Publications by authors named "Alberto Bresciani"

39 Publications

Identification of Potent and Long-Acting Single-Chain Peptide Mimetics of Human Relaxin-2 for Cardiovascular Diseases.

J Med Chem 2021 Feb 8;64(4):2139-2150. Epub 2021 Feb 8.

Cardio-Vascular and metabolism, Sanofi R&D, 1 rue Pierre Brossolette, Chilly Mazarin 91385, France.

The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (, ). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and efficacy (, ).
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http://dx.doi.org/10.1021/acs.jmedchem.0c01533DOI Listing
February 2021

Analysis of mutant and total huntingtin expression in Huntington's disease murine models.

Sci Rep 2020 12 17;10(1):22137. Epub 2020 Dec 17.

Department of Translational Biology, IRBM S.p.A., via Pontina Km 30, 600, Pomezia, Rome, Italy.

Huntington's disease (HD) is a monogenetic neurodegenerative disorder that is caused by the expansion of a polyglutamine region within the huntingtin (HTT) protein, but there is still an incomplete understanding of the molecular mechanisms that drive pathology. Expression of the mutant form of HTT is a key aspect of diseased tissues, and the most promising therapeutic approaches aim to lower expanded HTT levels. Consequently, the investigation of HTT expression in time and in multiple tissues, with assays that accurately quantify expanded and non-expanded HTT, are required to delineate HTT homeostasis and to best design and interpret pharmacodynamic readouts for HTT lowering therapeutics. Here we evaluate mutant polyglutamine-expanded (mHTT) and polyglutamine-independent HTT specific immunoassays for validation in human HD and control fibroblasts and use to elucidate the CSF/brain and peripheral tissue expression of HTT in preclinical HD models.
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http://dx.doi.org/10.1038/s41598-020-78790-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746729PMC
December 2020

NRF2 activation by reversible KEAP1 binding induces the antioxidant response in primary neurons and astrocytes of a Huntington's disease mouse model.

Free Radic Biol Med 2021 Jan 21;162:243-254. Epub 2020 Oct 21.

Department of Translational and Discovery Research, IRBM S.p.A., Via Pontina Km 30,600, 00071, Pomezia, Roma, Italy. Electronic address:

Oxidative stress has been associated with pathogenesis in several diseases including Huntington's disease (HD), a neurodegenerative disorder caused by a mutation in the huntingtin gene. Oxidative stress induced reactive oxygen species (ROS) are normally controlled at the cellular level by the nuclear factor (erythroid-derived 2)-like 2 (NRF2) a transcription factor that regulates the expression of various antioxidants and detoxifying proteins. Normally NRF2 is largely inactivated in the cytoplasm by the Kelch-like ECH-associated protein 1 (KEAP1)/Cullin-3 (CUL3) mediated ubiquitination and subsequent proteosomal degradation. In the presence of ROS, KEAP1 sensor cysteines are directly or indirectly engaged resulting in NRF2 release, nuclear translocation, and activation of its target genes. Consequently the activation of NRF2 by a small-molecule drug may have the therapeutic potential to control oxidative stress by upregulation of the endogenous antioxidant responses. Here we attempted to validate the use of a reversible non-acidic KEAP1 binder (Compound 2) to activate NRF2 with better cellular activity than similar acidic compounds. When tested head to head with sulforaphane, a covalent KEAP1 binder, Compound 2 had a similar ability to induce the expression of genes known to be modulated by NRF2 in neurons and astrocytes isolated from wild-type rat, wild type mouse and zQ175 (an HD mouse model) embryos. However, while sulforaphane also negatively affected genes involved in neurotoxicity in these cells, Compound 2 showed a clean profile suggesting its mode of action has lower off-target activity. We show that Compound 2 was able to protect cells from an oxidative insult by preserving the ATP content and the mitochondrial potential of primary astrocytes, consistent with the hypothesis that neurotoxicity induced by oxidative stress can be limited by upregulation of innate antioxidant response.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.10.022DOI Listing
January 2021

Optimization of linear and cyclic peptide inhibitors of KEAP1-NRF2 protein-protein interaction.

Bioorg Med Chem 2020 11 30;28(21):115738. Epub 2020 Aug 30.

Department of Drug Discovery, IRBM Spa, Via Pontina km 30.600, 00071 Pomezia, Rome, Italy.

