scholarly journals Synergy between Readthrough and Nonsense Mediated Decay Inhibition in a Murine Model of Cystic Fibrosis Nonsense Mutations

2020 ◽  
Vol 22 (1) ◽  
pp. 344
Author(s):  
Daniel R. McHugh ◽  
Calvin U. Cotton ◽  
Craig A. Hodges
Keyword(s):  
Protein Function ◽  
Therapeutic Option ◽  
Genetic Disorders ◽  
Therapeutic Strategies ◽  
Primary Cells ◽  
Functional Protein ◽  
Nonsense Mutations ◽  
Nonsense Mediated Decay ◽  
Premature Termination Codons ◽  
Cftr Protein

Many heritable genetic disorders arise from nonsense mutations, which generate premature termination codons (PTCs) in transcribed mRNA. PTCs ablate protein synthesis by prematurely terminating the translation of mutant mRNA, as well as reducing mutant mRNA quantity through targeted degradation by nonsense-mediated decay (NMD) mechanisms. Therapeutic strategies for nonsense mutations include facilitating ribosomal readthrough of the PTC and/or inhibiting NMD to restore protein function. However, the efficacy of combining readthrough agents and NMD inhibitors has not been thoroughly explored. In this study, we examined combinations of known NMD inhibitors and readthrough agents using functional analysis of the CFTR protein in primary cells from a mouse model carrying a G542X nonsense mutation in Cftr. We observed synergy between an inhibitor of the NMD component SMG-1 (SMG1i) and the readthrough agents G418, gentamicin, and paromomycin, but did not observe synergy with readthrough caused by amikacin, tobramycin, PTC124, escin, or amlexanox. These results indicate that treatment with NMD inhibitors can increase the quantity of functional protein following readthrough, and that combining NMD inhibitors and readthrough agents represents a potential therapeutic option for treating nonsense mutations.

scholarly journals Comprehensive Analysis of Combinatorial Pharmacological Treatments to Correct Nonsense Mutations in the CFTR Gene

2021 ◽  
Vol 22 (21) ◽  
pp. 11972
Author(s):  
Arianna Venturini ◽  
Anna Borrelli ◽  
Ilaria Musante ◽  
Paolo Scudieri ◽  
Valeria Capurro ◽  
...  
Keyword(s):  
Comprehensive Analysis ◽  
Cftr Gene ◽  
Loss Of Function ◽  
Pharmacological Treatments ◽  
Protein Maturation ◽  
Nonsense Mutations ◽  
Nonsense Mediated Decay ◽  
Cftr Mutations ◽  
Cftr Protein ◽  
And Function

Cystic fibrosis (CF) is caused by loss of function of the CFTR chloride channel. A substantial number of CF patients carry nonsense mutations in the CFTR gene. These patients cannot directly benefit from pharmacological correctors and potentiators that have been developed for other types of CFTR mutations. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD), G418 and ELX-02 for readthrough, VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with W1282X and R553X being the mutations most and least sensitive to pharmacological treatments, respectively. In particular, W1282X-CFTR was highly responsive to NMD suppression by SMG1i but also required treatment with VX-445 corrector to show function. In contrast, G542X-CFTR required treatment with readthrough agents and VX-809. Importantly, we never found cooperativity between the NMD inhibitor and readthrough compounds. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation.

scholarly journals CRISPR-pass: Gene rescue of nonsense mutations using adenine base editors

2019 ◽  
Author(s):  
Choongil Lee ◽  
Dong Hyun Jo ◽  
Gue-Ho Hwang ◽  
Jihyeon Yu ◽  
Jin Hyoung Kim ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Dna Sequence ◽  
Clinical Utility ◽  
Premature Termination ◽  
Genetic Disorders ◽  
Nonsense Mutations ◽  
Premature Termination Codons ◽  
Potential Clinical Utility ◽  
Clinically Significant ◽  
Adenine Base

AbstractA nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read-through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database, rescues protein synthesis in patient-derived fibroblasts, suggesting potential clinical utility.

