Advances in therapeutic use of a drug-stimulated translational readthrough of premature termination codons

Advances in therapeutic use of a drug-stimulated translational readthrough of premature... Premature termination codons (PTCs) in the coding regions of mRNA lead to the incorrect termination of translation and generation of non-functional, truncated proteins. Translational readthrough of PTCs induced by pharmaceutical compounds is a promising way of restoring functional protein expression and reducing disease symptoms, without affecting the genome or transcriptome of the patient. While in some cases proven effective, the clinical use of readthrough-inducing compounds is still associated with many risks and difficulties. This review focuses on problems directly associated with compounds used to stimulate PTC readthrough, such as their interactions with the cell and organism, their toxicity and bioavailability (cell permeability; tissue deposition etc.). Various strategies designed to overcome these problems are presented. Keywords: Translational readthrough, Stop codon suppression, Nonsense suppression, Premature termination codon, Genetic diseases Background Main text Most of the reviews on the premature termination co- Premature termination codons dons (PTCs) readthrough studies focus on the efficiency An extensive meta-analysis study, based on the Human of the PTC suppression-stimulating agents, without ex- Gene Mutation Database, has revealed that 12% of all ploring the associated problems of these agents’ toxicity described gene lesions causing human inherited diseases and bioavailability. The present review, by providing a is caused by nonsense mutations (Mort et al., 2008). These different perspective, aims to contribute to understand- mutations, by changing an amino acid coding triplet into ing of the problems associated with using PTC read- a stop codon, introduce premature termination codons, through strategies. The risks and difficulties associated PTCs, into the protein-coding gene sequence. PTCs may with compounds used to stimulate PTC readthrough, also be caused by other types of mutations, such as frame- mostly related with their interaction with the cell and or- shifts (insertion or deletion other than multiple-of-three ganism, and concerning their toxicity and bioavailability base pairs) or mutations in the conserved splice-site se- (cell permeability; tissue deposition etc.) are discussed. In quences (leading to a defective intron removal from the addition, strategies to overcome these problems are dem- pre-mRNA) (Mort et al., 2008; Mendell & Dietz, 2001). onstrated. We also present outcomes of recently finished PTCs in mRNAs can even occur independently of any clinical trials on the readthrough compounds. The aspect changes in DNA, through the aberrant mRNA processing of PTC identity and its nucleotide context has been re- (alternative splicing) (Pan et al., 2006; Lewis et al., 2003). cently reviewed (Dabrowski et al., 2015)and is notexten- In any case, PTCs located in the coding regions of sively discussed here; other problems, related to the mRNA lead to the premature termination of translation molecular side of PTC-RT, deserve a separate review and and, as a consequence, to the production of truncated are only briefly mentioned. proteins (Mendell & Dietz, 2001). In most cases, this re- duces the amount of a full-length protein in a recessive- negative manner, whereby mutations of both alleles are * Correspondence: ewa.zietkiewicz@igcz.poznan.pl necessary to result in a deleterious phenotype (Khajavi et Institute of Human Genetics; Polish Academy of Sciences, Poznan, Poland © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dabrowski et al. Molecular Medicine (2018) 24:25 Page 2 of 15 al., 2006). However, PTCs can also exert dominant- Not all stop codons can be read through with the same negative effect (Inoue et al., 2004). Phenotypic manifest- efficacy. The level of “leakiness” of the stop codons can be ation usually involves loss-of-function effects, but there ranked as UGA > UAG > UAA (Manuvakhova et al., 2000). are examples of gain-of-function events (Manuvakhova et Also the sequence of nucleotides upstream and down- al., 2000). For example, in β-thalassemia, truncated pro- stream to the stop codon has the effect on the efficacy of teins derived from PTC-bearing transcripts are respon- the readthrough process. The majority of evidence indicates sible for generation of insoluble globin chains, which are that a nucleotide immediately following the termination toxic to the cell (Thein et al., 1990). codoninthe 3′ direction (position + 4, when the first nu- The synthesis of deleterious C-terminally truncated cleotide of the stop codon is marked as + 1), is in- proteins in eukaryotic organisms is reduced due to the volved in the interactions between mRNA and translational specialized mRNA surveillance mechanisms. One of them machinery (McCaughan et al., 1995;Jungreisetal., 2011; is the nonsense-mediated mRNA decay (NMD) pathway Loughran et al., 2014). This suggests that the actual (Lejeune, 2017a). This conserved quality-control process translation termination signal consists of a tetranu- detects and degrades transcripts containing abnormalities cleotide sequence, rather than only the stop codon itself such as: PTCs, introns downstream of the termination (Brown et al., 1990). Recently, it has been proposed, based codon, long 3′ untranslated regions (3′UTRs) or upstream on genetic interaction studies in yeast, that + 4 cytosine open reading frames (uORFs) (Mühlemann, 2008;Hogg & compromises the ability of eRF1 to recognize appropri- Goff, 2010;Miller & Pearce, 2014;Lejeune, 2017b). ately a stop codon, thus providing precedence for the im- Many efforts have been made to develop therapeutic portance of nucleotide positioning for eRF1-mediated strategies that would counteract negative effects of PTCs. termination efficiency (Beznosková et al., 2016). In addition, One of them relies on exploring the natural phenomenon the upstream sequence, immediately 5′ of the stop codon of termination codon suppression through the transla- (positions −1to − 3) also exerts an effect; this effect is con- tional readthrough mechanism. sideredtobemoresubtlethanthe downstream sequence context of the transcript. Studies suggest that even relatively distant nucleotides (positions + 5, + 6, + 9) may also influence Basal translational readthrough the translational readthrough (for a review, see Dabrowski et Effective termination of protein translation in eukaryotes al., 2015, RNA Biology) (Dabrowski et al., 2015). requires recognition of the stop codon, localized in the A- It has been estimated that basal readthrough of normal site of the ribosome, by a release factor, eRF1. The eRF1 termination codons (NTCs) occurs in 0.001 to 0.1% of further interacts with another release factor – eRF3, and total rounds of translation of a given transcript (Keeling with GTP. This ternary termination complex (eRF1-eRF3- et al., 2012). In the case of PTCs, basal readthrough GTP) interacts with the poly(A)-binding protein (PABP) levels are higher and can range from 0.01% to even 1% present at the 3′-UTR of mRNA; this leads to efficient hy- (Manuvakhova et al., 2000; Bonetti et al., 1995; Cassan & drolysis of GTP and cleavage of the peptidyl-tRNA bond Rousset, 2001). It is not known, why the basal read- (Bulygin et al., 2017). As a consequence, the newly synthe- through levels differ so much. It has been hypothesized, sized polypeptide chain is released from the ribosome. that efficient translation termination requires the close With the termination of translation being based on the contact between the release factors interacting with steric match between the stop codon and the eRF1, this NTC and PABPs (Lejeune, 2017a; Cosson et al., 2002; process is not 100% efficient. Stop codons in mRNA can Celik et al., 2015). Presence of a PTC in the transcript, be suppressed (recoded) through the natural mechanism usually more distant from the 3’UTR, could limit the of basal translational readthrough (Dabrowski et al., 2015; interaction between eRFs and PABPs, leading to the less Fearon et al., 1994). In this process, a near-cognate tRNA efficient action of eRF3 and the delayed release of the (nc-tRNA) outcompetes eRF1 at the A-site of the ribo- transcript from the ribosome (Ivanov et al., 2008). The some. Nc-tRNAs have anticodons, which are complemen- prolonged presence of the translational machinery at the tary to only two of the three positions of a nonsense PTC would increase the susceptibility to the basal transla- codon in mRNA. The recent studies indicate, that the tional readthrough of the PTC (PTC-RT) (Amrani et al., interaction between PTC and a nc-tRNA anticodon oc- 2004). curs by mispairing at either position 3 or 1 of the stop codon (Roy et al., 2015; Roy et al., 2016). If the nc-tRNA Drug-induced translational readthrough interacts with the PTC at the A-site, the amino acid A number of studies have demonstrated that certain transported by such nc-tRNA is incorporated into the low-molecular-mass drugs can stimulate recoding of a synthesized polypeptide chain, and translation – instead PTC by the translation machinery (Lee & Dougherty, of being terminated – continues until the next in-frame 2012). These findings have opened the way to new thera- stop codon (Fig. 1). peutic approaches to nonsense mutations in genes Dabrowski et al. Molecular Medicine (2018) 24:25 Page 3 of 15 Fig. 1 PTC-RT process. a Ribosome encounters premature termination codon (PTC); the site is recognized by the translation termination machinery and the polypeptide elongation is prematurely terminated. b After addition of readthrough compound, translational machinery decodes PTC (PTC is recognized by nc- tRNAs), and translation continues until the normal termination codon (NTC). It allows translating a full-length protein. The NMD surveillance mechanism may detect and degrade PTC-bearing transcripts. If the NMD process is inhibited, very low levels of the full-length protein can be present even in the absence of stimulating agents, as the result of the endogeneous suppression of PTC associated with genetic diseases. However, the efficient already in 1960s, first papers indicating readthrough use of this approach in the clinic is still hampered by a potential of AAGs in bacteria have been published number of problems (Fig. 2). (Davies et al., 1964;Andersonet al., 1965). In 1985, this Aminoglycoside antibiotics (AAGs) are among the process was demonstrated in mammalian cells (Burke & most studied drugs capable of inducing PTC-RT - Mogg, 1985). AAGs are oligosaccharides consisting of Fig. 2 Different problems associated with the drug-stimulated PTC-RT therapy. Upper part – problems associated with the PTC-RT- stimulating drugs. Lower part – problems associated with the biology of PTC-RT process Dabrowski et al. Molecular Medicine (2018) 24:25 Page 4 of 15 streptidine or 2-deoxystreptidine as the molecular core, effect is reversible after stopping AAGs administration with a variable number of sugar rings and ammonium (Mingeot-Leclercq et al., 1999). The negative influence groups (François et al., 2005). Commonly used to treat of AAGs on the renal function in patients can be also di- Gram-negative bacterial infections, they bind with the minished by the appropriate hydration therapy and ap- seven-nucleotide loop structure in the decoding center of plication of dialysis, when necessary (Lopez-Novoa et al., the bacterial ribosome and interfere with its function 2011). The ototoxicity induced by AAGs manifests as ir- (Fan-Minogue & Bedwell, 2008). This enables effective reversible, symmetric, bilateral sensorineural hearing misincorporation of the nc-tRNAs, resulting in extensive loss. Due to the very slow removal of AAGs from the translational misreading, followed by a complete inhib- inner ear fluids, the onset of hearing loss can occur days, ition of protein synthesis in bacteria. In eukaryotes, a or even weeks, after finishing the AAG administration small difference in the rRNA nucleotide sequence signifi- (Huth et al., 2011). Inside the hair cell, AAGs cause cantly lowers the efficiency of this interaction (Lynch & damage, either directly or indirectly, first by causing dis- Puglisi, 2001). Nonetheless, in the case of PTC, the impact array of stereocilia, and ultimately by activating apop- of AAGs on the translational apparatus is often sufficient tosis (Abi-Hachem et al., 2010). to reduce discrimination between cognate and nc-tRNAs, The selective toxicity observed in the inner ear and and to enhance translational readthrough. kidneys is associated with the cellular uptake of AAGs The therapeutic potential of AAGs as PTC readthrough- through megalin, an endocytic receptor localized in the ap- stimulating compounds has been tested in many models, ical membrane of epithelial cells present in the proximal from the in vitro transcription and translation system renal tubules and in the hair cells of the inner ear (Manuvakhova et al., 2000;Du et al., 2002; Keeling & (Moestrup et al., 1995). Themechanism,bywhich AAGs Bedwell, 2002;Lai et al., 2004), through dual reporters in induce apoptosis, both in the renal cells and in the hair cell lines (Howard et al., 2004; Bidou et al., 2004; Floquet et cells, is to date not well-defined. Some evidence indicates a al., 2011; Bukowy-Bieryllo et al., 2016), patient derived cells role of reactive oxygen species (ROS) (Huth et al., 2011). (Salvatori et al., 2009;Nakamuraet al., 2012; Cogan et al., Positively-charged AAGs are able to interact with various 2014;Dündaretal., 2017). The first in vivo demonstration negatively-charged cellular components such as phospho- of the therapeutic AAG properties has been in mdx mice, lipids, phospholipases, and metal ions. AAGs bounded to an animal model of Duchenne muscular dystrophy (DMD), phospholipids form complexes, which can aggregate in the where gentamicin treatment restored 10–20% of normal internal lysosomal membranes, causing phospholipidosis, a dystrophin levels in the skeletal muscles (Barton-Davis et condition often associated with nephro- and ototoxicity al., 1999). Since then, a large variety of AAGs has been (Couture et al., 1994; Reasor & Kacew, 2001). More import- tested for their PTC-RT-stimulating properties, both in ani- antly, the AAG-phospholipid interaction leads to the gener- mal models (Arakawa et al., 2003;Duet al., 2006;Guerinet ation of ROS, which affect membrane fluidity and al., 2008; Yukihara et al., 2011;Roweet al., 2011;Gunnet permeability, disrupt the activity of enzymes, ion chan- al., 2014) and clinical trials (Clancy et al., 2001; Wilschanski nels and receptors, and finally direct the cell to the et al., 2003; Politano et al., 2003; Sermet-Gaudelus et al., apoptotic pathway (Xie et al., 2011). Another explanation 2007; Malik et al., 2010). The latest pilot clinical study for the cell apoptosis related to AAGs administration sug- (ClinicalTrials.gov; id: NCT02698735) has shown positive gests involvement of the decoding site of the ribosome of results of gentamicin use in recessive dystrophic epidermo- mitochondrial ribosomes, which closely resembles the lysis bullosa patients with nonsense mutation in the gene A-site of bacterial ribosomes. The interaction of encoding type VII collagen. Topical or intradermal adminis- AAGs with the 12S rRNA at the A-site causes mis- tration of gentamicin increased the level of full-length colla- translation, then inhibition of protein synthesis, ultimately gen VII from 20 to 165% of the wild-type level, the leading to cell death (Hobbie et al., 2008). AAG-mediated expression of the protein persisted for 3 months. In oxidative damage of the mitochondrial enzyme – aconitase addition, a significant reduction of disease symptoms was can also cause an increase in superoxide overproduction observed (improved epidermal-dermal adherence, reduced from the free ferrous iron in mitochondria, that in turn blister formation, and enhanced wound closure) (Woodley increases the amount of hydroxyl radicals via the Fenton et al., 2017). reaction, and finally induces the cell apoptosis (Shulman et al., 2014). Toxicity of AAGs In spite of these adverse effects, one study has demon- Even though AAGs have been commonly used in clinics strated that AAGs used to stimulate PTC-RT can be for decades, they may exert strong oto- and nephrotoxic relatively safe. The six month-long gentamicin therapy effects on the organism. Accumulation of AAGs in the in DMD patients was completed safely, with no impair- renal epithelial cells leads to apoptosis and necrosis of ment of either renal or hearing function. The negative these cells (Lopez-Novoa et al., 2011). However, this effects of AAGs were avoided thanks to the informed Dabrowski et al. Molecular Medicine (2018) 24:25 Page 5 of 15 choice of the study cohort and a careful study design; all stimulating efficacy of AAGs. This strategy allowed signifi- subjects with the A1555G mutation in 12S rRNA gene cant lowering of the therapeuticdoseof AAGs, thus redu- of mtDNA, known to predispose to gentamicin-induced cing the AAG-related toxicity. For example, CDX5–1 ototoxicity, had been excluded from the treatment, while administered together with G418 not only increased the in the remaining patients a strict regimen of gentamicin level of PTC-RT up to 180-fold, but also accelerated syn- administration was obeyed (Malik et al., 2010). thesis of the full-length protein (the full-length product was Although the clinical use of AAGs for a prolonged detected 10 times faster). CDX5–1 action did not rely on time is associated with many risks, the benefits of a pos- stimulating PTC-RT; nor did it inhibit the NMD process sible PTC-RT-based therapy are indisputable. To allevi- (Baradaran-Heravi et al., 2016). The mechanism by which ate the problems resulting from AAGs toxicity, various this compound potentiates PTC-RT activity of AAGs, as attempts have been undertaken to mitigate the toxicity well as its potential interference with other cellular pro- or to select other compounds with PTC-RT-inducing cesses, remains to be elucidated. potential but without undesirable side effects of AAGs. Other approaches aiming to reduce AAG toxicity related to their positive electrostatic charge involve the use of Overcoming cytotoxic effects of AAGs liposome-encapsulated AAGs. Liposomes containing en- One approach tested for reducing the cytotoxicity of capsulated gentamicin, used to stimulate PTC-RT in DMD AAGs involves their co-administration with different mice, were more effective and caused 10-fold lower ototox- compounds, which are able to reduce interaction of icity than the traditionally administered gentamicin. Cre- AAGs with different cellular targets. This strategy was atinine concentrations (an indicator of renal malfunction) shown to be successful for tobramycin or gentamicin co- in mice treated with these compounds were normal, sug- administrated with a lipopeptic antibiotic, daptomycin gesting that encapsulation of AAG also reduced nephrotox- (Beauchamp et al., 1990; Thibault et al., 1994). Nega- icity (Yukihara et al., 2011; Schiffelers et al., 2001). tively charged daptomycin complexed AAGs through an electrostatic interaction, preventing them from binding Chemical modification of AAGs - AAG derivatives to phospholipids and other intracellular targets (Couture A different approach to the therapeutic use of PTC-RT et al., 1994). Co-administration of gentamicin with an- involves modification of the chemical structure of AAGs other compound, the negatively charged poly-L-aspartic with a proven nonsense suppressing ability, to obtain acid (PAA), was shown to protect rats against the develop- compounds with a reduced toxicity and retained or in- ment of kidney nephrotoxicity (Gilbert et al., 1989; creased therapeutic potential. An example of this ap- Ramsammy et al., 1989). It has been suggested that PAA, proach comes from the studies on artificially designed similarly to daptomycin, binds AAGs and prevents phos- paromomycin derivatives, where NB30 represents the pholipidosis, which would otherwise lead to nephrotox- first, and NB54 – the second generation. The modification icity (Kishore et al., 1990). In addition, PAA increased the of the paromomycin structure effectively reduced toxicity intracellular concentration of gentamicin, contributing to of the derivative compounds. For example, NB30 was 15- a higher level of PTC-RT. It also slowed-down elimination fold less toxic than gentamicin, while both compounds of AAGs from cytosol, extending the time period during showed nonsense suppression at the levels similar to those which readthrough stimulation occurred. Similar results induced by gentamicin and paromomycin (Nudelman et al., were obtained in the mouse model of cystic fibrosis 2009). The compounds were able to induce statistically sig- (Du et al., 2009a). nificant PTC-RT levels in cell lines from patients with dif- Another approach used to mitigate the toxic effects of ferent PTC-caused diseases, such as: Rett syndrome AAGs relies on their co-administration with antioxidants. (Brendel et al., 2011; Vecsler et al., 2011), cystic fibrosis D-methionine and melatonin were shown to reduce the (Rowe et al., 2011), mucopolysaccharidosis type I – Hurler harmful ROS formation caused by the interaction of syndrome (MPS I-H) (Wang et al., 2012; Kamei et al., AAGs with the cell components (Campbell et al., 2007; 2013) and Usher syndrome type 1 (Rebibo-Sabbah et Reiter et al., 2011). Melatonin turned out to be particularly al., 2007;Goldmannetal., 2012). effective; compared to a mixture of other antioxidants, it Modification of the structure of another AAG, G418, was 150 times more effective in reducing ototoxic effects resulted in the third generation of PTC-RT-stimulating of gentamicin or tobramycin. AAG derivatives, NB74 and NB84. These compounds Recently, a new approach to reduce AAG toxicity has were several-fold more active than the previous genera- been tested, whereby AAGs were co-administered with tions (Nudelman et al., 2010). Importantly, modifications small molecular mass molecules, derivatives of phthalimide also highly reduced the toxicity of the compounds. The (e.g. CDX5–1) (Baradaran-Heravi et al., 2016). When used capacity of NB74 and NB84 to stimulate PTC-RT was alone, these compounds did not induce PTC-RT, but analyzed in the mouse model of MPS I-H caused by a applied with AAGs, they efficiently potentiated PTC-RT PTC mutation in the α-L-iduronidase gene. The level of Dabrowski et al. Molecular Medicine (2018) 24:25 Page 6 of 15 PTC-RT induced by NB84 was higher than that caused known example of this group is a small molecule by gentamicin or by the first and second generation of drug 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]-benzoic AAG derivatives, and the LC50 values were 9–10 times acid, also known as PTC124, Ataluren or Translarna. Identi- higher than for G418 (Shulman et al., 2014). A long- fied during a high-throughput screen, PTC124 has been term (28-weeks) treatment of MPS I-H mice with NB84 selected as the most potent readthrough promoting drug resulted in an increased activity of α-L-iduronidase in from over 800,000 compounds screened (Welch et al., different tissues, including the brain, heart and bone. 2007). The level of the enzyme was sufficient to significantly re- PTC-RT stimulating efficiency of PTC124 was success- duce disease symptoms in the tissues, while no visible fully demonstrated in animals (Goldmann et al., 2012; toxicity was observed; moreover both the NB84 and Welch et al., 2007). It was also tested in different in vitro NB74 compounds were shown to be able to cross the and ex vivo models of PTC-mediated diseases, including blood-brain barrier (Gunn et al., 2014). The newest rep- cystic fibrosis (Du et al., 2008), Duchenne muscular resentative of the third generation of chemically modi- dystrophy (Yukihara et al., 2011; Finkel et al., 2013), fied AAGs, NB124, turned out to be even more Miyoshi myopathy (Wang et al., 2010), Usher syndrome promising. Its therapeutic index (comparison of the (Goldmann et al., 2012) and Batten disease (Sarkar et al., amount of a compound that causes the therapeutic ef- 2011). Phase I and phase II clinical trials have shown that fect to the amount that causes toxicity) was 10 times its adverse effects were mild or moderate and similar to better than that obtained for gentamicin and other non- the placebo-treated group; therefore, PTC124 has been modified AAGs. Moreover, a series of cell and animal deemed safe for therapeutic use in humans (Hirawat et al., based experiments using the CF models have shown that 2007; Kerem et al., 2008), and a number of clinical trials NB124 restored up to 10% of the wild-type CFTR func- have been undertaken. Unfortunately, despite many posi- tion (Xue et al., 2014). In the most recent study, using tive results from different experimental models, the effects reporter vectors in the transiently transfected cell lines, of PTC124 in patients remain inconclusive. NB124 was shown to efficiently decode common PTC In CF patients with PTC mutations in the CFTR gene, mutations in tumor suppressor genes (p53 and APC) in treatment with PTC124 slightly increased chloride chan- the murine cell line (NIH3T3), resulting in over 20% of nel activity, resulting in an improvement of clinical pa- the PTC-RT. In the HDQ-P1 cell line (derived from hu- rameters (Kerem et al., 2008); however, not all patients man primary breast carcinoma), the restoration of p53 responded positively to the drug. In a similar clinical and APC tumor suppressor proteins expression by trial with pediatric CF patients, an improvement in NB124 induced apoptosis in 38% of the cells, whereas chloride channel activity was observed again, and the untreated cells displayed no signs of apoptosis. In presence of CFTR protein in nasal epithelium was con- addition, similarly to G418, NB124 also affected the firmed by immunofluorescence; nevertheless, no signifi- NMD process, inhibiting the degradation of p53 tran- cant therapeutic improvement was proved (Sermet- scripts (Bidou et al., 2017). Gaudelus et al., 2010). In the phase III clinical trials, Another modified AAG is a pyranmycin (TC007), a some improvement in lung function and reduction in derivative of another aminoglycoside antibiotic, neomy- exacerbation frequency were reported after 48 weeks of cin (Chang et al., 2002). In the fibroblasts from patients PTC124 therapy, although statistical significance in the with spinal muscular atrophy (SMA) caused by the pre- overall patient population was not reached (Kerem et al., mature termination of the SMN protein, stimulation 2014). The main outcome of another phase III clinical with TC007 resulted in the 10-fold increase in the level study (ClinicalTrials.gov; id: NCT02139306, finished in of full-length SMN protein. In a mouse model of SMA, March 2017), has been announced via the PTC Thera- injection of TC007 into the central nervous system peutics press release, and at the time of writing this (brain ventricles) induced higher expression SMN pro- review, the full data were still unpublished. The tein, led to a longer survival of the motor neurons and announcement reported a failure to reach the expected was associated with a 27% increase in the lifespan of the primary and secondary endpoints. In light of these SMA mice (Mattis et al., 2009; Mattis et al., 2012). discouraging results, PTC Therapeutics has recently decided to discontinue their clinical development of PTC124 for CF and withdraw its application for Readthrough compounds marketing authorization in Europe (PR Newswire). Apart from modification of AAG structure, a number PTC124-induced suppression of PTC was also tested in of studies have searched for readthrough-inducing clinical trials with DMD patients. In the phase IIA trial, an compounds, chemically not related to AAG, but pos- increased expression of the full-length dystrophin was sessing the potential to suppress nonsense mutations in shown in patients’ biopsies after 28 days of oral adminis- mammalian cells (Lee & Dougherty, 2012). The best tration of