Novel small molecules potentiate premature termination codon readthrough by aminoglycosides

Baradaran‐Heravi, Alireza; Balgi, Aruna D.; Zimmerman, Carla; Choi, Kunho; Shidmoossavee, Fahimeh S.; Tan, Jason Seng Chong; Bergeaud, Célia; Krause, Alexandra; Flibotte, Stéphane; Shimizu, Yoko; Anderson, Hilary; Mouly, Vincent; Jan, Eric; Pfeifer, Tom; Jaquith, James B.; Roberge, Michel

doi:10.1093/nar/gkw638

Cited by 63 publications

(91 citation statements)

References 65 publications

(61 reference statements)

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“…Western blotting for SIOD fibroblasts was performed as previously described (7). RDEB keratinocytes were treated with various concentrations of gentamicin B1 or gentamicin for 72 h, lysed, and 20 μg protein from each lysate was separated on a 5% polyacrylamide gel and electroblotted onto a nitrocellulose membrane.…”

Section: Methodsmentioning

confidence: 99%

“…This conformation is also produced when G418, another aminoglycoside that can induce PTC readthrough, binds rRNA helix 44 (13). The production of full-length p53 (FL-p53) and truncated p53 (TR-p53) in HDQ-P1 cells exposed for 72 h to different concentrations of three gentamicin batches or B1 was determined by automated capillary electrophoresis Western analysis and the results are displayed as pseudoblots (7). Vinculin was used as a protein loading control.…”

Section: Significancementioning

confidence: 99%

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Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity

Baradaran‐Heravi

Niesser

Balgi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Nonsense mutations underlie about 10% of rare genetic disease cases. They introduce a premature termination codon (PTC) and prevent the formation of full-length protein. Pharmaceutical gentamicin, a mixture of several related aminoglycosides, is a frequently used antibiotic in humans that can induce PTC readthrough and suppress nonsense mutations at high concentrations. However, testing of gentamicin in clinical trials has shown that safe doses of this drug produce weak and variable readthrough activity that is insufficient for use as therapy. In this study we show that the major components of pharmaceutical gentamicin lack PTC readthrough activity but the minor component gentamicin B1 (B1) is a potent readthrough inducer. Molecular dynamics simulations reveal the importance of ring I of B1 in establishing a ribosome configuration that permits pairing of a near-cognate complex at a PTC. B1 induced readthrough at all three nonsense codons in cultured cancer cells with TP53 (tumor protein p53) mutations, in cells from patients with nonsense mutations in the TPP1 (tripeptidyl peptidase 1), DMD (dystrophin), SMARCAL1 (SWI/SNF-related, matrixassociated, actin-dependent regulator of chromatin, subfamily a-like 1), and COL7A1 (collagen type VII alpha 1 chain) genes, and in an in vivo tumor xenograft model. The B1 content of pharmaceutical gentamicin is highly variable and major gentamicins suppress the PTC readthrough activity of B1. Purified B1 provides a consistent and effective source of PTC readthrough activity to study the potential of nonsense suppression for treatment of rare genetic disorders.gentamicin B1 | nonsense mutation | premature stop codon readthrough | rare genetic diseases | cancer

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Section: Methodsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity

Baradaran‐Heravi

Niesser

Balgi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…We attribute this to different mechanisms of action, as amlexanox has been identified as an inhibitor of NMD (Gonzalez-Hilarion et al, 2012) while gentamicin, as an aminoglycoside, is thought to act on the ribosome (Yoshizawa et al, 1998). Indeed, mRNA data at 24 hours after treatment (Figure 3b) identifies increase with amlexanox and not gentamicin, in keeping with previous studies showing gentamicin induces transcript increase after 48 hours (Baradaran-Heravi et al, 2016), presumably due to feedback as a result of read-through.…”

