Publications by authors named "Alfonso Carotenuto"

81 Publications

Synthesis and Characterization of Bis-Triazolyl-Pyridine Derivatives as Noncanonical DNA-Interacting Compounds.

Int J Mol Sci 2021 Nov 4;22(21). Epub 2021 Nov 4.

Department of Pharmacy, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy.

Besides the well-known double-helical conformation, DNA is capable of folding into various noncanonical arrangements, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. Particularly, previous studies have reported that G4 and iM structures may play different roles in controlling gene transcription. Anyway, molecular tools able to simultaneously stabilize/destabilize those structures are still needed to shed light on what happens at the biological level. Herein, a multicomponent reaction and a click chemistry functionalization were combined to generate a set of 31 bis-triazolyl-pyridine derivatives which were initially screened by circular dichroism for their ability to interact with different G4 and/or iM DNAs and to affect the thermal stability of these structures. All the compounds were then clustered through multivariate data analysis, based on such capability. The most promising compounds were subjected to a further biophysical and biological characterization, leading to the identification of two molecules simultaneously able to stabilize G4s and destabilize iMs, both in vitro and in living cells.
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http://dx.doi.org/10.3390/ijms222111959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8584640PMC
November 2021

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands.

J Biol Chem 2021 09 10;297(3):101057. Epub 2021 Aug 10.

Institut National de la Recherche Scientifique, Centre Armand-Frappier Santé Biotechnologie, Groupe de Recherche en Ingénierie des Peptides et en Pharmacothérapie (GRIPP), Université du Québec, Ville de Laval, Québec, Canada. Electronic address:

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp, Leu]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp, Leu]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK phosphorylation and, to a lesser extent, IP production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.
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http://dx.doi.org/10.1016/j.jbc.2021.101057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8424217PMC
September 2021

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

J Med Chem 2021 08 23;64(15):11675-11694. Epub 2021 Jul 23.

Department of Pharmacy, University of Naples "Federico II", Naples 80131, Italy.

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.
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http://dx.doi.org/10.1021/acs.jmedchem.1c01033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8389922PMC
August 2021

A novel β-hairpin peptide derived from the ARC repressor selectively interacts with the major groove of B-DNA.

Bioorg Chem 2021 07 22;112:104836. Epub 2021 Mar 22.

Department of Pharmacy, University of Chieti-Pescara "G. d'Annunzio", Via dei Vestini 31, 66100 Chieti, Italy. Electronic address:

Transcription factors (TFs) have a remarkable role in the homeostasis of the organisms and there is a growing interest in how they recognize and interact with specific DNA sequences. TFs recognize DNA using a variety of structural motifs. Among those, the ribbon-helix-helix (RHH) proteins, exemplified by the MetJ and ARC repressors, form dimers that insert antiparallel β-sheets into the major groove of DNA. A great chemical challenge consists of using the principles of DNA recognition by TFs to design minimized peptides that maintain the DNA affinity and specificity characteristics of the natural counterparts. In this context, a peptide mimic of an antiparallel β-sheet is very attractive since it can be obtained by a single peptide chain folding in a β-hairpin structure and can be as short as 14 amino acids or less. Herein, we designed eight linear and two cyclic dodeca-peptides endowed with β-hairpins. Their DNA binding properties have been investigated using fluorescence spectroscopy together with the conformational analysis through circular dichroism and solution NMR. We found that one of our peptides, peptide 6, is able to bind DNA, albeit without sequence selectivity. Notably, it shows a topological selectivity for the major groove of the DNA which is the interaction site of ARC and many other DNA-binding proteins. Moreover, we found that a type I' β-hairpin folding pattern is a favorite peptide structure for interaction with the B-DNA major groove. Peptide 6 is a valuable lead compound for the development of novel analogs with sequence selectivity.
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http://dx.doi.org/10.1016/j.bioorg.2021.104836DOI Listing
July 2021

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

Cancers (Basel) 2020 Aug 24;12(9). Epub 2020 Aug 24.

Institute of Genetics and Biophysics "Adriano Buzzati Traverso", National Research Council, 80131 Naples, Italy.

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 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.
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http://dx.doi.org/10.3390/cancers12092404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564779PMC
August 2020

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.

Chemistry 2020 Aug 20;26(44):10113-10125. Epub 2020 Jul 20.

