Evaluation of Readthrough Efficiency of Negamycin Derivatives against Nonsense Mutations in Muscular Dystrophy Genes

Abstract

Recently, “readthrough compounds” have attracted attention as a promising approach to treat human hereditary diseases caused by nonsense mutations. These compounds enable ribosomes to bypass a premature termination codon (PTC) introduced into mRNA by a nonsense mutation, thereby restoring the expression of full-length functional proteins. We performed a structure–activity relationship study focusing on (+)-negamycin, a known readthrough compound, and identified potent derivatives, TCP-304 and TCP-306, featuring a cyclopropane moiety. In this study, we investigated how the nature of PTCs and their surrounding nucleotide sequences influence the readthrough activity of these negamycin derivatives, using nonsense mutation sequences derived from Duchenne muscular dystrophy and congenital muscular dystrophy genes. In cell-based reporter assay systems, TCP-306 exhibited potent readthrough efficiency against several nonsense mutation sequences containing the TGA-A. Moreover, its sequence preference differed from that of the aminoglycoside G418, a representative readthrough compound that preferentially induces readthrough at TGA-C sequences, suggesting that TCP-306 may serve as an alternative therapeutic option for muscular dystrophies associated with TGA-A nonsense mutations. Overall, this study provides valuable insights for the development of readthrough drugs for hereditary diseases such as muscular dystrophy caused by nonsense mutations.

INTRODUCTION

Nonsense mutations account for approximately 11% of the genetic mutations causing human hereditary diseases.¹⁾ In such diseases, a premature termination codon (PTC) is introduced into the gene coding sequence, resulting in the termination of translation and expression of truncated proteins without intact function. Among human hereditary diseases, Duchenne muscular dystrophy (DMD) is a common and serious X-linked recessive disorder, occurring in approximately every 1 in 5000 newborn boys.²⁾ At least 10% of DMD cases are caused by nonsense mutations in the dystrophin gene (DMD).³⁾ Dystrophin protein plays an essential role in maintaining the structural integrity of muscle cells, and its deficiency causes dysfunction and necrosis of these cells.⁴⁾ Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous neuromuscular disorder that occurs at birth (congenital) or an earlier stage during infancy. Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is the most common form of CMD, accounting for 30–40% of cases and is caused by mutations in the LAMA2 gene encoding the α-chain of laminin-211. Severe MDC1A is typically associated with truncating mutations or deletions that result in a complete loss of functional proteins. Notably, 20–30% of mutations identified in the LAMA2 gene are nonsense mutations, which lead to the expression of truncated, nonfunctional proteins.⁵⁾

Recently, small molecules that induce the translational readthrough, termed “readthrough compounds,” have attracted attention as potential treatments for hereditary diseases caused by nonsense mutations. These compounds enable ribosomes to bypass PTCs, thereby restoring the expression of full-length functional proteins.⁶⁾ Representative readthrough compounds are shown in Fig. 1. Among them, aminoglycoside antibiotics such as gentamicin (a mixture of gentamicin C1, C1a, C2, and C2a) and Geneticin (G418) have been reported to show readthrough activity⁷⁾ (Fig. 1). It is known that these antibiotics inhibit normal protein biosynthesis and promote reading mistakes during translation by interacting with the A site of ribosomal RNA.⁸⁾ Barton-Davis et al. reported that gentamicin restores the expression of full-length dystrophin protein in mdx mice, an animal model of DMD with a TAA-type nonsense mutation in the dystrophin gene.⁹⁾ However, long-term treatment with aminoglycosides is limited by serious side effects such as nephrotoxicity¹⁰⁾ and ototoxicity,¹¹⁾ as well as the emergence of drug-resistant bacteria.

