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

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs 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 reporter cell line and performed high-throughput screening (HTS) to identify potential 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 (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).

Activation of autosomal recessive Pseudohypoaldosteronism1 ENaC with aldosterone.

The mineralocorticoid hormone aldosterone stimulates sodium reabsorption in the collecting ducts by increasing the activity of the epithelial sodium channel (ENaC). Being a rate-liming channel the loss of function mutations caused Pseudohypoaldosteronism 1 (PHA1). Despite elevated plasma aldosterone in PHA 1 patients the modulation of PHA 1 causing ENaC mutants with hormone has never been studied. After recording control ENaC current in PHA1 causing ENaC stop codon mutants we demonstrated the activation of aldosterone in the whole cell as well as single channel patch clamp assays. Single channel recoding experiments demonstrated that aldosterone can increase the open probability of all analyzed PHA 1 stop codon mutants and WT. Additionally, we demonstrated by western blot experiments that aldosterone can increase the expression of WT and PHA 1 stop codon mutants. Extensive whole cell patch clamp experiments demonstrated that C-terminal γ ENaC domain is necessary for aldosterone to activate whole cell current in HEK-293 cells. This novel finding of γ ENaC C-terminus dependent activation of whole cell current by aldosterone could alter our understanding of ENaC-mediated sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN).

Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms.

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell's protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

Genome-wide effects of the antimicrobial peptide apidaecin on translation termination in bacteria.

Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here genome-wide analysis reveals that in bacteria, Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to the expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to the futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate the development of new medicines and research tools for genome exploration.

ELX-02 Generates Protein via Premature Stop Codon Read-Through without Inducing Native Stop Codon Read-Through Proteins.

ELX-02 is a clinical stage, small-molecule eukaryotic ribosomal selective glycoside acting to induce read-through of premature stop codons (PSCs) that results in translation of full-length protein. However, improved read-through at PSCs has raised the question of whether native stop codon (NSC) fidelity would be impacted. Here, we compare read-through by ELX-02 in PSC and NSC contexts. DMS-114 cells containing a PSC in the TP53 gene were treated with ELX-02 and tested for increased nuclear p53 protein expression while also monitoring two other proteins for NSC read-through. Additionally, blood samples were taken from healthy subjects pre- and post-treatment with ELX-02 (0.3-7.5 mg/kg). These samples were processed to collect white blood cells and then analyzed by western blot to identify native and potentially elongated proteins from NSC read-through. In a separate experiment, lymphocytes cultivated with vehicle or ELX-02 (20 and 100 µg/ml) were subjected to proteomic analysis. We found that ELX-02 produced significant read-through of the PSC found in TP53 mRNA in DMS-114 cells, resulting in increased p53 protein expression and consistent with decreased nonsense-mediated mRNA degradation. NSC read-through protein products were not observed in either DMS-114 cells or in clinical samples from subjects dosed with ELX-02. The number of read-through proteins identified by using proteomic analysis was lower than estimated, and none of the NSC read-through products identified with >2 peptides showed dose-dependent responses to ELX-02. Our results demonstrate significant PSC read-through by ELX-02 with maintained NSC fidelity, thus supporting the therapeutic utility of ELX-02 in diseases resulting from nonsense alleles. SIGNIFICANCE STATEMENT: ELX-02 produces significant read-through of premature stop codons leading to full-length functional protein, demonstrated here by using the R213X mutation in the TP53 gene of DMS-114 cells. In addition, three complementary techniques suggest that ELX-02 does not promote read-through of native stop codons at concentrations that lead to premature stop codon read-through. Thus, ELX-02 may be a potential therapeutic option for nonsense mutation-mediated genetic diseases.

Aminoglycosides are efficient reagents to induce readthrough of premature termination codon in mutant B4GALNT1 genes found in families of hereditary spastic paraplegia.

