Publications by authors named "Biagina Marrocco"

16 Publications

  • Page 1 of 1

A Tail-Based Mechanism Drives Nucleosome Demethylation by the LSD2/NPAC Multimeric Complex.

Cell Rep 2019 04;27(2):387-399.e7

Department of Biology and Biotechnology "Lazzaro Spallanzani," University of Pavia, via Ferrata 9, 27100 Pavia, Italy. Electronic address:

LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.
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http://dx.doi.org/10.1016/j.celrep.2019.03.061DOI Listing
April 2019

Development of alkyl glycerone phosphate synthase inhibitors: Structure-activity relationship and effects on ether lipids and epithelial-mesenchymal transition in cancer cells.

Eur J Med Chem 2019 Feb 28;163:722-735. Epub 2018 Nov 28.

Department of Chemistry and Technologies of Drugs, Sapienza University of Rome, P. le A. Moro 5, 00185, Rome, Italy. Electronic address:

In aggressive tumors, alkylglyceronephosphate synthase (AGPS) controls cellular ether phospholipid utilization and metabolism to promote cancer cell proliferation and motility. SAR studies on the first-in-class AGPS inhibitor 1, discovered by our group, led to the 2,6-difluoro analog 2i which showed higher binding affinity than 1in vitro. In 231MFP cancer cells, 2i reduced ether lipids levels and cell migration rate. When tested in PC-3 and MDA-MB-231 cancer cells, 2i specifically impaired epithelial to mesenchymal transition (EMT) by modulating E-cadherin, Snail and MMP2 expression levels. Moreover, the combination of siRNAs against AGPS and 2i provided no additive effect, confirming that the modulation of 2i on EMT specifically relies on AGPS inhibition. Finally, this compound also affected cancer cell proliferation especially in MDA-MB-231 cells expressing higher AGPS level, whereas it provided negligible effects on MeT5A, a non-tumorigenic cell line, thus showing cancer specificity.
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http://dx.doi.org/10.1016/j.ejmech.2018.11.050DOI Listing
February 2019

Pyrazole-based inhibitors of enhancer of zeste homologue 2 induce apoptosis and autophagy in cancer cells.

Philos Trans R Soc Lond B Biol Sci 2018 06;373(1748)

Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy

Novel pyrazole-based EZH2 inhibitors have been prepared through a molecular pruning approach from known inhibitors bearing a bicyclic moiety as a central scaffold. The hit compound (-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-methyl-1-phenyl-1-pyrazole-4-carboxamide) showed low micromolar EZH2/PRC2 inhibition and high selectivity towards a panel of other methyltransferases. Moreover, displayed cell growth arrest in breast MDA-MB231, leukaemia K562, and neuroblastoma SK-N-BE cancer cells joined to reduction of H3K27me3 levels and induction of apoptosis and autophagy.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.
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http://dx.doi.org/10.1098/rstb.2017.0150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915724PMC
June 2018

The histone methyltransferase EZH2 as a druggable target in SHH medulloblastoma cancer stem cells.

Oncotarget 2017 Sep 2;8(40):68557-68570. Epub 2017 Aug 2.

Department of Experimental Medicine, Sapienza University of Rome, Rome 00161, Italy.

The histone methyltransferase EZH2 plays a role in maintenance of the stem component of cancer, and its overexpression and/or mutation typically drives tumor aggressiveness, drug resistance and patients' poor prognosis. In this study, we use mouse and human medulloblastoma stem-like cells belonging to the Sonic Hedgehog subgroup (SHH MB-SLCs) and demonstrate that genetic suppression of EZH2 reduces the level of its histone mark H3K27me3 and lowers proliferation and self-renewal. We designed an EZH2 inhibitor (EZH2i) as a simplified analog of EPZ005687 and GSK2816126, MC3629, and we tested its biological activity in SHH MB-SLCs. Pharmacological inhibition of EZH2 impairs SHH MB cells proliferation and self-renewal, and induces apoptosis . Finally, we generated xenograft MB-SLCs orthotopic tumors in nude mice to test MC3629 . In treated mice, we observed impairment of tumor growth, together with induction of apoptosis and reduction of proliferation and stemness. Overall, these findings describe EZH2 as a druggable target in MB and provide insight into the biological activity of MC3629 as an EZH2i.
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http://dx.doi.org/10.18632/oncotarget.19782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620277PMC
September 2017

Polymyxins and quinazolines are LSD1/KDM1A inhibitors with unusual structural features.