Inhibition of KEAP1-NRF2 protein-protein interaction is considered a promising strategy to selectively and effectively activate NRF2, a transcription factor which is involved in several pathologies such as Huntington's disease (HD). A library of linear peptides based on the NRF2-binding motifs was generated on the nonapeptide lead Ac-LDEETGEFL-NH spanning residues 76-84 of the Neh2 domain of NRF2 with the aim to replace E78, E79 and E82 with non-acidic amino acids. A deeper understanding of the features and accessibility of the T80 subpocket was also targeted by structure-based design. Approaches to improve cell permeability were investigated using both different classes of cyclic peptides and conjugation to cell-penetrating peptides. This insight will guide future design of macrocycles, peptido-mimetics and, most importantly, small neutral brain-penetrating molecules to evaluate whether NRF2 activators have utility in HD.
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http://dx.doi.org/10.1016/j.bmc.2020.115738DOI Listing
November 2020

Drug effects on metabolic profiles of Schistosoma mansoni adult male parasites detected by 1H-NMR spectroscopy.

PLoS Negl Trop Dis 2020 10 12;14(10):e0008767. Epub 2020 Oct 12.

Institute of Biochemistry and Cell Biology, National Research Council, Campus A. Buzzati-Traverso, Monterotondo (Rome) Italy.

Schistosomiasis is one of the most devastating neglected tropical parasitic diseases caused by trematodes of the genus Schistosoma. Praziquantel (PZQ) is today the only drug used in humans and animals for the treatment of schistosomiasis but unfortunately it is poorly effective on larval and juvenile stages of the parasite. Therefore, it is urgent the discovery of new drug targets and compounds. We have recently showed that the anti-anginal drug perhexiline maleate (PHX) is very active on multiple developmental stages of Schistosoma mansoni in vitro. It is well known that PHX impacts the lipid metabolism in mammals, but the final target on schistosomes still remains unknown. The aim of this study was to evaluate the ability of 1H nuclear magnetic resonance (NMR) spectroscopy in revealing metabolic perturbations due to PHX treatment of S. mansoni adult male worms. The effects of PHX were compared with the ones induced by vehicle and gambogic acid, in order to detect different metabolic profiles and specificity of the PHX action. Remarkably a list of metabolites associated to PHX-treatment was identified with enrichment in several connected metabolic pathways including also the Kennedy pathway mediating the glycerophospholipid metabolism. Our study represents the first 1H-NMR metabolomic approach to characterize the response of S. mansoni to drug treatment. The obtained "metabolic fingerprint" associated to PHX treatment could represent a strategy for displaying cellular metabolic changes for any given drug and to compare compounds targeting similar or distinct biochemical pathways.
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http://dx.doi.org/10.1371/journal.pntd.0008767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580944PMC
October 2020

Luminescence-Based, Low- and Medium-Throughput Assays for Drug Screening in Schistosoma mansoni Larval Stage.

Methods Mol Biol 2020 ;2151:219-227

National Research Council, Institute of Biochemistry and Cell Biology, Rome, Italy.

Schistosomiasis is one of the major parasitic diseases with more than  200 million people infected worldwide every year. Praziquantel is the drug of choice against the schistosomiasis although the use of a single drug to treat such a large amount of infected people appears particularly worrisome. For this reason, the search of new schistosomicidal compounds is viewed as an urgent goal and a number of screening campaigns have been carried out in the past years. The larval stage of Schistosoma (schistosomula) has been widely used in order to identify new compounds against the parasite. Here we describe detailed practical procedures for a luminescence-based assay proven to be highly effective for the selection of schistosomicidal compounds on small and medium-high scale. The assay is based on the quantitation of the parasite ATP, a good indicator of metabolically active cells, as measure of schistosomula viability. This assay is fast and reproducible, and it is suitable either for manual or for semiautomated screenings.
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http://dx.doi.org/10.1007/978-1-0716-0635-3_18DOI Listing
January 2020

Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme.

PLoS Negl Trop Dis 2020 05 21;14(5):e0008339. Epub 2020 May 21.

Department of Translational and Discovery Research, Pomezia (Roma) Italy.

Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461') and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections.
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http://dx.doi.org/10.1371/journal.pntd.0008339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269337PMC
May 2020

Combined Peptide and Small-Molecule Approach toward Nonacidic THIQ Inhibitors of the KEAP1/NRF2 Interaction.

ACS Med Chem Lett 2020 May 3;11(5):740-746. Epub 2020 Apr 3.

IRBM S.p.A., Via Pontina km 30.600, 00071 Pomezia, Rome, Italy.

