Publications by authors named "Salvatore Di Maro"

50 Publications

A novel smaller β-defensin-derived peptide is active against multidrug-resistant bacterial strains.

FASEB J 2021 12;35(12):e22026

Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy.

Antibiotic resistance is becoming a severe obstacle in the fight against acute and chronic infectious diseases that accompany most degenerative illnesses from neoplasia to osteo-arthritis and obesity. Currently, the race is on to identify pharmaceutical molecules or combinations of molecules able to prevent or reduce the insurgence and/or progression of infectivity. Attempts to substitute antibiotics with antimicrobial peptides have, thus far, met with little success against multidrug-resistant (MDR) bacterial strains. During the last decade, we designed and studied the activity and features of human β-defensin analogs, which are salt-resistant, and hence active also under high salt concentrations as, for instance, in cystic fibrosis. Herein, we describe the design, synthesis, and major features of a new 21 aa long molecule, peptide γ2. The latter derives from the γ-core of the β-defensin natural molecules, a small fragment of these molecules still bearing high antibacterial activity. We found that peptide γ2, which contains only one disulphide bond, recapitulates most of the biological properties of natural human β-defensins and can also counteract both Gram-positive and Gram-negative MDR bacterial strains and biofilm formation. Moreover, it has great stability in human serum thereby enhancing its antibacterial presence and activity without cytotoxicity in human cells. In conclusion, peptide γ2 is a promising new weapon also in the battle against intractable infectious diseases.
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http://dx.doi.org/10.1096/fj.202002330RRDOI Listing
December 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

Halting the Spread of Herpes Simplex Virus-1: The Discovery of an Effective Dual αvβ6/αvβ8 Integrin Ligand.

J Med Chem 2021 05 7;64(10):6972-6984. Epub 2021 May 7.

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

Over recent years, αvβ6 and αvβ8 Arg-Gly-Asp (RGD) integrins have risen to prominence as interchangeable co-receptors for the cellular entry of herpes simplex virus-1 (HSV-1). In fact, the employment of subtype-specific integrin-neutralizing antibodies or gene-silencing siRNAs has emerged as a valuable strategy for impairing HSV infectivity. Here, we shift the focus to a more affordable pharmaceutical approach based on small RGD-containing cyclic pentapeptides. Starting from our recently developed αvβ6-preferential peptide [RGD-Chg-E]-CONH (), a small library of N-methylated derivatives (-) was indeed synthesized in the attempt to increase its affinity toward αvβ8. Among the novel compounds, [RGD-Chg-(Me)E]-CONH () turned out to be a potent αvβ6/αvβ8 binder and a promising inhibitor of HSV entry through an integrin-dependent mechanism. Furthermore, the renewed selectivity profile of was fully rationalized by a NMR/molecular modeling combined approach, providing novel valuable hints for the design of RGD integrin ligands with the desired specificity profile.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8279406PMC
May 2021

Lead Discovery of SARS-CoV-2 Main Protease Inhibitors through Covalent Docking-Based Virtual Screening.

J Chem Inf Model 2021 04 30;61(4):2062-2073. Epub 2021 Mar 30.

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

During almost all 2020, coronavirus disease 2019 (COVID-19) pandemic has constituted the major risk for the worldwide health and economy, propelling unprecedented efforts to discover drugs for its prevention and cure. At the end of the year, these efforts have culminated with the approval of vaccines by the American Food and Drug Administration (FDA) and the European Medicines Agency (EMA) giving new hope for the future. On the other hand, clinical data underscore the urgent need for effective drugs to treat COVID-19 patients. In this work, we embarked on a virtual screening campaign against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M chymotrypsin-like cysteine protease employing our in-house database of peptide and non-peptide ligands characterized by different types of warheads acting as Michael acceptors. To this end, we employed the AutoDock4 docking software customized to predict the formation of a covalent adduct with the target protein. verification of the inhibition properties of the most promising candidates allowed us to identify two new lead inhibitors that will deserve further optimization. From the computational point of view, this work demonstrates the predictive power of AutoDock4 and suggests its application for the screening of large chemical libraries of potential covalent binders against the SARS-CoV-2 M enzyme.
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http://dx.doi.org/10.1021/acs.jcim.1c00184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8029447PMC
April 2021

The organometallic ferrocene exhibits amplified anti-tumor activity by targeted delivery via highly selective ligands to αvβ3, αvβ6, or α5β1 integrins.