DiSTABiF, University of Campania "Luigi Vanvitelli", Via Vivaldi 43, 81100, Caserta, Italy.

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.
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http://dx.doi.org/10.1002/chem.202002468DOI Listing
August 2020

Antimicrobial peptide Temporin-L complexed with anionic cyclodextrins results in a potent and safe agent against sessile bacteria.

Int J Pharm 2020 Jun 21;584:119437. Epub 2020 May 21.

Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Naples, Italy. Electronic address:

Concern over antibiotic resistance is growing, and new classes of antibiotics, particularly against Gram-negative bacteria, are needed. Antimicrobial peptides (AMPs) have been proposed as a new class of clinically useful antimicrobials. Special attention has been devoted to frog-skin temporins. In particular, temporin L (TL) is strongly active against Gram-positive, Gram-negative bacteria and yeast strains. With the aim of overcoming some of the main drawbacks preventing the widespread clinical use of this peptide, i.e. toxicity and unfavorable pharmacokinetics profile, we designed new formulations combining TL with different types of cyclodextrins (CDs). TL was associated to a panel of neutral or negatively charged, monomeric and polymeric CDs. The impact of CDs association on TL solubility, as well as the transport through bacterial alginates was assessed. The biocompatibility on human cells together with the antimicrobial and antibiofilm properties of TL/CD systems was explored.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119437DOI Listing
June 2020

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

ACS Med Chem Lett 2020 May 20;11(5):1047-1053. Epub 2020 Feb 20.

Department of Pharmacy, University of Naples "Federico II", 80131 Naples, Italy.

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.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236535PMC
May 2020

Constrained Peptides with Fine-Tuned Flexibility Inhibit NF-Y Transcription Factor Assembly.

Angew Chem Int Ed Engl 2019 11 17;58(48):17351-17358. Epub 2019 Oct 17.

Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, 44227, Dortmund, Germany.

Protein complex formation depends on the interplay between preorganization and flexibility of the binding epitopes involved. The design of epitope mimetics typically focuses on stabilizing a particular bioactive conformation, often without considering conformational dynamics, which limits the potential of peptidomimetics against challenging targets such as transcription factors. We developed a peptide-derived inhibitor of the NF-Y transcription factor by first constraining the conformation of an epitope through hydrocarbon stapling and then fine-tuning its flexibility. In the initial set of constrained peptides, a single non-interacting α-methyl group was observed to have a detrimental effect on complex stability. Biophysical characterization revealed how this methyl group affects the conformation of the peptide in its bound state. Adaption of the methylation pattern resulted in a peptide that inhibits transcription factor assembly and subsequent recruitment to the target DNA.
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http://dx.doi.org/10.1002/anie.201907901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900064PMC
November 2019

The Outcomes of Decorated Prolines in the Discovery of Antimicrobial Peptides from Temporin-L.

ChemMedChem 2019 07 3;14(13):1283-1290. Epub 2019 Jun 3.

Department of Pharmacy, University of Naples "Federico II", Naples, 80131, Italy.

Previously, we identified a potent antimicrobial analogue of temporin L (TL), [Pro ]TL, in which glutamine at position 3 was substituted with proline. In this study, a series of analogues in which position 3 is substituted with non-natural proline derivatives, was investigated for correlations between the conformational properties of the compounds and their antibacterial, cytotoxic, and hemolytic activities. Non-natural proline analogues with substituents at position 4 of the pyrrolidine ring were considered. Structure-activity relationship (SAR) studies of these analogues were performed by means of antimicrobial and cytotoxicity assays along with circular dichroism (CD) and NMR spectroscopic analyses for selected compounds. The most promising peptides were additionally evaluated for their activity against some representative veterinary microbial strains to compare with those from human strains. We identified novel analogues with interesting properties that make them attractive lead compounds.
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http://dx.doi.org/10.1002/cmdc.201900221DOI Listing
July 2019

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

J Med Chem 2019 02 23;62(3):1455-1467. Epub 2019 Jan 23.

Department of Pharmacy , University of Naples "Federico II" , via D. Montesano 49 , Naples 80131 , Italy.

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-II (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.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01601DOI Listing
February 2019

Studies of membranotropic and fusogenic activity of two putative HCV fusion peptides.

Biochim Biophys Acta Biomembr 2019 01 19;1861(1):50-61. Epub 2018 Oct 19.

