Generating SM(a)RTer Compounds for Translation Termination Suppression in A-T and Other Genetic Disorders

Reading of the genetic code through nonsense mutations to restore protein function is a concept that dates back 50 years, with the discovery that streptomycin was capable of correcting defective genotypes.¹ In the intervening years, suppression of premature termination codons (PTCs) by aminoglycoside antibiotics and other compounds has been employed to express full-length normal protein in a range of disease states. This approach has met with success in restoring at least partial function, and several of these compounds are at an advanced stage in clinical trials. Important targets include genes encoding the cystic fibrosis (CF) fibrosis transmembrane conductance regulator (CFTR) and dystrophin, which is defective in the X-linked neuromuscular disorder Duchenne muscular dystrophy. However, this approach can be applied to many less common genetic disorders caused by nonsense mutations that lead to premature truncation of a protein.² In this issue, Du et al. build on previous efforts using high-throughput screening of small-molecule libraries to identify novel readthrough compounds (RTCs) that suppress all three stop codons (TGA, TAG, and TAA) in ataxia telangiectasia mutated (ATM), which is defective in the human genetic disorder ataxia telangiectasia (A-T).³ The authors refer to these as small-molecular readthrough (SMRT) compounds and reveal that they have structures that are quite different from those of two previously described compounds (RTC 13 and RTC 14)⁴ but are comparable or superior to them in efficiency in their readthrough capacity for ATM. Although successful treatment of this rare genetic disorder is unlikely to have a major impact on community health, the authors point to a “one drug fits all” model that would have broad translational potential for several other disorders.

A-T, an autosomal recessive ataxia with an incidence of 1/300,000 among live births, is recorded worldwide. It is characterized by immunodeficiency, predisposition to lymphoid tumors, progressive cerebellar atrophy, radiosensitivity, and defective cell cycle control.⁵ ATM is a serine protein kinase that is recruited and activated by DNA double-strand breaks that arise during maturation of T and B cells or as a consequence of DNA damage.⁶ ATM can also be activated in response to alterations in chromatin structure and by oxidative stress.⁷ The most debilitating aspect of A-T is progressive neurodegeneration that results in loss of mobility, uncoordinated movement, aspiration, and infection, as well as defects in swallowing that affect nutritional status. The neurodegeneration is characterized by loss of Purkinje cells and, to a lesser extent, granule cells located in the cerebellum. How the genetic lesion in ATM leads to this cell loss remains unknown. Although A-T is a multisystem disease, halting or slowing the progress of the neurodegeneration would greatly improve patients' quality of life.

One of the earliest reported uses of aminoglycoside antibiotics to suppress premature stop mutations was to restore CFTR expression and the cyclic adenosine monophosphate–activated chloride currents.⁸ It is believed that the aminoglycosides distorted the structure of the ribosome RNA complex, leading to a misreading of the termination codon, causing the ribosome to “skip” over the stop sequence and to continue with the normal elongation and production of the CFTR protein. Aminoglycosides and nonantibiotic termination suppressors such as PCT124 (Ataluren; PTC Therapeutics, South Plainfield, NJ) have been tested in several clinical trials of suppression therapy for Duchenne muscular dystrophy and CF, although only a fraction of patients showed a therapeutic benefit.² PTC124 has been reported to selectively induce ribosomal readthrough of premature but not normal termination codons, although this mechanism has been called into question by recent studies.⁹ The long-term effects of PTC124 in patients with nonsense mutation dystrophinopathy are being investigated in a multicenter phase III trial (ClinicalTrials.gov NCT01247207).²

Aminoglycosides bind to the decoding site in the eukaryotic 18S ribosomal RNA (rRNA) with weaker affinity, as compared with prokaryotic 16S rRNA, to suppress translation termination.¹⁰ The efficacy of a readthrough compound is influenced by several factors. Among these is the extent of activation of nonsense-mediated messenger RNA (mRNA) decay (NMD), which degrades mRNAs containing PTCs. Rather than leading to shortened polypeptide products in vivo, PTCs tend to lead to activation of NMD to degrade the affected mRNA. However, a previous study showed no evidence of appreciable operation by NMD on ATM mRNA.¹¹ Other factors include sequence context of the stop codon, nonfunctional amino acid incorporation at PTCs, and, in the present context, the capacity to cross the blood–brain barrier. As indicated above, although several regions of the brain are affected in A-T, major targets for readthrough to address the progressive neurodegeneration are the Purkinje and associated cells in the cerebellum.¹²

