1,721,040 research outputs found
COMBINING TRANSLATION READTHROUGH INDUCING DRUGS AND NONSENSE MEDIATED DECAY PATWHAY INHIBITION TO THE CFTR RESCUE IN CYSTIC FIBROSIS CELL MODEL SYSTEM
Full text linkNonsense mutations affect 10% of patients with cystic
fibrosis and produce a premature termination codon in CFTR
(Cystic Fibrosis Transmembrane Conductance Regulator)
mRNA causing early termination of translation and leading to
lack of CFTR function. A potential therapy for nonsense mutations provides the use of small molecules able to overcome the
premature stop codon (PTC) by a readthrough mechanism that
lead to synthesis a complete CFTR protein. Despite the good
results obtained from this approach, TRIDs efficiency is considerably reduced by the poor amount of target transcript, that is
the mRNA containing the PTC. The readthrough, indeed, does
not occur on the totality of target transcripts because of their
degradation due to the nonsense mediated decay pathway
(NMD). This pathway provides the degradation of mRNA harboring premature stop codon to prevent the production of
altered polypeptides. In contrast, the activity of this pathway
interferes with the effectiveness of the readthrough drugs, limiting the mRNA concentration of the target protein. Thus, a
promising strategy for nonsense mutation treatment is a combined use of readthrough agents and factors that attenuate the
nonsense mRNA decay. By silencing the UPF1 mRNA/protein, the activity of the NMD pathway was reduced, in FRT
cells CFTR W1282X. Alternatively, caffeine was used as specific
inhibitor of the UPF1 activity, to increase the efficiency of
readthrough molecules (NV848 and NV914) in FRT cells
CFTRW1282X cells. In both cases, the combined treatment:
NV848 or NV914/caffeine and NV848 or
NV914/UPF1siRNA caused an increase of CFTRW1282X
mRNA level followed by the rescue of the CFTR expression
and functionality. However, unexpectedly, despite the higher
CFTRW1282X mRNA level in caffeine treated samples, both
expression and functionality CFTR rescue resulted slightly
lower than the recovery achieved by UPF1 silencing. Our
results indicate that modulation of NMD pathway, although
still to be optimized, could be a promising approach in order
to increase TRIDs effects in presence of stop mutations
Functionalization of Graphene with Molecules and/or Nanoparticles for Advanced Applications
No full textGraphene is considered the material of the third millennium, due to its extraordinary electronic and mechanical properties, and due to the possibility to modulate its conductivity, flexibility, elasticity, transparency, and biocompatibility by bottom-up approach. The possibility to gather the proper- ties of graphene and graphene oxide with those of functional moieties or nanoparticles is herein reviewed. The synthetic approaches proposed, either covalent or noncovalent, are aimed to tune appropriately graphene’s properties for the realization of materials for advanced uses, such as bio- medical applications, sensors, catalysis, and energy devices. In particular, methods based on covalent linking and noncovalent functionalization by supramo- lecular interaction will be discussed, by elucidating main advantages and drawbacks, focusing on recent papers in developing advanced applications
Production of CHM R293X cell model system to study the rescue of the Rab Escort Protein-1 expression by TRIDs nonsense suppression activity
No full textChoroideremia is an inherited genetic disorder caused by several mutations in the CHM gene, which codifies for Rep1 protein strictly linked in intravesicular trafficking. The Rep1 lack causes choroid and photoreceptors degeneration, leading firstly to night blindness and at last to complete blindness. About 39% of mutations on the CHM gene are represented by nonsense mutations, which insert a premature termination codon in the reading frame of respective mRNA, with the production of truncated non-functional protein. Nowadays there is no cure for diseases caused by nonsense mutations, but a promising approach is the suppression therapy using TRIDs molecules (translational readthrough-inducing drugs). In our study, a CHMR293X/R293X cell model system was produced to evaluate the activity of three new optimized molecules (NV848, NV914, and NV930) in the rescue of the Rab-Escort-Protein-1 (Rep1) expression
Uno studio comparativo in silico sui possibili target di Ataluren e analoghi farmaci promotori di readthrough di codoni di stop prematuri