Fig. 1. Structure of Representative Readthrough Compounds

Recently, several non-aminoglycoside compounds have been developed as alternative readthrough agents. Translana (ataluren), an oxadiazole derivative identified by chemical library screening, was first reported by Sweeney and colleagues as a readthrough compound.¹²⁾ Ataluren was conditionally approved by the European Medicines Agency (EMA) for the treatment of DMD. Nevertheless, in 2024, the EMA reaffirmed its position not to renew this authorization, citing the lack of robust and conclusive evidence substantiating its therapeutic efficacy.¹³⁾ 2,6-Diaminopurine was identified by Trzaska et al. from an extract of the mushroom Lepista inversa in 2020.¹⁴⁾ This compound exhibits potent readthrough activity against TGA and TAA nonsense mutations by inhibiting putative ribosomal RNA methyltransferase 1 (FTSJ1), an enzyme involved in the modification of tRNA^Trp. Recently, Bidou et al. performed high-throughput screening of chemical libraries to identify new readthrough compounds and discovered that 1-(4-methylquinazolin-2-yl)guanidine (TLN468) promotes a high level of PTC readthrough without inducing the readthrough of a normal stop codon.¹⁵⁾

(+)-Negamycin (Fig. 1), a dipeptide-like antibiotic containing a hydrazide structure, was originally isolated from Streptomyces purpeofuscus in 1970 by Umezawa and colleagues and showed a strong antimicrobial activity against Gram-negative bacteria.¹⁶⁾ Arakawa et al. later reported that negamycin not only induced the expression of dystrophin protein in skeletal and cardiac muscles of mdx mice¹⁷⁾ but also had a 50% lethal dose (LD₅₀) 10 times lower than that of gentamicin,¹⁶⁾ suggesting a more favorable safety profile. These findings indicated that negamycin has the potential to be a preferable alternative to aminoglycosides as a readthrough compound for the development of treatments for hereditary diseases caused by nonsense mutations. To develop negamycin derivatives with potent readthrough activity, we performed a structure–activity relationship (SAR) study of negamycin. In a previous study, we reported the discovery of 3-epi-deoxynegamycin (TCP-107) and leucyl-3-epi-deoxynegamycin (TCP-126) as natural readthrough compounds¹⁸⁾ (Fig. 1). These negamycin analogues were originally isolated in 1977 from Streptomyces goshikiensis and displayed almost no antimicrobial activity compared to natural negamycin¹⁹⁾; their other biological activities remained unknown for over 40 years. Remarkably, chemically synthesized derivatives of these compounds exhibited a higher readthrough activity than negamycin itself.¹⁸⁾ These findings enabled us to clearly distinguish the 2 inherent activities of negamycin: antibacterial activity in prokaryotes and readthrough activity in eukaryotes. Encouraged by these findings, an additional SAR study was performed using TCP-107 as a lead scaffold. We found that TCP-112, a derivative with its main chain shortened by 1 carbon, exhibited approximately twice the readthrough activity of TCP-107.²⁰⁾ Moreover, by introducing l-α-aminoundecanoic acid to the 3-amino position of TCP-112, we identified a derivative, TCP-1109, that exhibited a 10-fold more potent activity than negamycin.²¹⁾ Conformational restriction with a cyclopropane scaffold has been reported to be an effective strategy for approximating the target-bound active conformation of flexible molecules.²²⁾ More recently, we developed conformationally restricted cyclopropane-based derivatives of TCP-107 and found that TCP-304 and TCP-306 possessed strong readthrough activity against TGA and TAG mutations.²³⁾ These findings indicated that the introduction of a cyclopropane moiety into the negamycin scaffold may contribute to conformational rigidity, which could be associated with enhanced readthrough activity. Although the precise mechanism underlying the readthrough phenomenon remains unknown, several general rules governing the readthrough efficiency have been reported: i) readthrough does not occur equally across the 3 types of PTCs; among them, UAA serves as the strongest translation terminator among the 3 types of PTCs^24–27); ii) nucleotide sequences both upstream and downstream of the PTC significantly influence readthrough efficiency.^24,26–31) In particular, the nucleotide immediately downstream of the PTC (referred to as the “+ 4 position”) plays a critical role in modulating readthrough activity.^24,26,27) These findings pertain to basal readthrough occurring in normal cells. However, the readthrough efficiency induced by compounds cannot be reliably predicted solely from the PTC and its surrounding nucleotide context, owing to the complexity of translational regulation. It is therefore important to clarify how each PTC and its neighboring nucleotide sequence affects compound-induced readthrough. Understanding sequence-specific readthrough efficiencies may provide valuable guidance for the rational design of effective readthrough therapies. Numerous studies have investigated the sequence dependence of aminoglycoside-induced readthrough, particularly with G418 and gentamicin.^{24,26–28,30)} For example, Manuvakhova et al. reported that the readthrough efficiency of G418 follows the order UGA > UAG > UAA and that sequences containing cytosine (C) at the +4 position of UGA exhibit particularly high readthrough efficiency.²⁴⁾ Floquet et al. similarly showed, through statistical analysis of 66 disease-derived stop codon sequences, that gentamicin-induced readthrough efficiency strongly depends on the +4 nucleotide.²⁷⁾ In contrast, the readthrough efficiency of negamycin derivatives against each PTC and the influence of the nucleotide sequences surrounding the PTC on the readthrough have not been systematically studied.

