Effect of small molecule eRF3 degraders on premature termination codon readthrough

Abstract

Premature termination codon (PTC) readthrough is considered a potential treatment for genetic diseases caused by nonsense mutations. High concentrations of aminoglycosides induce low levels of PTC readthrough but also elicit severe toxicity. Identifying compounds that enhance PTC readthrough by aminoglycosides or reduce their toxicity is a continuing challenge. In humans, a binary complex of eukaryotic release factors 1 (eRF1) and 3 (eRF3a or eRF3b) mediates translation termination. They also participate in the SURF (SMG1-UPF1-eRF1-eRF3) complex assembly involved in nonsense-mediated mRNA decay (NMD). We show that PTC readthrough by aminoglycoside G418 is considerably enhanced by eRF3a and eRF3b siRNAs and cereblon E3 ligase modulators CC-885 and CC-90009, which induce proteasomal degradation of eRF3a and eRF3b. eRF3 degradation also reduces eRF1 levels and upregulates UPF1 and selectively stabilizes TP53 transcripts bearing a nonsense mutation over WT, indicating NMD suppression. CC-90009 is considerably less toxic than CC-885 and it enhances PTC readthrough in combination with aminoglycosides in mucopolysaccharidosis type I-Hurler, late infantile neuronal ceroid lipofuscinosis, Duchenne muscular dystrophy and junctional epidermolysis bullosa patient-derived cells with nonsense mutations in the IDUA, TPP1, DMD and COL17A1 genes, respectively. Combination of CC-90009 with aminoglycosides such as gentamicin or ELX-02 may have potential for PTC readthrough therapy.

Robust W1282X-CFTR rescue by a small molecule GSPT1 degrader

“…We measured CFTR mRNA by qRT-PCR and found a significant increase in response to CC-90009 treatment (Figure 5E). This finding is consistent with the published observation that GSPT1 degradation stabilizes PTC-containing transcripts through a partial suppression of NMD (18). Overall, CC-90009 treatment significantly rescued CFTR function in a cohort of three W1282X homozygous patient-derived cell lines and primary nasal and bronchial epithelial cells.…”

“…CC-90009 has been previously studied at 10 μM concentrations and was reported to have negligible cytotoxicity (18). Yet in our culture system, the transepithelial resistance was reduced by ~50% in cultures treated with 10 μM CC-90009 (Supplemental Figure 5C).…”

“…CC-90009 rescued 22.8 ± 5.8% and 17.9 ± 5.6% wildtype CFTR function in the bronchial cell lines and primary cells, respectively (mean ± SD) (Figure 5A-D). Based on the literature, we suspect that the primary mechanism for W1282X-CFTR rescue is enhanced ribosomal readthrough (18). CFTR protein level is low in primary airway epithelial cells and made even lower by NMD in cells carrying PTC variants, making it challenging to assess.…”

“…Without a targetable protein, modulator therapies are ineffective, leaving these patients without treatment options. Recent studies illustrate that the cereblon E3 ubiquitin ligase modulator, CC-90009, promotes PTC readthrough of nonsense mutations associated with inherited diseases (18). Using one nasal and two bronchial Bmi-1/hTERT W1282X/W128X CFTR cell lines, we tested CC-90009 effects, again showing fidelity between cell lines and primary cells and importantly both predicted and unanticipated electrophysiological responses that would be beneficial in CF.…”

Clinical Perspectives of Gene-Targeted Therapies for Epidermolysis Bullosa

“…Additionally, molecules that act synergistically with aminoglycosides on translation, e.g., by inhibition of translation termination factors or nonsense-mediated decay (NMD) of PTC-bearing mRNAs, may enhance the efficacy of PTC readthrough. The potential of this approach was recently demonstrated in JEB-derived cells with COL17A1 nonsense mutations by combining gentamicin with drugs targeting the translation termination factor eRF3 in vitro [ 115 ]. In this context, the anti-inflammatory drug amlexanox has been associated with both NMD inhibition and PTC readthrough [ 116 , 117 ], and its application in nonsense mutated RDEB patient cells in vitro led to the generation of full-length type VII collagen [ 116 , 117 ].…”