scholarly journals Suppression of aminoglycoside-induced premature termination codon readthrough by the TRP channel inhibitor AC1903

2021 ◽  
Author(s):  
Alireza Baradaran-Heravi ◽  
Claudia C Bauer ◽  
Isabelle B Pickles ◽  
Sara Hosseini-Farahabadi ◽  
Aruna Balgi ◽  
...  
Keyword(s):  
Premature Termination ◽  
Genetic Disorders ◽  
Premature Termination Codon ◽  
Cation Channels ◽  
Trpc Channels ◽  
Nonsense Mutations ◽  
Variable Effect ◽  
Premature Termination Codons ◽  
Ic50 Values ◽  
Nonsense Mediated Mrna Decay

Nonsense mutations, which occur in ~11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as gentamicin and G418 permit PTC readthrough and so may address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we addressed the hypothesis that TRP non-selective cation channels contribute to the variable effect of aminoglycosides by controlling their cellular uptake. To attempt to identify the channel type involved, we tested AC1903, a 2-aminobenzimidazole derivative recently reported to selectively inhibit TRPC5 cation channels. AC1903 consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cell line and patient-derived JEB01 keratinocytes. In an effort to validate the suggested role of TRPC5, we tested an independent and more potent inhibitor called Pico145, which affects channels containing TRPC1, TRPC4 and TRPC5 but not other TRPCs or other channels. Unexpectedly, Pico145 was completely without effect, suggesting that AC1903 may work through other or additional targets. Consistent with this suggestion, AC1903 inhibited multiple TRPC channels including homomeric TRPC3, TRPC4, TRPC5, TRPC6 as well as concatemeric TRPC4-C1 and TRPC5-C1 channels, all with low micromolar IC50 values. It also inhibited TRPV4 channels but had weak or no effects on TRPV1 and no effect on another non-selective cation channel, PIEZO1. Overall, our study reveals a suppressor of aminoglycoside-mediated PTC readthrough (i.e., AC1903) but suggests that this compound has previously unrecognised effects. These effects require further investigation to determine the molecular mechanism by which AC1903 suppresses aminoglycoside uptake and PTC readthrough.

scholarly journals Strategies against Nonsense: Oxadiazoles as Translational Readthrough-Inducing Drugs (TRIDs)

2019 ◽  
Vol 20 (13) ◽  
pp. 3329 ◽  
Author(s):  
Ambra Campofelice ◽  
Laura Lentini ◽  
Aldo Di Leonardo ◽  
Raffaella Melfi ◽  
Marco Tutone ◽  
...  
Keyword(s):  
Small Molecules ◽  
Genetic Diseases ◽  
Nonsense Mutations ◽  
New Class ◽  
Premature Termination Codons ◽  
Translational Readthrough ◽  
Action Strategies ◽  
Oxadiazole Derivatives ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

This review focuses on the use of oxadiazoles as translational readthrough-inducing drugs (TRIDs) to rescue the functional full-length protein expression in mendelian genetic diseases caused by nonsense mutations. These mutations in specific genes generate premature termination codons (PTCs) responsible for the translation of truncated proteins. After a brief introduction on nonsense mutations and their pathological effects, the features of various classes of TRIDs will be described discussing differences or similarities in their mechanisms of action. Strategies to correct the PTCs will be presented, particularly focusing on a new class of Ataluren-like oxadiazole derivatives in comparison to aminoglycosides. Additionally, recent results on the efficiency of new candidate TRIDs in restoring the production of the cystic fibrosis transmembrane regulator (CFTR) protein will be presented. Finally, a prospectus on complementary strategies to enhance the effect of TRIDs will be illustrated together with a conclusive paragraph about perspectives, opportunities, and caveats in developing small molecules as TRIDs.

scholarly journals A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jyoti Sharma ◽  
Ming Du ◽  
Eric Wong ◽  
Venkateshwar Mutyam ◽  
Yao Li ◽  
...  
Keyword(s):  
Protein Function ◽  
Translation Termination ◽  
Nonsense Suppression ◽  
Nonsense Mutations ◽  
Bronchial Epithelial ◽  
Premature Termination Codons ◽  
Translational Readthrough ◽  
Nonsense Mediated Mrna Decay ◽  
Promising Treatment ◽  
And Function

AbstractPremature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.