PTC124; one third of the patients demonstrated Dabrowski et al. Molecular Medicine (2018) 24:25 Page 7 of 15 an increase in the post-treatment expression of dystrophin level of PTC-RT. In the mentioned studies, a full-lengh p53 appropriately localized in the sarcolemmal membrane of protein that was translated due to the readthrough was fully muscle cells (Finkel et al., 2013). In the phase IIb trial, the functional, both in the cell lines with PTC-containing alleles patients showed a slower decline in the walk test; no im- and in the tumor-bearing mice (Friesen et al., 2017). provement related to the higher dose of PTC124 was ob- Apart from PTC124, a number of other low-molecular- served, suggesting a bell-shaped dose-response curve mass compounds, capable of stimulating PTC-RT, have (Bushby et al., 2014). Unfortunately, phase III clinical study been identified to date. Screening of ~ 34,000 compounds completed in 2017, indicated that similarly to the CF studies, using a novel high-throughput screening assay identified the effects of PTC124 treatment did not differ significantly twelve most promising readthrough-stimulating com- from the placebo group (McDonald et al., 2017). pounds (Du et al., 2009b). The compounds displayed Despite the intensive studies, the exact mechanism of reduced toxicity in the mammalian cells and did not alter PTC124 action remains unknown. Computational model- the global protein expression patterns (Gatti, 2012). Two ing suggested that mRNAs containing PTC form stable leading first generation compounds, RTC13 and RTC14, complexes with PTC124, which may interfere with the were proven to suppress nonsense mutations in ataxia tel- recognition of PTC by eRF1 and suppress the termination angiectasia patients’ cell lines (A-T cells) and in myotube of translation; however, this interaction was shown to be cells from mdx mice (Du et al., 2009b;Kayaliet al., 2012). stable only for the UGA codon (Lentini et al., 2014). In More recent studies identified the further PTC-RT-stimu- cell lines transfected with different PTC-bearing con- lating compounds, namely GJ071 and GJ072, their an- structs, the PTC-RT-stimulating potential of PTC124 was alogs (RTC204 and RTC219), as well as non-related different for distinct stop codons; UGA suppression was compounds, BZ6 and BZ16 (Jung et al., 2011;Du et three times more effective than UAG, and 6 times more al., 2013). In the A-T cells, these compounds displayed effective than suppression of UAA (Welch et al., 2007). PTC-RT-stimulating efficiency comparable to RTC13, The latest evidence indicates, that PTC124 has a selectiv- RTC14 and PTC124, but with significantly lower toxicity. ity for the ribosomal A site and that it promotes insertion When tested in cell lines from patients with four different of nc-tRNAs at the PTC site (Roy et al., 2016). Tobra- lysosomal storage diseases, RTC13, RTC14, BZ6 and BZ16 mycin, a compound with an affinity for the ribosomal A showed ~ 1.5 fold increase in the amount of a relevant site, is a strong inhibitor of PTC124 (probably by competi- full-length mRNA compared to the previously used com- tion), what supports the expected interaction of PTC124 pounds (Gómez-Grau et al., 2015) and were able to sup- with the ribosome (Roy et al., 2016; Salian et al., 2012). press all three stop codons (Du et al., 2013). However, the Moreover, in a post-hoc analysis of the recent clinical trial, mechanism of their action remains unknown. it was observed that CF patients with nonsense mutations New readthrough compounds have also been found failed to show a significant response to administered among non-AAG antibiotics. A dipeptide antibiotic, PTC124, if they were concurrently treated with the in- negamycin, structurally not related to AAG, affects the haled tobramycin (Kerem et al., 2014). process of ribosomal decoding in a way similar to AAGs Clitocine [6-amino-5-nitro-4-(β-D-ribofuranosylamino) (Arakawa et al., 2003). However, in contrast to AAG, nega- pyrimidine] has been identified in the same high- mycin exhibits much lower cytotoxicity and no ototoxicity. throughput screen as PTC124 (Welch et al., 2000). This Negamycin was shown to promote PTC-RT in the dys- compound is an adenosine nucleoside analog origin- trophingenebothinvivointhe mousemodel of DMDand ally isolated from the mushroom Clitocybe inverse in the cultured mdx myotubes (Arakawa et al., 2003). It (Kubo et al., 1986). Clitocine is incorporated into mRNA was also tested in congenital muscular dystrophy: PTC-RT during transcription as an adenosine substitute; unlike induced by negamycin in the transfected NIH3T3 AAGs that act on the translation machinery, clitocine fa- cells was several-fold higher than that induced by AAGs cilitates decoding of the nonsense codon just by its pres- (Allamand et al., 2008). ence in the transcript (Friesen et al., 2017). Clitocine can Another group of compounds capable of inducing be incorporated instead of the adenine in each of the PTC-RT are macrolides, antibiotics often used to treat stop codons. The order of readthrough susceptibility Gram-positive bacterial infections. Induction of PTC- of the stop codons in the presence of clitocine is RT by tylosin, josamycin, and spiramycin was demon- UAA> > UGA > UAG. Surprisingly, the UAA codon is the strated in colorectal cancer models (mutations in the APC most leaky, probably because of the incorporation of clito- gene) (Zilberberg et al., 2010). The effect was also con- cine in both the second and third position of the codon firmed in a xenograft mouse model – treatment with (Friesen et al., 2017). Stop codons with the incorporated macrolides resulted in reduced tumor growth and led to a clitocine are poorly recognized by the eRF1 translation notable increase in the lifespan of the mice (Zilberberg et termination factor; this prevents efficient termination, en- al., 2010). These data were later extended by the flow hances nc-tRNA recruitment and therefore increases the cytometry-based reporter assay, in which erythromycin Dabrowski et al. Molecular Medicine (2018) 24:25 Page 8 of 15 and its derivative, azithromycin, were used (Caspi et al., incorporation of amino acid that does not necessarily cor- 2015). Macrolides were analyzed in cell lines derived from respond to the wild-type), suppressor tRNAs do not intro- patients with mutations in a number of genes; ATM duce missense mutations, and thus lead to the synthesis of in ataxia-telangiectasia (A-T), MeCP2 in RTT syndrome, the functional protein (Bordeira-Carriço et al., 2014). How- SMN in the SMA syndrome, APC in familial adenomatous ever, a clinical use of suppressor tRNAs faces many chal- polyposis (FAP). In all these cases, macrolides induced lenges, especially regarding the efficiency of delivery and in PTC-RT; additionally, azithromycin worked at a 100-fold vivo expression of these systems, with minimal toxicity to lower concentration than any other compound tested so the organism. far, enabling a significant reduction of the therapeutic dose (Caspi et al., 2015). PTC-RT-stimulating potential of Bioavailability of the drugs used to stimulate PTC-RT azithromycin was also confirmed in SMA mouse model When drug-stimulated PTC-RT is considered in the after intracerebroventricular administration of this drug context of its possible therapeutic application, bioavail- (Osman & Iii, 2017). The mechanism of macrolide action ability of the readthrough compounds, both at the level in PTC-RT differs from that of AAGs: according to stud- of the cell as that of the organism, is an important issue. ies on bacteria, macrolides are thought to influence the mechanism of protein synthesis termination, either by re- The cell membrane permeability to readthrough ducing binding of the RF1 or RF2 to the A-site of the ribo- compounds some, or by inhibiting the release of a peptide from the PTC-RT may be limited by the permeability of the cells to ribosome after binding of the release factors is completed readthrough compounds. Uptake of the drugs, especially of (Thompson et al., 2004). AAGs, has been explored for many years, but still no un- Identification of novel readthrough compounds among equivocal conclusions have been reached. Even less is antibiotics has inspired the search for readthrough- known about the mechanisms of the cell penetrance by the stimulating properties among other clinically approved AAG derivatives or non-AAG compounds with PTC-RT- drugs, which already have an existing safety profile. A stimulating potential. screen of 1600 clinically approved drugs identified an Nearly all mammalian cells take up AAGs; these drugs herbal anti-inflammatory drug, escin (Mutyam et al., 2016). may cross cellular membranes via endocytosis or non- Escin induced a significant PTC-RT, both in the in vitro endocytotic pathways (Steyger, 2005). One of the best tests and in the cell lines from a CF patient. It also stabi- studied AAGs uptake mechanism is through the multi- lized transcripts by inhibiting NMD, which resulted in a ligand endocytic receptor – megalin. Megalin has been significant increase of the functional protein level in the localized in the apical membrane of the epithelial cells of cells. No side effects have been reported so far, and escin the proximal renal tubules and in the hair cells of the inner seems to be a promising PTC-RT-stimulating compound. ear, and is related to the nephro- and ototoxic effects of However, further functional tests need to be performed be- AAGs (Tauris et al., 2009; Christensen et al., 2012). How- fore it can be used in clinical trials. ever, at low temperature, when endocytosis is significantly slowed down, the presence of Texas Red-tagged gentami- Readthrough induced by the use of suppressor tRNAs cin (GTTR) in the cytoplasm or the gentamicin-related There is an alternative for the stop suppression stimulated ROS production occurred within seconds (Myrdal et al., by readthrough compounds - the suppressor tRNAs. 2005;Hirose et al., 1997). This suggests that AAGs uptake Suppressor tRNAs are altered aminoacylated-tRNAs with involves different mechanisms than endocytosis alone. anticodons complementary to stop codons (Beier & A potential non-endocytic route of AAGs uptake is Grimm, 2001). They do not stimulate translational ma- through non-selective cation channels (NSCCs), such as chinery like others readthrough compounds, but they out- TRP channels (Marcotti et al., 2005; Lee et al., 2013; compete translation termination factors (like nc-tRNAs), Stepanyan et al., 2011). Low Ca2+ level in the extracellular and drive incorporation of an amino acid, being therefore fluid, hyperpolarization of the cell membrane or applica- readthrough agents per se. Suppressor tRNAs have been tion of NTSCC regulators (e.g. TRP agonists: resinifera- shown to restore the expression of functional proteins in toxin and anandamide) activate NSCCs opening, induce PTC-carrying human cell models of beta-thalassemia influx of cations along with the influx of positively charged (Temple et al., 1982), xeroderma pigmentosum (Panchal compounds. Factors inducing activation of NSCCs open- et al., 1999), Ulrich disease (Sako et al., 2006) and, more ing also favor AAGs uptake (Steyger, 2005). Similar to recently, cancer (Bordeira-Carriço et al., 2014). The PTC megalin, TRP channels are present in the membrane of suppression using suppressor tRNA was also shown in kidney epithelial cells and of inner ear sensory cells, thus mice, albeit with very low efficiency (Buvoli et al., 2000). may also be involved in oto- and nephrotoxic effects of In contrast to the readthrough compounds, which stimu- AAGs. Moreover, AAGs uptake may also be stimulated by late PTC recognition by nc-tRNAs (resulting in the loop diuretics, like bumetanide and furosemide. These Dabrowski et al. Molecular Medicine (2018) 24:25 Page 9 of 15 drugs, which hyperpolarize the cell membrane, were the most effective regimen of drug administration for a shown to increase the influx of a cationic AAG (GTTR) given disease remains challenging. through NSCCs, enhancing GTTR uptake by 60% (Wang et al., 2013). Other factors influencing therapeutic potential of drug-induced PTC-RT The efficiency of PTC-RT stimulation, the toxicity and the Effective dose of readthrough compounds bioavailability of stimulating compounds are important is- Regarding the modes of administration, the choice of sues to be addressed. As presented above, these problems doses and duration of therapy is important. The stimula- are potentially manageable, mainly through the search for tion of PTC-RT is a transient phenomenon, and the new, more efficient and less toxic compounds, for the therapeutic use of drugs requires a repetitive, prolonged, means to enhance cellular uptake of the drugs and for the usually life-long administration of the compounds. Most efficient ways to deliver these drug to proper tissues. studies in animal models indicate that the action of Other PTC-RT-related problems are even more elusive AAGs follows a peak-driven mode. For example, in the and finding ways to overcome them may be difficult, if mdx mice, maximal levels of intravenously injected genta- possible at all. micin in serum were observed 20 min after administra- The availability of a PTC-bearing transcript, which can tion; after 4 h, the drug level rapidly decreased. They also affect the efficiency of the nonsense suppression, is one showed that a constant pump-driven administration of such issue. In the cell, mRNA availability is strictly re- low-concentrations of gentamicin did not result in read- lated to the efficiency of the NMD process (Miller & through (Barton-Davis et al., 1999). It was confirmed in Pearce, 2014). When NMD is efficient, the level of mu- retinitis pigmentosa mice, that a better response to the tant mRNA is noticeably reduced and, even if potent treatment was obtained with repeated single injections of nonsense suppressing drugs are provided, the amount of a higher dose than with continuous delivery of small doses functional protein is very low (Kuzmiak & Maquat, of AAGs (Guerin et al., 2008). In the clinical trials with CF 2006). In