Biomaterials 2021 04 12;271:120754. Epub 2021 Mar 12.

Department of Nuclear Medicine, University Hospital Klinikum rechts der Isar, Technical University Munich, Munich, Germany; Central Institute for Translational Cancer Research (TranslaTUM), Technical University of Munich, Munich, Germany; Department of Chemistry, Technical University Munich, Munich, Germany. Electronic address:

High levels of reactive oxygen species (ROS) in tumors have been shown to exert anti-tumor activity, leading to the concept of ROS induction as therapeutic strategy. The organometallic compound ferrocene (Fc) generates ROS through a reversible one-electron oxidation. Incorporation of Fc into a tumor-targeting, bioactive molecule can enhance its therapeutic activity and enable tumor specific delivery. Therefore, we conjugated Fc to five synthetic, Arg-Gly-Asp (RGD)-based integrin binding ligands to enable targeting of the cell adhesion and signaling receptor integrin subtypes αvβ3, α5β1, or αvβ6, which are overexpressed in various, distinct tumors. We designed and synthesized a library of integrin-ligand-ferrocene (ILF) derivatives and showed that ILF conjugates maintained the high integrin affinity and selectivity of their parent ligands. A thorough biological characterization allowed us to identify the two most promising ligands, an αvβ3 (L2b) and an αvβ6 (L3b) targeting ILF, which displayed selective integrin-dependent cell uptake and pronounced ferrocene-mediated anti-tumor effects in vitro, along with increased ROS production and DNA damage. Hence, ILFs are promising candidates for the selective, tumor-targeted delivery of ferrocene to maximize its anti-cancer efficacy and minimize systemic toxicity, thereby improving the therapeutic window of ferrocene compared to currently used non-selective anti-cancer drugs.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120754DOI Listing
April 2021

Novel Peptide-Based PET Probe for Non-invasive Imaging of C-X-C Chemokine Receptor Type 4 (CXCR4) in Tumors.

J Med Chem 2021 03 4;64(6):3449-3461. Epub 2021 Mar 4.

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

The recently reported CXCR4 antagonist (Ac-Arg-Ala-[DCys-Arg-2Nal-His-Pen]-COH) was investigated as a molecular scaffold for a CXCR4-targeted positron emission tomography (PET) tracer. Toward this end, was functionalized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononanetriacetic acid (NOTA). On the basis of convincing affinity data, both tracers, [Ga]NOTA analogue ([Ga]-) and [Ga]DOTA analogue ([Ga]-), were evaluated for PET imaging in "" models of CHO-hCXCR4 and Daudi lymphoma cells. PET imaging and biodistribution studies revealed higher CXCR4-specific tumor uptake and high tumor/background ratios for the [Ga]NOTA analogue ([Ga]-) than for the [Ga]DOTA analogue ([Ga]-) in both models. Moreover, [Ga]- and [Ga]- displayed rapid clearance and very low levels of accumulation in all nontarget tissues but the kidney. Although the high tumor/background ratios observed in the mouse xenograft model could partially derive from the hCXCR4 selectivity of [Ga]-, our results encourage its translation into a clinical context as a novel peptide-based tracer for imaging of CXCR4-overexpressing tumors.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00066DOI Listing
March 2021

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

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

Pyrrolyl Pyrazoles as Non-Diketo Acid Inhibitors of the HIV-1 Ribonuclease H Function of Reverse Transcriptase.

ACS Med Chem Lett 2020 May 5;11(5):798-805. Epub 2020 Mar 5.

Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti, "Sapienza" Università di Roma, p.le Aldo Moro 5, I-00185 Roma, Italy.