The NRF2-ARE pathway is an intrinsic mechanism of defense against oxidative stress. Inhibition of the interaction between NRF2 and its main negative regulator KEAP1 is an attractive strategy toward neuroprotective agents. We report here the identification of nonacidic tetrahydroisoquinolines (THIQs) that inhibit the KEAP1/NRF2 protein-protein interaction. Peptide SAR at one residue is utilized as a tool to probe structural changes within a specific pocket of the KEAP1 binding site. We used structural information from peptide screening at the P2 pocket, noncovalent small-molecules inhibitors, and the outcome from an explorative SAR at position 5 of THIQs to identify a series of neutral THIQ analogs that bind to KEAP1 in the low micromolar range. These analogs establish new H-bond interactions at the P3 and P2 pockets allowing the replacement of the carboxylic acid functionality by a neutral primary carboxamide. X-ray crystallographic studies reveal the novel binding mode of these molecules to KEAP1.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236277PMC
May 2020

Adipogenesis of skeletal muscle fibro/adipogenic progenitors is affected by the WNT5a/GSK3/β-catenin axis.

Cell Death Differ 2020 Oct 7;27(10):2921-2941. Epub 2020 May 7.

Department of Biology, University of Rome "Tor Vergata", 00133, Rome, Italy.

Fibro/Adipogenic Progenitors (FAPs) are muscle-interstitial progenitors mediating pro-myogenic signals that are critical for muscle homeostasis and regeneration. In myopathies, the autocrine/paracrine constraints controlling FAP adipogenesis are released causing fat infiltrates. Here, by combining pharmacological screening, high-dimensional mass cytometry and in silico network modeling with the integration of single-cell/bulk RNA sequencing data, we highlighted the canonical WNT/GSK/β-catenin signaling as a crucial pathway modulating FAP adipogenesis triggered by insulin signaling. Consistently, pharmacological blockade of GSK3, by the LY2090314 inhibitor, stabilizes β-catenin and represses PPARγ expression abrogating FAP adipogenesis ex vivo while limiting fatty degeneration in vivo. Furthermore, GSK3 inhibition improves the FAP pro-myogenic role by efficiently stimulating, via follistatin secretion, muscle satellite cell (MuSC) differentiation into mature myotubes. Combining, publicly available single-cell RNAseq datasets, we characterize FAPs as the main source of WNT ligands inferring their potential in mediating autocrine/paracrine responses in the muscle niche. Lastly, we identify WNT5a, whose expression is impaired in dystrophic FAPs, as a crucial WNT ligand able to restrain the detrimental adipogenic differentiation drift of these cells through the positive modulation of the β-catenin signaling.
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http://dx.doi.org/10.1038/s41418-020-0551-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492278PMC
October 2020

Janus effect of glucocorticoids on differentiation of muscle fibro/adipogenic progenitors.

Sci Rep 2020 03 24;10(1):5363. Epub 2020 Mar 24.

Department of Biology, University of Rome "Tor Vergata", Rome, Italy.

Muscle resident fibro-adipogenic progenitors (FAPs), support muscle regeneration by releasing cytokines that stimulate the differentiation of myogenic stem cells. However, in non-physiological contexts (myopathies, atrophy, aging) FAPs cause fibrotic and fat infiltrations that impair muscle function. We set out to perform a fluorescence microscopy-based screening to identify compounds that perturb the differentiation trajectories of these multipotent stem cells. From a primary screen of 1,120 FDA/EMA approved drugs, we identified 34 compounds as potential inhibitors of adipogenic differentiation of FAPs isolated from the murine model (mdx) of Duchenne muscular dystrophy (DMD). The hit list from this screen was surprisingly enriched with compounds from the glucocorticoid (GCs) chemical class, drugs that are known to promote adipogenesis in vitro and in vivo. To shed light on these data, three GCs identified in our screening efforts were characterized by different approaches. We found that like dexamethasone, budesonide inhibits adipogenesis induced by insulin in sub-confluent FAPs. However, both drugs have a pro-adipogenic impact when the adipogenic mix contains factors that increase the concentration of cAMP. Gene expression analysis demonstrated that treatment with glucocorticoids induces the transcription of Gilz/Tsc22d3, an inhibitor of the adipogenic master regulator PPARγ, only in anti-adipogenic conditions. Additionally, alongside their anti-adipogenic effect, GCs are shown to promote terminal differentiation of satellite cells. Both the anti-adipogenic and pro-myogenic effects are mediated by the glucocorticoid receptor and are not observed in the presence of receptor inhibitors. Steroid administration currently represents the standard treatment for DMD patients, the rationale being based on their anti-inflammatory effects. The findings presented here offer new insights on additional glucocorticoid effects on muscle stem cells that may affect muscle homeostasis and physiology.
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http://dx.doi.org/10.1038/s41598-020-62194-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093513PMC
March 2020

Discovery of 4-((1-(1H-imidazol-2-yl)alkoxy)methyl)pyridines as a new class of Trypanosoma cruzi growth inhibitors.