the study using gentamicin as the PTC-RT- patients, different regimens of the administered gentami- stimulating drug in CF patients carrying the same cin were used (Clancy et al., 2001; Sermet-Gaudelus et al., W1282X nonsense mutation, the response to gentamicin 2007; Malik et al., 2010). In the Clancy’s study, the scheme was enhanced in the individuals, in whom the level of of intravenous administration of gentamicin (2.5 mg/kg mutant mRNA was high due to low NMD efficiency three times a day) was adjusted to achieve peak concentra- (Linde et al., 2007). These results suggest that the level tion in the serum from 8 to 10 μg/ml (Clancy et al., 2001). of PTC-bearing transcripts might be a limiting factor in In Sermet-Gaudelus study, gentamicin was adminis- the response to gentamicin treatment. Some reports sug- tered at 10 mg/kg once daily for 15 days; gentamicin gested that even a moderate induction of PTC-RT (e.g. peak concentration was achieved 30 min after the in- by using AAGs) may promote a stabilization of mutant fusion (Sermet-Gaudelus et al., 2007). In another long transcripts and counteract the NMD process (Bedwell et term study, gentamicin was administered intraven- al., 1997). This hypothesis has been later supported by ously (7.5 mg/kg) once or twice per week for several reports (Floquet et al., 2011; Salvatori et al., 6 months; gentamicin efficacy was limited and its tox- 2009; Bellais et al., 2009). icity precluded administration of higher doses of this Since the level of mutated transcript expression is of a drug (Malik et al., 2010). paramount importance for PTC-RT therapy, blocking NMD Effective dose of the best studied non-aminoglycoside by pharmaceutical agents has been considered as the solu- compound, PTC124 was shown on healthy volunteers tion to the problem, and a number of studies have explored and CF patients in phase I and II clinical trials. PTC124 this path (Usuki et al., 2004;Usuki et al., 2006;Durand etal., was characterized by rapid oral absorption and dose- 2007; Keeling et al., 2013;Gothamet al., 2016). proportional increases in pharmacokinetic parameters. Inhibitors of hSMG-1 kinase, which is an essential Peak concentration of this compound was achieved at protein for the regulation of NMD process, form one of approximately 2 h after dosing and its half-life ranged the groups of tested compounds (represented by wort- between 3 to 6 h (Hirawat et al., 2007). Similarly to mannin and caffeine) (Usuki et al., 2004). In the experi- AAGs, the best response for the treatment was observed ments performed in fibroblasts derived from patients after a multi-dose administration, however without toxic with Ullrich’s disease, caused by PTC in the collagen VI drug accumulation or metabolic auto-induction. The dos- gene, the use of these agents resulted in a correct assem- age used in clinical trials varied from 16 mg/kg/day in a bly of collagen VI, despite its truncated C-terminus and 14-day treatment period for the first studies to the 40 mg/ formation of a partially functional extracellular matrix kg/day in three divided doses for 48 weeks (Kerem et al., (Usuki et al., 2004; Usuki et al., 2006). Another molecule, 2008; Sermet-Gaudelus et al., 2010). Generally, choosing a small polycyclic indole derivative, NMDI-1, was shown Dabrowski et al. Molecular Medicine (2018) 24:25 Page 10 of 15 to block the NMD process by interfering with the induced readthrough can vary considerably for different interaction between key NMD factors, hSMG5 and PTC introducing mutations, such that only a subset of hUPF1. This led to the stabilization of the hyperpho- patients would be likely to profit from the therapy sphorylated form of hUPF1 and its sequestering in (Malik et al., 2010; Woodley et al., 2017;Finkelet al., 2013; cytoplasmic P-bodies (Durand et al., 2007). NMDI-1 Nagel-Wolfrum et al., 2016). This variability depends on was shown to be ~ 1500-fold more effective than caf- the stop codon itself and/or on the sequence context of feine in attenuating NMD; at the same time, it did PTC (Dabrowski et al., 2015). The mechanism of the in- not have any influence on cell growth and no signifi- duced stop codon suppression still remains not fully under- cant effect on protein synthesis (Keeling et al., 2013). stood, complicating the prediction of PTC-RT efficiency Short term studies in a mouse model of mucopolysacchar- based on the nucleotide context of the PTC mutation. To idosis (MPS I-H) have shown that a co-administration of some extent, this problem may be solved if the efficient pair NMDI-1 with gentamicin restored 50% more α-L-iduro- of the PTC and a specific readthrough compound is found. nidase activity than AAG administered alone (Keeling This however has to be done in an empirical way –to pre- et al., 2013). Chemical synthesis of NMDI-1 is compli- dict which patients are likely to take benefits from the cated and poorly efficient; however, there were made at- PTC-RT therapy, it is necessary to experimentally deter- tempts to synthesize a close NMDI-1 analog, VG1, with mine the PTC-RT level of each nonsense mutation in pre- similar NMD inhibiting potential and significantly less clinical settings (Floquet et al., 2012). Moreover, one has to complicated synthesis protocol (only 6 synthesis steps in- keep in mind that some of the PTCs, in specific sequence stead of 13 reported previously) (Keeling et al., 2013; context, may be resistant to the drug-induced PTC-RT Gotham et al., 2016). (Bukowy-Bieryllo et al., 2016). Another promising NMD inhibitor, amlexanox, is a Even if the full-length protein is produced after drug- long-used drug with anti-allergic and anti-inflammatory induced PTC-RT, the important question about the properties (Makino et al., 1987). Amlexanox has been identity of the amino acid incorporated during decoding approved by the FDA for the treatment of canker sores, of the premature stop still remains. If it is the same aphthous ulcers and asthma; it is also currently in a amino acid as that present in the normal protein, the phase II clinical trial for the treatment of diabetes question can be disregarded. However, if the amino acid (ClinicalTrials.gov; id: NCT01842282). The recent studies is different from the wild-type, it can lead to synthe- showed that amlexanox can inhibit NMD process and sizing a potentially non-functional protein, with differ- promote synthesis of PTC-bearing mRNAs (Gonzalez- ent biochemical properties or impaired stability. A Hilarion et al., 2012). The increase in the amount of number of studies showed that, even if the full-length pro- PTC-bearing mRNA does not affect general transla- teins were produced after administration of PTC-RT- tion or expression of potential NMD substrates (Gon- inducing drugs, their functional activity and subcellular zalez-Hilarion et al., 2012). In the cell lines from CF localization were often impaired (Schulz et al., 2002;Yao patients, amlexanox alone caused an increase of et al., 2009; Simon et al., 2010; Brumm et al., 2012). cAMP-dependent halide efflux, suggesting presence of Some preferences for the amino acid incorporated the functional CFTR protein, probably due to the during the PTC decoding can be predicted from the suppression of PTC in the mutated CFTR transcript. near-cognate anticodons (Dabrowski et al., 2015). How- The increase in CFTR activity after treatment with ever, this is not the unequivocal indicator. In vitro stud- amlexanox alone was ~ 3 times higher than after ies in rabbit reticulocyte lysates identified Trp, Arg, and treatment with other PTC-RT inducing compounds, Cys incorporated at the UGA codon (Feng et al., 1990). G418 or PTC124. Although the initial results for Studies addressing the insertion of amino acids at UAA these agents seem promising, long-term safety and and UAG codons in viral mRNAs revealed only the pres- effectivity studies are required before the clinical use ence of Gln (Feng et al., 1990; Yoshinaka et al., 1985). Re- of these compounds becomes possible. Efficacy of cent readthrough studies using mass-spectrometry shed amlexanox as a readthrough compound and a potent more light on this issue (Roy et al., 2015; Roy et al., 2016; NMD inhibitor was confirmed recently in cells de- Xue et al., 2017). Sequence analysis of full-length read- rived from patients with recessive dystrophic epider- through products in the yeast system (with reporter vec- molysis bullosa, where a significant increase in a full- tors containing different stop codons) has shown that Gln, length protein level translated from PTC-bearing tran- Lys, and Tyr incorporated at UAA and UAG, whereas scripts was observed (8–80%), compared do the wild- Trp, Arg, and Cys were inserted at UGA. This was irre- type protein level (Atanasova et al., 2017). The recov- spective of whether readthrough is endogenous, or in- ered protein was also functional and stable. duced. However, the frequency of individual amino acids The inherent problem associated with the PTC-RT- insertion differed for specific nonsense codons and read- based therapies is related to the fact that the level of the through compound; Tyr and Gln were incorporated with Dabrowski et al. Molecular Medicine (2018) 24:25 Page 11 of 15 Table 1 Amino acids inserted during readthrough Basal PTC-RT UGA UAA UAG Arg (CGA) Trp (UGG) Cys (UGU/C) Tyr (UAU/C) Gln (CAA) Tyr (UAU/C) Gln (CAG) Trp (UGG) (58.6 ± 2.3%) (41.4 ± 2.3%) - (67.5 ± 8.9%) (32.4 ± 8.9%) (73.1 + 15%) (25.2 ± 13.7%) (1.3 ± 1.4%) Stimulated PTC-RT PTC124 (30 uM) UGA UAA UAG Arg (CGA) Trp (UGG) Cys (UGU/C) Tyr (UAU/C) Gln (CAA) Tyr (UGG) Gln (CAG) Trp (UGG) (69.7 ± 11.3%) (28.8 ± 11.4%) (0.7 ± 0.7%) (57.9 ± 11.3%) (39.9 ± 11.7%) (43.9 ± 20.9%) (53.2 ± 20.3%) (1.8 ± 1.9%) Stimulated PTC-RT G418 (150 uM) UGA UAA UAG Arg (CGA) Trp (UGG) Cys (UGU/C) Tyr (UGG) Gln (CAA) Tyr (UAU/C) Gln (CAG) Lys (AAG) (64.5 ± 11.8%) (17.9 + 6.8%) (17.7 ± 8.0%) (47.9 ± 14.1%) (52 ± 14.2%) (10.8 ± 7.0%) (86.5 ± 8.3%) (2.0 ± 0.8%) The frequency of amino acids inserted during either basal or stimulated readthrough (PTC124 or G418) in the human cell line transfected with reported vectors containing different stop codons (based on Roy et al., 2016) comparable frequency at UAA (~ 45% and ~ 55%, respect- health or significant alleviation of the disease phenotype. ively), Gln dominated at UAG (~ 80%) and Trp at UGA The establishment of a truly efficient PTC-RT-based (~ 86%). Interestingly, for all these amino acids, the nc- therapy requires new, more potent compounds with tRNAs mispaired at position 1 or 3 of nonsense codons. less toxicity. It can be achieved by the modification of Slightly different results were obtained a human cell line available drugs or by searching for completely new transfected with reporter vectors containing different stop compounds with higher nonsense suppression poten- codons (Roy et al., 2016)(Table 1). The frequencies of the tial. Additional enhancement might be ensured by co- incorporated amino acids were different and mispairing at administration of readthrough compounds with some position 2 of nonsense codons was allowed. Nevertheless, enhancers, like inhibitors of NMD process or amino- the set of possible amino acids incorporated at PTCs was glycoside potentiators, such as CDX5–1. Despite the similar to that in the yeast system. The newest evidence promising results of the basic research and clinical indicates that, after stimulation with G418, identity of trials, plenty of work is still needed to better under- inserted amino acids depends on the nucleotide context of stand/predict ADME (absorption, distribution, metab- the same stop codon (UGA). This confirms, that mRNA olism and excretion) characteristics, pharmacokinetics sequence context plays a key role in near-cognate tRNA (PK) and pharmacodynamics (PD) of a drug, before selection during PTC-RT (Beznosková et al., 2016;Xue et these new drugs can be considered as a clinical thera- al., 2017). peutic option (Lee & Dougherty, 2012). An important issue in PTC-RT based therapies is the Abbreviations minimal amount of the full-length protein that should AAGs: Aminoglycoside antibiotics; eRF1/3: Eukaryotic release factor 1/3; be restored to achieve the functional and – hopefully – GTTR: Texas Red-tagged gentamicin; N c-tRNA: Near-cognate tRNA; therapeutic effects. In each disease, this level is different: NMD: Nonsense-mediated mRNA decay; NTC: Normal termination codon; PAA: Poly-L-aspartic acid; PABP: Poly(A)-binding proteins; PTC: Premature in CF, 30–35% of normal CFTR activity is sufficient; in termination codons; PTC-RT: Premature termination codons readthrough; DMD – 20-30% of the full-length dystrophin (but ac- ROS: Reactive oxygen species; RT: STOP codon readthrough cording to others, even 1% is sufficient); in MPS I-H, even 0.4–1% of the α-L-iduronidase level is enough to Funding alleviate disease symptoms (Hoffman et al., 1988; Keel- This work was supported by the National Science Centre, Poland; grants no. 2013/09/D/NZ4/01692 (ZBB); 2016/20/T/NZ4/00525 (MD) and ing et al., 2014). Unfortunately, the effective amount of 2016/23/N/NZ4/03228 (MD). the full-length protein has to be examined for each gene and disease separately. Authors’ contributions MD - a major contributor in writing of the manuscript, ZBB - editorial and substantive corrections, EZ - manuscript supervisor. All authors read and Conclusions approved the final manuscript. To date, more than 100 studies have shown that the stimulation of PTC-RT may result in the partial res- Ethics approval and consent to participate toration of the expression of deficient proteins that Not applicable underlie ~ 40 different genetic diseases (Lee & Dougherty, 2012; Nagel-Wolfrum et al., 2016). However, no study has Competing interests demonstrated a remarkable improvement of patients’ The authors declare that they have no competing interests. Dabrowski et al. Molecular Medicine (2018) 24:25 Page 12 of 15 Publisher’sNote Bukowy-Bieryllo Z, Dabrowski M, Witt M, Zietkiewicz E. 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Advances in therapeutic use of a drug-stimulated translational readthrough of premature termination codons