Due to the biological liability of diketo acid (DKA) chain, we transferred this element of our previously reported anti-HIV-1 pyrrolyl derivatives to a non-DKA scaffold, obtaining a series of pyrrolyl-pyrazole carboxylic acids as new RNase H inhibitors. Among the newly synthesized derivatives, oxyphenylpyrrolyl-pyrazoles demonstrated inhibitory activities within the low micromolar/submicromolar range with compound being the most potent. Interestingly, all tested compounds showed up to 2 orders of magnitude of selectivity for RNase H vs integrase. Docking studies within the RNase H catalytic site, coupled with site-directed mutagenesis, showed the key structural features that could confer the ability to establish specific interactions within RNase H. Furthermore, they proved the ability of our compounds to interact with amino acids highly conserved among HIV-1 subspecies isolated among patients carrying drug-resistant variants. In the end, the newly discovered pyrazole carboxylic acid derivatives feature promising serum stability with respect to their corresponding DKAs.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236236PMC
May 2020

Click-Chemistry (CuAAC) Trimerization of an α β Integrin Targeting Ga-68-Peptide: Enhanced Contrast for in-Vivo PET Imaging of Human Lung Adenocarcinoma Xenografts.

Chembiochem 2020 10 9;21(19):2836-2843. Epub 2020 Jun 9.

Institute of Pathology, Technische Universität München, Trogerstrasse 18, 81675, München, Germany.

α β Integrin is an epithelial transmembrane protein that recognizes latency-associated peptide (LAP) and primarily activates transforming growth factor beta (TGF-β). It is overexpressed in carcinomas (most notably, pancreatic) and other conditions associated with α β integrin-dependent TGF-β dysregulation, such as fibrosis. We have designed a trimeric Ga-68-labeled TRAP conjugate of the α β -specific cyclic pentapeptide SDM17 (cyclo[RGD-Chg-E]-CONH ) to enhance α β integrin affinity as well as target-specific in-vivo uptake. Ga-68-TRAP(SDM17) showed a 28-fold higher α β affinity than the corresponding monomer Ga-68-NOTA-SDM17 (IC of 0.26 vs. 7.4 nM, respectively), a 13-fold higher IC -based selectivity over the related integrin α β (factors of 662 vs. 49), and a threefold higher tumor uptake (2.1 vs. 0.66 %ID/g) in biodistribution experiments with H2009 tumor-bearing SCID mice. The remarkably high tumor/organ ratios (tumor-to-blood 11.2; -to-liver 8.7; -to-pancreas 29.7) enabled high-contrast tumor delineation in PET images. We conclude that Ga-68-TRAP(SDM17) holds promise for improved clinical PET diagnostics of carcinomas and fibrosis.
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http://dx.doi.org/10.1002/cbic.202000200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586803PMC
October 2020

Two novel SIRT1 activators, SCIC2 and SCIC2.1, enhance SIRT1-mediated effects in stress response and senescence.

Epigenetics 2020 Jun - Jul;15(6-7):664-683. Epub 2020 Jan 16.

Dipartimento di Medicina di Precisione, Università degli Studi della Campania "Luigi Vanvitelli" Napoli , Napoli, Italy.

SIRT1, a NAD-dependent deacetylase, is the most well-studied member of class III histone deacetylases. Due to its wide range of activities and substrate targets, this enzyme has emerged as a major regulator of different physiological processes. However, SIRT1-mediated alterations are also implicated in the pathogenesis of several conditions, including metabolic and neurodegenerative disorders, and cancer. Current evidence highlights the potential role of SIRT1 as an attractive therapeutic target for disease prevention and treatment strategies, thus propelling the development of new pharmacological agents. By high-throughput screening of a large library of compounds, we identified SCIC2 as an effective SIRT1 activator. This small molecule showed enzymatic activity of 135.8% at 10 μM, an AC value of 50 ± 1.8 µM, and bound SIRT1 with a K of 26.4 ± 0.6 μM. In order to potentiate its SIRT1-activating ability, SCIC2 was subjected to modelling studies, leading to the identification of a more potent derivative, SCIC2.1. SCIC2.1 displayed higher SIRT1 activity (175%; AC = 36.83 ± 2.23 µM), stronger binding to SIRT1, and greater cell permeability than SCIC2. At cellular level, both molecules did not alter the cell cycle progression of cancer cells and normal cells, and were able to strengthen SIRT1-mediated effects in stress response. Finally, SCIC2 and SCIC2.1 attenuated induction of senescence by reducing senescence-associated β-galactosidase activity. Our findings warrant further investigation of these two novel SIRT1 activators in and human studies.
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http://dx.doi.org/10.1080/15592294.2019.1704349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574383PMC
May 2021