Full text linkE’ noto in letteratura che Ataluren (acido 5-(fluorofenil)-1,2,4-ossadiazolil-benzoico) sia in grado di sopprimere le mutazioni non senso favorendo il readthrough dei codoni di stop prematuri, anche se il suo meccanismo di azione non risulta ancora chiaro. La probabile interazione tra Ataluren e CTFR-mRNA è stata precedentemente studiata mediante dinamica molecolare. In questo studio1, abbiamo esteso il modeling del probabile meccanismo di azione di Ataluren mediante approcci computazionali completementari, quali Induced Fit Docking (IFD), Quantum Polarized Ligand Docking (QPLD), metodi MM-GBSA e mutagenesi computazionale. Oltre a considerare il CTFR-mRNA, sono stati presi in considerazione altri target implicati nel processo di traduzione, quali la subunità 16S dell’rRNA batterico e la subunità 18S dell’rRNA eucariotico, che sono target comprovati di molti aminoglicosidi noti per la loro capacità di sopprimere l’attività di correzione svolta normalmente dal ribosoma; il fattore di rilascio eucariotico eRF1, per valutare la potenziale influenza di Ataluren sulla fine del processo di traduzione. Inoltre, è stato effettuato un confronto tra Ataluren, un suo nuovo promettente analogo NV2445 (acido 4-(5-(o-tolil)-1,3,4-ossadiazol-2-il)benzoico)2 e una serie di antibiotici aminoglicosidici. I risultati hanno confermato che mRNA è il più probabile target per Ataluren e i suoi derivati. I calcoli di energia libera di legame effettuati in seguito alla mutagenesi computazionale, hanno mostrato che il legame tra Ataluren e il codone di stop prematuro è fortemente influenzato dalla presenza di nucleotidi ausiliari nell’intorno genico
OXADIAZOLE DERIVATIVES FOR THE TREATMENT OF GENETIC DISEASES DUE TO NONSENSE MUTATIONS
No full textAre disclosed oxadiazole derivatives, their use as medicaments and in particular for the treatment of diseases associated with the presence of a nonsense mutation in the gene or a premature stop codon in the mRNA, pharmaceutical formulation comprising said oxadiazole derivatives and prodrug or mixture thereof and the methods for the preparation of said Oxadiazole derivatives
PERSONALIZED MEDICINE FIGHTING NONSENSE DISEASES: EXPLORATION OF NV MOLECULES MECHANISM OF ACTION IN PURE-LITE SYSTEM AND THEIR READTHROUGH ACTIVITY IN CHOROIDEREMIA NONSENSE MODEL
No full textPrecision medicine represents a new approach in genetic medicine for treating a patient's mutation profile. Nonsense mutations cause 11% of inherited genetic diseases, including Choroideremia (CHM), Cystic fibrosis (CF), Duchenne Muscular Dystrophy (DMD), and some Cancers. Choroideremia is an X-linked disease associated with many retinal disorders. This retinal dystrophy causes the progressive degeneration of the choriocapillaris, photoreceptors, and retinal pigment epithelium. The CHM gene codifies for a 95 kDa, known as rab escort protein 1 (Rep1), involved in the intracellular trafficking and prenylation of polypeptides, a post-translational modification fundamental for the correct functionality of specific proteins. Nonsense mutations represent about 34% of mutations in the CHM gene. These mutations prematurely introduce a premature termination codon (PTC: TGA, TAG, and TAA) in the mRNA frame, causing a truncated and non-functional protein that will be eliminated from intracellular surveillance pathways. Nowadays there is no cure for diseases caused by nonsense mutations, but in the last decades, promising results have come from a pharmacological approach, called suppression therapy. This approach uses specific drugs having readthrough activity, known as TRIDs (Translational Readthrough Inducing Drugs), which permit the insertion of a near-cognate-tRNA in correspondence with a PTC during protein translation, allowing the correct development of a functional full-length protein. Our study is focused on investigating three molecules, NV848, NV914 and NV930, patented by Pibiri-Lentini group, that have shown readthrough activity in different nonsense model diseases. Specifically, elucidating their Mechanism of Action in the PURE-LITE system and exploring their readthrough activity in a choroideremia nonsense model system have been the goals of our efforts. PURE-LITE system is a highly purified, eukaryotic cell-free protein synthesis system, that allows the investigation of both the termination of protein synthesis in the P-site catalyzed by the RF complex (eRF1/eRF3/ribosome) and the readthrough via mispairing at the termination codon by a near-cognate tRNA when a PTC enter into the 40S A-site ribosome subunit. Simultaneously, chronic treatment and protein expression analyses was performed on Choroideremia nonsense cell model system to study their readthrough activity. Our results have shown both that the mechanism of action of these molecules is different by their precursor Ataluren, and in addition their readthrough activity with the rescue of Rep1 protein
Rescuing CFTR Protein Function: 1,3,4-oxadiazoles versus 1,2,4-oxadiazoles as readthrough inducing drugs
Full text linkIn Cystic fibrosis (CF) disease nonsense mutations in the CFTR gene cause the absence of the CFTR protein expression and a more severe form of the disease. About 10% of patient affected by CF show a nonsense mutation. A potential treatment of this alteration is to promote translational readthrough of premature termination codons (PTCs) by translational readthrough inducing drugs such as Ataluren (1). We reported a rationale for Ataluren promoted readthrough of PTCs by computational approach and GFP-reporter cell-based assay (2) and the observed enhancement of readthrough activity by some Ataluren derivatives (3, 4).