To address this issue, we analyzed the readthrough efficiencies of TCP-304 and TCP-306 against the 3 types of PTCs using a dual-luciferase reporter plasmid. These derivatives were selected for this study because they are optimized derivatives exhibiting substantially higher readthrough activity than the parent compound, negamycin, as demonstrated in our previous SAR study.²³⁾ We then examined the effect of the nucleotide immediately downstream of the PTC (+4 position; PTC-X mutation) on the readthrough efficiency of these negamycin derivatives. Finally, we evaluated the readthrough efficiency against various nonsense sequences derived from DMD and MDC1A and found that these compounds were effective across a broad range of sequences, including TGA mutations, although the efficiency varied depending on the nucleotide context surrounding the PTC.

MATERIALS AND METHODS

Chemicals

Geneticin (G418) solution was purchased from Roche Diagnostics (Basel, Switzerland). TCP-304 and TCP-306 were synthesized according to previously reported procedures.²³⁾

Cell Line and Cell Culture

COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Nichirei Biosciences Inc., Tokyo, Japan). Cells were incubated at 37°C in a humidified 5% CO₂ atmosphere.

Plasmid Constructs

Previously reported plasmids²³⁾ were used to evaluate the readthrough efficiency (%) against a UGA-type PTC derived from the nonsense mutation observed in the DMD model mdx mice (Table 1). Previously reported plasmids²¹⁾ were used to evaluate the readthrough efficiency against DMD R3381X (TGA) and LAMA2 C1546X (TGA). Additional constructs were generated according to a previously reported method.²¹⁾ Briefly, the linear vector of the pGL4.75 backbone was generated by inverse PCR using 1 set of primers (Supplementary Table S1). After agarose gel electrophoresis, the linear vector was purified using the Qiagen Gel Extraction Kit (Qiagen North American Holdings Inc., Germantown, MD, U.S.A.). The firefly luciferase gene containing peripheral nucleotide sequences with PTCs, which were selected from several nonsense mutations in DMD and MDC1A instead of the original stop codon of firefly luciferase, was amplified from pGL4.38 using KOD-Plus- (TOYOBO Inc., Tokyo, Japan) and the corresponding primer set (Supplementary Table S1). After purification using a Qiagen Gel Extraction Kit, the desired luciferase fragment was cloned into the linearized pGL4.75 vector using an In-Fusion HD Cloning Kit (TaKaRa Bio USA, Inc., San Jose, CA, U.S.A.) according to the manufacturer’s protocol. To measure the readthrough efficiency of each mutation, a control plasmid was constructed in which the PTC was replaced with the corresponding sense codon in the wild-type gene. All plasmids were purified using a Qiagen Midiprep Kit (Qiagen North American Holdings Inc.). The sequences of all the constructs were confirmed by Sanger sequencing (Eurofins Japan, Tokyo, Japan).