Bioorg Med Chem Lett 2020 04 22;30(8):127052. Epub 2020 Feb 22.

Departments of Chemistry and Biology, IRBM Spa, Via Pontina km 30, 600, 00071 Pomezia, Rome, Italy.

The identification of a new series of growth inhibitors of Trypanosoma cruzi, the causative agent of Chagas' disease, is described. In vitro screening of a subset of compounds from our in-house compound collection against the parasite led to the identification of hit compound 1 with low micromolar inhibition of T. cruzi growth. SAR exploration on the hit compound led to the identification of compounds that show nanomolar parasite growth inhibition (T. cruzi EC ≤ 100 nM) and no cytotoxicity in human cells (HeLa CC > 50 μM). Further investigation identified CYP51 inhibition (compound 11 CYP51 IC 52 nM) as a possible mechanism of action of this new class of anti-parasitic agents.
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http://dx.doi.org/10.1016/j.bmcl.2020.127052DOI Listing
April 2020

Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits.

Cells 2020 01 10;9(1). Epub 2020 Jan 10.

National Institute of Molecular Genetics, "Fondazione Romeo ed Enrica Invernizzi", INGM, Via Francesco Sforza 35, 20122 Milan, Italy.

Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.
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http://dx.doi.org/10.3390/cells9010172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017188PMC
January 2020

sp. nov., a halotolerant species isolated from olive brine.

Int J Syst Evol Microbiol 2019 Sep;69(9):2899-2906

Mycotoxin Prevention and Applied Microbiology Research Unit, Agricultural Research Service, US Department of Agriculture, Peoria 61604, USA.

A facultative halo-tolerant strain was isolated from olive brine waste, the effluent from the debittering process of table olives. Phenotypic and molecular characteristics showed clearly that the isolate represents a novel species. Based on the source of isolation, the new species has been named . It was found tolerant to high concentrations of NaCl (15 %) or sucrose (60 %) and it exhibits substantial growth under these conditions. Although the new species grew profusely at 37 °C, no growth was observed at 40 °C, conidia were avellaneous on all media. The description of the new species brings the total species of sect. to 15. The type strain of sp. nov. is NRRL 66783 (CCF 6208), its whole genome has been deposited as PRJNA498048.
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http://dx.doi.org/10.1099/ijsem.0.003575DOI Listing
September 2019

Development and characterization of cellular biosensors for HTS of erythroid differentiation inducers targeting the transcriptional activity of γ-globin and β-globin gene promoters.

Anal Bioanal Chem 2019 Nov 4;411(29):7669-7680. Epub 2019 Jul 4.

Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121, Ferrara, Italy.

There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of β-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and β-globin genes by FACS with efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the β-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from β-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of β-thalassemia and sickle cell anemia. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-019-01959-zDOI Listing
November 2019

Inhibiting pathologically active ADAM10 rescues synaptic and cognitive decline in Huntington's disease.

J Clin Invest 2019 05 6;129(6):2390-2403. Epub 2019 May 6.

Department of Biosciences, University of Milan, Milan, Italy.

A disintegrine and metalloproteinase 10 (ADAM10) is implicated in synaptic function through its interaction with postsynaptic receptors and adhesion molecules. Here, we report that levels of active ADAM10 are increased in Huntington's disease (HD) mouse cortices and striata and in human postmortem caudate. We show that, in the presence of polyglutamine-expanded (polyQ-expanded) huntingtin (HTT), ADAM10 accumulates at the postsynaptic densities (PSDs) and causes excessive cleavage of the synaptic protein N-cadherin (N-CAD). This aberrant phenotype is also detected in neurons from HD patients where it can be reverted by selective silencing of mutant HTT. Consistently, ex vivo delivery of an ADAM10 synthetic inhibitor reduces N-CAD proteolysis and corrects electrophysiological alterations in striatal medium-sized spiny neurons (MSNs) of 2 HD mouse models. Moreover, we show that heterozygous conditional deletion of ADAM10 or delivery of a competitive TAT-Pro-ADAM10709-729 peptide in R6/2 mice prevents N-CAD proteolysis and ameliorates cognitive deficits in the mice. Reduction in synapse loss was also found in R6/2 mice conditionally deleted for ADAM10. Taken together, these results point to a detrimental role of hyperactive ADAM10 at the HD synapse and provide preclinical evidence of the therapeutic potential of ADAM10 inhibition in HD.
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http://dx.doi.org/10.1172/JCI120616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546448PMC
May 2019

Improved Selective Class I HDAC and Novel Selective HDAC3 Inhibitors: Beyond Hydroxamic Acids and Benzamides.