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Biomedicine; Molecular Medicine
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Abstract

Premature termination codons (PTCs) in the coding regions of mRNA lead to the incorrect termination of translation and generation of non-functional, truncated proteins. Translational readthrough of PTCs induced by pharmaceutical compounds is a promising way of restoring functional protein expression and reducing disease symptoms, without affecting the genome or transcriptome of the patient. While in some cases proven effective, the clinical use of readthrough-inducing compounds is still associated with many risks and difficulties. This review focuses on problems directly associated with compounds used to stimulate PTC readthrough, such as their interactions with the cell and organism, their toxicity and bioavailability (cell permeability; tissue deposition etc.). Various strategies designed to overcome these problems are presented. Keywords: Translational readthrough, Stop codon suppression, Nonsense suppression, Premature termination codon, Genetic diseases Background Main text Most of the reviews on the premature termination co- Premature termination codons dons (PTCs) readthrough studies focus on the efficiency An extensive meta-analysis study, based on the Human of the PTC suppression-stimulating agents, without ex- Gene Mutation Database, has revealed that 12% of all ploring the associated problems of these agents’ toxicity described gene lesions causing human inherited diseases and bioavailability. The present review, by providing a is caused by nonsense mutations (Mort et al., 2008). These different perspective, aims to contribute to understand- mutations, by changing an amino acid coding triplet into ing of the problems associated with using PTC read- a stop codon, introduce premature termination codons, through strategies. The risks and difficulties associated PTCs, into the protein-coding gene sequence. PTCs may with compounds used to stimulate PTC readthrough, also be caused by other types of mutations, such as frame- mostly related with their interaction with the cell and or- shifts (insertion or deletion other than multiple-of-three ganism, and concerning their toxicity and bioavailability base pairs) or mutations in the conserved splice-site se- (cell permeability; tissue deposition etc.) are discussed. In quences (leading to a defective intron removal from the addition, strategies to overcome these problems are dem- pre-mRNA) (Mort et al., 2008; Mendell & Dietz, 2001). onstrated. We also present outcomes of recently finished PTCs in mRNAs can even occur independently of any clinical trials on the readthrough compounds. The aspect changes in DNA, through the aberrant mRNA processing of PTC identity and its nucleotide context has been re- (alternative splicing) (Pan et al., 2006; Lewis et al., 2003). cently reviewed (Dabrowski et al., 2015)and is notexten- In any case, PTCs located in the coding regions of sively discussed here; other problems, related to the mRNA lead to the premature termination of translation molecular side of PTC-RT, deserve a separate review and and, as a consequence, to the production of truncated are only briefly mentioned. proteins (Mendell & Dietz, 2001). In most cases, this re- duces the amount of a full-length protein in a recessive- negative manner, whereby mutations of both alleles are * Correspondence: ewa.zietkiewicz@igcz.poznan.pl necessary to result in a deleterious phenotype (Khajavi et Institute of Human Genetics; Polish Academy of Sciences, Poznan, Poland © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dabrowski et al. Molecular Medicine (2018) 24:25 Page 2 of 15 al., 2006). However, PTCs can also exert dominant- Not all stop codons can be read through with the same negative effect (Inoue et al., 2004). Phenotypic manifest- efficacy. The level of “leakiness” of the stop codons can be ation usually involves loss-of-function effects, but there ranked as UGA > UAG > UAA (Manuvakhova et al., 2000). are examples of gain-of-function events (Manuvakhova et Also the sequence of nucleotides upstream and down- al., 2000). For example, in β-thalassemia, truncated pro- stream to the stop codon has the effect on the efficacy of teins derived from PTC-bearing transcripts are respon- the readthrough process. The majority of evidence indicates sible for generation of insoluble globin chains, which are that a nucleotide immediately following the termination toxic to the cell (Thein et al., 1990). codoninthe 3′ direction (position + 4, when the first nu- The synthesis of deleterious C-terminally truncated cleotide of the stop codon is marked as + 1), is in- proteins in eukaryotic organisms is reduced due to the volved in the interactions between mRNA and translational specialized mRNA surveillance mechanisms. One of them machinery (McCaughan et al., 1995;Jungreisetal., 2011; is the nonsense-mediated mRNA decay (NMD) pathway Loughran et al., 2014). This suggests that the actual (Lejeune, 2017a). This conserved quality-control process translation termination signal consists of a tetranu- detects and degrades transcripts containing abnormalities cleotide sequence, rather than only the stop codon itself such as: PTCs, introns downstream of the termination (Brown et al., 1990). Recently, it has been proposed, based codon, long 3′ untranslated regions (3′UTRs) or upstream on genetic interaction studies in yeast, that + 4 cytosine open reading frames (uORFs) (Mühlemann, 2008;Hogg & compromises the ability of eRF1 to recognize appropri- Goff, 2010;Miller & Pearce, 2014;Lejeune, 2017b). ately a stop codon, thus providing precedence for the im- Many efforts have been made to develop therapeutic portance of nucleotide positioning for eRF1-mediated strategies that would counteract negative effects of PTCs. termination efficiency (Beznosková et al., 2016). In addition, One of them relies on exploring the natural phenomenon the upstream sequence, immediately 5′ of the stop codon of termination codon suppression through the transla- (positions −1to − 3) also exerts an effect; this effect is con- tional readthrough mechanism. sideredtobemoresubtlethanthe downstream sequence context of the transcript. Studies suggest that even relatively distant nucleotides (positions + 5, + 6, + 9) may also influence Basal translational readthrough the translational readthrough (for a review, see Dabrowski et Effective termination of protein translation in eukaryotes al., 2015, RNA Biology) (Dabrowski et al., 2015). requires recognition of the stop codon, localized in the A- It has been estimated that basal readthrough of normal site of the ribosome, by a release factor, eRF1. The eRF1 termination codons (NTCs) occurs in 0.001 to 0.1% of further interacts with another release factor – eRF3, and total rounds of translation of a given transcript (Keeling with GTP. This ternary termination complex (eRF1-eRF3- et al., 2012). In the case of PTCs, basal readthrough GTP) interacts with the poly(A)-binding protein (PABP) levels are higher and can range from 0.01% to even 1% present at the 3′-UTR of mRNA; this leads to efficient hy- (Manuvakhova et al., 2000; Bonetti et al., 1995; Cassan & drolysis of GTP and cleavage of the peptidyl-tRNA bond Rousset, 2001). It is not known, why the basal read- (Bulygin et al., 2017). As a consequence, the newly synthe- through levels differ so much. It has been hypothesized, sized polypeptide chain is released from the ribosome. that efficient translation termination requires the close With the termination of translation being based on the contact between the release factors interacting with steric match between the stop codon and the eRF1, this NTC and PABPs (Lejeune, 2017a; Cosson et al., 2002; process is not 100% efficient. Stop codons in mRNA can Celik et al., 2015). Presence of a PTC in the transcript, be suppressed (recoded) through the natural mechanism usually more distant from the 3’UTR, could limit the of basal translational readthrough (Dabrowski et al., 2015; interaction between eRFs and PABPs, leading to the less Fearon et al., 1994). In this process, a near-cognate tRNA efficient action of eRF3 and the delayed release of the (nc-tRNA) outcompetes eRF1 at the A-site of the ribo- transcript from the ribosome (Ivanov et al., 2008). The some. Nc-tRNAs have anticodons, which are complemen- prolonged presence of the translational machinery at the tary to only two of the three positions of a nonsense PTC would increase the susceptibility to the basal transla- codon in mRNA. The recent studies indicate, that the tional readthrough of the PTC (PTC-RT) (Amrani et al., interaction between PTC and a nc-tRNA anticodon oc- 2004). curs by mispairing at either position 3 or 1 of the stop codon (Roy et al., 2015; Roy et al., 2016). If the nc-tRNA Drug-induced translational readthrough interacts with the PTC at the A-site, the amino acid A number of studies have demonstrated that certain transported by such nc-tRNA is incorporated into the low-molecular-mass drugs can stimulate recoding of a synthesized polypeptide chain, and translation – instead PTC by the translation machinery (Lee & Dougherty, of being terminated – continues until the next in-frame 2012). These findings have opened the way to new thera- stop codon (Fig. 1). peutic approaches to nonsense mutations in genes Dabrowski et al. Molecular Medicine (2018) 24:25 Page 3 of 15 Fig. 1 PTC-RT process. a Ribosome encounters premature termination codon (PTC); the site is recognized by the translation termination machinery and the polypeptide elongation is prematurely terminated. b After addition of readthrough compound, translational machinery decodes PTC (PTC is recognized by nc- tRNAs), and translation continues until the normal termination codon (NTC). It allows translating a full-length protein. The NMD surveillance mechanism may detect and degrade PTC-bearing transcripts. If the NMD process is inhibited, very low levels of the full-length protein can be present even in the absence of stimulating agents, as the result of the endogeneous suppression of PTC associated with genetic diseases. However, the efficient already in 1960s, first papers indicating readthrough use of this approach in the clinic is still hampered by a potential of AAGs in bacteria have been published number of problems (Fig. 2). (Davies et al., 1964;Andersonet al., 1965). In 1985, this Aminoglycoside antibiotics (AAGs) are among the process was demonstrated in mammalian cells (Burke & most studied drugs capable of inducing PTC-RT - Mogg, 1985). AAGs are oligosaccharides consisting of Fig. 2 Different problems associated with the drug-stimulated PTC-RT therapy. Upper part – problems associated with the PTC-RT- stimulating drugs. Lower part – problems associated with the biology of PTC-RT process Dabrowski et al. Molecular Medicine (2018) 24:25 Page 4 of 15 streptidine or 2-deoxystreptidine as the molecular core, effect is reversible after stopping AAGs administration with a variable number of sugar rings and ammonium (Mingeot-Leclercq et al., 1999). The negative influence groups (François et al., 2005). Commonly used to treat of AAGs on the renal function in patients can be also di- Gram-negative bacterial infections, they bind with the minished by the appropriate hydration therapy and ap- seven-nucleotide loop structure in the decoding center of plication of dialysis, when necessary (Lopez-Novoa et al., the bacterial ribosome and interfere with its function 2011). The ototoxicity induced by AAGs manifests as ir- (Fan-Minogue & Bedwell, 2008). This enables effective reversible, symmetric, bilateral sensorineural hearing misincorporation of the nc-tRNAs, resulting in extensive loss. Due to the very slow removal of AAGs from the translational misreading, followed by a complete inhib- inner ear fluids, the onset of hearing loss can occur days, ition of protein synthesis in bacteria. In eukaryotes, a or even weeks, after finishing the AAG administration small difference in the rRNA nucleotide sequence signifi- (Huth et al., 2011). Inside the hair cell, AAGs cause cantly lowers the efficiency of this interaction (Lynch & damage, either directly or indirectly, first by causing dis- Puglisi, 2001). Nonetheless, in the case of PTC, the impact array of stereocilia, and ultimately by activating apop- of AAGs on the translational apparatus is often sufficient tosis (Abi-Hachem et al., 2010). to reduce discrimination between cognate and nc-tRNAs, The selective toxicity observed in the inner ear and and to enhance translational readthrough. kidneys is associated with the cellular uptake of AAGs The therapeutic potential of AAGs as PTC readthrough- through megalin, an endocytic receptor localized in the ap- stimulating compounds has been tested in many models, ical membrane of epithelial cells present in the proximal from the in vitro transcription and translation system renal tubules and in the hair cells of the inner ear (Manuvakhova et al., 2000;Du et al., 2002; Keeling & (Moestrup et al., 1995). Themechanism,bywhich AAGs Bedwell, 2002;Lai et al., 2004), through dual reporters in induce apoptosis, both in the renal cells and in the hair cell lines (Howard et al., 2004; Bidou et al., 2004; Floquet et cells, is to date not well-defined. Some evidence indicates a al., 2011; Bukowy-Bieryllo et al., 2016), patient derived cells role of reactive oxygen species (ROS) (Huth et al., 2011). (Salvatori et al., 2009;Nakamuraet al., 2012; Cogan et al., Positively-charged AAGs are able to interact with various 2014;Dündaretal., 2017). The first in vivo demonstration negatively-charged cellular components such as phospho- of the therapeutic AAG properties has been in mdx mice, lipids, phospholipases, and metal ions. AAGs bounded to an animal model of Duchenne muscular dystrophy (DMD), phospholipids form complexes, which can aggregate in the where gentamicin treatment restored 10–20% of normal internal lysosomal membranes, causing phospholipidosis, a dystrophin levels in the skeletal muscles (Barton-Davis et condition