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Structure of aminoglycoside bound to rRNA. Paromomycin (red) binds to helix 44 in 16S rRNA. Adenine in position 1408 is critical for high-affinity interaction with bacterial ribosome, with other residues critical for codon–anticodon cognition also indicated. Atomic coordinates are from 1IBK.
Source publication
A third of inherited diseases result from premature termination codon mutations. Aminoglycosides have emerged as vanguard pharmacogenetic agents in treating human genetic disorders due to their unique ability to suppress gene translation termination induced by nonsense mutations. In preclinical and pilot clinical studies, this therapeutic approach...
Context in source publication
Context 1
... are widely used in clinical practice as bactericidal antibiotics with established effects on transla- tional accuracy or efficiency. 8 Their utility as antibacterial agents arises from binding to the highly conserved 16S ribosomal RNA (rRNA) at the decoding center (Figure 1). This center normally facilitates accurate codon-anticodon pairing. ...
Citations
... This finding is particularly noteworthy because PTC bypass can have profound implications for both normal cellular functioning and disease state, where a nonsense mutation can lead to aberrant protein synthesis. Conditions such as Duchenne Muscular Dystrophy (DMD) [56,57], Cystic Fibrosis [58], Beta-Thalassemia [59], Spinal Muscular Atrophy (SMA) [60], Hurler Syndrome [61], Ataxia Telangiectasia [62], and certain form of cancer [63] are prime examples. In these diseases, PTCs lead to the synthesis of truncated, typically non-functional proteins. ...
Maintaining translation fidelity is a critical step within the process of gene expression. It requires the involvement of numerous regulatory elements to ensure the synthesis of functional proteins. The efficient termination of protein synthesis can play a crucial role in preserving this fidelity. Here, we report on investigating a protein of unknown function, YNR069C (also known as BSC5), for its activity in the process of translation. We observed a significant increase in the bypass of premature stop codons upon the deletion of YNR069C. Interestingly, the genomic arrangement of this ORF suggests a compatible mode of expression reliant on translational readthrough, incorporating the neighboring open reading frame. We also showed that the deletion of YNR069C results in an increase in the rate of translation. Based on our results, we propose that YNR069C may play a role in translation fidelity, impacting the overall quantity and quality of translation. Our genetic interaction analysis supports our hypothesis, associating the role of YNR069C to the regulation of protein synthesis.
... Наиболее тяжелые проявления врожденного БЭ ассоциируются с нонсенс-мутациями. Так, от 10 до 25% вызывающих заболевание мутаций гена COL7A1, кодирующего коллаген VII типа, -основной компонент якорных фибрилл, структур, отвечающих за прикрепление эпидермиса к дерме в дермо-эпидермальном соединении, представляют собой нонсенсмутации [5,6]. Эти мутации приводят к появлению стоп-кодона и преждевременной терминации синтеза белка, необходимого для построения якорных фибрилл. ...
... функционального белка. Согласно базе данных мутаций генов человека (The Human Gene Mutation Database), 12% всех зарегистрированных мутаций являются нонсенс-мутациями[5].В целом ряде исследований in vitro и in vivo показано, что аминогликозидные антибиотики могут подавлять преждевременную терминацию и способствовать синтезу первичного полноразмерного белка при таких генетических заболеваниях, как муковисцидоз, Нежелательных реакций зарегистрировано не было. Результаты наружного применения гентамицина продемонстрировали улучшение заживления, уменьшение числа эрозий в сравнении с плацебо.В настоящее время в США проводится клиническое исследование «Внутривенная терапия гентамицином при рецессивном дистрофическом БЭ (Intravenous Gentamicin Therapy for Recessive Dystrophic Epidermolysis Bullosa)»[26]. ...
Epidermolysis bullosa is a group of rare hereditary skin diseases based on mutations in the genes of structural proteins of the epidermis and the dermal-epidermal junction. Clinically, epidermolysis bullosa is characterized by the appearance of erosions and blisters on the skin and mucous membranes in response to any minor impact. Currently, the treatment of epidermolysis bullosa is only symptomatic. Pathogenetic methods of the epidermolysis bullosa therapy are under development. One of the new possible pathogenetic directions in the treatment of epidermolysis bullosa is aminoglycoside antibiotics (gentamicin, geneticin, paromomycin). A number of studies have shown the ability of gentamicin to promote readthrough terminating codon and resume the synthesis of type VII collagen in keratinocytes and fibroblasts in patients with epidermolysis bullosa with nonsense mutations in the COL7A1. The review presented the possibilities of gentamicin therapy for patients with epidermolysis bullosa, describes the mechanism of its action, summarizes data from clinical trials.