Sci Adv 2016 09 9;2(9):e1601017. Epub 2016 Sep 9.

Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy.

Because of its involvement in the progression of several malignant tumors, the histone lysine-specific demethylase 1 (LSD1) has become a prominent drug target in modern medicinal chemistry research. We report on the discovery of two classes of noncovalent inhibitors displaying unique structural features. The antibiotics polymyxins bind at the entrance of the substrate cleft, where their highly charged cyclic moiety interacts with a cluster of positively charged amino acids. The same site is occupied by quinazoline-based compounds, which were found to inhibit the enzyme through a most peculiar mode because they form a pile of five to seven molecules that obstruct access to the active center. These data significantly indicate unpredictable strategies for the development of epigenetic inhibitors.
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http://dx.doi.org/10.1126/sciadv.1601017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017823PMC
September 2016

The growing structural and functional complexity of the LSD1/KDM1A histone demethylase.

Curr Opin Struct Biol 2016 12 26;41:135-144. Epub 2016 Jul 26.

Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy. Electronic address:

LSD1 was the first discovered histone demethylase. Using a flavin-dependent oxidative mechanism, LSD1 demethylates the N-terminal tail of histone H3 in the context of a variety of developmental processes. This functional complexity involves the association with nuclear factors and non-coding RNAs. A number of exciting studies are uncovering the bases of these specific and diverse molecular interactions, which occur both at catalytic and non-catalytic regions of the enzyme. Alternative splicing and post-translation modifications represent further layers for modulating this complex molecular network. By combining structural methods with the usage of chemically modified histones, it is becoming possible to visualize how LSD1 and associated co-repressors recognize the nucleosome. The enzyme clamps the nucleosomal particle through multivalent interactions mediated by the non-catalytic domains, which represent prospective sites for drug design.
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http://dx.doi.org/10.1016/j.sbi.2016.07.011DOI Listing
December 2016

Histone deacetylase inhibitors restore IL-10 expression in lipopolysaccharide-induced cell inflammation and reduce IL-1β and IL-6 production in breast silicone implant in C57BL/6J wild-type murine model.

Autoimmunity 2016 Jan 20:1-11. Epub 2016 Jan 20.

c Dipartimento Organi di Senso , Sapienza Università di Roma , Roma , Italy.

Among epigenetic enzymes, histone deacetylases (HDACs) are responsible for regulating the expression of an extensive array of genes by reversible deacetylation of nuclear histones as well as a large number of non-histone proteins. Initially proposed for cancer therapy, recently the interest for HDAC inhibitors (HDACi) as orally active, safe, and anti-inflammatory agents is rising due to their ability in reducing the severity of inflammatory and autoimmune diseases. In particular, selective HDAC3, HDAC6, and HDAC8 inhibitors have been described to downregulate the expression of pro-inflammatory cytokines (TNF-α, TGF-β, IL-1β, and IL-6). Herein, using KB31, C2C12, and 3T3-J2 cell lines, we demonstrated that, under lipopolysaccharide-induced in vitro inflammation, HDAC3/6/8 inhibitor MC2625 and HDAC6-selective inhibitor MC2780 were effective at a concentration of 30 ng/mL to downregulate mRNA expression of pro-inflammatory cytokines (IL-1β and IL-6) and to promote the transcription of IL-10 gene, without affecting the cell viability. Afterwards, we investigated by immunohistochemistry the activity of MC2625 and MC2780 at a concentration of 60 ng/kg animal weight to regulate silicone-triggered immune response in C57BL/6J female mice. Our findings evidenced the ability of such inhibitors to reduce host inflammation in silicone implants promoting a thickness reduction of peri-implant fibrous capsule, upregulating IL-10 expression, and reducing the production of both IL-1β and IL-6. These results underline the potential application of MC2625 and MC2780 in inflammation-related diseases.
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http://dx.doi.org/10.3109/08916934.2015.1134510DOI Listing
January 2016

Discovery of a Novel Inhibitor of Histone Lysine-Specific Demethylase 1A (KDM1A/LSD1) as Orally Active Antitumor Agent.

J Med Chem 2016 Feb 7;59(4):1501-17. Epub 2016 Jan 7.

Department of Experimental Oncology, Academic Drug Discovery, European Institute of Oncology , Via Adamello 16, 20139 Milan, Italy.

