Remdesivir was shown to inhibit RNA-dependent RNA-polymerases (RdRp) from distinct viral families such as from Filoviridae (Ebola) and Coronaviridae (SARS-CoV, SARS-CoV-2, MERS). In this study, we tested the ability of remdesivir to inhibit RdRps from the Flaviviridae family. Instead of remdesivir, we used the active species that is produced in cells from remdesivir, the appropriate triphosphate, which could be directly tested in vitro using recombinant flaviviral polymerases. Our results show that remdesivir can efficiently inhibit RdRps from viruses causing severe illnesses such as Yellow fever, West Nile fever, Japanese and Tick-borne encephalitis, Zika and Dengue. Taken together, this study demonstrates that remdesivir or its derivatives have the potential to become a broad-spectrum antiviral agent effective against many RNA viruses.
- MeSH
- adenosintrifosfát analogy a deriváty chemie farmakologie MeSH
- antivirové látky chemie farmakologie MeSH
- Betacoronavirus účinky léků enzymologie MeSH
- COVID-19 MeSH
- Flavivirus účinky léků enzymologie MeSH
- inhibiční koncentrace 50 MeSH
- koronavirové infekce farmakoterapie virologie MeSH
- lidé MeSH
- pandemie MeSH
- RNA-dependentní RNA-polymerasa antagonisté a inhibitory metabolismus MeSH
- RNA-viry účinky léků enzymologie MeSH
- SARS-CoV-2 MeSH
- virová pneumonie farmakoterapie virologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We synthesized a small library of eighteen 5-substituted pyrimidine or 7-substituted 7-deazapurine nucleoside triphosphates bearing methyl, ethynyl, phenyl, benzofuryl or dibenzofuryl groups through cross-coupling reactions of nucleosides followed by triphosphorylation or through direct cross-coupling reactions of halogenated nucleoside triphosphates. We systematically studied the influence of the modification on the efficiency of T7 RNA polymerase catalyzed synthesis of modified RNA and found that modified ATP, UTP and CTP analogues bearing smaller modifications were good substrates and building blocks for the RNA synthesis even in difficult sequences incorporating multiple modified nucleotides. Bulky dibenzofuryl derivatives of ATP and GTP were not substrates for the RNA polymerase. In the case of modified GTP analogues, a modified procedure using a special promoter and GMP as initiator needed to be used to obtain efficient RNA synthesis. The T7 RNA polymerase synthesis of modified RNA can be very efficiently used for synthesis of modified RNA but the method has constraints in the sequence of the first three nucleotides of the transcript, which must contain a non-modified G in the +1 position.
- MeSH
- adenosintrifosfát analogy a deriváty metabolismus MeSH
- bakteriofág T7 enzymologie MeSH
- cytidintrifosfát analogy a deriváty metabolismus MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- purinové nukleosidy chemie metabolismus MeSH
- puriny chemie metabolismus MeSH
- pyrimidinové nukleosidy chemie metabolismus MeSH
- RNA chemie metabolismus MeSH
- substrátová specifita MeSH
- uridintrifosfát analogy a deriváty metabolismus MeSH
- virové proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
We describe the expression and purification of an active recombinant Zika virus RNA-dependent RNA polymerase (RdRp). Next, we present the development and optimization of an in vitro assay to measure its activity. We then applied the assay to selected triphosphate analogs and discovered that 2'-C-methylated nucleosides exhibit strong inhibitory activity. Surprisingly, also carbocyclic derivatives with the carbohydrate locked in a North-like conformation as well as a ribonucleotide with a South conformation exhibited strong activity. Our results suggest that the traditional 2'-C-methylated nucleosides pursued in the race for anti-HCV treatment can be superseded by brand new scaffolds in the case of the Zika virus.