In this context we aimed to compare the 1,2,4-oxadiazole core of Ataluren with a slightly different scaffold, the 1,3,4-oxadiazole core. By a validated protocol consisting of computational screening, synthesis and biological tests we identified, a new small molecule with 1,3,4-oxadiazole core showing high readthrough activity. Moreover, we evaluated quantitatively the CFTR functionality after treatment with our new lead in CF model systems and in cells expressing a nonsense-CFTR-mRNA. Finally, we studied the supramolecular interactions among readthrough inducing drugs and CFTR mRNA to assess the biological target and hypothesized mechanism and further we calculated and compared the ADME properties of our new lead to Atalure
AN APPROACH AGAINST CANCER: TRANSLATIONAL READTHROUGH INDUCING DRUGS (TRIDs) FOR RESTORING P53 EXPRESSION IN STOP MUTATED CELLS
Full text linkStop mutations are gene mutations characterized by the substitution of a single nucleotide in the coding sequence of a gene, which causes the onset of a premature stop codon (PTC) within the reading frame of the mRNA, resulting in the formation of a truncated and non-functional protein. This type of mutation accounts for approximately 11% of genetic diseases, including conditions such as Cystic Fibrosis, Duchenne Muscular Dystrophy, Choroideremia, Schwachman-Diamond syndrome, and certain types of hereditary cancers involving mutations in the TP53 gene. About 10% of TP53 mutations are stop mutations [1, 2]. TP53 encodes a protein made up of 393 amino acid residues called p53, which acts mainly as a transcription factor, regulating numerous pathways such as the cell cycle arrest, DNA damage repair, apoptosis, autophagy, and metabolism when cells are under certain stress conditions. TP53 mutations create a favorable environment for tumor formation, and mutant p53 may exhibit loss of function, dominant-negative repression, or gain of oncogenic function, contributing to tumor stability and progression [2]. Today there is no therapy for the pathologies caused by this type of mutation, but an approach that has proven to be particularly effective is represented by molecules with readthrough activity (TRIDs; Translational Readthrough Inducing Drugs) which intervene on the ribosome allowing the overcoming of the PTC and the restoration of the synthesis and subsequent functionality of the protein [3]. In this work, we investigate the effects of TRID molecules with readthrough activity on the TP53 gene in tumor cells, which harbors the PTC R213X, the most common TP53 stop mutation, that generates a truncated and non-functional p53. We analyzed the restoration of p53 protein expression before and after induction of DNA damage by Western blot, its nuclear localization with fluorescence microscopy, the mRNA expression of p53, and its targets p21 and GADD45 to evaluate the functionality of the protein by Real-Time RT PCR. After 24 hours of treatment with TRIDs, we observed a partial nuclear localization of p53, an increase in mRNA expression of its targets and a restoration of protein expression after the induction of DNA damage. These results represent a promising path for developing targeted cancer therapies against stop mutations, a new approach to impede tumor proliferation, and a solid foundation for the formulation of novel personalized therapy modalities not only against cancer
Synthesis, characterization and study of covalently modified triazole LAPONITE® edges
Full text linkLAPONITE® (Lap) clay mineral was successful functionalized by triazole groups in a two steps procedure. First, the Lap edges were modified with 3-azidopropyltrimethoxysilane by traditional heating and microwave irradiation. Microwave irradiation allowed us to obtain high loading onto the Lap edges in lower times compared to those obtained through conventional method. Afterwards, the triazole moieties were obtained by reaction be- tween azido functionalized Lap and propargyl alcohol. The successful functionalization of Lap was proved by thermogravimetric analysis, FT-IR spectroscopy, dynamic light scattering and ζ-potential measurements. Finally, the effects of the surface modification on the gel formation ability of Lap were studied by gelation tests and the morphology of the gel phases was investigated by polarized optical microscopy measurements and diffusion experiments
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