ACS Med Chem Lett 2019 Apr 27;10(4):481-486. Epub 2018 Nov 27.

IRBM Science Park, Via Pontina km 30,600, 00071 Pomezia, Rome, Italy.

The application of class I HDAC inhibitors as cancer therapies is well established, but more recently their development for nononcological indications has increased. We report here on the generation of improved class I selective human HDAC inhibitors based on an ethylketone zinc binding group (ZBG) in place of the hydroxamic acid that features the majority of HDAC inhibitors. We also describe a novel set of HDAC3 isoform selective inhibitors that show stronger potency and selectivity than the most commonly used HDAC3 selective tool compound RGFP966. These compounds are again based on an alternative ZBG with respect to the -anilide that is featured in HDAC3 selective compounds reported to date.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466521PMC
April 2019

Fish-derived antimicrobial peptides: Activity of a chionodracine mutant against bacterial models and human bacterial pathogens.

Dev Comp Immunol 2019 07 18;96:9-17. Epub 2019 Feb 18.

Department for Innovation in Biological, Agrofood and Forest Systems, University of Tuscia, Viterbo, Italy. Electronic address:

The increasing resistance to conventional antibiotics is an urgent problem that can be addressed by the discovery of new antimicrobial drugs such as antimicrobial peptides (AMPs). AMPs are components of innate immune system of eukaryotes and are not prone to the conventional mechanisms that are responsible of drug resistance. Fish are an important source of AMPs and, recently, we have isolated and characterized a new 22 amino acid residues peptide, the chionodracine (Cnd), from the Antarctic icefish Chionodraco hamatus. In this paper we focused on a new Cnd-derived mutant peptide, namely Cnd-m3a, designed to improve the selectivity against prokaryotic cells and the antimicrobial activity against human pathogens of the initial Cnd template. Cnd-m3a was used for immunization of rabbits, which gave rise to a polyclonal antibody able to detect the peptide. The interaction kinetic of Cnd-m3a with the Antarctic bacterium Psychrobacter sp. (TAD1) was imaged using a transmission electron microscopy (TEM) immunogold method. Initially the peptide was associated with the plasma membrane, but after 180 min of incubation, it was found in the cytoplasm interacting with a DNA target inside the bacterial cells. Using fluorescent probes we showed that the newly designed mutant can create pores in the outer membrane of the bacteria E. coli and Psychrobacter sp. (TAD1), confirming the results of TEM analysis. Moreover, in vitro assays demonstrated that Cnd-m3a is able to bind lipid vesicles of different compositions with a preference toward negatively charged ones, which mimics the prokaryotic cell. The Cnd-m3a peptide showed quite low hemolytic activity and weak cytotoxic effect against human primary and tumor cell lines, but high antimicrobial activity against selected Gram - human pathogens. These results highlighted the high potential of the Cnd-m3a peptide as a starting point for developing a new human therapeutic agent.
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http://dx.doi.org/10.1016/j.dci.2019.02.012DOI Listing
July 2019

Identification of novel multi-stage histone deacetylase (HDAC) inhibitors that impair Schistosoma mansoni viability and egg production.

Parasit Vectors 2018 Dec 27;11(1):668. Epub 2018 Dec 27.

National Research Council, Institute of Cell Biology and Neurobiology, Campus A. Buzzati-Traverso, Monterotondo (Roma), Italy.

Background: Novel anti-schistosomal multi-stage drugs are needed because only a single drug, praziquantel, is available for the treatment of schistosomiasis and is poorly effective on larval and juvenile stages of the parasite. Schistosomes have a complex life-cycle and multiple developmental stages in the intermediate and definitive hosts. Acetylation and deacetylation of histones play pivotal roles in chromatin structure and in the regulation of transcription in eukaryotic cells. Histone deacetylase (HDAC) inhibitors modulate acetylation of several other proteins localized both in the nucleus and in the cytoplasm and therefore impact on many signaling networks and biological processes. Histone post-translational modifications may provide parasites with the ability to readily adapt to changes in gene expression required for their development and adaptation to the host environment. The aim of the present study was to screen a HDAC class I inhibitor library in order to identify and characterize novel multi-stage hit compounds.

Methods: We used a high-throughput assay based on the quantitation of ATP in the Schistosoma mansoni larval stage (schistosomula) and screened a library of 1500 class I HDAC inhibitors. Subsequently, a few hits were selected and further characterized by viability assays and phenotypic analyses on adult parasites by carmine red and confocal microscopy.

