Publications by authors named "Stefano Tomassi"

31 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

Polycomb Repressive Complex 2 Modulation through the Development of EZH2-EED Interaction Inhibitors and EED Binders.

J Med Chem 2021 08 5;64(16):11774-11797. Epub 2021 Aug 5.

Department of Chemistry and Technology of Drugs, "Sapienza" University of Rome, P.le A. Moro 5, 00185 Rome, Italy.

Epigenetics is nowadays a well-accepted area of research. In the last years, tremendous progress was made regarding molecules targeting EZH2, directly or indirectly. Recently tazemetostat hit the market after FDA-approval for the treatment of lymphoma. However, the impairment of EZH2 activity by orthosteric intervention has proven to be effective only in a limited subset of cancers. Considering the multiproteic nature of the PRC2 complex and the marked dependence of EZH2 functions on the other core subunits such as EED, in recent years, a new targeting approach ascended to prominence. The possibility to cripple the function of the PRC2 complex by interfering with its multimeric integrity fueled the interest in developing EZH2-EED protein-protein interaction and EED inhibitors as indirect modulators of PRC2-dependent methyltransferase activity. In this Perspective, we aim to summarize the latest findings regarding the development and the biological activity of these emerging classes of PRC2 modulators from a medicinal chemist's viewpoint.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8404197PMC
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

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

Properly Substituted Cyclic Bis-(2-bromobenzylidene) Compounds Behaved as Dual p300/CARM1 Inhibitors and Induced Apoptosis in Cancer Cells.

Molecules 2020 Jul 8;25(14). Epub 2020 Jul 8.

Dipartimento di Chimica e Tecnologie del Farmaco, 'Sapienza' Università di Roma, 00185 Roma, Italy.

Bis-(3-bromo-4-hydroxy)benzylidene cyclic compounds have been reported by us as epigenetic multiple ligands, but different substitutions at the two wings provided analogues with selective inhibition. Since the 1-benzyl-3,5-bis(()-3-bromobenzylidene)piperidin-4-one displayed dual p300/EZH2 inhibition joined to cancer-selective cell death in a panel of tumor cells and in in vivo xenograft models, we prepared a series of bis(()-2-bromobenzylidene) cyclic compounds - to test in biochemical (p300, PCAF, SIRT1/2, EZH2, and CARM1) and cellular (NB4, U937, MCF-7, SH-SY5Y) assays. The majority of - exhibited potent dual p300 and CARM1 inhibition, sometimes reaching the submicromolar level, and induction of apoptosis mainly in the tested leukemia cell lines. The most effective compounds in both enzyme and cellular assays carried a 4-piperidone moiety and a methyl (), benzyl (), or acyl (-) substituent at N1 position. Elongation of the benzyl portion to 2-phenylethyl () and 3-phenylpropyl () decreased the potency of compounds at both the enzymatic and cellular levels, but the activity was promptly restored by introduction of a ketone group into the phenylalkyl substituent (-). Western blot analyses performed in NB4 and MCF-7 cells on selected compounds confirmed their inhibition of p300 and CARM1 through decrease of the levels of acetyl-H3 and acetyl-H4, marks for p300 inhibition, and of H3R17me2, mark for CARM1 inhibition.
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http://dx.doi.org/10.3390/molecules25143122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397249PMC
July 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

Characterization of water-soluble esters of nitrobenzoxadiazole-based GSTP1-1 inhibitors for cancer treatment.

Biochem Pharmacol 2020 08 29;178:114060. Epub 2020 May 29.

Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", Rome, Italy; The NAST Centre for Nanoscience and Nanotechnology and Innovative Instrumentation, University of Rome "Tor Vergata", Rome, Italy. Electronic address:

The 7-nitrobenzo[c][1,2,5]oxadiazole (NBD) derivative NBDHEX (compound 1) and its analogue MC3181 (compound 2) have been found to be potent inhibitors of tumor cell growth in vitro and therapeutically active and safe in mice bearing human melanoma xenografts. To enhance the aqueous solubility of these compounds, we synthesized the hemisuccinate of 1 (compound 3) and the phosphate monoesters of 1 and 2 (compound 4 and 5, respectively). These novel NBD derivatives displayed a solubility in the conventional phosphate-buffered saline up to 150-fold higher than that of 1, and up to 4-fold higher than that of 2. Notably, solubility of phosphates 4 and 5 in a potassium phosphate buffer at pH 7.4, was up to 500-fold higher than that of 1, and ~10-fold higher than that of 2. Compounds 3-5 retained high cytotoxicity towards cultured human melanoma and osteosarcoma cells and were cleaved in vitro by both human and murine hydrolases, thus releasing the corresponding parent compound (i.e., 1 or 2). Interestingly, esters 3-5 displayed high inhibitory activity towards the glutathione transferase (GST) isoform GSTP1-1 and showed a reactivity towards reduced glutathione comparable to that of the respective parent compound. Finally, both 4 and 5 were safe and effective when administered intravenously or orally as an aqueous solution to mice xenografted with A375 human melanoma tumors. Collectively, these results and the previously observed synergistic interaction between 1 and 2 and various approved anticancer drugs, suggest the possible utility of phosphates 4 and 5 as single agents and in combination regimens in cancers with unmet medical need, including melanoma.
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http://dx.doi.org/10.1016/j.bcp.2020.114060DOI 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

From PARP1 to TNKS2 Inhibition: A Structure-Based Approach.

ACS Med Chem Lett 2020 May 3;11(5):862-868. Epub 2020 Feb 3.

Department of Chemistry and Technology of Drugs, ″Sapienza" University of Rome, 00185 Rome, Italy.

Tankyrases (TNKSs) have recently gained great consideration as potential targets in Wnt/β-catenin pathway-dependent solid tumors. Previously, we reported the 2-mercaptoquinazolin-4-one MC2050 as a micromolar PARP1 inhibitor. Here we show how the resolution of the X-ray structure of PARP1 in complex with MC2050, combined with the computational investigation of the structural differences between TNKSs and PARP1/2 active sites, provided the rationale for a structure-based drug design campaign that with a limited synthetic effort led to the discovery of the bis-quinazolinone as a picomolar and selective TNKS2 inhibitor, endowed with antiproliferative effects in a colorectal cancer cell line (DLD-1) where the Wnt pathway is constitutively activated.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236224PMC
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

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

Discovery of Pyrido[3',2':5,6]thiopyrano[4,3-]pyrimidine-Based Antiproliferative Multikinase Inhibitors.

ACS Med Chem Lett 2019 Apr 17;10(4):457-462. Epub 2019 Jan 17.

Dipartimento di Scienze del Farmaco, Università di Padova, Via Marzolo 5, 35131 Padova, Italy.

Protein kinases dysregulation is extremely common in cancer cells, and the development of new agents able to simultaneously target multiple kinase pathways involved in angiogenesis and tumor growth may offer several advantages in the treatment of cancer. Herein we report the discovery of new pyridothiopyranopyrimidine derivatives (-) showing high potencies in VEGFR-2 KDR inhibition as well as antiproliferative effect on a panel of human tumor cell lines. Investigation on the selectivity profile of the representative 2-anilino-substituted compounds , , and revealed a multiplicity of kinase targets that should account for the potent antiproliferative effect produced by these pyridothiopyranopyrimidine derivatives.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466516PMC
April 2019

Combined HAT/EZH2 modulation leads to cancer-selective cell death.

Oncotarget 2018 May 22;9(39):25630-25646. Epub 2018 May 22.

Centro Nacional de Análisis Genómico, Barcelona, Spain.

Epigenetic alterations have been associated with both pathogenesis and progression of cancer. By screening of library compounds, we identified a novel hybrid epi-drug MC2884, a HAT/EZH2 inhibitor, able to induce cancer-selective cell death in both solid and hematological cancers , and xenograft models. Anticancer action was due to an epigenome modulation by H3K27me3, H3K27ac, H3K9/14ac decrease, and to caspase-dependent apoptosis induction. MC2884 triggered mitochondrial pathway apoptosis by up-regulation of cleaved-BID, and strong down-regulation of BCL2. Even aggressive models of cancer, such as p53 or TET2 cells, responded to MC2884, suggesting MC2884 therapeutic potential also for the therapy of TP53 or TET2-deficient human cancers. MC2884 induced massive apoptosis in human primary leukemia blasts with poor prognosis , by targeting BCL2 expression. MC2884-treatment reduced acetylation of the BCL2 promoter at higher level than combined p300 and EZH2 inhibition. This suggests a key role for BCL-2 reduction in potentiating responsiveness, also in combination therapy with BCL2 inhibitors. Finally, we identified both the mechanism of MC2884 action as well as a potential therapeutic scheme of its use. Altogether, this provides proof of concept for the use of epi-drugs coupled with epigenome analyses to 'personalize' precision medicine.
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http://dx.doi.org/10.18632/oncotarget.25428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986654PMC
May 2018

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

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

New insights in the structure-activity relationships of 2-phenylamino-substituted benzothiopyrano[4,3-d]pyrimidines as kinase inhibitors.