Table 1. Readthrough Efficiency (%) against the 3 Types of PTCs

Compound	Readthrough efficiency (%)a)
	-UGA-	-UAG-	-UAA-
Basal (without compound)	0.32 ± 0.02	0.25 ± 0.01	0.22 ± 0.02
G418	1.71 ± 0.58b)	1.08 ± 0.07	0.57 ± 0.24
TCP-304	1.18 ± 0.27	0.35 ± 0.04	0.17 ± 0.02
TCP-306	2.88 ± 0.28b)	0.45 ± 0.07	0.16 ± 0.01

a) Cell-based readthrough efficiency (%) was calculated using the equation shown in Fig. 2. b) The readthrough efficiencies of G418 and TCP-306 against UGA were obtained from our recent study.²³⁾ All compounds were evaluated at a concentration of 200 µM. Each value represents the mean ± S.D. (n = 3).

Dual-Luciferase Reporter Assay

According to our previous report,²¹⁾ the readthrough efficiencies of the compounds were evaluated. The procedure is briefly described as follows. COS-7 cells were seeded in 96-well plates at 8000 cells/well. After incubation at 37°C for 12 h, cells were transfected with the dual-luciferase plasmid using the FuGENE^® HD transfection reagent (Promega, Madison, WI, U.S.A.) according to the manufacturer’s protocol. The culture medium was removed from the wells, and fresh medium containing the test compounds at a concentration of 200 µM was added to the wells. Medium without the compounds was also added as a control. The cells were incubated at 37°C for 48 h, collected, and lysed with a passive lysis buffer (Promega). Firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s protocol. The readthrough efficiency (%) of each compound at 200 µM or control without the compound was calculated using the equation shown in Fig. 2.

Fig. 2. Equation for the Calculation of Readthrough Efficiency (%)

Statistical Analysis

All data were expressed as means ± standard deviation (S.D.). Data were analyzed by one-way ANOVA followed by Tukey’s test for multiple comparisons. p < 0.05 was considered statistically significant. All analyses were performed using JMP Pro version 16.2.0 (SAS Institute Inc., Cary, NC, U.S.A.).

RESULTS

Effect of Stop Codon Type on Readthrough Efficiency

We used a dual-luciferase reporter plasmid containing 27 nucleotide bases derived from mdx mice [DMD mdx (Exon 23)] centered on different stop codons for the quantification of cell-based readthrough efficiency (%) against 3 types of PTCs, UGA, UAG, and UAA, in the absence or presence of compounds (Fig. 3). The wild-type plasmid, containing a sense codon (CAA) instead of a PTC, was used as a control for quantification of the readthrough. As shown in Table 1, the basal readthrough efficiency (without compound) increased in the order UGA > UAG > UAA, although it was consistently low, ranging from 0.22% (UAA) to 0.32% (UGA). This trend was consistent with the efficiency of translation termination previously described for the 3 stop codons (UAA > UAG > UGA),^24–27) supporting the validity of our assay system. A similar preference among the 3 PTCs was observed in the presence of G418, TCP-304, and TCP-306. These derivatives exhibited the highest selectivity for UGA among the 3 types of PTCs (G418: 1.71%²³⁾; TCP-304: 1.18%; and TCP-306: 2.88%).²³⁾ Notably, TCP-306 demonstrated a higher readthrough efficiency for the UGA codon than for G418, although its efficiency for the UAG codon was approximately half that of G418 (0.45 vs. 1.08%). The efficiencies of TCP-304 and TCP-306 against the UAA codon (0.17 and 0.16%, respectively) were slightly lower than at the basal level (0.22%).

