Identification of Potent and Long-Acting Single-Chain Peptide Mimetics of Human Relaxin-2 for Cardiovascular Diseases
IRBM & University of Rome publishes in PLOS on new chemotype with antiparasitic activity for treating sleeping sickness
Trypanosoma brucei is a parasite responsible for human African trypanosomiasis, also known as sleeping sickness, a disease endemic in sub-Saharan Africa, with 70 million people at risk of infection. Current treatments are limited by their toxicity, administration in endemic countries and treatment resistance. Therapies against infectious diseases typically rely on targeting components of the parasite that are not present in humans. Here, to identify novel inhibitors, we aimed at targeting the Trypanosoma brucei trypanothione reductase (TR), an enzyme that synthetizes a key molecule for preserving the parasite redox balance. This enzyme does not exist in humans. Through use of a recent, higher quality, TR activity assay to test a collection of compounds previously reported to have antiparasitic activity, we identified and validated a new chemotype with a unique mode of TR inhibition. The chemotype’s central spiro scaffold is suitable for brain active compounds in humans, providing a drug-like starting point for treatment of the CNS stage of T. brucei infections.
IRBM publishes on optimizing a new series of Trypanosoma brucei growth inhibitors, the parasite responsible of the sleeping sickness
Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic disease that causes significant mortality in sub- Saharan Africa. A previous publication from IRBM laboratory reported the identification of 2-(1H-imidazo-2-yl)piperazines as a new series of potent T. brucei growth inhibitors. This work describes the structure–activity relationship (SAR) around the hit compound 1, which led to the identification of the optimized compound 18, a single digit nanomolar inhibitor, not cytotoxic and with optimal in vivo profile that made it a suitable candidate for efficacy studies in animal models mimicking the second stage of disease.
IRBM & University of Rome publishes in Nature on crucial signaling pathway in muscle homeostasis and regeneration
IRBM, working with the University of Rome’s Department of Biology, examined signaling pathways involved with the regulation of Fibro/Adipogenic Progenitors (FAPs). FAPs are muscle-interstitial progenitors mediating pro-myogenic signals that are critical for muscle homeostasis and regeneration. In myopathies, FAP adipogenesis goes awry, causing fat infiltrates and muscle degeneration. By combining pharmacological screening, high-dimensional mass cytometry and in silico network modelling with the integration of single-cell/bulk RNA sequencing data, the canonical WNT/GSK/β-catenin signaling was highlighted as a crucial pathway modulating FAP adipogenesis triggered by insulin signaling. This suggests FAPs have potential in mediating autocrine/paracrine responses in the muscle niche.
IRBM’s publishes in Cells on establishing a human blood-brain barrier model for use in drug discovery for neurodegenerative diseases such as Huntington’s
The blood-brain barrier (BBB) is responsible for the homeostasis between the cerebral vasculature and the brain. It has a key role in regulating the influx and efflux of substances, in healthy and diseased states. Stem cell technology offers the opportunity to use human brain-specific cells to establish in vitro BBB models. These are valuable tools for studying mechanistic aspects of drug transport to the brain and developing strategic solutions to circumvent the BBB and allow passage of central nervous system (CNS) therapeutics for neurodegenerative diseases such as Huntington’s. This paper describes the establishment of a human BBB model in a two-dimensional monolayer culture, derived from human induced pluripotent stem cells. The model maintained the functionality of major endothelial transporter proteins and receptors. Some proprietary molecules from IRBM’s CNS programs were evaluated revealing comparable permeability in the human model and in the model from primary porcine brain endothelial cells. Given the high attrition rate of CNS drugs in clinical trials, such a model should be valuable for optimizing drug selection and predicting human brain exposure.
Combined Peptide and Small-Molecule Approach Toward Nonacidic THIQ Inhibitors of the KEAP1/NRF2 Interaction
The NRF2/ARE signalling pathway is a key mediator in oxidative stress and therefore a potential target for neuroprotective agents. The publication describes the identification of nonacidic tetrahydroisoquinolines (THIQs) that inhibit the interaction between NRF2 and its main negative regulator KEAP1. The approach utilized structural information from peptide screening at the P2 pocket, noncovalent small-molecule 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. X-ray crystallography studies reveal the novel binding mode of these molecules to KEAP1.