often associated with nephro- and ototoxicity al., 1999). Since then, a large variety of AAGs has been (Couture et al., 1994; Reasor & Kacew, 2001). More import- tested for their PTC-RT-stimulating properties, both in ani- antly, the AAG-phospholipid interaction leads to the gener- mal models (Arakawa et al., 2003;Duet al., 2006;Guerinet ation of ROS, which affect membrane fluidity and al., 2008; Yukihara et al., 2011;Roweet al., 2011;Gunnet permeability, disrupt the activity of enzymes, ion chan- al., 2014) and clinical trials (Clancy et al., 2001; Wilschanski nels and receptors, and finally direct the cell to the et al., 2003; Politano et al., 2003; Sermet-Gaudelus et al., apoptotic pathway (Xie et al., 2011). Another explanation 2007; Malik et al., 2010). The latest pilot clinical study for the cell apoptosis related to AAGs administration sug- (ClinicalTrials.gov; id: NCT02698735) has shown positive gests involvement of the decoding site of the ribosome of results of gentamicin use in recessive dystrophic epidermo- mitochondrial ribosomes, which closely resembles the lysis bullosa patients with nonsense mutation in the gene A-site of bacterial ribosomes. The interaction of encoding type VII collagen. Topical or intradermal adminis- AAGs with the 12S rRNA at the A-site causes mis- tration of gentamicin increased the level of full-length colla- translation, then inhibition of protein synthesis, ultimately gen VII from 20 to 165% of the wild-type level, the leading to cell death (Hobbie et al., 2008). AAG-mediated expression of the protein persisted for 3 months. In oxidative damage of the mitochondrial enzyme – aconitase addition, a significant reduction of disease symptoms was can also cause an increase in superoxide overproduction observed (improved epidermal-dermal adherence, reduced from the free ferrous iron in mitochondria, that in turn blister formation, and enhanced wound closure) (Woodley increases the amount of hydroxyl radicals via the Fenton et al., 2017). reaction, and finally induces the cell apoptosis (Shulman et al., 2014). Toxicity of AAGs In spite of these adverse effects, one study has demon- Even though AAGs have been commonly used in clinics strated that AAGs used to stimulate PTC-RT can be for decades, they may exert strong oto- and nephrotoxic relatively safe. The six month-long gentamicin therapy effects on the organism. Accumulation of AAGs in the in DMD patients was completed safely, with no impair- renal epithelial cells leads to apoptosis and necrosis of ment of either renal or hearing function. The negative these cells (Lopez-Novoa et al., 2011). However, this effects of AAGs were avoided thanks to the informed Dabrowski et al. Molecular Medicine (2018) 24:25 Page 5 of 15 choice of the study cohort and a careful study design; all stimulating efficacy of AAGs. This strategy allowed signifi- subjects with the A1555G mutation in 12S rRNA gene cant lowering of the therapeuticdoseof AAGs, thus redu- of mtDNA, known to predispose to gentamicin-induced cing the AAG-related toxicity. For example, CDX5–1 ototoxicity, had been excluded from the treatment, while administered together with G418 not only increased the in the remaining patients a strict regimen of gentamicin level of PTC-RT up to 180-fold, but also accelerated syn- administration was obeyed (Malik et al., 2010). thesis of the full-length protein (the full-length product was Although the clinical use of AAGs for a prolonged detected 10 times faster). CDX5–1 action did not rely on time is associated with many risks, the benefits of a pos- stimulating PTC-RT; nor did it inhibit the NMD process sible PTC-RT-based therapy are indisputable. To allevi- (Baradaran-Heravi et al., 2016). The mechanism by which ate the problems resulting from AAGs toxicity, various this compound potentiates PTC-RT activity of AAGs, as attempts have been undertaken to mitigate the toxicity well as its potential interference with other cellular pro- or to select other compounds with PTC-RT-inducing cesses, remains to be elucidated. potential but without undesirable side effects of AAGs. Other approaches aiming to reduce AAG toxicity related to their positive electrostatic charge involve the use of Overcoming cytotoxic effects of AAGs liposome-encapsulated AAGs. Liposomes containing en- One approach tested for reducing the cytotoxicity of capsulated gentamicin, used to stimulate PTC-RT in DMD AAGs involves their co-administration with different mice, were more effective and caused 10-fold lower ototox- compounds, which are able to reduce interaction of icity than the traditionally administered gentamicin. Cre- AAGs with different cellular targets. This strategy was atinine concentrations (an indicator of renal malfunction) shown to be successful for tobramycin or gentamicin co- in mice treated with these compounds were normal, sug- administrated with a lipopeptic antibiotic, daptomycin gesting that encapsulation of AAG also reduced nephrotox- (Beauchamp et al., 1990; Thibault et al., 1994). Nega- icity (Yukihara et al., 2011; Schiffelers et al., 2001). tively charged daptomycin complexed AAGs through an electrostatic interaction, preventing them from binding Chemical modification of AAGs - AAG derivatives to phospholipids and other intracellular targets (Couture A different approach to the therapeutic use of PTC-RT et al., 1994). Co-administration of gentamicin with an- involves modification of the chemical structure of AAGs other compound, the negatively charged poly-L-aspartic with a proven nonsense suppressing ability, to obtain acid (PAA), was shown to protect rats against the develop- compounds with a reduced toxicity and retained or in- ment of kidney nephrotoxicity (Gilbert et al., 1989; creased therapeutic potential. An example of this ap- Ramsammy et al., 1989). It has been suggested that PAA, proach comes from the studies on artificially designed similarly to daptomycin, binds AAGs and prevents phos- paromomycin derivatives, where NB30 represents the pholipidosis, which would otherwise lead to nephrotox- first, and NB54 – the second generation. The modification icity (Kishore et al., 1990). In addition, PAA increased the of the paromomycin structure effectively reduced toxicity intracellular concentration of gentamicin, contributing to of the derivative compounds. For example, NB30 was 15- a higher level of PTC-RT. It also slowed-down elimination fold less toxic than gentamicin, while both compounds of AAGs from cytosol, extending the time period during showed nonsense suppression at the levels similar to those which readthrough stimulation occurred. Similar results induced by gentamicin and paromomycin (Nudelman et al., were obtained in the mouse model of cystic fibrosis 2009). The compounds were able to induce statistically sig- (Du et al., 2009a). nificant PTC-RT levels in cell lines from patients with dif- Another approach used to mitigate the toxic effects of ferent PTC-caused diseases, such as: Rett syndrome AAGs relies on their co-administration with antioxidants. (Brendel et al., 2011; Vecsler et al., 2011), cystic fibrosis D-methionine and melatonin were shown to reduce the (Rowe et al., 2011), mucopolysaccharidosis type I – Hurler harmful ROS formation caused by the interaction of syndrome (MPS I-H) (Wang et al., 2012; Kamei et al., AAGs with the cell components (Campbell et al., 2007; 2013) and Usher syndrome type 1 (Rebibo-Sabbah et Reiter et al., 2011). Melatonin turned out to be particularly al., 2007;Goldmannetal., 2012). effective; compared to a mixture of other antioxidants, it Modification of the structure of another AAG, G418, was 150 times more effective in reducing ototoxic effects resulted in the third generation of PTC-RT-stimulating of gentamicin or tobramycin. AAG derivatives, NB74 and NB84. These compounds Recently, a new approach to reduce AAG toxicity has were several-fold more active than the previous genera- been tested, whereby AAGs were co-administered with tions (Nudelman et al., 2010). Importantly, modifications small molecular mass molecules, derivatives of phthalimide also highly reduced the toxicity of the compounds. The (e.g. CDX5–1) (Baradaran-Heravi et al., 2016). When used capacity of NB74 and NB84 to stimulate PTC-RT was alone, these compounds did not induce PTC-RT, but analyzed in the mouse model of MPS I-H caused by a applied with AAGs, they efficiently potentiated PTC-RT PTC mutation in the α-L-iduronidase gene. The level of Dabrowski et al. Molecular Medicine (2018) 24:25 Page 6 of 15 PTC-RT induced by NB84 was higher than that caused known example of this group is a small molecule by gentamicin or by the first and second generation of drug 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]-benzoic AAG derivatives, and the LC50 values were 9–10 times acid, also known as PTC124, Ataluren or Translarna. Identi- higher than for G418 (Shulman et al., 2014). A long- fied during a high-throughput screen, PTC124 has been term (28-weeks) treatment of MPS I-H mice with NB84 selected as the most potent readthrough promoting drug resulted in an increased activity of α-L-iduronidase in from over 800,000 compounds screened (Welch et al., different tissues, including the brain, heart and bone. 2007). The level of the enzyme was sufficient to significantly re- PTC-RT stimulating efficiency of PTC124 was success- duce disease symptoms in the tissues, while no visible fully demonstrated in animals (Goldmann et al., 2012; toxicity was observed; moreover both the NB84 and Welch et al., 2007). It was also tested in different in vitro NB74 compounds were shown to be able to cross the and ex vivo models of PTC-mediated diseases, including blood-brain barrier (Gunn et al., 2014). The newest rep- cystic fibrosis (Du et al., 2008), Duchenne muscular resentative of the third generation of chemically modi- dystrophy (Yukihara et al., 2011; Finkel et al., 2013), fied AAGs, NB124, turned out to be even more Miyoshi myopathy (Wang et al., 2010), Usher syndrome promising. Its therapeutic index (comparison of the (Goldmann et al., 2012) and Batten disease (Sarkar et al., amount of a compound that causes the therapeutic ef- 2011). Phase I and phase II clinical trials have shown that fect to the amount that causes toxicity) was 10 times its adverse effects were mild or moderate and similar to better than that obtained for gentamicin and other non- the placebo-treated group; therefore, PTC124 has been modified AAGs. Moreover, a series of cell and animal deemed safe for therapeutic use in humans (Hirawat et al., based experiments using the CF models have shown that 2007; Kerem et al., 2008), and a number of clinical trials NB124 restored up to 10% of the wild-type CFTR func- have been undertaken. Unfortunately, despite many posi- tion (Xue et al., 2014). In the most recent study, using tive results from different experimental models, the effects reporter vectors in the transiently transfected cell lines, of PTC124 in patients remain inconclusive. NB124 was shown to efficiently decode common PTC In CF patients with PTC mutations in the CFTR gene, mutations in tumor suppressor genes (p53 and APC) in treatment with PTC124 slightly increased chloride chan- the murine cell line (NIH3T3), resulting in over 20% of nel activity, resulting in an improvement of clinical pa- the PTC-RT. In the HDQ-P1 cell line (derived from hu- rameters (Kerem et al., 2008); however, not all patients man primary breast carcinoma), the restoration of p53 responded positively to the drug. In a similar clinical and APC tumor suppressor proteins expression by trial with pediatric CF patients, an improvement in NB124 induced apoptosis in 38% of the cells, whereas chloride channel activity was observed again, and the untreated cells displayed no signs of apoptosis. In presence of CFTR protein in nasal epithelium was con- addition, similarly to G418, NB124 also affected the firmed by immunofluorescence; nevertheless, no signifi- NMD process, inhibiting the degradation of p53 tran- cant therapeutic improvement was proved (Sermet- scripts (Bidou et al., 2017). Gaudelus et al., 2010). In the phase III clinical trials, Another modified AAG is a pyranmycin (TC007), a some improvement in lung function and reduction in derivative of another aminoglycoside antibiotic, neomy- exacerbation frequency were reported after 48 weeks of cin (Chang et al., 2002). In the fibroblasts from patients PTC124 therapy, although statistical significance in the with spinal muscular atrophy (SMA) caused by the pre- overall patient population was not reached (Kerem et al., mature termination of the SMN protein, stimulation 2014). The main outcome of another phase III clinical with TC007 resulted in the 10-fold increase in the level study (ClinicalTrials.gov; id: NCT02139306, finished in of full-length SMN protein. In a mouse model of SMA, March 2017), has been announced via the PTC Thera- injection of TC007 into the central nervous system peutics press release, and at the time of writing this (brain ventricles) induced higher expression SMN pro- review, the full data were still unpublished. The tein, led to a longer survival of the motor neurons and announcement reported a failure to reach the expected was associated with a 27% increase in the lifespan of the primary and secondary endpoints. In light of these SMA mice (Mattis et al., 2009; Mattis et al., 2012). discouraging results, PTC Therapeutics has recently decided to discontinue their clinical development of PTC124 for CF and withdraw its application for Readthrough compounds marketing authorization in Europe (PR Newswire). Apart from modification of AAG structure, a number PTC124-induced suppression of PTC was also tested in of studies have searched for readthrough-inducing clinical trials with DMD patients. In the phase IIA trial, an compounds, chemically not related to AAG, but pos- increased expression of the full-length dystrophin was sessing the potential to suppress nonsense mutations in shown in patients’ biopsies after 28 days of oral adminis- mammalian cells (Lee & Dougherty, 2012). The best tration of PTC124; one third of the patients