scholarly journals 2-Guanidino-quinazoline promotes the readthrough of nonsense mutations underlying human genetic diseases

2021 ◽  
Author(s):  
Laure Bidou ◽  
Olivier Bugaud ◽  
Goulven Merer ◽  
Matthieu Coupet ◽  
Isabelle Hatin ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
High Throughput Screening ◽  
Therapeutic Strategy ◽  
Genetic Diseases ◽  
Medical Applications ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Nonsense Mutations ◽  
Premature Termination Codons ◽  
Clinical Potential

Premature termination codons (PTCs) account for 10% to 20% of genetic diseases in humans. The gene inactivation resulting from PTC can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a new reporter cell line and performed high-throughput screening (HTS) to identify potential new readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy. We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of natural stop codons.

scholarly journals Efficient Suppression of Endogenous CFTR Nonsense Mutations Using Anticodon Engineered Transfer RNAs

2021 ◽  
Author(s):  
Wooree Ko ◽  
Joseph J. Porter ◽  
Matthew T. Sipple ◽  
Katherine M. Edwards ◽  
John D. Lueck
Keyword(s):  
Cystic Fibrosis ◽  
Mammalian Cells ◽  
Genetic Diseases ◽  
Protein Translation ◽  
Nonsense Mutations ◽  
Channel Function ◽  
Associated Diseases ◽  
Highly Active ◽  
Premature Termination Codons ◽  
Efficient Suppression

Nonsense mutations or premature termination codons (PTCs) comprise ~11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases, however their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells. Here we investigated the ability of anticodon edited (ACE)-tRNAs to suppress cystic fibrosis (CF) causing PTCs in the cystic fibrosis transmembrane regulator (CFTR) gene in gene-edited immortalized human bronchial epithelial (16HBEge) cells. Delivery of ACE-tRNAs to 16HBEge cells harboring three common CF mutations G542X-, R1162X- and W1282X-CFTR PTCs significantly inhibited NMD and rescued endogenous mRNA expression. Furthermore, delivery of our highly active leucine encoding ACE-tRNA resulted in rescue of W1282X-CFTR channel function to levels that significantly exceed the necessary CFTR channel function for therapeutic relevance. This study establishes the ACE-tRNA approach as a potential stand-alone therapeutic for nonsense-associated diseases due to its ability to rescue both mRNA and full-length protein expression from PTC containing endogenous genes.

scholarly journals Development and utilization of human decidualization reporter cell line uncovers new modulators of female fertility

2019 ◽  
Vol 116 (39) ◽  
pp. 19541-19551
Author(s):  
Meade Haller ◽  
Yan Yin ◽  
Liang Ma
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Patient Specific ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
A Genome ◽  
Decidual Cells

Failure of embryo implantation accounts for a significant percentage of female infertility. Exquisitely coordinated molecular programs govern the interaction between the competent blastocyst and the receptive uterus. Decidualization, the rapid proliferation and differentiation of endometrial stromal cells into decidual cells, is required for implantation. Decidualization defects can cause poor placentation, intrauterine growth restriction, and early parturition leading to preterm birth. Decidualization has not yet been systematically studied at the genetic level due to the lack of a suitable high-throughput screening tool. Herein we describe the generation of an immortalized human endometrial stromal cell line that uses yellow fluorescent protein under the control of the prolactin promoter as a quantifiable visual readout of the decidualization response (hESC-PRLY cells). Using this cell line, we performed a genome-wide siRNA library screen, as well as a screen of 910 small molecules, to identify more than 4,000 previously unrecognized genetic and chemical modulators of decidualization. Ontology analysis revealed several groups of decidualization modulators, including many previously unappreciated transcription factors, sensory receptors, growth factors, and kinases. Expression studies of hits revealed that the majority of decidualization modulators are acutely sensitive to ovarian hormone exposure. Gradient treatment of exogenous factors was used to identify EC50 values of small-molecule hits, as well as verify several growth factor hits identified by the siRNA screen. The high-throughput decidualization reporter cell line and the findings described herein will aid in the development of patient-specific treatments for decidualization-based recurrent pregnancy loss, subfertility, and infertility.

scholarly journals Development of a Cell-Based High-Throughput Screening Assay to Identify Porcine Host Defense Peptide-Inducing Compounds

2018 ◽  
Vol 2018 ◽  
pp. 1-13 ◽  
Author(s):  
Zhuo Deng ◽  
Jing Wang ◽  
Wentao Lyu ◽  
Xuwen Wieneke ◽  
Robert Matts ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Hdac Inhibitors ◽  
Luciferase Reporter ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
A Cell

