COMBINING TRANSLATION READTHROUGH INDUCING DRUGS AND NONSENSE MEDIATED DECAY PATWHAY INHIBITION TO THE CFTR RESCUE IN CYSTIC FIBROSIS CELL MODEL SYSTEM

Nonsense mutations affect 10% of patients with cystic fibrosis and produce a premature termination codon in CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) mRNA causing early termination of translation and leading to lack of CFTR function. A potential therapy for nonsense mutations provides the use of small molecules able to overcome the premature stop codon (PTC) by a readthrough mechanism that lead to synthesis a complete CFTR protein. Despite the good results obtained from this approach, TRIDs efficiency is considerably reduced by the poor amount of target transcript, that is the mRNA containing the PTC. The readthrough, indeed, does not occur on the totality of target transcripts because of their degradation due to the nonsense mediated decay pathway (NMD). This pathway provides the degradation of mRNA harboring premature stop codon to prevent the production of altered polypeptides. In contrast, the activity of this pathway interferes with the effectiveness of the readthrough drugs, limiting the mRNA concentration of the target protein. Thus, a promising strategy for nonsense mutation treatment is a combined use of readthrough agents and factors that attenuate the nonsense mRNA decay. By silencing the UPF1 mRNA/protein, the activity of the NMD pathway was reduced, in FRT cells CFTR W1282X. Alternatively, caffeine was used as specific inhibitor of the UPF1 activity, to increase the efficiency of readthrough molecules (NV848 and NV914) in FRT cells CFTRW1282X cells. In both cases, the combined treatment: NV848 or NV914/caffeine and NV848 or NV914/UPF1siRNA caused an increase of CFTRW1282X mRNA level followed by the rescue of the CFTR expression and functionality. However, unexpectedly, despite the higher CFTRW1282X mRNA level in caffeine treated samples, both expression and functionality CFTR rescue resulted slightly lower than the recovery achieved by UPF1 silencing. Our results indicate that modulation of NMD pathway, although still to be optimized, could be a promising approach in order to increase TRIDs effects in presence of stop mutations

New Insight on Archaeological Metal Finds, Nails and Lead Sheathings of the Punic Ship from Battle of the Egadi Islands

: The wreck of the Punic ship exhibited at the Archaeological Park of Lilybaeum (Marsala, Italy) is a unique example in the world. In this paper, the investigation of some metal finds (30 nails and 3 fragments of sheathings) belonging to the wreck of the Punic ship is reported. Portable X-ray fluorescence and Raman spectroscopy allowed us to identify the elements and compounds constituting them and make some deductions about their composition. X-ray diffractometry, polarised optical microscopy and scanning electron microscopy of the collected micro-samples allowed us to explain the degradation that occurred in the underwater environment

Caffeine boosts Ataluren's readthrough activity

The readthrough of nonsense mutations by small molecules like Ataluren is considered a novel therapeutic approach to overcome the gene defect in several genetic diseases as cystic fibrosis (CF). This pharmacological approach suppresses translation termination at premature termination codons (PTCs readthrough) thus restoring the expression of a functional protein. However, readthrough might be limited by the nonsense-mediated mRNA decay (NMD), a cell process that reduces the amount/level of PTCs containing mRNAs. Here we investigate the combined action of Ataluren and caffeine to enhance the readthrough of PTCs. IB3.1 CF cells with a nonsense mutation were treated with caffeine to attenuate the Nonsense-Mediated mRNA Decay (NMD) activity and thus enhance the stability of the nonsense (ns)-CFTR-mRNA to be targeted by Ataluren. Our results show that NMD attenuation by caffeine enhances mRNA stability and more importantly when combined with Ataluren increase the recovery of the full-length CFTR protein

Fluorinated heterocyclic compounds. A photochemical approach to a synthesis of fluorinated quinazolin-4-ones

An efficient and generalized photochemical methodology for the preparation of fluorinated quinazolin-4-ones is described. Depending on the starting substrate, quinazolin-4-ones bearing a perfluoroalkyl- or perfluoroaryl- substituent in position 2 or fluorine atoms on any positions of the benzo-fused moiety can easily be obtained. 5-Aryl-3-perfluoroalkylpentafluorophenyl- or 5-polyfluoroaryl-3-phenyl(methyl)-1,2,4-oxadiazoles, respectively, can be considered as ideal precursors that can be transformed into the target quinazolin-4-ones by irradiation in the presence of triethylamine (TEA) (at λ = 313 nm) or pyrene (at λ = 365 nm) in dry methanol or acetonitrile as solvent. Some mechanistic considerations confirm the involvement of a photoinduced electron transfer process