Inhibition of Histone Demethylases LSD1 and UTX Regulates ERα Signaling in Breast Cancer.

Cancers (Basel) 2019 Dec 16;11(12). Epub 2019 Dec 16.

Department of Precision Medicine, University of Campania "Luigi Vanvitelli", 80138 Naples, Italy.

In breast cancer, Lysine-specific demethylase-1 (LSD1) and other lysine demethylases (KDMs), such as Lysine-specific demethylase 6A also known as Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), are co-expressed and co-localize with estrogen receptors (ERs), suggesting the potential use of hybrid (epi)molecules to target histone methylation and therefore regulate/redirect hormone receptor signaling. Here, we report on the biological activity of a dual-KDM inhibitor (MC3324), obtained by coupling the chemical properties of tranylcypromine, a known LSD1 inhibitor, with the 2OG competitive moiety developed for JmjC inhibition. MC3324 displays unique features not exhibited by the single moieties and well-characterized mono-pharmacological inhibitors. Inhibiting LSD1 and UTX, MC3324 induces significant growth arrest and apoptosis in hormone-responsive breast cancer model accompanied by a robust increase in H3K4me2 and H3K27me3. MC3324 down-regulates ERα in breast cancer at both transcriptional and non-transcriptional levels, mimicking the action of a selective endocrine receptor disruptor. MC3324 alters the histone methylation of ERα-regulated promoters, thereby affecting the transcription of genes involved in cell surveillance, hormone response, and death. MC3324 reduces cell proliferation in ex vivo breast cancers, as well as in breast models with acquired resistance to endocrine therapies. Similarly, MC3324 displays tumor-selective potential in vivo, in both xenograft mice and chicken embryo models, with no toxicity and good oral efficacy. This epigenetic multi-target approach is effective and may overcome potential mechanism(s) of resistance in breast cancer.
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http://dx.doi.org/10.3390/cancers11122027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966629PMC
December 2019

Boosting Fmoc Solid-Phase Peptide Synthesis by Ultrasonication.

Org Lett 2019 08 30;21(16):6378-6382. Epub 2019 Jul 30.

DiSTABiF , Università degli Studi della Campania "Luigi Vanvitelli" , via A. Vivaldi 43 , 81100 Caserta , Italy.

We investigated the ultrasonication-mediated effects on the Fmoc-based solid-phase peptide synthesis (SPPS). Our study culminated with the development of an ultrasound-assisted strategy (US-SPPS) that allowed for the synthesis of different biologically active peptides (up to 44-mer), with a remarkable savings of material and reaction time. Noteworthy, ultrasonic irradiation did not exacerbate the main side reactions and improved the synthesis of peptides endowed with "difficult sequences", placing the US-SPPS among the current high-efficient peptide synthetic strategies.
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http://dx.doi.org/10.1021/acs.orglett.9b02283DOI Listing
August 2019

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

Nutrients 2019 Mar 13;11(3). Epub 2019 Mar 13.

Department of Pharmacy, School of Medicine and Surgery, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy.

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 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 and 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 and . These results could have an important impact on the therapeutic potential of MBCP in helping to restore the intestinal epithelium integrity damaged by inflammation.
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http://dx.doi.org/10.3390/nu11030610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471453PMC
March 2019

Selective Targeting of Integrin αvβ8 by a Highly Active Cyclic Peptide.

J Med Chem 2019 02 6;62(4):2024-2037. Epub 2019 Feb 6.

