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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.

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 that generate a premature termination codon (PTC), thus synthesizing a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, which is the ribosome’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 investigate 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.

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 mutations affecting the CFTR gene are "stop" mutations, which generate a Premature Termination Codon (PTC), thus resulting in the synthesis of a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, that is the capacity of the ribosome to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough and for some of them the mechanism of action is still under debate. By in silico analysis as well as in vitro studies, we investigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914 and NV930, could exert their readthrough activity. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase. In addition, we report that our TRIDs do not exert readthrough on natural termination codons.

Objective Nonsense mutations are responsible for 15% of Cystic Fibrosis (CF) patients due to the introduction of a premature stop codon (PTC) in the mRNA and the production of a truncated CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein1. A promising therapeutic approach for stop mutations is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs) to restore the expression of the protein2,3. Recently three new TRIDS (NV848, NV914, NV930) have been proposed and validated by several assays. Our work was focused on TRIDs NV848, NV914, NV930. Important aspects of TRIDs to be evaluated are their specificity towards PTC, to demonstrate that TRIDs do not interfere with the normal translation process4 in vitro, and their in vivo toxicity in mice models, to assess their tolerability at high doses. Methods For the toxicity study, mice were treated with an acute dose as reported in standard protocols5, monitored and then sacrificed to collect organs. To study the effects of the TRIDs on natural termination codons (NTC) we have examined the molecular weight and functional activity of a panel of proteins. Results Acute toxicity studies evidenced that all three molecules used have been well tolerated in all subjects treated. Histological analyses have not shown any tissue damage. So, we can assess TRIDs are safe in vivo also at the high doses tested. About NTC preservation, experiments have not shown any impairment or dysfunction in all samples. Indeed, no aberrant protein form has been detected. Conclusions In conclusion, our experiments identified the acute dosage for each TRIDs and their tolerability also at elevated doses, encouraging to test their efficacy in in vivo CF models. Accordingly, the in vitro analyses have suggested that these TRIDs do not lead to jeopardise NTC integrity, assessing their promising use for PTC readthrough. Consequently, our results could be indicative of TRIDs safety in in vitro models, laying the basis for further investigations. Bibliography 1. Giordani B, Amato A, Majo F, Ferrari G, Quattrucci S, Minicucci L, Padoan R, Floridia G, Salvatore D, Carnovale V, Puppo Fornaro G, Taruscio D, Salvatore M; Gruppo di lavoro RIFC. Italian Cystic Fibrosis Registry (ICFR). Report 2015-2016]. Epidemiol Prev. 2019 Jul-Aug;43(4S1):1-36. 2. Pibiri I., A., Tutone, M., Lentini, L., Melfi, R., & DI LEONARDO, A. et al. Oxadiazole Derivatives For The Treatment Of Genetic Diseases Due To Nonsense Mutations, PCT Int. Appl. (2019), WO 2019/101709 A1 20190531. 3. Pibiri I, Melfi R, Tutone M, Di Leonardo A, Pace A, Lentini L. Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems. Int J Mol Sci. 2020 Sep 3;21(17):6420. doi: 10.3390/ijms21176420. PMID: 32899265; PMCID: PMC7504161 4. Grover R, Candeias MM, Fåhraeus R, Das S. p53 and little brother p53/47: linking IRES activities with protein functions. Oncogene. 2009 Jul 30;28(30):2766-72. doi: 10.1038/onc.2009.138. Epub 2009 Jun 1. PMID: 19483723. 5. OECD (1992). OECD Guidelines for the Testing of Chemicals No. 420: Acute Oral Toxicity Fixed Dose Method, 7pp. Paris, France: OECD.

The involvement of renin-angiotensin system in the modulation of gut motility and age-related changes in mRNA expression of angiotensin (Ang II) receptors (ATR) are well accepted. We aimed to characterize, in vitro, the contractile responses induced by Ang II, in jejunum from young (3–6 weeks old) and old rats (≥ 1 year old), to evaluate possible functional differences associated to changes in receptor expression. Mechanical responses to Ang II were examined in vitro as changes in isometric tension. ATR expression was assessed by qRT-PCR. Ang II induced a contractile effect, antagonized by losartan, AT1R antagonist, and increased by PD123319, AT2R antagonist, as well by neural blocker ω-conotoxin and by nitric oxide (NO) synthase inhibitor. No difference in the response was observed between young and old groups. AT1 receptor-mediated contractile response was decreased by U-73122, phospholipase C (PLC) inhibitor; or 2-aminoethoxy-diphenylborate (2-APB), inositol triphosphate (IP3) receptor inhibitor; or nifedipine, l-type calcium channel blocker. Age-related changes in the expression of both AT1 receptor subtypes, AT1a and AT1b, and of AT2 receptors were detected. In conclusion, Ang II modulates the spontaneous contractility of rat jejunum via postjunctional AT1 receptors, involving Ca2+ mobilization from intracellular stores, via PLC/IP3 pathway, and Ca2+ influx from extracellular space, via l-type channels. Prejunctional AT2 receptors would counteract AT1 receptor effects, via NO synthesis. The observed age-related differences in the expression of all AT receptor subtypes are not reflected in the muscular contractile response to Ang II.