Fluorinated heterocyclic compounds. A photochemical approach to a synthesis of polyfluoroaryl-1,2,4-triazoles

The reaction of some fluorinated 1,2,4-oxadiazoles in the presence of methylamine or propylamine has been investigated. The irradiation in methanol or acetonitrile leads with acceptable yields to the corresponding fluorinated 1-methyl- or 1-propyl-1,2,4-triazole

Synthesis of platinum complexes with 2-(5-perfluoroalkyl-1,2,4-oxadiazol-3yl)-pyridine and 2-(3-perfluoroalkyl-1-methyl-1,2,4-triazole-5yl)-pyridine ligands and their in vitro antitumor activity

Five new mononuclear Pt(II) complexes with 5-perfluoroalkyl-1,2,4-oxadiazolyl-pyridine and 3-perfluoroalkyl-1,2,4-triazolyl-pyridine ligands are reported. The ligands 2-(5-perfluoroheptyl-1,2,4-oxadiazole-3yl)-pyridine (pfhop), 2-(5-perfluoropropyl)-1,2,4-oxadiazole-3yl)-pyridine (pfpop), 2-(3-perfluoroheptyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfhtp), 2-(3-perfluoropropyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfptp) and their complexes [PtCl2(pfhop)(2)]center dot 1.5 DMSO (2a), [PtCl2(pfpop)(2)]center dot 1.5 DMSO (3a), [PtCl2(pfhtp)(2)]center dot 1.5 DMSO (4a), PtCl2(pfhtp) (4b), [PtCl2(PfPtP)(2)]center dot 1.5 DMSO (5a) have been synthesized and structurally characterized. The complexes 2a, 3a, 4a and 5a have the same chemical environment of Pt(II) where PtCl2 moieties coordinate two molecules of ligand via N1 atom of pyridine in the case of pfhop and pfpop, and N2 atom of 1,2,4-triazole in the case of pfhtp and pfptp. For 4b, pfhtp behaves as bidentate ligand, coordinating Pt(II) ion via N4 atom of triazole and N1 atom of pyridine. All complexes have been tested in vitro by 3-(4,5-dimethyl-2-thiazolyl)bromide-2,5-diphenyl-2 H-tetrazolium (MTT) test on four tumor cell lines MCF-7 (human breast cancer), HepG2 (human hepatocellular carcinoma), HCT116 (human colorectal carcinoma). Compounds 2a and 4b showed a dose-dependent anti-proliferative effect against the three tumor cell lines whereas did not affect viability of intestinal normal-like differentiated Caco-2 cells. The cell death of HepG2, MCF-7 and HCT116 induced by the compounds, was considered to be apoptotic by measuring the exposure of phosphatidylserine to the outer membrane and observing the typical apoptotic morphological change by acridine orange (AO)/ethidium bromide (EB) stainin

Halogen bond directionality translates tecton geometry into self-assembled architecture geometry

The structures of halogen-bonded infinite chains involving two diiodoperfluoroalkanes and a bent bis(pyrid-4′-yl)oxadiazole show that the geometry of the pyridyl pendant rings is translated into the angle between the formed halogen bonds. © 2013 The Royal Society of Chemistry

Investigating the inhibition of FTSJ1 a tryptophan tRNA-specific 2’-O-methyltransferase by NV TRIDs, as a mechanism of readthrough in nonsense mutated CFTR

Abstract: Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10% of the CFTR gene mutations are "stop" mutations, which generate a Premature Termination Codon (PTC), thus synthesizing a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, which is the ri-bosome’s capacity to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough; for some, the mechanism of action is still under debate. We in-vestigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914, and NV930, could exert their readthrough activity by in silico analysis and in vitro studies. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase

Readthrough Approach Using NV Translational Readthrough-Inducing Drugs (TRIDs): A Study of the Possible Off-Target Effects on Natural Termination Codons (NTCs) on TP53 and Housekeeping Gene Expression

Nonsense mutations cause several genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, and Shwachman–Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named “translational readthrough” and restore the synthesis of full-length and potentially functional proteins. The new oxadiazole-core TRIDs NV848, NV914, and NV930 (NV) showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecule treatment induces NTC readthrough: (1) a study of the translational-induced p53 molecular weight and functionality; (2) the evaluation of two housekeeping proteins’ (Cys-C and β2M) molecular weights. Our results showed that the treatment with NV848, NV914, or NV930 did not induce any translation alterations in both experimental systems. The data suggested that NV molecules have a specific action for the PTCs and an undetectable effect on the NTCs