Preparation and Characterization of β-glucosidase Films for Stabilization and Handling in Dry Configurations

2020 ◽  
Vol 21 (8) ◽  
pp. 741-747
Author(s):  
Liguang Zhang ◽  
Yanan Shen ◽  
Wenjing Lu ◽  
Lengqiu Guo ◽  
Min Xiang ◽  
...  
Keyword(s):  
Tensile Strength ◽  
Protein Function ◽  
Protein Stabilization ◽  
Functional Protein ◽  
Elongation At Break ◽  
Gelatin Films ◽  
The Stability ◽  
And Function ◽  
General Method

Background: Although the stability of proteins is of significance to maintain protein function for therapeutical applications, this remains a challenge. Herein, a general method of preserving protein stability and function was developed using gelatin films. Method: Enzymes immobilized onto films composed of gelatin and Ethylene Glycol (EG) were developed to study their ability to stabilize proteins. As a model functional protein, β-glucosidase was selected. The tensile properties, microstructure, and crystallization behavior of the gelatin films were assessed. Result: Our results indicated that film configurations can preserve the activity of β-glucosidase under rigorous conditions (75% relative humidity and 37°C for 47 days). In both control films and films containing 1.8 % β-glucosidase, tensile strength increased with increased EG content, whilst the elongation at break increased initially, then decreased over time. The presence of β-glucosidase had a negligible influence on tensile strength and elongation at break. Scanning electron-microscopy (SEM) revealed that with increasing EG content or decreasing enzyme concentrations, a denser microstructure was observed. Conclusion: In conclusion, the dry film is a promising candidate to maintain protein stabilization and handling. The configuration is convenient and cheap, and thus applicable to protein storage and transportation processes in the future.

scholarly journals Nonaminoglycoside compounds induce readthrough of nonsense mutations

2009 ◽  
Vol 206 (10) ◽  
pp. 2285-2297 ◽  
Author(s):  
Liutao Du ◽  
Robert Damoiseaux ◽  
Shareef Nahas ◽  
Kun Gao ◽  
Hailiang Hu ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Genetic Disorders ◽  
Disease Model ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mdx Mouse ◽  
Nonsense Mutations ◽  
Atm Kinase ◽  
Atm Protein ◽  
Dystrophin Protein ◽  
Leading Compounds

Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)–enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model. This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS. The assay is luciferase independent. We screened ∼34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein.

scholarly journals Breakthroughs in Preclinical Development of Ataluren (PTC124) As Therapeutic Option for Patients Affected By Shwachman-Diamond Syndrome: Towards the First Clinical Trial

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 451-451
Author(s):  
Valentino Bezzerri ◽  
Antonio Vella ◽  
Elisabetta D'Aversa ◽  
Martina Api ◽  
Marisole Allegri ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Stem Cells ◽  
Bone Marrow ◽  
Therapeutic Option ◽  
Bone Marrow Failure ◽  
Severe Neutropenia ◽  
Control Group ◽  
Nonsense Mutations ◽  
Colony Forming Unit ◽  
Shwachman Diamond Syndrome