Dihydroxypyrimidine Scaffold to Target HIV‑1 Nucleocapsid Protein
IRBM and their collaborators’ at the universities of Strasbourg and Siena have recently published a paper in the ACS Medicinal Chemistry Letters which describes their approach to the identification of a 5-dihydroxypyrimidine-6-carboxamide substructure as a privileged scaffold of a new class of HIV-1 NC inhibitors. The HIV-1 nucleocapsid (NC) protein is a target of great interest because it is essential for viral replication. A bioisosteric catechol replacement approach led to identification the 5-dihydroxypyrimidine-6-carboxamide scaffold and subsequent hit validation efforts generated optimized analogs showing improved NC inhibition and antiviral activity as well as good ADME and PK properties.
IRBM publishes in Nature Scientific Reports on the Janus effect of glucocorticoids on the differentiation of muscle fibro/adipogenic progenitors
Fibro-adipogenic progenitors (FAPs) are known to cause muscle impairment in non-physiological settings, such as aging and myopathies, by promoting fibrosis and fat infiltration. IRBM and their collaborators at the University of Rome Tor Vergata, screened a library of 1,120 FDA/EMEA approved drugs using high content screening (HCS) to identify potential inhibitors of adipogenic differentiation of FAPs. The resulting hits contained a remarkable number of glucocorticoids (GCs) – drugs known to promote adipogenesis. Three of the GCs identified by the screen were further investigated by different approaches. The findings showed that GCs have additional effects to their known anti-inflammatory properties, affecting muscle homeostasis and physiology.
A novel member of Prame family, Gm12794c, counteracts retinoic acid differentiation through the methyltransferase activity of PRC2
By Napolitano Giuliana; Fusco Salvatore; Cirillo Carmine; De Martino Ilaria; Addeo Martina; Falco Geppino; Tagliaferri Daniela; Mazzone Pellegrino; Russo Nicola Antonino; Falco Geppino; et al
From Cell death and differentiation (2020), 27(1), 345-362, Language: English, Database: MEDLINE
Embryonic stem cells (ESCs) fluctuate among different levels of pluripotency defined as metastates. Sporadically, metastable cellular populations convert to a highly pluripotent metastate that resembles the preimplantation two-cell embryos stage (defined as 2C stage) in terms of transcriptome, DNA methylation, and chromatin structure. Recently, we found that the retinoic acid (RA) signaling leads to a robust increase of cells specifically expressing 2C genes, such as members of the Prame family. Here, we show that Gm12794c, one of the most highly upregulated Prame members, and previously identified as a key player for the maintenance of pluripotency, has a functional role in conferring ESCs resistance to RA signaling. In particular, RA-dependent expression of Gm12794c induces a ground state-like metastate, as evaluated by activation of 2C-specific genes, global DNA hypomethylation and rearrangement of chromatin similar to that observed in naive totipotent preimplantation epiblast cells and 2C-like cells. Mechanistically, we demonstrated that Gm12794c inhibits Cdkn1A gene expression through the polycomb repressive complex 2 (PRC2) histone methyltransferase activity. Collectively, our data highlight a molecular mechanism employed by ESCs to counteract retinoic acid differentiation stimuli and contribute to shed light on the molecular mechanisms at grounds of ESCs naive pluripotency-state maintenance.
Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits
By Pesce Elisa; Miluzio Annarita; Cirino Delia; Calamita Piera; Manfrini Nicola; Oliveto Stefania; Ricciardi Sara; Grifantini Renata; Biffo Stefano; Turcano Lorenzo; et al
From Cells (2020), 9(1), , Language: English, Database: MEDLINE
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.
Discovery of the First Human Arylsulfatase A Reversible Inhibitor Impairing Mouse Oocyte Fertilization
By Caroselli Silvia; Canipari Rita; Zwergel Clemens; Pirolli Adele; Sabatino Manuela; Mai Antonello; Valente Sergio; Ragno Rino; Zwergel Clemens; Pirolli Adele; et al
From ACS chemical biology (2020), , Language: English, Database: MEDLINE
Arylsulfatase A (ARSA) plays a crucial role in the reproduction of mammals due to its involvement in the specific gamete interaction preceding sperm and egg fusion leading to fertilization. Recently, it has been shown that zona pellucida (ZP) sperm binding and in vivo fertilization in mice are markedly hampered by using a specific anti-ARSA antibody. Herein, the design and discovery of the first ARSA small molecule inhibitor based on a coumarin-containing polycycle are presented. Through a structure-based approach applied on our in-house library, compound 1r was identified as an ARSA reversible inhibitor (ARSAi); then its activity was validated through both surface plasmon resonance and biochemical inhibition experiments, the first providing a KD value of 21 μM and the latter an IC50 value of 13.2 μM. Further investigations highlighted that compound 1r induced 20% sperm death at 25 μM and also impaired sperm motility; nevertheless both the effects were mediated by ROS production, since they were rescued by the cotreatment of 1r and N-acetyl cysteine (NAC). Interestingly, while 1r was not able to hamper the ZP/sperm binding, it markedly decreased the in vitro oocyte fertilization by mouse sperm up to 60%. Notably, this effect was not hampered by 1r/NAC coadministration, hence allowing the ruling out of an ROS-dependent mechanism. In conclusion, herein is reported the first ever hit of ARSAi as a chemical tool that will enable better exploration of ARSA’s biological role in fertilization as well as provide a starting point for developing 1r structure optimization aimed at increasing enzyme inhibition potency but also providing a deeper understanding of the involvement of ARSA in the fertilization pathway mechanism.