Section: Discussionsupporting

confidence: 90%

Amlexanox Enhances Premature Termination Codon Read-Through in COL7A1 and Expression of Full Length Type VII Collagen: Potential Therapy for Recessive Dystrophic Epidermolysis Bullosa

Atanasova

Jiang

Prisco

et al. 2017

Journal of Investigative Dermatology

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Recessive dystrophic epidermolysis bullosa (RDEB) is a rare monogenic blistering disorder caused by lack of functional type VII collagen, leading to skin fragility and subsequent trauma-induced separation of the epidermis from the underlying dermis. 46% of RDEB patients harbor at least one premature termination codon (PTC) mutation in COL7A1 and previous studies have shown that aminoglycosides are able to overcome RDEB PTC mutations by inducing “read-through” and incorporation of an amino acid at the PTC site. However, aminoglycoside toxicity will likely prevent widespread clinical application. Here the FDA-approved drug amlexanox was tested for its ability to read-through PTC mutations in RDEB patient derived cells. Eight of 12 different PTC alleles responded to treatment and produced full length protein, in some cases over 50% relative to normal controls. Read-through type VII collagen was readily detectable in cell culture media and also localized to the dermal-epidermal junction in organotypic skin culture. Amlexanox increased COL7A1 transcript and the phosphorylation of UPF-1, an RNA helicase associated with nonsense mediated mRNA decay (NMD), suggesting that amlexanox inhibits NMD in RDEB patient cells that read-through. This pre-clinical study demonstrates the potential of re-purposing amlexanox for treatment of RDEB patients harboring PTC mutation in COL7A1.

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“…There have been a number of previous clinical and translational studies that examined the applicability of the read‐through approach to a clinical setting . Though some of these studies focused on the cancer arena, none resulted in clinical trials . Therefore, the ability to induce read‐through of the APC full‐length protein by antibiotic treatment represents a new potential strategy to delay or even prevent cancer formation in FAP patients.…”

Section: Discussionmentioning

confidence: 99%

Resorting the function of the colorectal cancer gatekeeper adenomatous polyposis coli

Kariv

Caspi

Fliss-Isakov³

et al. 2019

Intl Journal of Cancer

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As a large number of cancers are caused by nonsense mutations in key genes, read‐through of these mutations to restore full‐length protein expression is a potential therapeutic strategy. Mutations in the adenomatous polyposis coli (APC) gene initiate the majority of both sporadic and hereditary colorectal cancers (CRC) and around 30% of these mutations are nonsense mutations. Our goal was to test the feasibility and effectiveness of APC nonsense mutation read‐through as a potential chemo‐preventive therapy in Familial Adenomatous Polyposis (FAP), an inherited CRC syndrome patients. Ten FAP patients harboring APC nonsense mutations were treated with the read‐through inducing antibiotic erythromycin for 4 months. Endoscopic assessment of the adenomas was performed at baseline, after 4 and after 12 months. Adenoma burden was documented in terms of adenoma number, maximal polyp size and cumulative polyp size per procedure. Tissue samples were collected and subjected to molecular and genetic analyses. Our results show that in the majority of patients the treatment led to a decrease in cumulative adenoma burden, median reduction in cumulative adenoma size and median reduction in adenoma number. Molecular and genetic analyses of the adenomas revealed that the treatment led to a reduced number of somatic APC mutations, reduced cellular proliferation and restoration of APC tumor‐suppressing activity. Together, our findings show that induced read‐through of APC nonsense mutations leads to promising clinical results and should be further investigated to establish its therapeutic potential in FAP and sporadic CRCs harboring nonsense APC mutations.

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Novel small molecules potentiate premature termination codon readthrough by aminoglycosides

Cited by 63 publications

References 65 publications

Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity

Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity

Amlexanox Enhances Premature Termination Codon Read-Through in COL7A1 and Expression of Full Length Type VII Collagen: Potential Therapy for Recessive Dystrophic Epidermolysis Bullosa

Resorting the function of the colorectal cancer gatekeeper adenomatous polyposis coli

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