Results: Three compounds (SmI-124, SmI-148 and SmI-558) that had an effect on the viability of both the schistosomula larval stage and the adult worm were identified. Treatment with sub-lethal doses of SmI-148 and SmI-558 also decreased egg production. Moreover, treatment of adult parasites with SmI-148, and to a lesser extent Sm-124, was associated with histone hyperacetylation. Finally, SmI-148 and SmI-558 treatments of worm pairs caused a phenotype characterized by defects in the parasite reproductive system, with peculiar features in the ovary. In addition, SmI-558 induced oocyte- and vitelline cell-engulfment and signs of degeneration in the uterus and/or oviduct.

Conclusions: We report the screening of a small HDAC inhibitor library and the identification of three novel compounds which impair viability of the S. mansoni larval stage and adult pairs. These compounds are useful tools for studying deacetylase activity during parasite development and for interfering with egg production. Characterization of their specificity for selected S. mansoni versus human HDAC could provide insights that can be used in optimization and compound design.
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http://dx.doi.org/10.1186/s13071-018-3268-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307185PMC
December 2018

Discovery of 2-(1H-imidazo-2-yl)piperazines as a new class of potent and non-cytotoxic inhibitors of Trypanosoma brucei growth in vitro.

Bioorg Med Chem Lett 2018 12 22;28(23-24):3689-3692. Epub 2018 Oct 22.

Departments of Chemistry and Biology, IRBM Science Park, Via Pontina km 30,600, 00071 Pomezia, Rome, Italy.

The identification of a new series of growth inhibitors of Trypanosoma brucei rhodesiense, causative agent of Human African Trypanosomiasis (HAT), is described. A selection of compounds from our in-house compound collection was screened in vitro against the parasite leading to the identification of compounds with nanomolar inhibition of T. brucei growth. Preliminary SAR on the hit compound led to the identification of compound 34 that shows low nanomolar parasite growth inhibition (T. brucei EC 5 nM), is not cytotoxic (HeLa CC > 25,000 nM) and is selective over other parasites, such as Trypanosoma cruzi and Plasmodium falciparum (T. cruzi EC 8120 nM, P. falciparum EC 3624 nM).
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http://dx.doi.org/10.1016/j.bmcl.2018.10.028DOI Listing
December 2018

Identification and binding mode of a novel Leishmania Trypanothione reductase inhibitor from high throughput screening.

PLoS Negl Trop Dis 2018 11 26;12(11):e0006969. Epub 2018 Nov 26.

Istituto di Biologia e Patologia Molecolari-CNR, and Dipartimento di Scienze Biochimiche, "Sapienza" Università di Roma P.le A. Moro, Roma, Italy.

Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against Leishmaniasis. This enzyme is fundamental for parasite survival in the host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize hydrogen peroxide produced by host macrophages during infection. In order to identify new lead compounds against Leishmania we developed and validated a new luminescence-based high-throughput screening (HTS) assay that allowed us to screen a library of 120,000 compounds. We identified a novel chemical class of TR inhibitors, able to kill parasites with an IC50 in the low micromolar range. The X-ray crystal structure of TR in complex with a compound from this class (compound 3) allowed the identification of its binding site in a pocket at the entrance of the NADPH binding site. Since the binding site of compound 3 identified by the X-ray structure is unique, and is not present in human homologs such as glutathione reductase (hGR), it represents a new target for drug discovery efforts.
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http://dx.doi.org/10.1371/journal.pntd.0006969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283646PMC
November 2018

Development of a Broadly Applicable Assay for Measurement of Glycan-Directed Enzymatic Activity.

SLAS Discov 2018 10 22;23(9):941-950. Epub 2018 Jun 22.

2 Retrophin, Inc., San Diego, CA, USA.

Glycosylation is a key posttranslational modification that tags protein to membranes, organelles, secretory pathways, and degradation. Aberrant protein glycosylation is present both in acquired diseases, such as cancer and neurodegeneration, and in congenital disorders of glycosylation (CDGs). Consequently, the ability to interrogate the activity of enzymes that can modify protein glycan moieties is key for drug discovery projects aimed at finding modulators of these enzymes. To date, low-throughput technologies such as SDS-PAGE and mass spectrometry have been used, which are not suitable for compound screening in drug discovery. In the present work, a broadly applicable time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed that can determine the activity of endoglycosidase enzymes in high-throughput formats. The assay was validated using PNGaseF and EndoH as tool deglycosylases. Even though the current setup is based on the recognition of glycans that bind concanavalin A (ConA), the assay concept can be adapted to glycans that bind other lectins.
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http://dx.doi.org/10.1177/2472555218782625DOI Listing
October 2018

Peptidomimetic nitrile inhibitors of malarial protease falcipain-2 with high selectivity against human cathepsins.

