/4 and α5ß1/αvß3 blocker. In p53-wild type glioma cells, 9 arrested cell cycle and proliferation and strongly reduced cell invasiveness, emerging as the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in subpopulations of patients with glioblastoma expressing a functional p53 concomitantly with a high level of α5ß1 integrin.

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.