Laboratoire de Chimie Biologique, University of Cergy-Pontoise, 5 mail Gay-Lussac, Cergy-Pontoise, France. Electronic address:

Over the past decades, membranotropic peptides such as positively charged cell-penetrating peptides (CPPs) or amphipathic antimicrobial peptides (AMPs) have received increasing interest in order to improve therapeutic agent cellular uptake. As far as we are concerned, we were interested in studying HCV fusion peptides as putative anchors. Two peptides, HCV6 and HCV7, were identified and conjugated to a fluorescent tag NBD and tested for their interaction with liposomes as model membranes. DSC and spectrofluorescence analyses demonstrate HCV7 propensity to insert or internalize in vesicles containing anionic lipids DMPG whereas no activity was observed with zwitterionic DMPC. This behavior could be explained by the peptide sequence containing a cationic arginine residue. On the contrary, HCV6 did not exhibit any membranotropic activity but was the only sequence able to induce liposomes' fusion or aggregation monitored by spectrofluorescence and DLS. This two peptides mild activity was related to their inefficient structuration in contact with membrane mimetics, which was demonstrated by CD and NMR experiments. Altogether, our data allowed us to identify two promising membrane-active peptides from E1 and E2 HCV viral proteins, one fusogenic (HCV6) and the other membranotropic (HCV7). The latter was also confirmed by fluorescence microscopy with CHO cells, indicating that HCV7 could cross the plasma membrane via an endocytosis process. Therefore, this study provides new evidences supporting the identification of HCV6 as the HCV fusion peptide as well as insights on a novel membranotropic peptide from the HCV-E2 viral protein.
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http://dx.doi.org/10.1016/j.bbamem.2018.10.011DOI Listing
January 2019

Investigation on side-product formation during the synthesis of a lactoferrin-derived lactam-bridged cyclic peptide.

Amino Acids 2018 Oct 4;50(10):1367-1375. Epub 2018 Jul 4.

Dipartimento di Farmacia, Università degli Studi di Salerno, 84084, Fisciano, SA, Italy.

Bovine lactoferrin C-lobe is able to prevent both influenza virus hemagglutination and cell infection. In particular, it was demonstrated that the fragment SKHSSLDCVLRP is a potent antiviral peptide. Therefore, we tried to increase the stability of this fragment through side-chain lactam cyclization of the peptide, S[KHSSLD]CVLRP (1). However, classic strategy involving solid-supported cyclization of the linear precursor, containing orthogonal allyl/alloc-based protection for the key amino and carboxyl residues, did not provide the desired cyclic peptide. Here, we report the identification of problematic stretches during the sequence assembly process and the optimization of the different parameters involved in the construction of 1. Results indicated a significant influence of β-protecting group of both aspartic acid and adjacent cysteine residues on the formation of side products. Therefore, the identification of suitable β-protecting groups of these residues allowed us to optimize the synthesis of designed lactam-bridged cyclic peptide.
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http://dx.doi.org/10.1007/s00726-018-2612-9DOI Listing
October 2018

Development of Macrocyclic Peptidomimetics Containing Constrained α,α-Dialkylated Amino Acids with Potent and Selective Activity at Human Melanocortin Receptors.

J Med Chem 2018 05 25;61(9):4263-4269. Epub 2018 Apr 25.

Department of Chemistry and Biochemistry , University of Arizona , 1306 E. University Boulevard , Tucson , Arizona 85721 , United States.

We report the development of macrocyclic melanocortin derivatives of MT-II and SHU-9119, achieved by modifying the cycle dimension and incorporating constrained amino acids in ring-closing. This study culminated in the discovery of novel agonists/antagonists with an unprecedented activity profile by adding pieces to the puzzle of the melanocortin receptor selectivity. Finally, the resulting 19- and 20-membered rings represent a suitable frame for the design of further therapeutic ligands as selective modulators of the melanocortin system.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5999404PMC
May 2018

Ligand-Based NMR Study of C-X-C Chemokine Receptor Type 4 (CXCR4)-Ligand Interactions on Living Cancer Cells.

J Med Chem 2018 04 19;61(7):2910-2923. Epub 2018 Mar 19.

Dipartimento di Farmacia, Università di Napoli Federico II , 80131 Naples , Italy.