... However, gentamicin, a wellknown and widely used antibiotic, has been shown to restore affected protein translation in several genetic diseases caused by premature termination codons (PTCs), also referred to as nonsense mutations. 5 This feature has been termed PTC suppression. Moreover, PTCs is a rather common cause of severe EB, [6][7][8][9] and has been reported to account for 10%-25% of cases with RDEB. 9 Topical gentamicin treatment has already been reported to improve wound healing through PTC suppression in patients with PTC-caused EB. [9][10][11][12][13] Although these results have been promising, the presented cases are still relatively few and the patients therein have received a rather intensive treatment on a limited area. ...
Background
Severe epidermolysis bullosa (EB) is caused by premature termination codons (PTCs) in a significant minority of the patients. Topical gentamicin has been shown to increase wound healing and can restore protein translation in patients with EB through PTC suppression. However, only intensive treatment of limited wound areas have been reported previously.
Objectives
The objectives of this study is to investigate whether nonintensive topical gentamicin therapy is an effective wound‐healing treatment in patients with severe EB caused by PTCs.
Methods
A randomized, open‐controlled, single‐centre, single‐arm clinical trial designed to determine effectiveness at an individual level was performed. Each patient served as their own control and received topical 0.1% gentamicin ointment once daily for 6 weeks on all wounds on one body half. Wound areas were measured at Weeks 0 and 6. Change in wound areas at Week 6 compared with baseline was calculated and the changes in treated areas were compared with control areas. Immunohistochemistry was performed at Weeks 0 and 6.
Results
Four patients were included. One patient withdrew before completion. No significant effect on wound healing was seen. Immunohistochemistry analysis supported successful PTC suppression in two patients.
Conclusions
Our data suggest that nonintensive topical treatment with 0.1% gentamicin ointment for 6 weeks does not improve wound healing.
... Even when only a low amount of full-length protein is produced, they may have an enormous influence on the patient's disease phenotype. This is particularly important for recessive disorders, in which low amounts of full-length functional protein are able to restore a clinically less severe phenotype [136]. Therefore, the use of aminoglycosides seems to offer a novel therapeutic for patients with nonsense mutations, particularly when used in short-term treatments, such as cancer diseases caused by nonsense mutations [137]. ...
About 11% of all human disease-associated gene lesions are nonsense mutations, resulting in the introduction of an in-frame premature translation-termination codon (PTC) into the protein-coding gene sequence. When translated, PTC-containing mRNAs originate truncated and often dysfunctional proteins that might be non-functional or have gain-of-function or dominant-negative effects. Therapeutic strategies aimed at suppressing PTCs to restore deficient protein function—the so-called nonsense suppression (or PTC readthrough) therapies—have the potential to provide a therapeutic benefit for many patients and in a broad range of genetic disorders, including cancer. These therapeutic approaches comprise the use of translational readthrough-inducing compounds that make the translational machinery recode an in-frame PTC into a sense codon. However, most of the mRNAs carrying a PTC can be rapidly degraded by the surveillance mechanism of nonsense-mediated decay (NMD), thus decreasing the levels of PTC-containing mRNAs in the cell and their availability for PTC readthrough. Accordingly, the use of NMD inhibitors, or readthrough-compound potentiators, may enhance the efficiency of PTC suppression. Here, we review the mechanisms of PTC readthrough and their regulation, as well as the recent advances in the development of novel approaches for PTC suppression, and their role in personalized medicine.
... It is not surprising that translation is the "Achilles heel" of tumor cells and actively propagat ing viruses [136,203,259,301,302]. The development of small molecule inhibitors for manipulating protein syn thesis is very important in the anticancer and immuno suppressive therapy, treatment of hereditary, viral and fungal diseases, neurology, parasitology and geriatrics, solving problems of lifespan extension, as well as agricul ture, veterinary, and other fields [8,27,132,148,151,155,214,259,261,301,302]. However, their clinical use is still limited due to the cytotoxicity related side effects. ...