Shwachman-Diamond syndrome (SDS) is one of the more common inherited bone marrow failure syndromes (IBMFS). Almost 90% of patients with SDS present mutations in the Shwachman-Bodian-Diamond syndrome gene (SBDS) which encodes for the homonymous small protein involved in ribogenesis. SDS is a multiple-organ disease mostly characterized by exocrine pancreas insufficiency, bone malformations, and more importantly bone marrow failure. Most patients with SDS present severe neutropenia, whereas thrombocytopenia and anemia are less frequent. Furthermore, 15-20% of patients develop myelodysplastic syndrome with high risk of acute myeloid leukemia (AML). STAT3 pathway is upregulated both in primary SDS leukocytes and immortalized B cells. Being STAT3 a key regulator of interleukin-6 (IL-6), we postulated that STAT3 hyper-activation could lead to a dysregulation of the IL-6 signaling cascade. Increased levels of IL-6 have been found in pediatric patients with AML and it has been associated with poorer outcomes in these patients, highlighting IL-6 as a cytokine potentially involved in the development of AML. Thus, our hypothesis is that STAT3-IL6 axis may contribute to leukemogenesis in SDS. Almost 55% of patients with SDS carry a specific nonsense mutations, namely the c.183-184TA>CT, which cause a premature termination codon (PTC). Ataluren (PTC124, PTC Therapeutics Inc, NJ) is a small PTC suppressor molecule already approved by the European Medicines Agency as a therapeutic option for Duchenne muscular dystrophy. Interestingly, we recently reported that ataluren can restore SBDS expression in bone marrow progenitors and in peripheral blood mononuclear cells isolated from patients with SDS. Moreover, we have shown that ataluren can reduce mTOR hyper-phosphorylation and excessive apoptotic rate observed in SDS leukocytes. More importantly, we reported that ataluren can improve myeloid differentiation in a small cohort of patients (Bezzerri et al, Am J Hematol 2018). In this further analysis considering an enlarged cohort of 20 SDS patients carrying nonsense mutations we found the following: Ataluren can significantly improve both myeloid colony-forming unit-granulocyte/macrophage (CFU-GM) and colony-forming unit granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM) generation from bone marrow mononuclear stem cells obtained from an enlarged cohort of 20 patients with SDS carrying nonsense mutations. Ataluren indeed almost doubled the number of CFU-GM and CFU-GEMM after 7 and 14 days of treatment.Colony-forming unit erythroid (CFU-E) generation was not affected by the treatment.Ataluren induces neutrophil maturation in SDS bone marrow mononuclear stem cells (mean increase of 61% CD16+ CD11b+ cells over untreated controls) after 24-48 hours of treatment.Consistently with STAT3 hyper-activation observed in SDS cells, here we show that patients with SDS present a significantly increased level of IL-6 in plasma (4.3-fold higher expression than the healthy control group). Also lymphoblastoid cell lines (LCL) and primary bone marrow mesenchymal stromal cells (MSC) obtained from patients with SDS show increased IL-6 release in culture supernatants compared to healthy controls (2.5-fold and 6.8-fold higher levels, respectively).Of note, ataluren can reduce IL-6 expression in SDS cells restoring normal levels both in LCL and MSC. In conclusion, these new data support the enrollment of patients for the first clinical trial for this drug in SDS. Furthermore, this study could pave the way for the use of ataluren for other nonsense mutation-mediated IBMFS where STAT3-IL6 axis and similar pro-leukemic pathways are involved. Disclosures Bezzerri: Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome". Cipolli:Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome".

scholarly journals Neurogenesis: Regulation by Alternative Splicing and Related Posttranscriptional Processes

2016 ◽  
Vol 23 (5) ◽  
pp. 466-477 ◽  
Author(s):  
Enrique Lara-Pezzi ◽  
Manuel Desco ◽  
Alberto Gatto ◽  
María Victoria Gómez-Gaviro
Keyword(s):  
Alternative Splicing ◽  
Protein Function ◽  
Posttranscriptional Regulation ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Degradation ◽  
Protein Isoforms ◽  
Mammalian Brain ◽  
Nonsense Mediated Decay

The complexity of the mammalian brain requires highly specialized protein function and diversity. As neurons differentiate and the neuronal circuitry is established, several mRNAs undergo alternative splicing and other posttranscriptional changes that expand the variety of protein isoforms produced. Recent advances are beginning to shed light on the molecular mechanisms that regulate isoform switching during neurogenesis and the role played by specific RNA binding proteins in this process. Neurogenesis and neuronal wiring were recently shown to also be regulated by RNA degradation through nonsense-mediated decay. An additional layer of regulatory complexity in these biological processes is the interplay between alternative splicing and long noncoding RNAs. Dysregulation of posttranscriptional regulation results in defective neuronal differentiation and/or synaptic connections that lead to neurodevelopmental and psychiatric disorders.

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