The readthrough of premature termination codon (PTC) by ribosome sometimes produces full-length proteins. We previously reported a readthrough of PTC of glycosyltransferase gene B4GALNT1 with hereditary spastic paraplegia (HSP). Here we featured the readthrough of B4GALNT1 of two mutants, M4 and M2 with PTC by immunoblotting and flow cytometry after transfection of B4GALNT1 cDNAs into cells. Immunoblotting showed a faint band of full-length mutant protein of M4 but not M2 at a similar position with that of wild-type B4GALNT1. AGC sequences at immediately before and after the PTC in M4 were critical for the readthrough. Treatment of cells transfected with mutant M4 cDNA with aminoglycosides resulted in increased readthrough of PTC. Furthermore, treatment of transfectants of mutant M2 cDNA with G418 also resulted in the induction of readthrough of PTC. Both M4 and M2 cDNA transfectants showed increased/induced bands in immunoblotting and GM2 expression in a dose-dependent manner of aminoglycosides. Results of mass spectrometry supported this effect. Here, we showed for the first time the induction and/or enhancement of the readthrough of PTCs of B4GALNT1 by aminoglycoside treatment, suggesting that aminoglycosides are efficient for patients with HSP caused by PTC of B4GALNT1, in which gradual neurological disorders emerged with aging.

Factors impacting the aminoglycoside-induced UGA stop codon readthrough in selenoprotein translation.

Aminoglycosides (AG) are oligosaccharide antibiotics that interfere with the small ribosomal subunit in aerobic, Gram-negative bacteria, causing pathogen-destructing error rates in their protein biosynthesis. Aminoglycosides also induce mRNA misinterpretation in eukaryotic cells, especially of the UGA (Opal)-stop codon, albeit to a lower extent. UGA recoding is essentially required for the incorporation of selenocysteine (Sec) into growing selenoproteins during translation. Selenocysteine incorporation requires the presence of a selenoprotein-specific stem-loop structure within the 3'-untranslated region of the mRNA, the so-called Sec-insertion sequence (SECIS) element. Interestingly, selenoprotein genes differ in their SECIS-element sequence and in their UGA base context. We hypothesized that the SECIS-element and the specific codon context synergize in controlling the effects of AG on stop codon readthrough. To this end, the SECIS-elements of glutathione peroxidase 1, glutathione peroxidase 4 and selenoprotein P transcripts were cloned into a reporter system and analyzed in combination with different UGA codon contexts. Our results indicate that a cytosine in position 4 (directly downstream of UGA) confers strongest effects on both the Se- and AG-dependent readthrough. Overall selenoprotein biosynthesis rate depends on the Se-status, AG concentration and the specific SECIS-element present in the transcript. These findings help to get a better understanding for the susceptibility of different transcripts towards AG-mediated interference with the biosynthesis of functional Se-containing selenoproteins, and highlight the importance of the Se-status for successful selenoprotein biosynthesis under antibiotic therapy.

Evaluation of Aminoglycoside and Non-Aminoglycoside Compounds for Stop-Codon Readthrough Therapy in Four Lysosomal Storage Diseases.