Institute for Advanced Study and Center of Integrated Protein Science (CIPSM), Department Chemie , Technische Universität München , Lichtenbergstraße 4 , 85748 Garching , Germany.

Integrins play important roles in physiological and pathophysiological processes. Among the RGD-recognizing integrin subtypes, the αvβ8 receptor is emerging as an attractive target because of its involvement in various illnesses, such as autoimmune diseases, viral infections, and cancer. However, its functions have, so far, not been investigated in living subjects mainly because of the lack of a selective αvβ8 ligand. Here, we report the design and potential medical applications of a cyclic octapeptide as the first highly selective small-molecule ligand for αvβ8. Remarkably, this compound displays low nanomolar αvβ8 binding affinity and a strong discriminating power of at least 2 orders of magnitude versus other RGD-recognizing integrins. Peptide functionalization with fluorescent or radioactive labels enables the selective imaging of αvβ8-positive cells and tissues. This new probe will pave the way for detailed characterization of the distinct (patho)physiological role of this relatively unexplored integrin, providing a basis to fully exploit the potential of αvβ8 as a target for molecular diagnostics and personalized therapy regimens.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01588DOI Listing
February 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

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

J Med Chem 2018 06 4;61(11):4791-4809. Epub 2018 Jun 4.

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

In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5β1 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 α5β1/αvβ3 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 α5β1/αvβ3 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 α5β1 integrin.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00004DOI Listing
June 2018

Antitumor efficacy of Kisspeptin in human malignant mesothelioma cells.

Oncotarget 2018 Apr 10;9(27):19273-19282. Epub 2018 Apr 10.

Oncologia Medica, Dipartimento di Internistica Clinica e Sperimentale "F. Magrassi", Università degli Studi della Campania Luigi Vanvitelli, Naples, Italy.

Purpose: Kisspeptin signaling, its receptors GPR54, could be an essential players in the inhibition of mesothelioma progression, invasion and metastasis formation. The loss of KiSS1 by tumor cells has been associated with a metastatic phenotype but the mechanistic insights of this process are still unknown.

Experimental Design: The blockade of the metastatic process at early stage is a hot topic in cancer research. We studied the role of KiSS1 on proliferation, invasiveness, migration abilities of mesothelioma cell lines focusing on the effect on epithelial-to-mesenchymal transition (EMT).

Results: Treatment with the KiSS1 peptide or with a synthesis peptide with longer half-life, the FTM080, significantly inhibited cell proliferation, migration and invasion of mesothelioma cell lines; the same treatment reduced the activity of MMP-2 and MMP-9 determining consequently a marked reduction in the invasiveness of primary tumors and metastases. Thespecificexpression of EMT markers, as E-caderin, Vimentin, Slug and Snail, suggested the inhibition of EMT after treatment with KiSS1 as well as the preservation of epithelial components.

Conclusion: Our results support anti-proliferative effect of KiSS1 in cancer cells and suggest that targeting the KiSS1/GPR54 system may represent a novel therapeutic approach for mesothelioma.
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http://dx.doi.org/10.18632/oncotarget.25018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922395PMC
April 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

From a Helix to a Small Cycle: Metadynamics-Inspired αvβ6 Integrin Selective Ligands.

Angew Chem Int Ed Engl 2018 10 2;57(44):14645-14649. Epub 2018 May 2.

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

The RGD-recognizing αvβ6 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 αvβ6 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 αvβ6-specific small peptides or even peptidomimetics. In vivo PET imaging studies demonstrated the potential use of 6 for medical applications.
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http://dx.doi.org/10.1002/anie.201803250DOI Listing
October 2018

Cationic nucleopeptides as novel non-covalent carriers for the delivery of peptide nucleic acid (PNA) and RNA oligomers.

Bioorg Med Chem 2018 05 7;26(9):2539-2550. Epub 2018 Apr 7.

Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania "Luigi Vanvitelli", Via Vivaldi 43, 81100 Caserta, Italy. Electronic address:

Cationic nucleopeptides belong to a family of synthetic oligomers composed by amino acids and nucleobases. Their capability to recognize nucleic acid targets and to cross cellular membranes provided the basis for considering them as novel non-covalent delivery agents for nucleic acid pharmaceuticals. Herein, starting from a 12-mer nucleopeptide model, the number of cationic residues was modulated in order to obtain new nucleopeptides endowed with high solubility in acqueous medium, acceptable bio-stability, low cytotoxicity and good capability to bind nucleic acid. Two candidates were selected to further investigate their potential as nucleic acid carriers, showing higher efficiency to deliver PNA in comparison with RNA. Noteworthy, this study encourages the development of nucleopeptides as new carriers to extend the known strategies for those nucleic acid analogues, especially PNA, that still remain difficult to drive into the cells.
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http://dx.doi.org/10.1016/j.bmc.2018.04.017DOI Listing
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

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

Targeting CXCR4 reverts the suppressive activity of T-regulatory cells in renal cancer.

Oncotarget 2017 Sep 19;8(44):77110-77120. Epub 2017 Aug 19.

Functional Genomics, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione "G. Pascale"-IRCCS, 80131 Naples, Italy.

With the intent to identify biomarkers in renal cell carcinoma (RCC) the functional status of T-regulatory cells (Tregs) was investigated in primary RCC. Tregs were isolated from tumoral-(TT), peritumoral tissue-(PT) and peripheral blood-(PB) of 42 primary RCC patients and function evaluated through effector T cells (Teff) proliferation, cytokines release and demethylation of Treg Specific Region (TSDR). The highest value of Tregs was detected in TT with the uppermost amount of effector-Tregs-(CD4CD25FOXP3CD45RA). PB-RCC Tregs efficiently suppress Teff proliferation compared to healthy donor (HD)-Tregs and, at the intrapatient evaluation, TT-derived Tregs were the most suppressive. Higher demethylation TSDR was detected in TT- and PB-RCC Tregs vs HD-Tregs ( <0,001). CXCR4 is highly expressed on Tregs, thus we wished to modulate Tregs function through CXCR4 inhibition. CXCR4 antagonism, elicited by a new peptidic antagonist, Peptide-R29, efficiently reversed Tregs suppression of Teff proliferation. Thus Tregs functional evaluation precisely reflects Tregs status and may be a reliable biomarker of tumoral immune response. In addition, treatment with CXCR4 antagonist, impairing Tregs function, could improve the anticancer immune response, in combination with conventional therapy and/or immunotherapy such as checkpoints inhibitors.
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http://dx.doi.org/10.18632/oncotarget.20363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652768PMC
September 2017

Dual MET and SMO Negative Modulators Overcome Resistance to EGFR Inhibitors in Human Nonsmall Cell Lung Cancer.

J Med Chem 2017 09 22;60(17):7447-7458. Epub 2017 Aug 22.

DiSTABiF, Università della Campania "Luigi Vanvitelli" , Via Vivaldi 43, 81100 Caserta, Italy.

Tyrosine kinase inhibitors (TKIs) of the EGF receptor (EGFR) have provided a significant improvement in the disease outcome of nonsmall cell lung cancer (NSCLC). Unfortunately, resistance to these agents frequently occurs, and it is often related to the activation of the Hedgehog (Hh) and MET signaling cascades driving the epithelial-to-mesenchymal transition (EMT). Because the concomitant inhibition of both Hh and MET pathways restores the sensitivity to anti-EGFR drugs, here we aimed at discovering the first compounds that block simultaneously MET and SMO. By using an "in silico drug repurposing" approach and by validating our predictions both in vitro and in vivo, we identified a set of compounds with the desired dual inhibitory activity and enhanced antiproliferative activity on EGFR TKI-resistant NSCLC. The identification of the known MET TKIs, glesatinib and foretinib, as negative modulators of the Hh pathway, widens their application in the context of NSCLC.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00794DOI Listing
September 2017

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

Nucleic Acids Res 2017 02;45(4):1820-1834

Oncogenomic and Epigenetic Unit, Regina Elena National Cancer Institute, Via Elio Chianesi 53, Rome 00144, Italy.