- MeSH
- adenosintrifosfát analogy a deriváty chemie MeSH
- antivirové látky farmakologie MeSH
- inhibitory enzymů farmakologie MeSH
- lidé MeSH
- molekulární konformace MeSH
- nukleosidy chemie farmakologie MeSH
- objevování léků MeSH
- RNA-dependentní RNA-polymerasa antagonisté a inhibitory genetika izolace a purifikace MeSH
- virus zika účinky léků enzymologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Various helicases and single-stranded DNA (ssDNA) binding proteins are known to destabilize G-quadruplex (GQ) structures, which otherwise result in genomic instability. Bulk biochemical studies have shown that Bloom helicase (BLM) unfolds both intermolecular and intramolecular GQ in the presence of ATP. Using single molecule FRET, we show that binding of RecQ-core of BLM (will be referred to as BLM) to ssDNA in the vicinity of an intramolecular GQ leads to destabilization and unfolding of the GQ in the absence of ATP. We show that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to ssDNA overhang, as modulated by the nucleotide state, ionic conditions, overhang length and overhang directionality. In particular, we observed enhanced GQ unfolding by BLM in the presence of non-hydrolysable ATP analogs, which has implications for the underlying mechanism. We also show that increasing GQ stability, via shorter loops or higher ionic strength, reduces BLM-mediated GQ unfolding. Finally, we show that while WRN has similar activity as BLM, RecQ and RECQ5 helicases do not unfold GQ in the absence of ATP at physiological ionic strength. In summary, our study points to a novel and potentially very common mechanism of GQ destabilization mediated by proteins binding to the vicinity of these structures.
- MeSH
- adenosindifosfát metabolismus MeSH
- adenosintrifosfát analogy a deriváty metabolismus MeSH
- G-kvadruplexy * MeSH
- helikasy RecQ chemie metabolismus MeSH
- jednovláknová DNA metabolismus MeSH
- lidé MeSH
- telomery chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Systémová mastocytóza (SM) je vzácné získané myeloproliferativní onemocnění. Podkladem je klonální proliferace mastocytů a jejich akumulace v tkáních. U většiny pacientů postižených SM je přítomna aktivující bodová mutace v genu kódujícím receptor c-KIT (substituce aspartát/valin v kodonu 816 – D816V). Systémová mastocytóza je na rozdíl od čistě kožní formy diagnostikována zejména v dospělosti. Část nemocných je zároveň postižena jiným klonálním hematologickým onemocněním, zejména myeloidní linie. Velmi vzácným podtypem systémové mastocytózy je mastocelulární leukemie. Symptomy onemocnění jsou dány jednak tkáňovou akumulací patologických mastocytů, jednak uvolněním mediátorů z těchto buněk. Při diagnostice onemocnění je zásadní histologické (s imunohistochemickým) a molekulárně genetické vyšetření postiženého orgánu (zejména kostní dřeně) a stanovení hladiny sérové tryptázy. Přirozený klinický průběh onemocnění je variabilní. Většina pacientů s indolentní formou zůstává bez progrese řadu let či desetiletí, zatímco agresivní forma onemocnění trvale progreduje do fatálního zakončení. Klasickou cytoredukční terapií je v současné době onemocnění nevyléčitelné. Díky detekci mutací do léčby pronikly cílově-specifické látky (tyrozin kinázové inhibitory), které mohou navodit kvalitní terapeutické odpovědi. U pacientů s přítomností mediátory podmíněných symptomů je třeba terapeuticky ovlivňovat i tyto.
Systemic mastocytosis (SM) is a rare and acquired myeloproliferative disorder characterized by a clonal proliferation of neoplastic mast cells and their accumulation in tissues. In most SM patients, the activating somatic mutation D816V (aspartate/valin substitution) of the c-KIT is detectable. In contrast to purely cutaneous form is systemic mastocytosis a disease of adulthood. A part of patients with SM presents with an associated non-mast cell clonal haematological disease, notably of myeloid line. The very rare variant of SM is mast cell leukemia. The symptoms of the disease are partly related to the organ infiltration, partly are caused by the release of mediators from neoplastic mast cells. The most important diagnostic tools are histology with immunohistochemistry of the infiltrated organ (particularly bone marrow), molecular analyses and the assesment of serum tryptase level. The natural clinical course of systemic mastocytosis is variable. Most patients, in particular those with indolent form, remain in an indolent stage over many years or decades, while others (in particular those with aggressive SM) show a progressive course, usually with a fatal outcome. Nowadays, using the classic cytoreductive treatment, there is no curative option available. However, concomitantly with the detection of some mutations of genes involved in the pathogenesis, the use of targeted drugs (tyrosine kinase inhibitors) is proposed with the possibility of reaching good responses. In patients with mediator-related symptoms, “mediator-targeting” drugs must be prescribed.