Peptide-binding G protein-coupled receptors (GPCRs) are key effectors in numerous pathological and physiological pathways. The assessment of the receptor-bound conformation of a peptidic ligand within a membrane receptor such as a GPCR is of great impact for a rational drug design of more potent analogues. In this work, we applied multiple ligand-based nuclear magnetic resonance (NMR) methods to study the interaction of peptide heptamers, derived from the C-X-C Motif Chemokine 12 (CXCL12), and the C-X-C Chemokine Receptor Type 4 (CXCR4) on membranes of human T-Leukemia cells (CCRF-CEM cells). This study represents the first structural investigation reporting the receptor-bound conformation of a peptide to a GPCR directly on a living cell. The results obtained in the field of CXCL12/CXCR4 are proofs of concept, although important information for researchers dealing with the CXCR4 field arises. General application of the presented NMR methodologies is possible and surely may help to boost the development of new therapeutic agents targeting GPCRs.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01830DOI Listing
April 2018

Urokinase receptor derived peptides as potent inhibitors of the formyl peptide receptor type 1-triggered cell migration.

Eur J Med Chem 2018 Jan 1;143:348-360. Epub 2017 Dec 1.

Department of Pharmacy, University of Naples 'Federico II', Naples 80131, Italy; Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPEB) University of Naples "Federico II" and DFM-Scarl, Institute of Biostructures and Bioimaging - CNR Via Mezzocannone 16, 80134 Naples, Italy.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration. We and others have previously documented that the uPAR(84-95) sequence, interacts with the formyl peptide receptors (FPR)s, henceforth inducing cell migration of several cell lines, including leukocytes, and the synthetic shorter peptide (Ser-Arg-Ser-Arg-Tyr, SRSRY) retains chemotactic activity in vitro and in vivo. Recently, we have developed the head-to-tail cyclic analog [SRSRY], a new potent and stable inhibitor of monocyte trafficking. This prompted us to develop novel cyclic and linear analogs of [SRSRY] with the aim to broaden the knowledge about structure-activity relationships of peptide [SRSRY]. Herein we report their synthesis, effects on cell migration, conformational and docking analyses which served to envisage a new pharmacophore model for inhibitors of FPR1-triggered cell migration.
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http://dx.doi.org/10.1016/j.ejmech.2017.11.030DOI Listing
January 2018

Structure-Activity Relationships and Biological Characterization of a Novel, Potent, and Serum Stable C-X-C Chemokine Receptor Type 4 (CXCR4) Antagonist.

J Med Chem 2017 12 20;60(23):9641-9652. Epub 2017 Nov 20.

Department of Pharmacy, University of Naples "Federico II" , Via D. Montesano 49, 80131 Naples, Italy.

In our ongoing pursuit of CXCR4 antagonists as potential anticancer agents, we recently developed a potent, selective, and plasma stable peptide, Ac-Arg-Ala-[d-Cys-Arg-Phe-Phe-Cys]-COOH (3). Nevertheless, this compound was still not potent enough (IC ≈ 53 nM) to enter preclinical studies. Thus, a lead-optimization campaign was here undertaken to further improve the binding affinity of 3 while preserving its selectivity and proteolytic stability. Specifically, extensive structure-activity relationships (SARs) investigations were carried out on both its aromatic and disulfide forming amino acids. One among the synthesized analogue, Ac-Arg-Ala-[d-Cys-Arg-Phe-His-Pen]-COOH (19), displayed subnanomolar affinity toward CXCR4, with a marked selectivity over CXCR3 and CXCR7. NMR and molecular modeling studies disclosed the molecular bases for the binding of 19 to CXCR4 and for its improved potency compared to the lead 3. Finally, biological assays on specific cancer cell lines showed that 19 can impair CXCL12-mediated cell migration and CXCR4 internalization more efficiently than the clinically approved CXCR4 antagonist plerixafor.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01062DOI Listing
December 2017

Glycine-replaced derivatives of [Pro,DLeu]TL, a temporin L analogue: Evaluation of antimicrobial, cytotoxic and hemolytic activities.

Eur J Med Chem 2017 Oct 21;139:750-761. Epub 2017 Aug 21.

