Hurler syndrome, a type of Mucopolysaccharidosis type I, is an inherited disorder caused by the accumulation of glycosaminoglycans (GAG) due to a deficiency in lysosomal α-L-iduronidase (IDUA), resulting in multiorgan dysfunction. In many patients with Hurler syndrome, IDUA proteins are not produced due to nonsense mutations in their genes; therefore, readthrough-inducing compounds, such as gentamycin, are expected to restore IDUA proteins by skipping the premature termination codon. In the present study, we synthesized a series of chromenopyridine derivatives to identify novel readthrough-inducing compounds. The readthrough-inducing activities of synthesized compounds were examined by measuring cellular IDUA activities and GAG concentrations in Hurler syndrome patient-derived cells. Compounds with a difluorophenyl group at the 2-position of chromenopyridine, a cyclobutyl group at the 3-position, and a basic side chain or basic fused ring exhibited excellent readthrough-inducing activities. KY-640, a chromenopyridine derivative with a tetrahydroisoquinoline sub-structure, increased the cellular IDUA activities of patient-derived cells by 3.2-fold at 0.3 µM and significantly reduced GAG concentrations, and also significantly increased enzyme activity in mouse models, suggesting its therapeutic potential in patients with Hurler syndrome.

The readthrough mechanism, which skips the premature termination codon and restores the biosynthesis of the defective enzyme, is an emerging therapeutic tactic for nonsense mutation-related diseases, such as Hurler syndrome, a type of mucopolysaccharidosis. In the present study, novel triaryl derivatives were synthesized and their readthrough-inducing activities were evaluated by a luciferase reporter assay with a partial α-L-iduronidase (IDUA) DNA sequence containing the Q70X nonsense mutation found in Hurler syndrome and by measuring the enzyme activity of IDUA knockout cells transfected with the mutant IDUA gene. KY-516, a representative compound in which the meta position carboxyl group of the left ring of the clinically used ataluren was converted to the para position sulfamoylamino group, the central ring to triazole, and the right ring to cyanobenzene, exhibited the most potent readthrough-inducing activity in the Q70X/luciferase reporter assay. In Q70X mutant IDUA transgenic cells, KY-516 significantly increased enzyme activity at 0.1 µM. After the oral administration of KY-516 (10 mg/kg), the highest plasma concentration of KY-516 was above 5 µM in rats. These results indicate that KY-516, a novel triaryl derivative, exhibits potent readthrough-inducing activity and has potential as a therapeutic agent for Hurler syndrome.

Suppressing translation termination at premature termination codons (PTCs), termed readthrough, is a potential therapy for genetic diseases caused by nonsense mutations. Ataluren is a compound that has shown promise for clinical use as a readthrough agent. However, some reports suggest that ataluren is ineffective at suppressing PTCs. To further evaluate the effectiveness of ataluren as a readthrough agent, we examined its ability to suppress PTCs in a variety of previously untested models. Using NanoLuc readthrough reporters expressed in two different cell types, we found that ataluren stimulated a significant level of readthrough. We also explored the ability of ataluren to suppress a nonsense mutation associated with Mucopolysaccharidosis I-Hurler (MPS I-H), a genetic disease that is caused by a deficiency of α-L-iduronidase that leads to lysosomal accumulation of glycosaminoglycans (GAGs). Using mouse embryonic fibroblasts (MEFs) derived from Idua-W402X mice, we found that ataluren partially rescued α-L-iduronidase function and significantly reduced GAG accumulation relative to controls. Two-week oral administration of ataluren to Idua-W402X mice led to significant GAG reductions in most tissues compared to controls. Together, these data reveal important details concerning the efficiency of ataluren as a readthrough agent and the mechanisms that govern its ability to suppress PTCs.
Ataluren promotes readthrough of PTCs in a wide variety of contexts. Ataluren reduces glycosaminoglyan storage in MPS I-H cell and mouse models. Ataluren has a bell-shaped dose-response curve and a narrow effective range.

Nonsense suppression therapy is a therapeutic approach aimed at treating genetic diseases caused by in-frame premature termination codons (PTCs; also commonly known as nonsense mutations). This approach utilizes compounds that suppress translation termination at PTCs, which allows translation to continue and partial levels of deficient protein function to be restored. We hypothesize that suppression therapy can attenuate the lysosomal storage disease mucopolysaccharidosis type I-Hurler (MPS I-H), the severe form of α-L-iduronidase deficiency. α-L-iduronidase participates in glycosaminoglycan (GAG) catabolism and its insufficiency causes progressive GAG accumulation and onset of the MPS I-H phenotype, which consists of multiple somatic and neurological defects. 60-80% of MPS I-H patients carry a nonsense mutation in the IDUA gene. We previously showed that 2-week treatment with the designer aminoglycoside NB84 restored enough α-L-iduronidase function via PTC suppression to reduce tissue GAG accumulation in the Idua(tm1Kmke) MPS I-H mouse model, which carries a PTC homologous to the human IDUA-W402X nonsense mutation. Here we report that long-term NB84 administration maintains α-L-iduronidase activity and GAG reduction in Idua(tm1Kmke) mice throughout a 28-week treatment period. An examination of more complex MPS I-H phenotypes in Idua(tm1Kmke) mice following 28-week NB84 treatment revealed significant moderation of the disease in multiple tissues, including the brain, heart and bone, that are resistant to current MPS I-H therapies. This study represents the first demonstration that long-term nonsense suppression therapy can moderate progression of a genetic disease.