We report the stereoselective synthesis and biological activity of a novel series of tranylcypromine (TCPA) derivatives (14a-k, 15, 16), potent inhibitors of KDM1A. The new compounds strongly inhibit the clonogenic potential of acute leukemia cell lines. In particular three molecules (14d, 14e, and 14g) showing selectivity versus MAO A and remarkably inhibiting colony formation in THP-1 human leukemia cells, were assessed in mouse for their preliminary pharmacokinetic. 14d and 14e were further tested in vivo in a murine acute promyelocytic leukemia model, resulting 14d the most effective. Its two enantiomers were synthesized: the (1S,2R) enantiomer 15 showed higher activity than its (1R,2S) analogue 16, in both biochemical and cellular assays. Compound 15 exhibited in vivo efficacy after oral administration, determining a 62% increased survival in mouse leukemia model with evidence of KDM1A inhibition. The biological profile of compound 15 supports its further investigation as a cancer therapeutic.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01209DOI Listing
February 2016

Design and Synthesis of Simplified Largazole Analogues as Isoform-Selective Human Lysine Deacetylase Inhibitors.

J Med Chem 2016 Feb 7;59(4):1613-33. Epub 2016 Jan 7.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine , 700 South Euclid Avenue, St. Louis, Missouri 63110, United States.

Selective inhibition of KDAC isoforms while maintaining potency remains a challenge. Using the largazole macrocyclic depsipeptide structure as a starting point for developing new KDACIs with increased selectivity, a combination of four different simplified largazole analogue (SLA) scaffolds with diverse zinc-binding groups (for a total of 60 compounds) were designed, synthesized, and evaluated against class I KDACs 1, 3, and 8, and class II KDAC6. Experimental evidence as well as molecular docking poses converged to establish the cyclic tetrapeptides (CTPs) as the primary determinant of both potency and selectivity by influencing the correct alignment of the zinc-binding group in the KDAC active site, providing a further basis for developing new KDACIs of higher isoform selectivity and potency.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01632DOI Listing
February 2016

Discovery of Inhibitors for the Ether Lipid-Generating Enzyme AGPS as Anti-Cancer Agents.

ACS Chem Biol 2015 Nov 4;10(11):2589-97. Epub 2015 Sep 4.

Department of Biology and Biotechnology, University of Pavia , via Ferrata 9, 27100 Pavia, Italy.

Dysregulated ether lipid metabolism is an important hallmark of cancer cells. Previous studies have reported that lowering ether lipid levels by genetic ablation of the ether lipid-generating enzyme alkyl-glycerone phosphate synthase (AGPS) lowers key structural and oncogenic ether lipid levels and alters fatty acid, glycerophospholipid, and eicosanoid metabolism to impair cancer pathogenicity, indicating that AGPS may be a potential therapeutic target for cancer. In this study, we have performed a small-molecule screen to identify candidate AGPS inhibitors. We have identified several lead AGPS inhibitors and have structurally characterized their interactions with the enzyme and show that these inhibitors bind to distinct portions of the active site. We further show that the lead AGPS inhibitor 1a selectively lowers ether lipid levels in several types of human cancer cells and impairs their cellular survival and migration. We provide here the first report of in situ-active pharmacological tools for inhibiting AGPS, which may provide chemical scaffolds for future AGPS inhibitor development for cancer therapy.
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http://dx.doi.org/10.1021/acschembio.5b00466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703096PMC
November 2015

Pure enantiomers of benzoylamino-tranylcypromine: LSD1 inhibition, gene modulation in human leukemia cells and effects on clonogenic potential of murine promyelocytic blasts.

Eur J Med Chem 2015 Apr 3;94:163-74. Epub 2015 Mar 3.

Department of Drug Chemistry and Technologies, Sapienza University of Roma, P.le A. Moro 5, 00185 Roma, Italy; Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Roma, P.le A. Moro 5, 00185 Roma, Italy. Electronic address:

The pure enantiomers of the N-(2-, 3-, and 4-(2-aminocyclopropyl)phenyl)benzamides hydrochlorides 11a-j were prepared and tested against LSD1 and MAO enzymes. The evaluation of the regioisomers 11a-j highlighted a net increase of the anti-LSD1 potency by shifting the benzamide moiety from ortho to meta and mainly to para position of tranylcypromine phenyl ring, independently from their trans or cis stereochemistry. In particular, the para-substituted 11a,b (trans) and 11g,h (cis) compounds displayed LSD1 and MAO-A inhibition at low nanomolar levels, while were less potent against MAO-B. The meta analogs 11c,d (trans) and 11i,j (cis) were in general less potent, but more efficient against MAO-A than against LSD1. In cellular assays, all the para and meta enantiomers were able to inhibit LSD1 by inducing Gfi-1b and ITGAM gene expression, with 11b,c and 11g-i giving the highest effects. Moreover, 11b and 11g,h strongly inhibited the clonogenic potential of murine promyelocytic blasts.
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http://dx.doi.org/10.1016/j.ejmech.2015.02.060DOI Listing
April 2015

Pure Diastereomers of a Tranylcypromine-Based LSD1 Inhibitor: Enzyme Selectivity and In-Cell Studies.