Fig. 3. Schematic Representation of the Dual-Luciferase Reporter Plasmid Containing Different Premature Termination Codons

Influence of the Nucleotide Sequence at the +4 Position of the PTC

Several previous studies have indicated that the nucleotide sequence at the +4 position affects the efficiency of PTC-readthrough.^24,26,27) To investigate this effect for negamycin derivatives, we evaluated their readthrough efficiency (%) by focusing on the nucleotide at this position. It has been reported that the UGA-C sequence promotes the highest level of readthrough, both in the absence of compounds (basal level) and in the presence of aminoglycosides.^24,26) However, the mechanism by which the nucleotide sequence at the +4 position affects the readthrough efficiency of negamycin derivatives has not been clarified. To address this, we constructed a series of plasmids based on the TGA mutant plasmid, DMD mdx (Exon 23), as shown in Table 1, in which the fourth base downstream of the stop codon was substituted from A to T, C, or G (Fig. 4A). These constructs were then used to quantify the readthrough efficiency using the same cell-based dual-luciferase reporter assay. Figure 4B summarizes the readthrough efficiencies in the absence of compound (basal) and in the presence of G418, TCP-304, or TCP-306, against sequences containing different nucleotide bases at the +4 position. Among the 4 sequences, the highest readthrough efficiency after treatment with G418 was observed for the UGA-C sequence (3.29%; Fig. 4B-ii). This result was consistent with a previous report describing that the hierarchy of aminoglycoside-induced readthrough levels for +4 nucleotides was C > U, A > G when the PTC was UGA.²⁶⁾ In contrast, as shown in Fig. 4B-iv, the negamycin derivative TCP-306 showed the highest efficiency for the mutant sequences containing A (2.88%)²³⁾ at the +4 position, followed by those for C (1.92%), U (1.18%), and G (0.66%). Although the readthrough efficiencies induced by TCP-304 for each sequence were lower than those induced by TCP-306, the efficiency followed the order A, C > U > G (Fig. 4B-iii).

Fig. 4. Readthrough Efficiency for TGA-Type Mutations with Different Nucleotides at the +4 Position

(A) Schematic structure of the dual-luciferase reporter plasmid containing a TGA-type mutation with different nucleotide bases at the +4 position downstream of the premature termination codon. B) Cell-based readthrough efficiency (%) for different nucleotide bases at the +4 position of TGA. a) The data for the UGA-A sequence correspond to the UGA (left column) data shown in Table 1. Compounds were evaluated at a concentration of 200 µM. Error bars represent SD (n = 3). ii) **p = 0.0010 vs. TGA-A, **p = 0.0014 vs. TGA-T, ***p = 0.0002 vs. TGA-G; iii) *p = 0.0121 vs. TGA-T, **p = 0.0013 vs. TGA-G, ***p = 0.0007 vs. TGA-T, ***p = 0.0001 vs. TGA-G; iv) ***p = 0.0002 vs. TGA-T, ** p = 0.0014 vs. TGA-C, ***p = 0.0001 vs. TGA-G, ***p = 0.0003 vs. TGA-T, ***p = 0.0009 vs. TGA-G.

Readthrough Efficiency against PTC-Containing Sequences Derived from Muscular Dystrophy

We next evaluated the readthrough efficiency of negamycin derivatives using a panel of 12 plasmids, each containing a PTC and its surrounding nucleotide sequences inserted between the same dual-reporter genes. Seven DMD-derived and 5 MDC1A-derived mutations were selected as representative sequences (Fig. 5A). The basal and compound-induced readthrough efficiencies for these 12 sequences are shown in Fig. 5B. In the absence of compound (Fig. 5B-i), basal readthrough efficiency ranged from 0.16% [for DMD R3381X (TGA-A)] to 1.41% [for DMD E931X (TAG-C)]. In the presence of 200 µM G418 (Fig. 5B-ii), efficiencies ranged from 0.66% [for DMD Q60X (TAA-A)] to 7.57% [for DMD S319X (TGA-C)]. The negamycin derivative TCP-304 induced moderate readthrough values, ranging from 0.27% [for LAMA2 R3085X (TGA-G)] to 2.77% [for LAMA2 C1546X (TGA-A)] (Fig. 5B-iii). TCP-306 displayed a broader distribution of readthrough efficiency, ranging from 0.25% [for LAMA2 Q1240X (TAA-T)] to 8.13% [for LAMA2 C1546X (TGA-A)] (Fig. 5B-iv). Overall, the responses to G418 and the negamycin derivatives followed a similar trend across mutations, with efficiencies in the order UGA > UAG > UAA (Table 1). The most responsive sequence in the TGA mutant after treatment with G418 was DMD S319X (7.57%), which had a C at the +4 position, consistent with the observations shown in Fig. 4B-ii. In contrast, the negamycin derivatives TCP-304 and TCP-306 induced lower efficiencies against the same mutation (1.24 and 2.94%, respectively). Notably, TCP-306 induced higher readthrough than G418 for 2 TGA-A sequences, LAMA2 C1546X and DMD R3381X (8.13 and 5.43%, respectively). Among the TAG mutation sequences, G418, the 2 negamycin derivatives, and the control (basal) showed a relatively higher readthrough efficiency against the DMD E931X (TAG-C) sequence in the range of 1.41–3.29%. The order was G418 > TCP-306 > basal, and TCP-304. The efficiencies of the negamycin derivatives for all the TAA mutants were lower than those for G418.