demonstrated Dabrowski et al. Molecular Medicine (2018) 24:25 Page 7 of 15 an increase in the post-treatment expression of dystrophin level of PTC-RT. In the mentioned studies, a full-lengh p53 appropriately localized in the sarcolemmal membrane of protein that was translated due to the readthrough was fully muscle cells (Finkel et al., 2013). In the phase IIb trial, the functional, both in the cell lines with PTC-containing alleles patients showed a slower decline in the walk test; no im- and in the tumor-bearing mice (Friesen et al., 2017). provement related to the higher dose of PTC124 was ob- Apart from PTC124, a number of other low-molecular- served, suggesting a bell-shaped dose-response curve mass compounds, capable of stimulating PTC-RT, have (Bushby et al., 2014). Unfortunately, phase III clinical study been identified to date. Screening of ~ 34,000 compounds completed in 2017, indicated that similarly to the CF studies, using a novel high-throughput screening assay identified the effects of PTC124 treatment did not differ significantly twelve most promising readthrough-stimulating com- from the placebo group (McDonald et al., 2017). pounds (Du et al., 2009b). The compounds displayed Despite the intensive studies, the exact mechanism of reduced toxicity in the mammalian cells and did not alter PTC124 action remains unknown. Computational model- the global protein expression patterns (Gatti, 2012). Two ing suggested that mRNAs containing PTC form stable leading first generation compounds, RTC13 and RTC14, complexes with PTC124, which may interfere with the were proven to suppress nonsense mutations in ataxia tel- recognition of PTC by eRF1 and suppress the termination angiectasia patients’ cell lines (A-T cells) and in myotube of translation; however, this interaction was shown to be cells from mdx mice (Du et al., 2009b;Kayaliet al., 2012). stable only for the UGA codon (Lentini et al., 2014). In More recent studies identified the further PTC-RT-stimu- cell lines transfected with different PTC-bearing con- lating compounds, namely GJ071 and GJ072, their an- structs, the PTC-RT-stimulating potential of PTC124 was alogs (RTC204 and RTC219), as well as non-related different for distinct stop codons; UGA suppression was compounds, BZ6 and BZ16 (Jung et al., 2011;Du et three times more effective than UAG, and 6 times more al., 2013). In the A-T cells, these compounds displayed effective than suppression of UAA (Welch et al., 2007). PTC-RT-stimulating efficiency comparable to RTC13, The latest evidence indicates, that PTC124 has a selectiv- RTC14 and PTC124, but with significantly lower toxicity. ity for the ribosomal A site and that it promotes insertion When tested in cell lines from patients with four different of nc-tRNAs at the PTC site (Roy et al., 2016). Tobra- lysosomal storage diseases, RTC13, RTC14, BZ6 and BZ16 mycin, a compound with an affinity for the ribosomal A showed ~ 1.5 fold increase in the amount of a relevant site, is a strong inhibitor of PTC124 (probably by competi- full-length mRNA compared to the previously used com- tion), what supports the expected interaction of PTC124 pounds (Gómez-Grau et al., 2015) and were able to sup- with the ribosome (Roy et al., 2016; Salian et al., 2012). press all three stop codons (Du et al., 2013). However, the Moreover, in a post-hoc analysis of the recent clinical trial, mechanism of their action remains unknown. it was observed that CF patients with nonsense mutations New readthrough compounds have also been found failed to show a significant response to administered among non-AAG antibiotics. A dipeptide antibiotic, PTC124, if they were concurrently treated with the in- negamycin, structurally not related to AAG, affects the haled tobramycin (Kerem et al., 2014). process of ribosomal decoding in a way similar to AAGs Clitocine [6-amino-5-nitro-4-(β-D-ribofuranosylamino) (Arakawa et al., 2003). However, in contrast to AAG, nega- pyrimidine] has been identified in the same high- mycin exhibits much lower cytotoxicity and no ototoxicity. throughput screen as PTC124 (Welch et al., 2000). This Negamycin was shown to promote PTC-RT in the dys- compound is an adenosine nucleoside analog origin- trophingenebothinvivointhe mousemodel of DMDand ally isolated from the mushroom Clitocybe inverse in the cultured mdx myotubes (Arakawa et al., 2003). It (Kubo et al., 1986). Clitocine is incorporated into mRNA was also tested in congenital muscular dystrophy: PTC-RT during transcription as an adenosine substitute; unlike induced by negamycin in the transfected NIH3T3 AAGs that act on the translation machinery, clitocine fa- cells was several-fold higher than that induced by AAGs cilitates decoding of the nonsense codon just by its pres- (Allamand et al., 2008). ence in the transcript (Friesen et al., 2017). Clitocine can Another group of compounds capable of inducing be incorporated instead of the adenine in each of the PTC-RT are macrolides, antibiotics often used to treat stop codons. The order of readthrough susceptibility Gram-positive bacterial infections. Induction of PTC- of the stop codons in the presence of clitocine is RT by tylosin, josamycin, and spiramycin was demon- UAA> > UGA > UAG. Surprisingly, the UAA codon is the strated in colorectal cancer models (mutations in the APC most leaky, probably because of the incorporation of clito- gene) (Zilberberg et al., 2010). The effect was also con- cine in both the second and third position of the codon firmed in a xenograft mouse model – treatment with (Friesen et al., 2017). Stop codons with the incorporated macrolides resulted in reduced tumor growth and led to a clitocine are poorly recognized by the eRF1 translation notable increase in the lifespan of the mice (Zilberberg et termination factor; this prevents efficient termination, en- al., 2010). These data were later extended by the flow hances nc-tRNA recruitment and therefore increases the cytometry-based reporter assay, in which erythromycin Dabrowski et al. Molecular Medicine (2018) 24:25 Page 8 of 15 and its derivative, azithromycin, were used (Caspi et al., incorporation of amino acid that does not necessarily cor- 2015). Macrolides were analyzed in cell lines derived from respond to the wild-type), suppressor tRNAs do not intro- patients with mutations in a number of genes; ATM duce missense mutations, and thus lead to the synthesis of in ataxia-telangiectasia (A-T), MeCP2 in RTT syndrome, the functional protein (Bordeira-Carriço et al., 2014). How- SMN in the SMA syndrome, APC in familial adenomatous ever, a clinical use of suppressor tRNAs faces many chal- polyposis (FAP). In all these cases, macrolides induced lenges, especially regarding the efficiency of delivery and in PTC-RT; additionally, azithromycin worked at a 100-fold vivo expression of these systems, with minimal toxicity to lower concentration than any other compound tested so the organism. far, enabling a significant reduction of the therapeutic dose (Caspi et al., 2015). PTC-RT-stimulating potential of Bioavailability of the drugs used to stimulate PTC-RT azithromycin was also confirmed in SMA mouse model When drug-stimulated PTC-RT is considered in the after intracerebroventricular administration of this drug context of its possible therapeutic application, bioavail- (Osman & Iii, 2017). The mechanism of macrolide action ability of the readthrough compounds, both at the level in PTC-RT differs from that of AAGs: according to stud- of the cell as that of the organism, is an important issue. ies on bacteria, macrolides are thought to influence the mechanism of protein synthesis termination, either by re- The cell membrane permeability to readthrough ducing binding of the RF1 or RF2 to the A-site of the ribo- compounds some, or by inhibiting the release of a peptide from the PTC-RT may be limited by the permeability of the cells to ribosome after binding of the release factors is completed readthrough compounds. Uptake of the drugs, especially of (Thompson et al., 2004). AAGs, has been explored for many years, but still no un- Identification of novel readthrough compounds among equivocal conclusions have been reached. Even less is antibiotics has inspired the search for readthrough- known about the mechanisms of the cell penetrance by the stimulating properties among other clinically approved AAG derivatives or non-AAG compounds with PTC-RT- drugs, which already have an existing safety profile. A stimulating potential. screen of 1600 clinically approved drugs identified an Nearly all mammalian cells take up AAGs; these drugs herbal anti-inflammatory drug, escin (Mutyam et al., 2016). may cross cellular membranes via endocytosis or non- Escin induced a significant PTC-RT, both in the in vitro endocytotic pathways (Steyger, 2005). One of the best tests and in the cell lines from a CF patient. It also stabi- studied AAGs uptake mechanism is through the multi- lized transcripts by inhibiting NMD, which resulted in a ligand endocytic receptor – megalin. Megalin has been significant increase of the functional protein level in the localized in the apical membrane of the epithelial cells of cells. No side effects have been reported so far, and escin the proximal renal tubules and in the hair cells of the inner seems to be a promising PTC-RT-stimulating compound. ear, and is related to the nephro- and ototoxic effects of However, further functional tests need to be performed be- AAGs (Tauris et al., 2009; Christensen et al., 2012). How- fore it can be used in clinical trials. ever, at low temperature, when endocytosis is significantly slowed down, the presence of Texas Red-tagged gentami- Readthrough induced by the use of suppressor tRNAs cin (GTTR) in the cytoplasm or the gentamicin-related There is an alternative for the stop suppression stimulated ROS production occurred within seconds (Myrdal et al., by readthrough compounds - the suppressor tRNAs. 2005;Hirose et al., 1997). This suggests that AAGs uptake Suppressor tRNAs are altered aminoacylated-tRNAs with involves different mechanisms than endocytosis alone. anticodons complementary to stop codons (Beier & A potential non-endocytic route of AAGs uptake is Grimm, 2001). They do not stimulate translational ma- through non-selective cation channels (NSCCs), such as chinery like others readthrough compounds, but they out- TRP channels (Marcotti et al., 2005; Lee et al., 2013; compete translation termination factors (like nc-tRNAs), Stepanyan et al., 2011). Low Ca2+ level in the extracellular and drive incorporation of an amino acid, being therefore fluid, hyperpolarization of the cell membrane or applica- readthrough agents per se. Suppressor tRNAs have been tion of NTSCC regulators (e.g. TRP agonists: resinifera- shown to restore the expression of functional proteins in toxin and anandamide) activate NSCCs opening, induce PTC-carrying human cell models of beta-thalassemia influx of cations along with the influx of positively charged (Temple et al., 1982), xeroderma pigmentosum (Panchal compounds. Factors inducing activation of NSCCs open- et al., 1999), Ulrich disease (Sako et al., 2006) and, more ing also favor AAGs uptake (Steyger, 2005). Similar to recently, cancer (Bordeira-Carriço et al., 2014). The PTC megalin, TRP channels are present in the membrane of suppression using suppressor tRNA was also shown in kidney epithelial cells and of inner ear sensory cells, thus mice, albeit with very low efficiency (Buvoli et al., 2000). may also be involved in oto- and nephrotoxic effects of In contrast to the readthrough compounds, which stimu- AAGs. Moreover, AAGs uptake may also be stimulated by late PTC recognition by nc-tRNAs (resulting in the loop diuretics, like bumetanide and furosemide. These Dabrowski et al. Molecular Medicine (2018) 24:25 Page 9 of 15 drugs, which hyperpolarize the cell membrane, were the most effective regimen of drug administration for a shown to increase the influx of a cationic AAG (GTTR) given disease remains challenging. through NSCCs, enhancing GTTR uptake by 60% (Wang et al., 2013). Other factors influencing therapeutic potential of drug-induced PTC-RT The efficiency of PTC-RT stimulation, the toxicity and the Effective dose of readthrough compounds bioavailability of stimulating compounds are important is- Regarding the modes of administration, the choice of sues to be addressed. As presented above, these problems doses and duration of therapy is important. The stimula- are potentially manageable, mainly through the search for tion of PTC-RT is a transient phenomenon, and the new, more efficient and less toxic compounds, for the therapeutic use of drugs requires a repetitive, prolonged, means to enhance cellular uptake of the drugs and for the usually life-long administration of the compounds. Most efficient ways to deliver these drug to proper tissues. studies in animal models indicate that the action of Other PTC-RT-related problems are even more elusive AAGs follows a peak-driven mode. For example, in the and finding ways to overcome them may be difficult, if mdx mice, maximal levels of intravenously injected genta- possible at all. micin in serum were observed 20 min after administra- The availability of a PTC-bearing transcript, which can tion; after 4 h, the drug level rapidly decreased. They also affect the efficiency of the nonsense suppression, is one showed that a constant pump-driven administration of such issue. In the cell, mRNA availability is strictly re- low-concentrations of gentamicin did not result in read- lated to the efficiency of the NMD process (Miller & through (Barton-Davis et al., 1999). It was confirmed in Pearce, 2014). When NMD is efficient, the level of mu- retinitis pigmentosa mice, that a better response to the tant mRNA is noticeably reduced and, even if potent treatment was obtained with repeated single injections of nonsense suppressing drugs are provided, the amount of a higher dose than with continuous delivery of small doses functional protein is very low (Kuzmiak & Maquat, of AAGs (Guerin et al., 2008). In the clinical trials with CF 2006). In the study using gentamicin