Luminescence-Based, Low- and Medium-Throughput Assays for Drug Screening in Schistosoma mansoni Larval Stage
By Guidi Alessandra; Gimmelli Roberto; Ruberti Giovina; Bresciani Alberto
From Methods in molecular biology (Clifton, N.J.) (2020), 2151219-227, Language: English, Database: MEDLINE
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.
Multiple Synthetic Routes to the Mini-Protein Omomyc and Coiled-Coil Domain Truncations
By Brown Zachary Z; Mapelli Claudio; McCracken Amy Bittner; Chen Kuanchang; Zhu Xiaohong; Garbaccio Robert M; Walji Abbas; Farasat Iman; Shoultz Alycia V; Canada Keith A; et al
From The Journal of organic chemistry (2020), 85(3), 1466-1475, Language: English, Database: MEDLINE
The Myc transcription factor represents an “undruggable” target of high biological interest due to its central role in various cancers. An abbreviated form of the c-Myc protein, called Omomyc, consists of the Myc DNA-binding domain and a coiled-coil region to facilitate dimerization of the 90 amino acid polypeptide. Here we present our results to evaluate the synthesis of Omomyc using three complementary strategies: linear Fmoc solid-phase peptide synthesis (SPPS) using several advancements for difficult sequences, native chemical ligation from smaller peptide fragments, and a high-throughput bacterial expression and assay platform for rapid mutagenesis. This multifaceted approach allowed access to up to gram quantities of the mini-protein and permitted in vitro and in vivo SAR exploration of this modality. DNA-binding results and cellular activity confirm that Omomyc and analogues presented here, are potent binders of the E-box DNA engaged by Myc for transcriptional activation and that this 90-amino acid mini-protein is cell permeable and can inhibit proliferation of Myc-dependent cell lines. We also present additional results on covalent homodimerization through disulfide formation of the full-length mini-protein and show the coiled-coil region can be truncated while preserving both DNA binding and cellular activity. Altogether, our results highlight the ability of advanced peptide synthesis to achieve SAR tractability in a challenging synthetic modality.
Ring Finger Protein 11 acts on ligand-activated EGFR via the direct interaction with the UIM region of ANKRD13 protein family
By Mattioni Anna; Castagnoli Luisa; Cesareni Gianni; Santonico Elena; Boldt Karsten; Ueffing Marius; Auciello Giulio; Komada Masayuki; Rappoport Joshua Z; Cesareni Gianni
From The FEBS journal (2020), , Language: English, Database: MEDLINE
RING finger protein 11 (RNF11) is an evolutionary conserved Really Interesting New Gene E3 ligase that is overexpressed in several human tumours. Although several reports have highlighted its involvement in crucial cellular processes, the mechanistic details underlying its function are still poorly understood. Utilizing stable isotope labelling by amino acids in culture (SILAC)-based proteomics analysis, we identified 51 proteins that co-immunoprecipitate with wild-type RNF11 and/or with its catalytically inactive mutant. We focused our attention on the interaction of RNF11 with Ankyrin repeat domain-containing protein 13 (ANKRD13)s family. Members of the ANKRD13 family contain ubiquitin-interacting motifs (UIM) that recognize the Lys-63-linked ubiquitin (Ub) chains appended to Epidermal growth factor receptor (EGFR) soon after ligand binding. We show that ANKRD13A, ANKRD13B and ANKRD13D form a complex with RNF11 in vivo and that the UIMs are required for complex formation. However, at odds with the conventional UIM binding mode, Ub modification of RNF11 is not required for the interaction with ANKRD13 proteins. We also show that the interaction between ANKRD13A and RNF11 is modulated by the EGF stimulus and that a complex formed by ANKRD13A, RNF11 and activated EGFR is transiently assembled in the early phases of receptor endocytosis. Moreover, loss of function of the E3 ligases Itchy E3 ubiquitin-protein ligase (ITCH) or RNF11, respectively, abrogates or increases the ubiquitination of endogenous ANKRD13A, affecting its ability to bind activated EGFR. We propose a model whereby the ANKRD13 proteins act as molecular scaffolds that promote the transient formation of a complex between the activated EGFR and the E3 ligases ITCH and RNF11. By regulating the ubiquitination status of ANKRD13A and consequently its endocytic adaptor function, RNF11 promotes sorting of the activated EGFR for lysosomal degradation.