Fig. 5. Readthrough Efficiency of PTC-Containing Sequences Derived from Muscular Dystrophy Genes

(A) Schematic representation of dual-luciferase reporter plasmid containing premature termination codon (PTC) sequences derived from DMD and MDC1A (caused by mutation in LAMA2 gene). The codon below each PTC sequence indicates the corresponding wild-type sequence. (B) Cell-based readthrough efficiency (%) against several nonsense mutation sequences derived from DMD and MDC1A. Compounds were evaluated at a concentration of 200 µM. Error bars represent S.D. (n = 3).

DISCUSSION

In this study, we investigated the readthrough effect of negamycin derivatives by quantifying the percentage of readthrough efficiency in mammalian cells against the 3 PTCs and the nucleotide sequences surrounding the PTCs. First, using the PTC-containing dual-luciferase reporter plasmid derived from DMD mdx (Exon 23), we demonstrated that the aminoglycoside G418 and negamycin derivatives TCP-304 and TCP-306 promoted readthrough in the order of UGA > UAG > UAA (Table 1). Notably, TCP-306 exhibited higher selectivity for UGA-type PTC than G418 (2.88 vs. 1.71%).

Secondly, we evaluated the readthrough efficiency of the compounds against TGA nonsense mutation sequences containing different nucleotides immediately downstream of the PTC (+4 position). This position was considered critical for the readthrough effect in a previous study.^24,26,27) Other groups have reported that the presence of a cytosine (C) at the +4 position of each PTC was the most effective in enhancing the readthrough levels induced by G418 and gentamicin.^24,26) As shown in Fig. 4B, our study revealed a similar trend, with G418 displaying the highest readthrough efficiency in the UGA-C context. In contrast, TCP-306 exhibited the highest efficiency in the UGA-A context. Similar results were observed in PTCs and the surrounding nucleotide sequences derived from inherited nonsense mutations in DMD and MDC1A (Fig. 5B). Among the 12 sequences tested, G418 exhibited the highest readthrough efficiency against DMD S319X, followed by LAMA2 R1549X; both mutations contain the UGA-C context. This tendency was consistent with gentamicin-induced readthrough efficiency.³⁰⁾ Moreover, Floquet et al. reported that the consensus sequence “T-PTC-C” is closely associated with gentamicin-induced readthrough. Based on these findings, our results suggest that DMD S319X, which contains the “T-PTC-C” consensus sequence, would respond more strongly to G418 treatment than LAMA2 R1549X, which carries the “C-PTC-C” sequence (Fig. 5). Interestingly, TCP-306 showed strong readthrough efficiency against LAMA2 R1549X, whereas only moderate efficiency was observed against DMD S319X. This result is consistent with a report by Allamand et al.⁵⁾ They conducted a dual-reporter assay to evaluate the readthrough activity against several nonsense mutations in the DMD and LAMA2 genes and found that the DMD S319X mutation was more sensitive to gentamicin than to negamycin.⁵⁾