Department of Pharmacy, University of Naples 'Federico II', Naples, 80131, Italy. Electronic address:

In this study we designed and synthesized a new library of antimicrobial peptides correlated to [Pro,DLeu]TL 1, a temporin L derivative devoid of cytolytic effects in vitro, and investigated the correlation between the α-helical content of the compounds and their antibacterial, cytotoxic and hemolytic activities. We systematically replaced Gly in position 10 of reference peptide with several amino acids. Structure-activity relationship studies of these analogues were performed by means of antimicrobial and cytotoxicity assays along with CD spectroscopy analyses. NMR analysis was also accomplished for compound 10. As well, the most promising peptides were additionally evaluated for their activity against some clinical strains isolated from human skin and for their mechanism of action by studying the kinetics of membrane perturbation of some representative microbial strains. We identified novel analogues with interesting properties that make them attractive lead compounds for potential topical applications.
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http://dx.doi.org/10.1016/j.ejmech.2017.08.040DOI Listing
October 2017

Cyclic Biphalin Analogues Incorporating a Xylene Bridge: Synthesis, Characterization, and Biological Profile.

ACS Med Chem Lett 2017 Aug 12;8(8):858-863. Epub 2017 Jul 12.

Dipartimento di Farmacia, Università di Chieti-Pescara "G. d'Annunzio", Via dei Vestini 31, 66100 Chieti, Italy.

In this work we enhanced the ring lipophilicity of biphalin introducing a xylene moiety, thus obtaining three cyclic regioisomers. Novel compounds have similar activity as the parent compound, but one of these () shows a remarkable increase of antinociceptive effect. Nociception tests have disclosed its significant high potency and the more prolonged effect in eliciting analgesia, higher than that of biphalin and of the disulfide-bridge-containing analogue ().
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http://dx.doi.org/10.1021/acsmedchemlett.7b00210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554896PMC
August 2017

Structure-Activity Relationship Studies, SPR Affinity Characterization, and Conformational Analysis of Peptides That Mimic the HNK-1 Carbohydrate Epitope.

ChemMedChem 2017 05 3;12(10):751-759. Epub 2017 May 3.

Laboratory of Peptide and Protein Chemistry and Biology, PeptLab, Via della Lastruccia 13, 50019, Sesto Fiorentino, Italy.

The design of molecules that mimic biologically relevant glycans is a significant goal for understanding important biological processes and may lead to new therapeutic and diagnostic agents. In this study we focused our attention on the trisaccharide human natural killer cell-1 (HNK-1), considered the antigenic determinant of myelin-associated glycoprotein and the target of clinically relevant auto-antibodies in autoimmune neurological disorders such as IgM monoclonal gammopathy and demyelinating polyneuropathy. We describe a structure-activity relationship study based on surface plasmon resonance binding affinities aimed at the optimization of a peptide that mimics the HNK-1 minimal epitope. We developed a cyclic heptapeptide that shows an affinity of 1.09×10  m for a commercial anti-HNK1 mouse monoclonal antibody. Detailed conformational analysis gave possible explanations for the good affinity displayed by this novel analogue, which was subsequently used as an immunological probe. However, preliminary screening indicates that patients' sera do not specifically recognize this peptide, showing that murine monoclonal antibodies cannot be used as a guide to select immunological probes for the detection of clinically relevant human auto-antibodies.
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http://dx.doi.org/10.1002/cmdc.201700042DOI Listing
May 2017

Structure- and conformation-activity studies of nociceptin/orphanin FQ receptor dimeric ligands.

Sci Rep 2017 04 6;7:45817. Epub 2017 Apr 6.

Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, 44121 Ferrara, Italy.

The peptide nociceptin/orphanin FQ (N/OFQ) and the N/OFQ receptor (NOP) constitute a neuropeptidergic system that modulates various biological functions and is currently targeted for the generation of innovative drugs. In the present study dimeric NOP receptor ligands with spacers of different lengths were generated using both peptide and non-peptide pharmacophores. The novel compounds (12 peptide and 7 nonpeptide ligands) were pharmacologically investigated in a calcium mobilization assay and in the mouse vas deferens bioassay. Both structure- and conformation-activity studies were performed. Results demonstrated that dimerization did not modify the pharmacological activity of both peptide and non-peptide pharmacophores. Moreover, when dimeric compounds were obtained with low potency peptide pharmacophores, dimerization recovered ligand potency. This effect depends on the doubling of the C-terminal address sequence rather than the presence of an additional N-terminal message sequence or modifications of peptide conformation.
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http://dx.doi.org/10.1038/srep45817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382891PMC
April 2017

DOTA-Derivatives of Octreotide Dicarba-Analogs with High Affinity for Somatostatin sst Receptors.