Nonsense mutations are quite prevalent in inherited diseases. Readthrough drugs could provide a therapeutic option for any disease caused by this type of mutation. Geneticin (G418) and gentamicin were among the first to be described. Novel compounds have been generated, but only a few have shown improved results. PTC124 is the only compound to have reached clinical trials. Here we first investigated the readthrough effects of gentamicin on fibroblasts from one patient with Sanfilippo B, one with Sanfilippo C, and one with Maroteaux-Lamy. We found that ARSB activity (Maroteaux-Lamy case) resulted in an increase of 2-3 folds and that the amount of this enzyme within the lysosomes was also increased, after treatment. Since the other two cases (Sanfilippo B and Sanfilippo C) did not respond to gentamicin, the treatments were extended with the use of geneticin and five non-aminoglycoside (PTC124, RTC13, RTC14, BZ6 and BZ16) readthrough compounds (RTCs). No recovery was observed at the enzyme activity level. However, mRNA recovery was observed in both cases, nearly a two-fold increase for Sanfilippo B fibroblasts with G418 and around 1.5 fold increase for Sanfilippo C cells with RTC14 and PTC124. Afterwards, some of the products were assessed through in vitro analyses for seven mutations in genes responsible for those diseases and, also, for Niemann-Pick A/B. Using the coupled transcription/translation system (TNT), the best results were obtained for SMPD1 mutations with G418, reaching a 35% recovery at 0.25 µg/ml, for the p.W168X mutation. The use of COS cells transfected with mutant cDNAs gave positive results for most of the mutations with some of the drugs, although to a different extent. The higher enzyme activity recovery, of around two-fold increase, was found for gentamicin on the ARSB p.W146X mutation. Our results are promising and consistent with those of other groups. Further studies of novel compounds are necessary to find those with more consistent efficacy and fewer toxic effects.

A new series of small molecular weight compounds induce read through of all three types of nonsense mutations in the ATM gene.

Chemical-induced read through of premature stop codons might be exploited as a potential treatment strategy for genetic disorders caused by nonsense mutations. Despite the promise of this approach, only a few read-through compounds (RTCs) have been discovered to date. These include aminoglycosides (e.g., gentamicin and G418) and nonaminoglycosides (e.g., PTC124 and RTC13). The therapeutic benefits of these RTCs remain to be determined. In an effort to find new RTCs, we screened an additional ~36,000 small molecular weight compounds using a high-throughput screening (HTS) assay that we had previously developed and identified two novel RTCs, GJ071, and GJ072. The activity of these two compounds was confirmed in cells derived from ataxia telangiectasia (A-T) patients with three different types of nonsense mutation in the ATM gene. Both compounds showed activity comparable to stop codons (TGA, TAG, and TAA) PTC124 and RTC13. Early structure-activity relationship studies generated eight active analogs of GJ072. Most of those analogs were effective on all three stop codons. GJ071 and GJ072, and some of the GJ072 analogs, appeared to be well tolerated by A-T cells. We also identified another two active RTCs in the primary screen, RTC204 and RTC219, which share a key structural feature with GJ072 and its analogs.

Development of generic immunoassay for the detection of a series of aminoglycosides with 6'-OH group for the treatment of genetic diseases in biological samples.

Over the last two decades, a growing number of scientific evidences highlighted the potential therapeutic value of several structures of aminoglycoside antibiotics (including gentamicin and G418) for the treatment of various genetic diseases caused by nonsense mutations. These findings resulted in a fast evolvement of synthetic derivatives of aminoglycosides which were shown to be more target specific and less toxic than the clinically used antibiotics. The emerging progress in drug design and development has necessitated the urge to develop a fast, easy and accurate procedure for the determination of these potential therapeutic agents in various biologically derived matrices. Here we describe the preparation of a generic polyclonal antibody that was used for the development of homologous and heterologous immunoassays for the detection of a wide range of natural and synthetic aminoglycoside derivatives, highlighted today as potential therapeutic agents for the treatment of various genetic diseases. A common two-ring scaffold, NB82, present in the majority of compounds exhibiting potent biological activity, was used as a generic immunization hapten for the immunization of two rabbits. By using a series of chemical steps, NB82 was selectively conjugated via the N-1 position through glutaric acid linker to a carrier protein. Sensitivity (I50) values for the recognition of three representative compounds NB82, NB84 and NB124 were determined to be 10 ± 3 ng mL⁻¹, 0.5 ± 0.04 µg mL⁻¹ and 1 ± 0.12 µg mL⁻¹, respectively. Limits of detection were determined to be 1 ± 0.3 ng mL⁻¹ for NB82, 20 ± 7 ng mL⁻¹ for NB84 and 15 ± 8 ng mL⁻¹ for NB124. The developed assays were further exploited for the in vivo monitoring of the therapeutic compounds in mice serum. Serum experimentations exhibited similar detection limits as observed for the PBS calibration experiments, demonstrating no interference with assays sensitivity, with rather high recovery ratios ranging from 92 to 107% in whole blood samples.