We observed potent readthrough efficiency of TCP-306 against TGA-A sequences such as DMD R3381X and LAMA2 C1546X. These efficiencies were approximately 2.4- and 3.5-fold higher, respectively, compared to those observed with G418. It has been reported that aminoglycosides induce translational misreading through interactions with the ribosomal RNA A site.⁸⁾ Although the precise mechanism of TCP-306 remains unclear, one possibility is that it exerts the readthrough activity by interacting with the ribosome in a manner different from that of aminoglycosides. However, despite containing a TGA-A sequence, the TCP-306-induced readthrough efficiency was reduced for DMD R1967X. These results indicate that the readthrough efficiency depends not only on the +4 nucleotide but also on downstream nucleotides, including the +5 position²⁸⁾ and other surrounding nucleotides near the PTC. Moreover, the readthrough level of TCP-306 against the LAMA2 R3085X (TGA-G) sequence was lower than those against the TGA-A and TGA-C sequences, strongly suggesting that the readthrough efficiency followed the order: UGA-A > UGA-C > UGA-U > UGA-G, as shown in Fig. 4B-iv. This tendency indicates that readthrough induced by negamycin derivatives may be influenced, at least in part, by intrinsic sequence-context effects on translation termination described for UGA stop codons in eukaryotic systems.³¹⁾ Despite sharing the same cyclopropane configuration and the RNA sequence preference, TCP-306 exhibits higher readthrough efficiency than TCP-304, indicating that structural differences primarily modulate the magnitude of readthrough efficiency.

In the DMD E931X (TAG-C) sequence, the groups treated with readthrough compounds showed relatively high readthrough efficiency. This sequence has previously been reported to respond relatively well to gentamicin treatment.^27,30) In our study, G418 demonstrated strong readthrough efficiency comparable to that observed in TGA mutants. TCP-306 also exhibited moderate readthrough efficiency against DMD E931X (TAG-C), although it did not reach the levels achieved by G418. Notably, the basal readthrough efficiency for this sequence was the highest among all mutated sequences tested. This finding suggests that readthrough at this PTC is more likely to occur even in the absence of readthrough compounds, despite the PTC being a UAG.

CONCLUSION

To elucidate the readthrough effect of negamycin derivatives, we examined the readthrough efficiency of TCP-304 and TCP-306 using 3 mutant plasmids and a wild-type plasmid containing the sequence derived from mdx mice, each consisting of a dual-reporter gene. We found that the hierarchy of TCP-306-induced readthrough efficiency was UGA > UAG > UAA. In addition, we found that the A residue immediately downstream of the TGA mutation (TGA-A) was the most efficient sequence, increasing the negamycin-induced readthrough. Furthermore, we evaluated the readthrough efficiency of the negamycin derivatives using 12 plasmids containing various nonsense mutation-containing sequences derived from muscular dystrophies, DMD, and MDC1A. TCP-306 showed potent readthrough efficiency in TGA-A sequences such as LAMA2 C1546X and DMD R3381X. We demonstrated that the negamycin derivative showed a sequence-specific readthrough efficiency distinct from that of the aminoglycosides, indicating a different spectrum of applications. TCP-306 may be an effective alternative to aminoglycosides against muscular dystrophies associated with TGA-A nonsense mutations. Therefore, this study provides valuable insights for the future development of a negamycin-based readthrough drug for hereditary diseases caused by nonsense mutations.

Acknowledgments

The authors thank Ms. Ayaka Ema, Tokyo University of Pharmacy and Life Sciences, for the technical assistance. This work was supported by a Nippon Shinyaku Research Grant (A. Taguchi) and the Japan Society for the Promotion of Science (JSPS), KAKENHI, including a Grant-in-Aid for JSPS Research Fellow JP21J23350 (N.O.) and an Intramural Research Grant (2-5) for Neurological and Psychiatric Disorders of NCNP (Y.H.).

DECLARATIONS

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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