Front Chem 2017 23;5. Epub 2017 Feb 23.

Department of Pharmacy, University of Naples "Federico II" Naples, Italy.

somatostatin receptor scintigraphy is a valuable method for the visualization of human endocrine tumors and their metastases. In fact, peptide ligands of somatostatin receptors (sst's) conjugated with chelating agents are in clinical use. We have recently developed octreotide dicarba-analogs, which show interesting binding profiles at sst's. In this context, it was mandatory to explore the possibility that our analogs could maintain their activity also upon conjugation with DOTA. In this paper, we report and discuss the synthesis, binding affinity and conformational preferences of three DOTA-conjugated dicarba-analogs of octreotide. Interestingly, two conjugated analogs exhibited nanomolar affinities on sst and sst somatostatin receptor subtypes.
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http://dx.doi.org/10.3389/fchem.2017.00008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324734PMC
February 2017

Exploring the N-Terminal Region of C-X-C Motif Chemokine 12 (CXCL12): Identification of Plasma-Stable Cyclic Peptides As Novel, Potent C-X-C Chemokine Receptor Type 4 (CXCR4) Antagonists.

J Med Chem 2016 Sep 9;59(18):8369-80. Epub 2016 Sep 9.

Dipartimento di Farmacia, Università degli Studi di Napoli "Federico II" , via D. Montesano 49, 80131 Naples, Italy.

We previously reported the discovery of a CXCL12-mimetic cyclic peptide (2) as a selective CXCR4 antagonist showing promising in vitro and in vivo anticancer activity. However, further development of this peptide was hampered by its degradation in biological fluids as well as by its low micromolar affinity for the receptor. Herein, extensive chemical modifications led to the development of a new analogue (10) with enhanced potency, specificity, and plasma stability. A combined approach of Ala-amino acid scan, NMR, and molecular modeling unraveled the reasons behind the improved binding properties of 10 vs 2. Biological investigations on leukemia (CEM) and colon (HT29 and HCT116) cancer cell lines showed that 10 is able to impair CXCL12-mediated cell migration, ERK-phosphorylation, and CXCR4 internalization. These outcomes might pave the way for the future preclinical development of 10 in CXCR4 overexpressing leukemia and colon cancer.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00695DOI Listing
September 2016

Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase.

Front Microbiol 2016 10;7:845. Epub 2016 Jun 10.

Department of Pharmacy, Medicicnal Chemistry and Toxicologic, University of Naples Federico II Napoli, Italy.

The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1-50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1.
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http://dx.doi.org/10.3389/fmicb.2016.00845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901077PMC
July 2016

Structure-Activity Study of the Peptides P5U and Urantide by the Development of Analogues Containing Uncoded Amino Acids at Position 9.

ChemMedChem 2016 08 9;11(16):1856-64. Epub 2016 Apr 9.

Department of Pharmacy, University of Naples "Federico II", via D. Montesano 49, 80131, Naples, Italy.

Previous modifications of the peptide sequence of human urotensin-II (U-II) led to the identification of two well-known ligands: P5U and urantide. These derivatives are considered to be the most representative agonist and antagonist, respectively, at the human urotensin receptor (UT). Optimization of P5U and urantide was carried out to stabilize specific conformations that may suggest new elements for discriminating agonist versus antagonist activity. We studied novel derivatives containing uncoded amino acids. In particular, the Tyr(9) residue of both P5U and urantide was replaced with nonaromatic hydrophobic bulky residues, as well as conformationally constrained aromatic moieties to generate eight novel derivatives. These analogues further contributed to determining the influence of such residues on binding affinity for and biological activity at UT. One of these eight peptides was also investigated by NMR spectroscopy and docking studies owing to its peculiar conformational properties and mode of interaction with UT. This structure-activity study is aimed at a more thorough examination of the role of tyrosine in modulating the agonism/antagonism of human U-II.
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http://dx.doi.org/10.1002/cmdc.201500607DOI Listing
August 2016

Screening Platform toward New Anti-HIV Aptamers Set on Molecular Docking and Fluorescence Quenching Techniques.

Anal Chem 2016 Feb 5;88(4):2327-34. Epub 2016 Feb 5.

University of Naples Federico II , Department of Pharmacy, Napoli, 80131, Italy.