Eukaryotic ribosome and cap-dependent translation are attractive targets in the antitumor, antiviral, anti-inflammatory, and antiparasitic therapies. Currently, a broad array of small-molecule drugs is known that specifically inhibit protein synthesis in eukaryotic cells. Many of them are well-studied ribosome-targeting antibiotics that block translocation, the peptidyl transferase center or the polypeptide exit tunnel, modulate the binding of translation machinery components to the ribosome, and induce miscoding, premature termination or stop codon readthrough. Such inhibitors are widely used as anticancer, anthelmintic and antifungal agents in medicine, as well as fungicides in agriculture. Chemicals that affect the accuracy of stop codon recognition are promising drugs for the nonsense suppression therapy of hereditary diseases and restoration of tumor suppressor function in cancer cells. Other compounds inhibit aminoacyl-tRNA synthetases, translation factors, and components of translation-associated signaling pathways, including mTOR kinase. Some of them have antidepressant, immunosuppressive and geroprotective properties. Translation inhibitors are also used in research for gene expression analysis by ribosome profiling, as well as in cell culture techniques. In this article, we review well-studied and less known inhibitors of eukaryotic protein synthesis (with the exception of mitochondrial and plastid translation) classified by their targets and briefly describe the action mechanisms of these compounds. We also present a continuously updated database (http://eupsic.belozersky.msu.ru) that currently contains information on 370 inhibitors of eukaryotic protein synthesis.
... Тем не менее полностью избежать побочных эффектов при применении аминогликозидов всё же не удаётся: долговременная терапия связана с рисками нефро и ототоксичности [111,[153][154][155]. Поэтому большие усилия направлены на поиск индукторов сквозного прочтения не аминогликозидной природы. ...
... Неудиви тельно, что биосинтез белка является «ахилле совой пятой» опухолевых клеток и активно размножающихся в клетке вирусов [136,203,259,301,302]. Разработка методов специфично го воздействия на биосинтез белка с помощью низкомолекулярных ингибиторов имеет боль шое значение для борьбы с раком, в иммуносуп рессорной терапии, лечении наследственных, вирусных и грибковых заболеваний, в невроло гии, паразитологии и гериатрии, в решении за дач продления жизни, а также в сельском хозяй стве, ветеринарии и других областях [8,27,132,148,151,155,214,259,261,301,302]. Тем не ме нее в медицине их использование пока ограни чено из за побочных эффектов, вызываемых цитотоксичностью этих препаратов. ...
Эукариотическая рибосома и аппарат кеп-зависимой трансляции являются привлекательными мишенями для противоопухолевой, антивовирусной, противовоспалительной и антипаразитарной терапии. В настоящее время известен широкий спектр низкомолекулярных ингибиторов, специфично подавляющих биосинтез белка в клетках эукариот. Большое количество таких веществ обнаруживается среди хорошо изученных антибиотиков, чьё действие направлено на рибосому. Они включают ингибиторы транслокации и пептидилтрансферазного центра, блокаторы рибосомного пептидного туннеля, индукторы ошибок декодирования, преждевременной терминации и сквозного прочтения стоп-кодонов, а также модуляторы связывания компонентов трансляционного аппарата с рибосомой. Отдельного внимания заслуживают низкомолекулярные ингибиторы аминоацил-тРНК синтетаз, трансляционных факторов и сигнальных путей, ассоциированных с трансляцией, в том числе ингибиторы киназы mTOR. Рибосом-направленные ингибиторы широко применяются для анализа экспрессии генов методом рибосомного профайлинга, при селекции культивируемых клеток, используются в качестве фунгицидов в сельском хозяйстве, как противогрибковые и антигельминтные средства в медицине. С веществами, влияющими на точность распознавания стоп-кодона, связаны надежды в терапии наследственных заболеваний, вызываемых нонсенс-мутациями, и восстановлении функции онкосупрессоров в опухолях. Некоторые ингибиторы биосинтеза белка обнаруживают также свойства геропротекторов. В данном обзоре мы приводим список как хорошо изученных, так и малоизвестных ингибиторов эукариотической трансляции (не касаясь биосинтеза белка в митохондриях и пластидах), дополненный информацией об их непосредственных мишенях и краткой характеристикой механизмов действия. Мы также представляем обновляемую базу данных, которая на данный момент содержит информацию о 260 ингибиторах. База данных размещена по адресу: http://eupsic.belozersky.msu.ru/.