Novel alternatives to antibiotics are needed for the swine industry, given increasing restrictions on subtherapeutic use of antibiotics. Augmenting the synthesis of endogenous host defense peptides (HDPs) has emerged as a promising antibiotic-alternative approach to disease control and prevention. To facilitate the identification of HDP inducers for swine use, we developed a stable luciferase reporter cell line, IPEC-J2/PBD3-luc, through permanent integration of a luciferase reporter gene driven by a 1.1 kb porcine β-defensin 3 (PBD3) gene promoter in porcine IPEC-J2 intestinal epithelial cells. Such a stable reporter cell line was employed in a high-throughput screening of 148 epigenetic compounds and 584 natural products, resulting in the identification of 41 unique hits with a minimum strictly standardized mean difference (SSMD) value of 3.0. Among them, 13 compounds were further confirmed to give at least a 5-fold increase in the luciferase activity in the stable reporter cell line, with 12 being histone deacetylase (HDAC) inhibitors. Eight compounds were subsequently observed to be comparable to sodium butyrate in inducing PBD3 mRNA expression in parental IPEC-J2 cells in the low micromolar range. Six HDAC inhibitors including suberoylanilide hydroxamine (SAHA), HC toxin, apicidin, panobinostat, SB939, and LAQ824 were additionally found to be highly effective HDP inducers in a porcine 3D4/31 macrophage cell line. Besides PBD3, other HDP genes such as PBD2 and cathelicidins (PG1–5) were concentration-dependently induced by those compounds in both IPEC-J2 and 3D4/31 cells. Furthermore, the antibacterial activities of 3D4/31 cells were augmented following 24 h exposure to HDAC inhibitors. In conclusion, a cell-based high-throughput screening assay was developed for the discovery of porcine HDP inducers, and newly identified HDP-inducing compounds may have potential to be developed as alternatives to antibiotics for applications in swine and possibly other animal species.

scholarly journals Translational Read-Through Therapy of RPGR Nonsense Mutations

2020 ◽  
Vol 21 (22) ◽  
pp. 8418
Author(s):  
Christine Vössing ◽  
Marta Owczarek-Lipska ◽  
Kerstin Nagel-Wolfrum ◽  
Charlotte Reiff ◽  
Christoph Jüschke ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Western Blot ◽  
Primary Cilium ◽  
Genetic Diseases ◽  
The Novel ◽  
Rt Pcr ◽  
Nonsense Mutations ◽  
Premature Termination Codons ◽  
New Treatment ◽  
First Time

X-chromosomal retinitis pigmentosa (RP) frequently is caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We evaluated the potential of PTC124 (Ataluren, TranslamaTM) treatment to promote ribosomal read-through of premature termination codons (PTC) in RPGR. Expression constructs in HEK293T cells showed that the efficacy of read-through reagents is higher for UGA than UAA PTCs. We identified the novel hemizygous nonsense mutation c.1154T > A, p.Leu385* (NM_000328.3) causing a UAA PTC in RPGR and generated patient-derived fibroblasts. Immunocytochemistry of serum-starved control fibroblasts showed the RPGR protein in a dot-like expression pattern along the primary cilium. In contrast, RPGR was no longer detectable at the primary cilium in patient-derived cells. Applying PTC124 restored RPGR at the cilium in approximately 8% of patient-derived cells. RT-PCR and Western blot assays verified the pathogenic mechanisms underlying the nonsense variant. Immunofluorescence stainings confirmed the successful PTC124 treatment. Our results showed for the first time that PTC124 induces read-through of PTCs in RPGR and restores the localization of the RPGR protein at the primary cilium in patient-derived cells. These results may provide a promising new treatment option for patients suffering from nonsense mutations in RPGR or other genetic diseases.

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 Molecular Insights into Determinants of Translational Readthrough and Implications for Nonsense Suppression Approaches

2020 ◽  
Vol 21 (24) ◽  
pp. 9449
Author(s):  
Silvia Lombardi ◽  
Maria Francesca Testa ◽  
Mirko Pinotti ◽  
Alessio Branchini
Keyword(s):  
Therapeutic Strategy ◽  
Low Frequency ◽  
External Factors ◽  
Full Length ◽  
Nonsense Suppression ◽  
Coagulation Disorders ◽  
Nonsense Mutations ◽  
Premature Termination Codons ◽  
Translational Readthrough ◽  
Molecular Determinants

The fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense and stop codons. However, premature termination codons (PTCs) arising from mutations may, at low frequency, be misrecognized and result in PTC suppression, named ribosome readthrough, with production of full-length proteins through the insertion of a subset of amino acids. Since some drugs have been identified as readthrough inducers, this fidelity drawback has been explored as a therapeutic approach in several models of human diseases caused by nonsense mutations. Here, we focus on the mechanisms driving translation in normal and aberrant conditions, the potential fates of mRNA in the presence of a PTC, as well as on the results obtained in the research of efficient readthrough-inducing compounds. In particular, we describe the molecular determinants shaping the outcome of readthrough, namely the nucleotide and protein context, with the latter being pivotal to produce functional full-length proteins. Through the interpretation of experimental and mechanistic findings, mainly obtained in lysosomal and coagulation disorders, we also propose a scenario of potential readthrough-favorable features to achieve relevant rescue profiles, representing the main issue for the potential translatability of readthrough as a therapeutic strategy.

scholarly journals A cell-based screen identifies HDAC inhibitors and PLK inhibitor as activators of RIG-I signaling

2021 ◽  
Author(s):  
Eugenia Fraile-Bethencourt ◽  
Marie H Foss ◽  
Dylan Nelson ◽  
Sanjay V Malhotra ◽  
Sudarshan Anand
Keyword(s):  
Nucleic Acid ◽  
Cell Line ◽  
High Throughput Screening ◽  
Hdac Inhibitors ◽  
Luciferase Reporter ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
High Throughput Screening Assay ◽  
A Cell ◽  
Acid Sensors

Enhancing the immune microenvironment in cancer by targeting the nucleic acid sensors is becoming a potent therapeutic strategy. Among the nucleic acid sensors, activation of the RNA sensor Retinoic Acid-inducible Gene (RIG-I) using small hairpin RNAs has been shown to elicit powerful innate and adaptive immune responses. Given the challenges inherent in pharmacokinetics and delivery of RNA based agonists, we set out to discover small molecule agonists of RIG-I using a cell-based assay. To this end, we established and validated a robust high throughput screening assay based on a commercially available HEK293 reporter cell line with a luciferase reporter downstream of tandem interferon stimulated gene 54 (ISG54) promoter elements. We first confirmed that the luminescence in this cell line is dependent on RIG-I and the interferon receptor using a hairpin RNA RIG-I agonist. We established a 96-well and a 384-well format HTS based on this cell line and performed a proof-of-concept screen using an FDA approved drug library of 1200 compounds. Surprisingly, we found two HDAC inhibitors Entinostat, Mocetinostat and the PLK1 inhibitor Volasertib significantly enhanced ISG-luciferase activity. This luminescence was substantially diminished in the null reporter cell line indicating the increase in signaling was dependent on RIG-I expression. Treatment of tumor cell lines with Entinostat, Mocetinostat or Volasertib induced interferon signature genes and increased RIG-I induced cell death in a mammary carcinoma cell line. Taken together, our data indicates an unexpected role for HDAC1,-3 inhibitors and PLK1 inhibitors in enhancing RIG-I signaling and highlight potential opportunities for therapeutic combinations.

scholarly journals From c-Photina® Mouse Embryonic Stem Cells to High-Throughput Screening of Differentiated Neural Cells via an Intermediate Step Enriched in Neural Precursor Cells

2010 ◽  
Vol 15 (9) ◽  
pp. 1132-1143 ◽  
Author(s):  
Silvia Cainarca ◽  
Simone Fenu ◽  
Silvia Bovolenta ◽  
Patrizia Arioli ◽  
Andrea Menegon ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
Precursor Cells ◽  
Neural Precursor Cells ◽  
Neural Precursor ◽  
Intermediate Step ◽  
Reporter Cell Line ◽  
Reporter Cell

The use of engineered mouse embryonic stem (mES) cells in high-throughput screening (HTS) can offer new opportunities for studying complex targets in their native environment, increasing the probability of discovering more meaningful hits. The authors have generated and developed a mouse embryonic stem cell line called c-Photina® mES stably expressing a Ca2+-activated photoprotein as a reporter gene. This reporter cell line retains the ability to differentiate into any cell lineage and can be used for miniaturized screening processes in 384-well microplates. The c-Photina® mES cell line is particularly well suited for the study of the pharmacological modulation of target genes that induce Ca2+ mobilization. The authors differentiated this mES reporter cell line into neuronal cells and screened the LOPAC1280™ library monitoring the agonistic or antagonistic activities of compounds. They also demonstrate the possibility to generate and freeze bulk preparations of cells at an intermediate stage of differentiation and enriched in neural precursor cells, which retain the ability to form fully functional neural networks once thawed. The proposed cell model is of high value for HTS purposes because it offers a more physiological environment to the targets of interest and the possibility of using frozen batches of neural precursor cells.

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