as the PTC-RT- patients, different regimens of the administered gentami- stimulating drug in CF patients carrying the same cin were used (Clancy et al., 2001; Sermet-Gaudelus et al., W1282X nonsense mutation, the response to gentamicin 2007; Malik et al., 2010). In the Clancy’s study, the scheme was enhanced in the individuals, in whom the level of of intravenous administration of gentamicin (2.5 mg/kg mutant mRNA was high due to low NMD efficiency three times a day) was adjusted to achieve peak concentra- (Linde et al., 2007). These results suggest that the level tion in the serum from 8 to 10 μg/ml (Clancy et al., 2001). of PTC-bearing transcripts might be a limiting factor in In Sermet-Gaudelus study, gentamicin was adminis- the response to gentamicin treatment. Some reports sug- tered at 10 mg/kg once daily for 15 days; gentamicin gested that even a moderate induction of PTC-RT (e.g. peak concentration was achieved 30 min after the in- by using AAGs) may promote a stabilization of mutant fusion (Sermet-Gaudelus et al., 2007). In another long transcripts and counteract the NMD process (Bedwell et term study, gentamicin was administered intraven- al., 1997). This hypothesis has been later supported by ously (7.5 mg/kg) once or twice per week for several reports (Floquet et al., 2011; Salvatori et al., 6 months; gentamicin efficacy was limited and its tox- 2009; Bellais et al., 2009). icity precluded administration of higher doses of this Since the level of mutated transcript expression is of a drug (Malik et al., 2010). paramount importance for PTC-RT therapy, blocking NMD Effective dose of the best studied non-aminoglycoside by pharmaceutical agents has been considered as the solu- compound, PTC124 was shown on healthy volunteers tion to the problem, and a number of studies have explored and CF patients in phase I and II clinical trials. PTC124 this path (Usuki et al., 2004;Usuki et al., 2006;Durand etal., was characterized by rapid oral absorption and dose- 2007; Keeling et al., 2013;Gothamet al., 2016). proportional increases in pharmacokinetic parameters. Inhibitors of hSMG-1 kinase, which is an essential Peak concentration of this compound was achieved at protein for the regulation of NMD process, form one of approximately 2 h after dosing and its half-life ranged the groups of tested compounds (represented by wort- between 3 to 6 h (Hirawat et al., 2007). Similarly to mannin and caffeine) (Usuki et al., 2004). In the experi- AAGs, the best response for the treatment was observed ments performed in fibroblasts derived from patients after a multi-dose administration, however without toxic with Ullrich’s disease, caused by PTC in the collagen VI drug accumulation or metabolic auto-induction. The dos- gene, the use of these agents resulted in a correct assem- age used in clinical trials varied from 16 mg/kg/day in a bly of collagen VI, despite its truncated C-terminus and 14-day treatment period for the first studies to the 40 mg/ formation of a partially functional extracellular matrix kg/day in three divided doses for 48 weeks (Kerem et al., (Usuki et al., 2004; Usuki et al., 2006). Another molecule, 2008; Sermet-Gaudelus et al., 2010). Generally, choosing a small polycyclic indole derivative, NMDI-1, was shown Dabrowski et al. Molecular Medicine (2018) 24:25 Page 10 of 15 to block the NMD process by interfering with the induced readthrough can vary considerably for different interaction between key NMD factors, hSMG5 and PTC introducing mutations, such that only a subset of hUPF1. This led to the stabilization of the hyperpho- patients would be likely to profit from the therapy sphorylated form of hUPF1 and its sequestering in (Malik et al., 2010; Woodley et al., 2017;Finkelet al., 2013; cytoplasmic P-bodies (Durand et al., 2007). NMDI-1 Nagel-Wolfrum et al., 2016). This variability depends on was shown to be ~ 1500-fold more effective than caf- the stop codon itself and/or on the sequence context of feine in attenuating NMD; at the same time, it did PTC (Dabrowski et al., 2015). The mechanism of the in- not have any influence on cell growth and no signifi- duced stop codon suppression still remains not fully under- cant effect on protein synthesis (Keeling et al., 2013). stood, complicating the prediction of PTC-RT efficiency Short term studies in a mouse model of mucopolysacchar- based on the nucleotide context of the PTC mutation. To idosis (MPS I-H) have shown that a co-administration of some extent, this problem may be solved if the efficient pair NMDI-1 with gentamicin restored 50% more α-L-iduro- of the PTC and a specific readthrough compound is found. nidase activity than AAG administered alone (Keeling This however has to be done in an empirical way –to pre- et al., 2013). Chemical synthesis of NMDI-1 is compli- dict which patients are likely to take benefits from the cated and poorly efficient; however, there were made at- PTC-RT therapy, it is necessary to experimentally deter- tempts to synthesize a close NMDI-1 analog, VG1, with mine the PTC-RT level of each nonsense mutation in pre- similar NMD inhibiting potential and significantly less clinical settings (Floquet et al., 2012). Moreover, one has to complicated synthesis protocol (only 6 synthesis steps in- keep in mind that some of the PTCs, in specific sequence stead of 13 reported previously) (Keeling et al., 2013; context, may be resistant to the drug-induced PTC-RT Gotham et al., 2016). (Bukowy-Bieryllo et al., 2016). Another promising NMD inhibitor, amlexanox, is a Even if the full-length protein is produced after drug- long-used drug with anti-allergic and anti-inflammatory induced PTC-RT, the important question about the properties (Makino et al., 1987). Amlexanox has been identity of the amino acid incorporated during decoding approved by the FDA for the treatment of canker sores, of the premature stop still remains. If it is the same aphthous ulcers and asthma; it is also currently in a amino acid as that present in the normal protein, the phase II clinical trial for the treatment of diabetes question can be disregarded. However, if the amino acid (ClinicalTrials.gov; id: NCT01842282). The recent studies is different from the wild-type, it can lead to synthe- showed that amlexanox can inhibit NMD process and sizing a potentially non-functional protein, with differ- promote synthesis of PTC-bearing mRNAs (Gonzalez- ent biochemical properties or impaired stability. A Hilarion et al., 2012). The increase in the amount of number of studies showed that, even if the full-length pro- PTC-bearing mRNA does not affect general transla- teins were produced after administration of PTC-RT- tion or expression of potential NMD substrates (Gon- inducing drugs, their functional activity and subcellular zalez-Hilarion et al., 2012). In the cell lines from CF localization were often impaired (Schulz et al., 2002;Yao patients, amlexanox alone caused an increase of et al., 2009; Simon et al., 2010; Brumm et al., 2012). cAMP-dependent halide efflux, suggesting presence of Some preferences for the amino acid incorporated the functional CFTR protein, probably due to the during the PTC decoding can be predicted from the suppression of PTC in the mutated CFTR transcript. near-cognate anticodons (Dabrowski et al., 2015). How- The increase in CFTR activity after treatment with ever, this is not the unequivocal indicator. In vitro stud- amlexanox alone was ~ 3 times higher than after ies in rabbit reticulocyte lysates identified Trp, Arg, and treatment with other PTC-RT inducing compounds, Cys incorporated at the UGA codon (Feng et al., 1990). G418 or PTC124. Although the initial results for Studies addressing the insertion of amino acids at UAA these agents seem promising, long-term safety and and UAG codons in viral mRNAs revealed only the pres- effectivity studies are required before the clinical use ence of Gln (Feng et al., 1990; Yoshinaka et al., 1985). Re- of these compounds becomes possible. Efficacy of cent readthrough studies using mass-spectrometry shed amlexanox as a readthrough compound and a potent more light on this issue (Roy et al., 2015; Roy et al., 2016; NMD inhibitor was confirmed recently in cells de- Xue et al., 2017). Sequence analysis of full-length read- rived from patients with recessive dystrophic epider- through products in the yeast system (with reporter vec- molysis bullosa, where a significant increase in a full- tors containing different stop codons) has shown that Gln, length protein level translated from PTC-bearing tran- Lys, and Tyr incorporated at UAA and UAG, whereas scripts was observed (8–80%), compared do the wild- Trp, Arg, and Cys were inserted at UGA. This was irre- type protein level (Atanasova et al., 2017). The recov- spective of whether readthrough is endogenous, or in- ered protein was also functional and stable. duced. However, the frequency of individual amino acids The inherent problem associated with the PTC-RT- insertion differed for specific nonsense codons and read- based therapies is related to the fact that the level of the through compound; Tyr and Gln were incorporated with Dabrowski et al. Molecular Medicine (2018) 24:25 Page 11 of 15 Table 1 Amino acids inserted during readthrough Basal PTC-RT UGA UAA UAG Arg (CGA) Trp (UGG) Cys (UGU/C) Tyr (UAU/C) Gln (CAA) Tyr (UAU/C) Gln (CAG) Trp (UGG) (58.6 ± 2.3%) (41.4 ± 2.3%) - (67.5 ± 8.9%) (32.4 ± 8.9%) (73.1 + 15%) (25.2 ± 13.7%) (1.3 ± 1.4%) Stimulated PTC-RT PTC124 (30 uM) UGA UAA UAG Arg (CGA) Trp (UGG) Cys (UGU/C) Tyr (UAU/C) Gln (CAA) Tyr (UGG) Gln (CAG) Trp (UGG) (69.7 ± 11.3%) (28.8 ± 11.4%) (0.7 ± 0.7%) (57.9 ± 11.3%) (39.9 ± 11.7%) (43.9 ± 20.9%) (53.2 ± 20.3%) (1.8 ± 1.9%) Stimulated PTC-RT G418 (150 uM) UGA UAA UAG Arg (CGA) Trp (UGG) Cys (UGU/C) Tyr (UGG) Gln (CAA) Tyr (UAU/C) Gln (CAG) Lys (AAG) (64.5 ± 11.8%) (17.9 + 6.8%) (17.7 ± 8.0%) (47.9 ± 14.1%) (52 ± 14.2%) (10.8 ± 7.0%) (86.5 ± 8.3%) (2.0 ± 0.8%) The frequency of amino acids inserted during either basal or stimulated readthrough (PTC124 or G418) in the human cell line transfected with reported vectors containing different stop codons (based on Roy et al., 2016) comparable frequency at UAA (~ 45% and ~ 55%, respect- health or significant alleviation of the disease phenotype. ively), Gln dominated at UAG (~ 80%) and Trp at UGA The establishment of a truly efficient PTC-RT-based (~ 86%). Interestingly, for all these amino acids, the nc- therapy requires new, more potent compounds with tRNAs mispaired at position 1 or 3 of nonsense codons. less toxicity. It can be achieved by the modification of Slightly different results were obtained a human cell line available drugs or by searching for completely new transfected with reporter vectors containing different stop compounds with higher nonsense suppression poten- codons (Roy et al., 2016)(Table 1). The frequencies of the tial. Additional enhancement might be ensured by co- incorporated amino acids were different and mispairing at administration of readthrough compounds with some position 2 of nonsense codons was allowed. Nevertheless, enhancers, like inhibitors of NMD process or amino- the set of possible amino acids incorporated at PTCs was glycoside potentiators, such as CDX5–1. Despite the similar to that in the yeast system. The newest evidence promising results of the basic research and clinical indicates that, after stimulation with G418, identity of trials, plenty of work is still needed to better under- inserted amino acids depends on the nucleotide context of stand/predict ADME (absorption, distribution, metab- the same stop codon (UGA). This confirms, that mRNA olism and excretion) characteristics, pharmacokinetics sequence context plays a key role in near-cognate tRNA (PK) and pharmacodynamics (PD) of a drug, before selection during PTC-RT (Beznosková et al., 2016;Xue et these new drugs can be considered as a clinical thera- al., 2017). peutic option (Lee & Dougherty, 2012). An important issue in PTC-RT based therapies is the Abbreviations minimal amount of the full-length protein that should AAGs: Aminoglycoside antibiotics; eRF1/3: Eukaryotic release factor 1/3; be restored to achieve the functional and – hopefully – GTTR: Texas Red-tagged gentamicin; N c-tRNA: Near-cognate tRNA; therapeutic effects. In each disease, this level is different: NMD: Nonsense-mediated mRNA decay; NTC: Normal termination codon; PAA: Poly-L-aspartic acid; PABP: Poly(A)-binding proteins; PTC: Premature in CF, 30–35% of normal CFTR activity is sufficient; in termination codons; PTC-RT: Premature termination codons readthrough; DMD – 20-30% of the full-length dystrophin (but ac- ROS: Reactive oxygen species; RT: STOP codon readthrough cording to others, even 1% is sufficient); in MPS I-H, even 0.4–1% of the α-L-iduronidase level is enough to Funding alleviate disease symptoms (Hoffman et al., 1988; Keel- This work was supported by the National Science Centre, Poland; grants no. 2013/09/D/NZ4/01692 (ZBB); 2016/20/T/NZ4/00525 (MD) and ing et al., 2014). Unfortunately, the effective amount of 2016/23/N/NZ4/03228 (MD). the full-length protein has to be examined for each gene and disease separately. Authors’ contributions MD - a major contributor in writing of the manuscript, ZBB - editorial and substantive corrections, EZ - manuscript supervisor. All authors read and Conclusions approved the final manuscript. To date, more than 100 studies have shown that the stimulation of PTC-RT may result in the partial res- Ethics approval and consent to participate toration of the expression of deficient proteins that Not applicable underlie ~ 40 different genetic diseases (Lee & Dougherty, 2012; Nagel-Wolfrum et al., 2016). However, no study has Competing interests demonstrated a remarkable improvement of patients’ The authors declare that they have no competing interests. Dabrowski et al. Molecular Medicine (2018) 24:25 Page 12 of 15 Publisher’sNote Bukowy-Bieryllo Z, Dabrowski M, Witt M, Zietkiewicz E. 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Molecular MedicineSpringer Journals

Published: May 29, 2018

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