... The most promising are probably aminoglycoside antibiotics, the drugs inducing misincorporation of amino acids (see [47][48][49] and references therein). Taken in appropriate concentrations, they can induce read-through at PTCs, although at the same time reduce translation fidelity, which might cause nephrotoxicity and ototoxicity during long-term treatments [49][50][51][52]. Blasticidin S was reported to strongly inhibit peptidyl-tRNA hydrolysis in bacteria by expelling the RF1 GGQ motif from the peptidyl transferase center [53,54]. ...
Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.
... utišanje okvarjenega gena (29), uporaba AON (angl. antisense oligoribonucleotides) za preskakovanje mutiranih eksonov gena pri prepisovanju RNA v protein, aminoglikozide za preskok predčasnih stop kodonov (30) kot tudi uporaba različnih proteinov, ki režejo DNA na specifičnih zaporedjih (angl. gene editing), kot so ZFN-ji (angl. ...
Bulozna epidermoliza (EB) je dedna bolezen krhkosti kože, povezana z mutacijami 18 različnih genov, ki se izražajo v koži. Zaradi raznovrstnosti simptomov se razvršča v 4 osnovne tipe s 30 različnimi podtipi. Osnovni tipi EB so razdeljeni glede na plast kože, ki je z mutacijo prizadeta: EB simpleks, ki je vezana na epidermis, junkcijska EB, ki je vezana na bazalno membrano, ter distrofična EB, ki je vezana na dermis. Poznamo še Kindlerjev sindrom, ki je izredno redka oblika te bolezni. Pri vseh primerih je koža bolnikov zelo krhka, zato so osnovni simptomi mehurji in odprte rane, ki se težko celijo. Bolezen prizadene bolnike telesno in duševno, kronični vnetni procesi pa pogosto povzročijo nastanek agresivnih oblik ploščatoceličnega karcinoma kože. Gre za skupino redkih bolezni z incidenco okoli 1 : 40.000. V Sloveniji imamo 60 bolnikov z EB. V članku predstavljamo trenutno uveljavljen način oskrbe kroničnih ran bolnikov z EB in pregled najnovejših in najsodobnejših genetskih, regenerativnih in farmakoloških pristopov za razvoj načina zdravljenja te še vedno neozdravljive bolezni.
... It has been shown previously that the type of stop codon (UGA > UAG > UAA) as well as the nucleotides in the immediate vicinity of the codon determine the relative readthrough ability of any particular PTC mutation. 31,32 As presented in Supplemental Table 1, PT2 and PT3 harbor LAMA3 mutations with UAA stop codons and surrounding nucleotides that would predict a relatively poor response to gentamicin, while PT1 had a UGA stop codon in LAMB3, predicted to have the best readthrough, surrounded by a nucleotide context that would further support an optimal readthrough response in this particular stop codon. 31 While this study was limited by a small sample size, our current study does not support an apparent correlation between in vitro readthrough and the type of or nucleotide context surrounding each stop codon, in agreement with our previous studies. ...
Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal autosomal recessively inherited blistering skin disease caused by mutations in the LAMA3, LAMB3, or LAMC2 genes. The majority of these mutations are nonsense mutations that create premature termination codons which lead to impaired production of functional laminin 332, a protein needed for epidermal-dermal adherence. Gentamicin induces readthrough of nonsense mutations and restores the full-length protein in various genetic diseases. Using primary keratinocytes from three GS-JEB patients, we showed that gentamicin induced functional laminin 332 that reversed a JEB-associated, abnormal cell phenotype. In a subsequent open-label trial involving the same patients, we examined whether 0.5% gentamicin ointment applied topically to open skin wounds could promote nonsense mutation readthrough and create new laminin 332 in the patients’ skin. Gentamicin-treated wounds exhibited increased expression of laminin 332 at the dermal-epidermal junction for at least three months and was associated with improved wound closure. There were no untoward side effects from topical gentamicin. The newly induced-laminin 332 did not generate anti-laminin 332 autoantibodies in either the patients’ blood or skin. Gentamicin readthrough therapy may be a treatment for GS-JEB patients with nonsense mutations.
... 19 The consequence of a nonsense mutation is the premature termination of protein translation, causing the production of non-functional proteins. [20][21] Burke and Mogg 22 demonstrated that aminoglycosides could suppress the effect of a nonsense mutation by binding to the decoding site. The impact of this binding is the reduction of the translational fidelity by AG, allowing a random amino acid to incorporate at a premature termination codon in mammalian cells. ...