By using a new rapid screening platform set on molecular docking simulations and fluorescence quenching techniques, three new anti-HIV aptamers targeting the viral surface glycoprotein 120 (gp120) were selected, synthesized, and assayed. The use of the short synthetic fluorescent peptide V35-Fluo mimicking the V3 loop of gp120, as the molecular target for fluorescence-quenching binding affinity studies, allowed one to measure the binding affinities of the new aptamers for the HIV-1 gp120 without the need to obtain and purify the full recombinant gp120 protein. The almost perfect correspondence between the calculated Kd and the experimental EC50 on HIV-infected cells confirmed the reliability of the platform as an alternative to the existing methods for aptamer selection and measuring of aptamer-protein equilibria.
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http://dx.doi.org/10.1021/acs.analchem.5b04268DOI Listing
February 2016

An ancestral host defence peptide within human β-defensin 3 recapitulates the antibacterial and antiviral activity of the full-length molecule.

Sci Rep 2015 Dec 21;5:18450. Epub 2015 Dec 21.

CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Napoli, Italy.

Host defence peptides (HDPs) are critical components of innate immunity. Despite their diversity, they share common features including a structural signature, designated "γ-core motif". We reasoned that for each HDPs evolved from an ancestral γ-core, the latter should be the evolutionary starting point of the molecule, i.e. it should represent a structural scaffold for the modular construction of the full-length molecule, and possess biological properties. We explored the γ-core of human β-defensin 3 (HBD3) and found that it: (a) is the folding nucleus of HBD3; (b) folds rapidly and is stable in human serum; (c) displays antibacterial activity; (d) binds to CD98, which mediates HBD3 internalization in eukaryotic cells; (e) exerts antiviral activity against human immunodeficiency virus and herpes simplex virus; and (f) is not toxic to human cells. These results demonstrate that the γ-core within HBD3 is the ancestral core of the full-length molecule and is a viable HDP per se, since it is endowed with the most important biological features of HBD3. Notably, the small, stable scaffold of the HBD3 γ-core can be exploited to design disease-specific antimicrobial agents.
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http://dx.doi.org/10.1038/srep18450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4685272PMC
December 2015

Discovery of Novel Potent and Selective Agonists at the Melanocortin-3 Receptor.

J Med Chem 2015 Dec 16;58(24):9773-8. Epub 2015 Dec 16.

Dipartimento di Farmacia, Università di Napoli Federico II , Via D. Montesano, 49, 80131, Naples, Italy.

The melanocortin receptors 3 and 4 control energy homeostasis, food-intake behavior, and correlated pathophysiological conditions. The melanocortin-4 receptor (MC4R) has been broadly investigated. In contrast, the knowledge related to physiological roles of the melanocortin-3 receptor (MC3R) is lacking because of the limited number of known MC3R selective ligands. Here, we report the design, synthesis, biological activity, conformational analysis, and docking with receptors of two potent and selective agonists at the human MC3 receptor.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070805PMC
http://dx.doi.org/10.1021/acs.jmedchem.5b01285DOI Listing
December 2015

Nondenaturing polyacrylamide gel electrophoresis to study the dissociation of the p53·MDM2/X complex by potentially anticancer compounds.

Electrophoresis 2015 Dec 30;36(24):3101-4. Epub 2015 Oct 30.

Department of Movement Sciences and Wellness, University of Naples "Parthenope", Naples, Italy.

A new analytical method to study the dissociation of the complexes between the oncosuppressor p53 and its negative modulators murine double-minute protein 2 (MDM2) or MDMX, is proposed. This technique is reliable to determine the dissociative power exerted by small molecules on the complex taking advantage of the appearance of migrating MDM2 or MDMX in a native polyacrylamide gel, when inhibitors are added to the complex mixture. Therefore, we propose this new approach to easily screen library of compounds, with potential pharmacological anticancer activity.
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http://dx.doi.org/10.1002/elps.201500305DOI Listing
December 2015

Role of Lipoylation of the Immunodominant Epitope of Pyruvate Dehydrogenase Complex: Toward a Peptide-Based Diagnostic Assay for Primary Biliary Cirrhosis.

J Med Chem 2015 Aug 6;58(16):6619-29. Epub 2015 Aug 6.

Department of Chemistry "Ugo Schiff", University of Florence , Via della Lastruccia 13, 50019 Sesto Fiorentino, Italy.

Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00783DOI Listing
August 2015
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