Eur J Med Chem 2018 Apr 7;150:446-456. Epub 2018 Mar 7.

Dipartimento di Scienze del Farmaco, Università di Padova, Via Marzolo 5, 35131, Padova, Italy.

Inhibition of angiogenesis via blocking vascular endothelial growth factor receptor (VEGFR) signaling pathway emerged as an established approach in anticancer therapy. So far, many monoclonal antibodies and ATP-competitive small molecule inhibitors have been clinically validated and approved. In this study, structure-activity relationships (SAR) within the 2-phenylamino-substituted benzothiopyrano[4,3-d]pyrimidine class of kinase inhibitors were further refined by the synthesis and biological evaluation of new compounds 1-21 featuring different substitution patterns on the pendant phenyl moiety, combined with H, OCH, or Cl at 8-position. Most compounds showed a promising human kinase insert domain receptor (KDR) inhibition profile, with IC values in the submicromolar/low micromolar range, and promising antiproliferative activity on human umbilical vein endothelial cells (HUVECs) as well as on a panel of three human tumor cell lines. The angio-kinase selectivity profile was assessed for the most promising compound 16 against a set of six human kinases. Finally, computational studies allowed clarifying at molecular level the interaction pattern established by the compounds with KDR, highlighting key stable cation-π interactions, and thus providing the basis for further designing novel inhibitors.
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http://dx.doi.org/10.1016/j.ejmech.2018.03.013DOI Listing
April 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

Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS34.

Angew Chem Int Ed Engl 2017 07 12;56(28):8153-8157. Epub 2017 Jun 12.

Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227, Dortmund, Germany.

Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small-molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy-induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.
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http://dx.doi.org/10.1002/anie.201703738DOI Listing
July 2017

Indazole-Based Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor.

J Med Chem 2017 03 10;60(6):2361-2372. Epub 2017 Mar 10.

Faculty of Chemistry and Chemical Biology, TU Dortmund University , Otto-Hahn-Straße 4a, Dortmund D-44227, Germany.

The specific targeting of oncogenic mutant epidermal growth factor receptor (EGFR) is a breakthrough in targeted cancer therapy and marks a drastic change in the treatment of non-small cell lung cancer (NSCLC). The recurrent emergence of resistance to these targeted drugs requires the development of novel chemical entities that efficiently inhibit drug-resistant EGFR. Herein, we report the optimization process for a hit compound that has emerged from a phenotypic screen resulting in indazole-based compounds. These inhibitors are conformationally less flexible, target gatekeeper mutated drug-resistant EGFR-L858R/T790M, and covalently alkylate Cys797. Western blot analysis, as well as characterization of the binding kinetics and kinase selectivity profiling, substantiates our approach of targeting drug-resistant EGFR-L858R/T790M with inhibitors incorporating the indazole as hinge binder.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01626DOI Listing
March 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

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

Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach.

J Med Chem 2015 Sep 31;58(17):6844-63. Epub 2015 Aug 31.

Department of Chemistry and Chemical Biology, TU Dortmund University , Otto-Hahn-Straße 6, D-44227 Dortmund, Germany.

Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01082DOI Listing
September 2015

Quinoline-based p300 histone acetyltransferase inhibitors with pro-apoptotic activity in human leukemia U937 cells.

ChemMedChem 2014 Mar 6;9(3):542-8. Epub 2014 Feb 6.

Department of Drug Chemistry & Technologies, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome (Italy).

Chemical manipulations performed on 2-methyl-3-carbethoxyquinoline (1), a histone acetyltransferase inhibitor previously identified by our research group and active at the sub-millimolar/millimolar level, led to compounds bearing higher alkyl groups at the C2-quinoline or additional side chains at the C6-quinoline positions. Such compounds displayed at least threefold improved inhibitory potency toward p300 protein lysine acetyltransferase activity; some of them decreased histone H3 and H4 acetylation levels in U937 cells and induced high degrees of apoptosis (three compounds >10-fold higher than compound 1) after treatment of U937 cells.
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http://dx.doi.org/10.1002/cmdc.201300536DOI Listing
March 2014

Pan-histone demethylase inhibitors simultaneously targeting Jumonji C and lysine-specific demethylases display high anticancer activities.