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.
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http://dx.doi.org/10.1093/nar/gkw1202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389694PMC
February 2017

Switchable Protecting Strategy for Solid Phase Synthesis of DNA and RNA Interacting Nucleopeptides.

J Org Chem 2016 12 8;81(23):11612-11625. Epub 2016 Nov 8.

Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples , Via Vivaldi 43, 81100 Caserta, Italy.

Nucleopeptides are promising nucleic acid mimetics in which the peptide backbone bears nucleobases. They can recognize DNA and RNA targets modulating their biological functions. To date, the lack of an effective strategy for the synthesis of nucleopeptides prevents their evaluation for biological and biomedical applications. Herein, we describe an unprecedented approach that enables the synthesis of cationic both homo and heterosequence nucleopeptides wholly on solid support with high yield and purity. Spectroscopic studies indicate advantageous properties of the nucleopeptides in terms of binding, thermodynamic stability and sequence specific recognition. Biostability assay and laser scanning confocal microscopy analyses reveal that the nucleopeptides feature acceptable serum stability and ability to cross the cell membrane.
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http://dx.doi.org/10.1021/acs.joc.6b01829DOI Listing
December 2016

Neuropeptide S receptor ligands: a patent review (2005-2016).

Expert Opin Ther Pat 2017 Mar 9;27(3):347-362. Epub 2016 Nov 9.

c Department of Chemical and Pharmaceutical Sciences , University of Ferrara , Ferrara , Italy.

Introduction: Neuropeptide S (NPS) is a 20-residue peptide and endogenous ligand of the NPS receptor (NPSR). This receptor was a formerly orphan GPCR whose activation increases calcium and cyclic adenosine monophosphate levels. The NPS/NPSR system is expressed in several brain regions where it controls important biological functions including locomotor activity, arousal and sleep, anxiety, food intake, memory, pain, and drug addiction. Areas covered: This review furnishes an updated overview of the patent literature covering NPSR ligands since 2005, when the first example of an NPSR antagonist was disclosed. Expert opinion: Several potent NPSR antagonists are available as valuable pharmacological tools despite showing suboptimal pharmacokinetic properties in vivo. The optimization of these ligands is needed to speed up their potential clinical advancement as pharmaceuticals to treat drug addiction. In order to support the design of novel NPSR antagonists, we performed a ligand-based conformational analysis recognizing some structural requirements for NPSR antagonism. The identification of small-molecule NPSR agonists now represents an unmet challenge to be addressed. These molecules will allow investigation of the beneficial effects of selective NPSR activation in a large panel of psychiatric disorders and to foresee their therapeutic potential as anxiolytics, nootropics, and analgesics.
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http://dx.doi.org/10.1080/13543776.2017.1254195DOI Listing
March 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

Lead Optimization of 2-Phenylindolylglyoxylyldipeptide Murine Double Minute (MDM)2/Translocator Protein (TSPO) Dual Inhibitors for the Treatment of Gliomas.

J Med Chem 2016 05 3;59(10):4526-38. Epub 2016 May 3.

Department of Pharmacy, University of Pisa , Pisa 56126, Italy.

In glioblastoma multiforme (GBM), translocator protein (TSPO) and murine double minute (MDM)2/p53 complex represent two druggable targets. We recently reported the first dual binder 3 possessing a higher anticancer effect in GBM cells than the standards PK11195 1 or Nutlin-3 2 singularly applied. Herein, through a structure-activity relationship study, we developed derivatives 4-10 with improved potencies toward both TSPO and MDM2. As a result, compound 9: (i) reactivated the p53 functionality; (ii) inhibited the viability of two human GBM cells; (iii) impaired the proliferation of glioma cancer stem cells (CSCs), more resistant to chemotherapeutics and responsible of GBM recurrence; (iv) sensitized GBM cells and CSCs to the activity of temozolomide; (v) directed its effects preferentially toward tumor cells with respect to healthy ones. Thus, 9 may represent a promising cytotoxic agent, which is worthy of being further developed for a therapeutic approach against GBM, where the downstream p53 signaling is intact and TSPO is overexpressed.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01767DOI Listing
May 2016
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