In a previous study, Lai et al. employed aminoglycosides to induce the synthesis of full-length ATM protein in A-T cells with nonsense mutations.⁴ They further showed that the full-length protein was functional in that it corrected the radiosensitivity, cell cycle defects, and defective ATM-dependent signaling in the A-T cells.⁴ Workers from the same group subsequently developed a high-throughput protein transcription/translation enzyme-linked immunosorbent assay and screened approximately 34,000 compounds for readthrough of PTCs in ATM mRNA.¹³ In follow-up experiments on 12 low-molecular-weight nonaminoglycoside compounds, they identified two (RTC 13, RTC 14) that induced low levels of full-length functional ATM (Figure 1). Interestingly, both compounds also showed readthrough activity in Mdx mouse myotube harboring a nonsense mutation. However, because the efficiency of readthrough was <15% of normal for both compounds, their ability to provide clinical benefit was questionable. In addition, although the readthrough efficiency of RTC 14 was superior to that of RTC 13, it was more toxic. The authors therefore focused their attempts to generate more effective analogs on RTC 13. Analogs containing variations on both aryl and the thiazolidinone rings exhibited good readthrough activity (Figure 1a, bottom).¹⁴ In addition, they prepared compounds with a pyrimidinedione replacing the thiazolidinone ring; these were also excellent in PTC readthrough (Figure 1). The rationale for change in design was based in part on the supposition that RTC 13 functioned in a manner similar to aminoglycosides by interacting with the ribosome, but no mechanistic data were provided to support this hypothesis.

Figure 1.

Chemical structures of readthrough compounds identified by high-throughput screening. (a) Structures of RTC13 and RTC14 and derivatives of RTC13. Adapted from ref. 14. (b) Structures of SMRT compounds, GJ071 and GJ072, adapted fromf ref. 3.

The new work reported in this issue represents high-throughput screening of approximately 36,000 additional compounds in an attempt to identify novel RTCs.³ Parent compounds were identified that were capable of readthrough in an in vitro transcription and translation system driven by a plasmid containing the coding sequence for amino acids 403–1936 of ATM with a TGA mutation (c.5,623C→T; R1875X). These compounds were effective with all three types of stop codons and produced a functional product that responded to DNA damage and could phosphorylate a downstream substrate, and they also reduced the radiosensitivity of an A-T cell line. The structures of these compounds (Figure 1, GJ071 and GJ072) differ from those identified in the earlier screen, suggesting that the latter did not meet the expectations of the investigators. Analogs of one of these (GJ072) were similarly proficient for readthrough and had low cLogP (predictive of efficient absorption or permeation potential in vivo) values and good bioavailability. An additional water-soluble derivative was also described. The use of derivatives, of course, prompts the question as to whether the compounds also function at the ribosomal site for suppression by a different mechanism.

Du et al. have made a marathon effort in screening approximately 70,000 compounds to identify four parent candidate drugs and derivatives thereof, with very different structures, that suppress all three stop codons to produce functional ATM protein. Toxicity does not seem to be a problem in cultured cells, and at least one candidate is water-soluble, which augurs well for biodistribution and bioavailability. The authors refer to preliminary data suggesting that many of their SMRT drugs will reach the cerebellum but provide no details. Clearly the next step is to generate a knockin mouse model with a PTC. However, several Atm mutant (knockout and knockin) mouse models have been generated, and the consensus is that none of these displays the neurodegenerative phenotype seen in A-T patients. Nevertheless, there is evidence for behavioral defects in Atm mutant mice that could form the basis for a correction assay with potential for extrapolation to humans.¹⁵

In addition, although the emphasis has been on correcting the neurodegeneration, A-T is a multisystem disease associated with immunodeficiency, which if reduced could also contribute to patients' quality of life. Carriers of the A-T gene are also predisposed to breast cancer, and other cancers have been reported in males.¹⁶ Although the level of functional ATM may not be an issue, removing the potential for formation of an interfering truncated ATM in a heterozygote carrying a PTC could reduce the risk of cancer.¹⁶ Indeed. Du et al.³ propose the use of SMRT compounds as a long-term prophylactic therapy in cancer-prone individuals carrying highly penetrant nonsense mutations such as BRCA1, BRCA2, and CHEK2.

In considering the potential of SMRT compounds, it is important to reflect on the history of development of the aminoglycosides and nonglycoside compounds such as PTC124. The first application of these in CF cells dates back almost 20 years, and, although considerable progress has been made, it benefits only a fraction of patients with more common diseases.² Toxicity to the kidney and ear, which affects up to 25% of patients, has also been reported for aminoglycosides. Similar challenges are in place for SMRT compounds, including definition of their mechanism of action. Another key issue is whether they are readily distributed into the brain and, if so, whether there is any toxicity to that or other organs. It also remains unclear how successful animal model studies are likely to be as a precursor for use in humans and ultimately what percentage of patients carry nonsense mutations. Despite these many unknowns, Du et al. are nevertheless to be commended for their efforts to reach first base.