J Med Chem 2014 Jan 19;57(1):42-55. Epub 2013 Dec 19.

Department of Drug Chemistry and Technologies, Sapienza University of Rome , P. le A. Moro 5, 00185 Rome, Italy.

In prostate cancer, two different types of histone lysine demethylases (KDM), LSD1/KDM1 and JMJD2/KDM4, are coexpressed and colocalize with the androgen receptor. We designed and synthesized hybrid LSD1/JmjC or "pan-KDM" inhibitors 1-6 by coupling the skeleton of tranylcypromine 7, a known LSD1 inhibitor, with 4-carboxy-4'-carbomethoxy-2,2'-bipyridine 8 or 5-carboxy-8-hydroxyquinoline 9, two 2-oxoglutarate competitive templates developed for JmjC inhibition. Hybrid compounds 1-6 are able to simultaneously target both KDM families and have been validated as potential antitumor agents in cells. Among them, 2 and 3 increase H3K4 and H3K9 methylation levels in cells and cause growth arrest and substantial apoptosis in LNCaP prostate and HCT116 colon cancer cells. When tested in noncancer mesenchymal progenitor (MePR) cells, 2 and 3 induced little and no apoptosis, respectively, thus showing cancer-selective inhibiting action.
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http://dx.doi.org/10.1021/jm4012802DOI Listing
January 2014

tert-Butylcarbamate-containing histone deacetylase inhibitors: apoptosis induction, cytodifferentiation, and antiproliferative activities in cancer cells.

ChemMedChem 2013 May 25;8(5):800-11. Epub 2013 Mar 25.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, P.le A. Moro 5, 00185 Roma, Italy.

Herein we report novel pyrrole- and benzene-based hydroxamates (8, 10) and 2'-aminoanilides (9, 11) bearing the tert-butylcarbamate group at the CAP moiety as histone deacetylase (HDAC) inhibitors. Compounds 8 b and 10 c selectively inhibited HDAC6 at the nanomolar level, whereas the other hydroxamates effected an increase in acetyl-α-tubulin levels in human acute myeloid leukemia U937 cells. In the same cell line, compounds 8 b and 10 c elicited 18.4 and 21.4 % apoptosis, respectively (SAHA: 16.9 %), and the pyrrole anilide 9 c displayed the highest cytodifferentiating effect (90.9 %). In tests against a wide range of various cancer cell lines to determine its antiproliferative effects, compound 10 c exhibited growth inhibition from sub-micromolar (neuroblastoma LAN-5 and SH-SY5Y cells, chronic myeloid leukemia K562 cells) to low-micromolar (lung H1299 and A549, colon HCT116 and HT29 cancer cells) concentrations. In HT29 cells, 10 c increased histone H3 acetylation, and decreased the colony-forming potential of the cancer cells by up to 60 %.
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http://dx.doi.org/10.1002/cmdc.201300005DOI Listing
May 2013

Identification of PR-SET7 and EZH2 selective inhibitors inducing cell death in human leukemia U937 cells.

Biochimie 2012 Nov 16;94(11):2308-13. Epub 2012 Jun 16.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, P.le A. Moro 5, 00185 Roma, Italy.

Chemical manipulations undertaken on some bis(bromo- and dibromo-phenol) compounds previously reported by us as wide-spectrum epigenetic inhibitors let us to identify bis (bromo- and dibromo-methoxyphenyl) derivatives highly selective for PR-SET7 and EZH2 (compounds 4, 5, 9, and 10). Western blot analyses were carried out in U937 cells to determine the effects of such compounds on the methyl marks related to the tested enzymes (H3K4me1, H3K9me2, H4H20me1, and H3K27me3). The 1,5-bis(3-bromo-4-methoxyphenyl)penta-1,4-dien-3-one 4 (EC(50) vs EZH2 = 74.9 μM), tested in U937 cells at 50 μM, induced massive cell death and 28% of granulocytic differentiation, highlighting the potential use of EZH2 inhibitors in cancer.
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http://dx.doi.org/10.1016/j.biochi.2012.06.003DOI Listing
November 2012
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