ACS Med Chem Lett 2015 Feb 8;6(2):173-7. Epub 2014 Dec 8.

Department of Drug Chemistry and Technologies, Sapienza University of Roma , P.le A. Moro 5, 00185 Roma, Italy ; Pasteur Institute-Cenci Bolognetti Foundation, Sapienza University of Roma , P.le A. Moro 5, 00185 Roma, Italy.

The pure four diastereomers (11a-d) of trans-benzyl (1-((4-(2-aminocyclopropyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate hydrochloride 11, previously described by us as LSD1 inhibitor, were obtained by enantiospecific synthesis/chiral HPLC separation method. Tested in LSD1 and MAO assays, 11b (S,1S,2R) and 11d (R,1S,2R) were the most potent isomers against LSD1 and were less active against MAO-A and practically inactive against MAO-B. In cells, all the four diastereomers induced Gfi-1b and ITGAM gene expression in NB4 cells, accordingly with their LSD1 inhibition, and 11b and 11d inhibited the colony forming potential in murine promyelocytic blasts.
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http://dx.doi.org/10.1021/ml500424zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329595PMC
February 2015

Exploring the role of 2-chloro-6-fluoro substitution in 2-alkylthio-6-benzyl-5-alkylpyrimidin-4(3H)-ones: effects in HIV-1-infected cells and in HIV-1 reverse transcriptase enzymes.

J Med Chem 2014 Jun 16;57(12):5212-25. Epub 2014 Jun 16.

Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma "La Sapienza" , P.le A. Moro 5, 00185 Roma, Italy.

A comparison of the effects of the 6-(2-chloro-6-fluorobenzyl)-2-(alkylthio)pyrimidin-4(3H)-ones (2-Cl-6-F-S-DABOs) 7-12 and the related 6-(2,6-difluorobenzyl) counterparts 13-15 in HIV-1 infected cells and in the HIV-1 reverse transcriptase (RT) assays is here described. The new 2-Cl-6-F-S-DABOs showed up to picomolar activity against wt HIV-1. Against clinically relevant HIV-1 mutants and in enzyme assays, the simultaneous C5(methyl)/C6(methyl/ethyl) substitution in the 2-Cl-6-F- and 2,6-F2-benzyl series furnished compounds with the highest, wide-spectrum inhibitory activity against HIV-1. Three representative 2-Cl-6-F-S-DABOs carrying two (9c, 10c) or one (10a) stereogenic centers were resolved into their individual stereoisomers and showed a significant diastereo- and enantioselectivity in HIV-1 inhibition, the highest antiviral activity well correlating with the R absolute configuration to the stereogenic center of the C6-benzylic position in both cellular and enzymatic tests. Application of previously reported COMBINEr protocol on 9c and 10c confirmed the influence of the stereogenic centers on their binding modes in the HIV-1 RT.
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http://dx.doi.org/10.1021/jm500284xDOI Listing
June 2014

Properly substituted analogues of BIX-01294 lose inhibition of G9a histone methyltransferase and gain selective anti-DNA methyltransferase 3A activity.

PLoS One 2014 8;9(5):e96941. Epub 2014 May 8.

Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Roma, IT; Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Roma, IT.

Chemical manipulations performed on the histone H3 lysine 9 methyltransferases (G9a/GLP) inhibitor BIX-01294 afforded novel desmethoxyquinazolines able to inhibit the DNA methyltransferase DNMT3A at low micromolar levels without any significant inhibition of DNMT1 and G9a. In KG-1 cells such compounds, when tested at sub-toxic doses, induced the luciferase re-expression in a stable construct controlled by a cytomegalovirus (CMV) promoter silenced by methylation (CMV-luc assay). Finally, in human lymphoma U-937 and RAJI cells, the N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine induced the highest proliferation arrest and cell death induction starting from 10 µM, in agreement with its DNMT3A inhibitory potency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096941PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014597PMC
June 2015

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