Bioorg Med Chem Lett 2018 05 26;28(9):1540-1544. Epub 2018 Mar 26.

Department of Chemistry, IRBM Science Park, Via Pontina km 30, 600, Pomezia 00071, Rome, Italy.

Falcipain-2 (FP2) is an essential enzyme in the lifecycle of malaria parasites such as Plasmodium falciparum, and its inhibition is viewed as an attractive mechanism of action for new anti-malarial agents. Selective inhibition of FP2 with respect to a family of human cysteine proteases (that include cathepsins B, K, L and S) is likely to be required for the development of agents targeting FP2. Here we describe a series of P2-modified aminonitrile based inhibitors of FP2 that provide a clear strategy toward addressing selectivity for the P. falciparum and show that it can provide potent FP2 inhibitors with strong selectivity against all four of these human cathepsin isoforms.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.069DOI Listing
May 2018

Quantifying autophagy using novel LC3B and p62 TR-FRET assays.

PLoS One 2018 19;13(3):e0194423. Epub 2018 Mar 19.

IRBM Promidis, Pomezia, Rome, Italy.

Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194423PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858923PMC
July 2018

Validation of Ultrasensitive Mutant Huntingtin Detection in Human Cerebrospinal Fluid by Single Molecule Counting Immunoassay.

J Huntingtons Dis 2017 ;6(4):349-361

IRBM Science Park, Pomezia, Rome, Italy.

Background: The measurement of disease-relevant biomarkers has become a major component of clinical trial design, but in the absence of rigorous clinical and analytical validation of detection methodology, interpretation of results may be misleading. In Huntington's disease (HD), measurement of the concentration of mutant huntingtin protein (mHTT) in cerebrospinal fluid (CSF) of patients may serve as both a disease progression biomarker and a pharmacodynamic readout for HTT-lowering therapeutic approaches. We recently published the quantification of mHTT levels in HD patient CSF by a novel ultrasensitive immunoassay-based technology and here analytically validate it for use.

Objective: This work aims to analytically and clinically validate our ultrasensitive assay for mHTT measurement in human HD CSF, for application as a pharmacodynamic biomarker of CNS mHTT lowering in clinical trials.

Methods: The single molecule counting (SMC) assay is an ultrasensitive bead-based immunoassay where upon specific recognition, dye-labeled antibodies are excited by a confocal laser and emit fluorescent light as a readout. The detection of mHTT by this technology was clinically validated following established Food and Drug Administration and European Medicine Agency guidelines.

Results: The SMC assay was demonstrated to be accurate, precise, specific, and reproducible. While no matrix influence was detected, a list of interfering substances was compiled as a guideline for proper collection and storage of patient CSF samples. In addition, a set of recommendations on result interpretation is provided.

Conclusions: This SMC assay is a robust and ultrasensitive method for the relative quantification of mHTT in human CSF.
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http://dx.doi.org/10.3233/JHD-170269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757651PMC
July 2018

Discovery by organism based high-throughput screening of new multi-stage compounds affecting Schistosoma mansoni viability, egg formation and production.

PLoS Negl Trop Dis 2017 Oct 6;11(10):e0005994. Epub 2017 Oct 6.

National Research Council, Institute of Cell Biology and Neurobiology, Campus A. Buzzati-Traverso, Monterotondo (Roma), Italy.

Schistosomiasis, one of the most prevalent neglected parasitic diseases affecting humans and animals, is caused by the Platyhelminthes of the genus Schistosoma. Schistosomes are the only trematodes to have evolved sexual dimorphism and the constant pairing with a male is essential for the sexual maturation of the female. Pairing is required for the full development of the two major female organs, ovary and vitellarium that are involved in the production of different cell types such as oocytes and vitellocytes, which represent the core elements of the whole egg machinery. Sexually mature females can produce a large number of eggs each day. Due to the importance of egg production for both life cycle and pathogenesis, there is significant interest in the search for new strategies and compounds not only affecting parasite viability but also egg production. Here we use a recently developed high-throughput organism-based approach, based on ATP quantitation in the schistosomula larval stage of Schistosoma mansoni for the screening of a large compound library, and describe a pharmacophore-based drug selection approach and phenotypic analyses to identify novel multi-stage schistosomicidal compounds. Interestingly, worm pairs treated with seven of the eight compounds identified show a phenotype characterized by defects in eggshell assemblage within the ootype and egg formation with degenerated oocytes and vitelline cells engulfment in the uterus and/or oviduct. We describe promising new molecules that not only impair the schistosomula larval stage but also impact juvenile and adult worm viability and egg formation and production in vitro.
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http://dx.doi.org/10.1371/journal.pntd.0005994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5646872PMC
October 2017

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) drug discovery: Biochemical toolbox to develop NRF2 activators by reversible binding of Kelch-like ECH-associated protein 1 (KEAP1).