Synthesis and Antiproliferative Activity of Nitric Oxide-Donor Largazole Prodrugs
By Borgini Matteo; Zamperini Claudio; Poggialini Federica; Botta Maurizio; Zamperini Claudio; Botta Maurizio; Ferrante Luca; Fabio Romano Di; Summa Vincenzo; Fabio Romano Di; et al
From ACS medicinal chemistry letters (2020), 11(5), 846-851, Language: English, Database: MEDLINE
The marine natural product Largazole is the most potent Class I HDAC inhibitor identified to date. Since its discovery, many research groups have been attracted by the structural complexity and the peculiar anticancer activity, due to its capability to discriminate between tumor cells and normal cells. Herein, we discuss the synthesis and the in vitro biological profile of hybrid analogues of Largazole, as dual HDAC inhibitor and nitric oxide (NO) donors, potentially useful as anticancer agents. In particular, the metabolic stability of the modified thioester moiety of Largazole, bearing the NO-donor function/s, the in vitro release of NO, and the antiproliferative activity in tumor cell lines are presented.
The miRNAs Role in Melanoma and in Its Resistance to Therapy
By Varrone Francesca; Caputo Emilia
From International journal of molecular sciences (2020), 21(3), , Language: English, Database: MEDLINE
Melanoma is the less common but the most malignant skin cancer. Since the survival rate of melanoma metastasis is about 10-15%, many different studies have been carried out in order to find a more effective treatment. Although the development of target-based therapies and immunotherapeutic strategies has improved chances for patient survival, melanoma treatment still remains a big challenge for oncologists. Here, we collect recent data about the emerging role of melanoma-associated microRNAs (miRNAs) currently available treatments, and their involvement in drug resistance. We also reviewed miRNAs as prognostic factors, because of their chemical stability and resistance to RNase activity, in melanoma progression. Moreover, despite miRNAs being considered small conserved regulators with the limitation of target specificity, we outline the dual role of melanoma-associated miRNAs, as oncogenic and/or tumor suppressive factors, compared to other tumors.
Ultrasensitive quantitative measurement of huntingtin phosphorylation at residue S13
By Cariulo, Cristina; Verani, Margherita; Martufi, Paola; Ingenito, Raffaele; Finotto, Marco; Deguire, Sean M.; Lavery, Daniel J.; Toledo-Sherman, Leticia; Lee, Ramee; Doherty, Elizabeth M.; et al
From Biochemical and Biophysical Research Communications (2020), 521(3), 549-554. Language: English, Database: CAPLUS, DOI:10.1016/j.bbrc.2019.09.097
Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by an expansion of a CAG triplet repeat (encoding for a polyglutamine tract) within the first exon of the huntingtin gene. Expression of the mutant huntingtin (mHTT) protein can result in the prodn. of N-terminal fragments with a robust propensity to form oligomers and aggregates, which may be causally assocd. with HD pathol. Several lines of evidence indicate that N17 phosphorylation or pseudophosphorylation at any of the residues T3, S13 or S16, alone or in combination, modulates mHTT aggregation, subcellular localization and toxicity. Consequently, increasing N17 phosphorylation has been proposed as a potential therapeutic approach. However, developing genetic/pharmacol. tools to quantify these phosphorylation events is necessary in order to subsequently develop tool modulators, which is difficult given the transient and incompletely penetrant nature of such post-translational modifications. Here we describe the first ultrasensitive sandwich immunoassay that quantifies HTT phosphorylated at residue S13 and demonstrate its utility for specific analyte detection in preclin. models of HD.