Pharmacological read-through of nonsense ARSB mutations as a potential therapeutic approach for mucopolysaccharidosis VI.

Mucopolysaccharidosis type VI (MPS VI) is a severe lysosomal storage disorder without central nervous system involvement caused by arylsulfatase B (ARSB) deficiency. MPS VI is characterized by dysostosis multiplex, corneal clouding, heart valve defects and urinary excretion of glycosaminoglycans (GAGs). The current treatment for MPS VI is enzyme replacement therapy (ERT) which has limited efficacy on bone, joints and heart valve disease, as well as high costs. A potential therapeutic approach for the subgroup of MPS VI patients that carry nonsense mutations is to enhance stop-codon read-through, using small molecules, to restore production of the full-length ARSB protein. In this study we investigated whether two compounds known to induce stop codon read-through, the aminoglycoside gentamicin and PTC124, can promote read-through of four different ARSB nonsense mutations (p.R315X, p.R327X, p.Q456X and p.Q503X) associated with MPS VI and enable the synthesis of full-length functional ARSB protein in patients fibroblast cell lines. Our study demonstrates that PTC124 but not gentamicin, increases the level of ARSB activity in three MPS VI patient fibroblast cell lines. In two of them the levels of ARSB activity obtained were significantly higher than in untreated cells, reaching ≤2.5 % of those detected in wild-type fibroblasts and resulting in significant reduction of lysosomal size. Since even small increases in enzyme activity can dramatically influence the clinical phenotype of MPS VI, our study suggests that pharmacological read-through may be combined with ERT potentially increasing therapeutic efficacy in those patients bearing nonsense ARSB mutations.

Treatment of a methylmalonyl-CoA mutase stopcodon mutation.

There are limited treatment options for the metabolic disorder methylmalonic aciduria. The disorder can be caused by nonsense mutations within the methylmalonyl-CoA mutase gene, resulting in the production of a truncated protein with little or no catalytic activity. We used a genomic reporter assay and mouse primary cell lines which carry a stop-codon mutation in the human methylmalonyl-CoA mutase gene to test the effects of gentamicin and PTC124 for stop-codon read-through potential. Fibroblast cell lines were established from methylmalonic aciduria knockout-stop codon mice. Addition of gentamicin to the culture medium caused a 1.5- to 2-fold increase in mRNA expression of the human methylmalonyl-CoA mutase gene. Without treatment the cells contained 19% of the normal levels of methylmalonyl-CoA mutase enzyme activity which increased to 32% with treatment, suggesting a functional improvement. Treatment with PTC124 increased the amount of human methylmalonyl-CoA mutase gene mRNA by 1.6±0.3-fold and a trend suggesting increased enzyme activity. The genomic reporter assay, BAC_MMA(∗)EGFP, expresses enhanced green fluorescent protein when read-through of the stop codon occurs. Using flow cytometry, RT-real-time PCR and enzyme assay, read-through was measured. Treatment with PTC124 at 20µmol/L resulted in a significant increase in enhanced green fluorescent protein, a 2-fold increase in mRNA expression and a trend to a slight increase in enzyme activity. The clinical relevance of these effects may be tested in mouse models of MMA carrying nonsense mutations in the methylmalonyl-CoA mutase gene. Pharmacological approaches have the advantage of providing a broader effect on multiple tissues, which will benefit many different disorders with similar nonsense mutations.

Statistical analysis of readthrough levels for nonsense mutations in mammalian cells reveals a major determinant of response to gentamicin.

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the -1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable.

Rescue of melanocortin 4 receptor (MC4R) nonsense mutations by aminoglycoside-mediated read-through.