Arch Biochem Biophys 2017 10 8;631:31-41. Epub 2017 Aug 8.

CHDI Management/CHDI Foundation, Los Angeles, CA, USA. Electronic address:

Mechanisms that activate innate antioxidant responses, as a way to mitigate oxidative stress at the site of action, hold much therapeutic potential in diseases, such as Parkinson's disease, Alzheimer's disease and Huntington's disease, where the use of antioxidants as monotherapy has not yielded positive results. The nuclear factor NRF2 is a transcription factor whose activity upregulates the expression of cell detoxifying enzymes in response to oxidative stress. NRF2 levels are modulated by KEAP1, a sensor of oxidative stress. KEAP1 binds NRF2 and facilitates its ubiquitination and subsequent degradation. Recently, compounds that reversibly disrupt the NRF2-KEAP1 interaction have been described, opening the field to a new era of safer NRF2 activators. This paper describes a set of new, robust and informative biochemical assays that enable the selection and optimization of non-covalent KEAP1 binders. These include a time-resolved fluorescence resonance energy transfer (TR-FRET) primary assay with high modularity and robustness, a surface plasmon resonance (SPR) based KEAP1 direct binding assay that enables the quantification and analysis of full kinetic binding parameters and finally a H-N heteronuclear single quantum coherence (HSQC) NMR assay suited to study the interaction surface of KEAP1 with residue-specific information to validate the interaction of ligands in the KEAP1 binding site.
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http://dx.doi.org/10.1016/j.abb.2017.08.003DOI Listing
October 2017

Polyglutamine expansion affects huntingtin conformation in multiple Huntington's disease models.

Sci Rep 2017 07 11;7(1):5070. Epub 2017 Jul 11.

IRBM Science Park, Via Pontina km 30.600, 00071, Pomezia, Rome, Italy.

Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington's disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine- and temperature-dependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperature- and polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients.
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http://dx.doi.org/10.1038/s41598-017-05336-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505970PMC
July 2017

Identification by High-Throughput Screening of Pseudomonas Acyl-Coenzyme A Synthetase Inhibitors.

SLAS Discov 2017 08 31;22(7):897-905. Epub 2017 Jan 31.

1 IRBM Science Park S.p.A, Pomezia, Italy.

Pseudomonas infections are common among hospitalized, immunocompromised, and chronic lung disease patients. These infections are recalcitrant to common antibacterial therapies due to inherent antibiotic resistance. To meet the need of new anti- Pseudomonas drugs, a sensitive, homogenous, and robust assay was developed with the aim of identifying inhibitors of acyl-coenzyme A synthetases (ACSs) from Pseudomonas. Given the importance of fatty acids for in vivo nutrition of Pseudomonas, such inhibitors might have the potential to reduce the bacterial fitness during infection. The assay, based on a coupled reaction between the Pseudomonas spp. ACS and the firefly luciferase, allowed the identification of three classes of inhibitors by screening of a diverse compound collection. These compounds were confirmed to reversibly bind ACS with potencies in the micromolar range. Two classes were found to compete with acyl-coenzyme A, while the third one was competitive with fatty acid binding. Although these compounds inhibit the bacterial ACS in cell-free assays, they show modest or no effect on Pseudomonas growth in vitro.
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http://dx.doi.org/10.1177/2472555216689283DOI Listing
August 2017

Vitamin B12 ameliorates the phenotype of a mouse model of DiGeorge syndrome.

Hum Mol Genet 2016 10;25(20):4369-4375

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

Abstract: Pathological conditions caused by reduced dosage of a gene, such as gene haploinsufficiency, can potentially be reverted by enhancing the expression of the functional allele. In practice, low specificity of therapeutic agents, or their toxicity reduces their clinical applicability. Here, we have used a high throughput screening (HTS) approach to identify molecules capable of increasing the expression of the gene Tbx1, which is involved in one of the most common gene haploinsufficiency syndromes, the 22q11.2 deletion syndrome. Surprisingly, we found that one of the two compounds identified by the HTS is the vitamin B12. Validation in a mouse model demonstrated that vitamin B12 treatment enhances Tbx1 gene expression and partially rescues the haploinsufficiency phenotype. These results lay the basis for preclinical and clinical studies to establish the effectiveness of this drug in the human syndrome.
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http://dx.doi.org/10.1093/hmg/ddw267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409220PMC
October 2016