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 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 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 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 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 Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR

2020 ◽  
pp. 247255522096200
Author(s):  
Emery Smith ◽  
Danijela Dukovski ◽  
Justin Shumate ◽  
Louis Scampavia ◽  
John P. Miller ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Membrane Potential ◽  
Small Molecule ◽  
High Throughput ◽  
Premature Termination ◽  
Target Area ◽  
Mrna Levels ◽  
Premature Termination Codons ◽  
Translational Readthrough ◽  
Cftr Protein

Cystic fibrosis (CF) is caused by a mutation of the Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR) gene, which disrupts an ion channel involved in hydration maintenance via anion homeostasis. Nearly 5% of CF patients possess one or more copies of the G542X allele, which results in a stop codon at residue 542, preventing full-length CFTR protein synthesis. Identifying small-molecule modulators of mutant CFTR biosynthesis that affect the readthrough of this and other premature termination codons to synthesize a fully functional CFTR protein represents a novel target area of drug discovery. We describe the implementation and integration for large-scale screening of a homogeneous, 1536-well functional G542X-CFTR readthrough assay. The assay uses HEK 293 cells engineered to overexpress the G542X-CFTR mutant, whose functional activity is monitored with a membrane potential dye. Cells are co-incubated with a CFTR amplifier and CFTR corrector to maximize mRNA levels and trafficking of CFTR to the cell surface. Compounds that allow translational readthrough and synthesis of functional CFTR chloride channels are reflected by changes in membrane potential in response to cAMP stimulation with forskolin and CFTR channel potentiation with genistein. Assay statistics yielded Z′ values of 0.69 ± 0.06. As further evidence of its suitability for high-throughput screening, we completed automated screening of approximately 666,000 compounds, identifying 7761 initial hits. Following secondary and tertiary assays, we identified 188 confirmed hit compounds with low and submicromolar potencies. Thus, this approach takes advantage of a phenotypic screen with high-throughput scalability to identify new small-molecule G542X-CFTR readthrough modulators.

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 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.

From Inhibition to Degradation: Targeting the Anti-apoptotic Protein Myeloid Cell Leukemia 1(MCL1)

2019 ◽  
Author(s):  
James Papatzimas ◽  
Evgueni Gorobets ◽  
Ranjan Maity ◽  
Mir Ishruna Muniyat ◽  
Justin L. MacCallum ◽  
...  
Keyword(s):  
Small Molecules ◽  
E3 Ligase ◽  
Myeloid Cell ◽  
Cell Leukemia ◽  
New Class ◽  
Apoptotic Protein ◽  
Family Protein ◽  
Ubiquitination Pathway

<div> <div> <div> <p>Here we show the development of heterobifunctional small molecules capable of selectively targeting MCL1 using a Proteolysis Targeting Chimera (PROTAC) methodology leading to successful degradation. We have confirmed the involvement of the E3 ligase CUL4A-DDB1 cereblon (CRBN) ubiquitination pathway, making these PROTACs a first step toward a new class of anti-apoptotic BCL-2 family protein degraders. </p> </div> </div> </div>

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications

2019 ◽  
Vol 26 (30) ◽  
pp. 5609-5624
Author(s):  
Dijana Saftić ◽  
Željka Ban ◽  
Josipa Matić ◽  
Lidija-Marija Tumirv ◽  
Ivo Piantanida
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Biomedical Applications ◽  
Protein Targets ◽  
New Class ◽  
Rna Targets ◽  
Covalently Linked ◽  
Structural Aspects

: Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder – nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

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