Aminoglycoside-mediated read-through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity-associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycoside-mediated functional rescue in vitro. We determined localization and amount of full-length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS-7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N-terminus), less successful for Q307X (C-terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full-length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood-brain barrier.

Stop codon read-through with PTC124 induces palmitoyl-protein thioesterase-1 activity, reduces thioester load and suppresses apoptosis in cultured cells from INCL patients.

Infantile neuronal ceroid lipofuscinosis (INCL), a lethal hereditary neurodegenerative lysosomal storage disorder, affects mostly children. It is caused by inactivating mutations in the palmitoyl-protein thioesterase-1(PPT1) gene. Nonsense mutations in a gene generate premature termination codons producing truncated,nonfunctional or deleterious proteins. PPT1 nonsense-mutations account for approximately 31% of INCL patients in the US. Currently, there is no effective treatment for this disease. While aminoglycosides such asgentamycin suppress nonsense mutations, inherent toxicity of aminoglycosides prohibits chronic use inpatients. PTC124 is a non-toxic compound that induces ribosomal read-through of premature termination codons. We sought to determine whether PTC124-treatment of cultured cells from INCL patients carrying nonsense mutations in the PPT1 gene would correct PPT1 enzyme-deficiency with beneficial effects. Our results showed that PTC124-treatment of cultured cells from INCL patients carrying PPT1 nonsense-mutations induced PPT1 enzymatic activity in a dose- and time-dependent manner. This low level of PPT1 enzyme activity induced by PTC124 is virtually identical to that induced by gentamycin-treatment. Even though only a modest increase in PPT1 activity was achieved by PTC124-treatment of INCL cells, this treatment reduced the levels of thioester (constituent of ceroid) load. Our results suggest that PTC124-treatment induces PPT1 enzymatic activity in cultured cells from INCL patients carrying PPT1 nonsense-mutations, and this modest enzymatic activity has demonstrable beneficial effects on these cells. The clinical relevance of these effects may be tested in animal models of INCL carrying nonsense mutations in the PPT1 gene.

Gentamicin-induced readthrough of stop codons in Duchenne muscular dystrophy.

OBJECTIVE: The objective of this study was to establish the feasibility of long-term gentamicin dosing to achieve stop codon readthrough and produce full-length dystrophin. Mutation suppression of stop codons, successfully achieved in the mdx mouse using gentamicin, represents an important evolving treatment strategy in Duchenne muscular dystrophy (DMD). METHODS: Two DMD cohorts received 14-day gentamicin (7.5mg/kg/day): Cohort 1 (n = 10) stop codon patients and Cohort 2 (n = 8) frameshift controls. Two additional stop codon DMD cohorts were gentamicin treated (7.5mg/kg) for 6 months: Cohort 3 (n = 12) dosed weekly and Cohort 4 (n = 4) dosed twice weekly. Pre- and post-treatment biopsies were assessed for dystrophin levels, as were clinical outcomes. RESULTS: In the 14-day study, serum creatine kinase (CK) dropped by 50%, which was not seen in frameshift DMD controls. After 6 months of gentamicin, dystrophin levels significantly increased (p = 0.027); the highest levels reached 13 to 15% of normal (1 in Cohort 3, and 2 in Cohort 4), accompanied by reduced serum CK favoring drug-induced readthrough of stop codons. This was supported by stabilization of strength and a slight increase in forced vital capacity. Pretreatment stable transcripts predicted an increase of dystrophin after gentamicin. Readthrough efficiency was not affected by the stop codon or its surrounding fourth nucleotide. In 1 subject, antigen-specific interferon-gamma enzyme-linked immunospot assay detected an immunogenic dystrophin epitope. INTERPRETATION: The results support efforts to achieve drug-induced mutation suppression of stop codons. The immunogenic epitope resulting from readthrough emphasizes the importance of monitoring T-cell immunity during clinical studies that suppress stop codons. Similar principles apply to other molecular strategies, including exon skipping and gene therapy.