- Klíčová slova
- Glivec, Sprycel,
- MeSH
- adenosinmonofosfát analogy a deriváty farmakologie MeSH
- adenosintrifosfát analogy a deriváty farmakologie MeSH
- financování organizované MeSH
- glukokortikoidy farmakologie MeSH
- interferon alfa farmakologie MeSH
- lidé MeSH
- mastocyty fyziologie imunologie sekrece MeSH
- monoklonální protilátky farmakologie MeSH
- systémová mastocytóza diagnóza patofyziologie terapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
The functional relevance of aromatic residues in the upper part of the transmembrane domain-1 of purinergic P2X receptors (P2XRs) was examined. Replacement of the conserved Tyr residue with Ala had a receptor-specific effect: the P2X1R was non-functional, the P2X2R, P2X4R, and P2X3R exhibited enhanced sensitivity to ATP and alphabeta-meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7R sensitivity for agonists was not affected, though decay of current was delayed. The replacement of the P2X4R-Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2R, P2X3R, and P2X4R function. Double mutation of ipsilateral Tyr42 and Trp46 P2X4R residues restored receptor function, whereas the corresponding P2X2R double mutant was not functional. In contrast, mutation of the contralateral Phe48 residue in the P2X4R-Y42A mutant had no effect. These results indicate that aromatic residues in the upper part of TM1 play important roles in the three-dimensional structure of the P2XRs and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating.
- MeSH
- adenosintrifosfát analogy a deriváty farmakologie MeSH
- aminokyseliny aromatické genetika metabolismus MeSH
- biofyzika MeSH
- elektrická stimulace MeSH
- financování organizované MeSH
- konformace proteinů MeSH
- lidé MeSH
- membránové potenciály genetika účinky léků MeSH
- metoda terčíkového zámku metody MeSH
- mutageneze genetika MeSH
- purinergní receptory P2 genetika klasifikace metabolismus MeSH
- sekvence aminokyselin MeSH
- signální transdukce fyziologie účinky léků MeSH
- terciární struktura proteinů genetika fyziologie MeSH
- transfekce metody MeSH
- transformované buněčné linie MeSH
- vazba proteinů fyziologie genetika MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zelené fluorescenční proteiny MeSH
- Check Tag
- lidé MeSH
- MeSH
- adenosintrifosfát analogy a deriváty analýza chemie MeSH
- cytidintrifosfát analogy a deriváty analýza chemie MeSH
- DNA-dependentní DNA-polymerasy chemie MeSH
- elektrochemie MeSH
- financování organizované MeSH
- kyseliny boronové chemie MeSH
- molekulární struktura MeSH
- oligonukleotidy chemická syntéza chemie MeSH
- uridintrifosfát analogy a deriváty analýza chemie MeSH
Transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a thermosensitive cation channel activated by noxious heat as well as a wide range of chemical stimuli. Although ATP by itself does not directly activate TRPV1, it was shown that intracellular ATP increases its activity by directly interacting with the Walker A motif residing on the C-terminus of TRPV1. In order to identify the amino acid residues that are essential for the binding of ATP to the TRPV1 channel, we performed the following point mutations of the Walker A motif: P732A, D733A, G734A, K735A, D736A, and D737A. Employing bulk fluorescence measurements, namely a TNP-ATP competition assay and FITC labelling and quenching experiments, we identified the key role of the K735 residue in the binding of the nucleotide. Experimental data was interpreted according to our molecular modelling simulations.
- MeSH
- adenosintrifosfát analogy a deriváty chemie MeSH
- aminokyseliny chemie MeSH
- arginin chemie MeSH
- finanční podpora výzkumu jako téma MeSH
- fluorescenční barviva chemie MeSH
- krystalografie rentgenová MeSH
- kyselina D-aspartová chemie MeSH
- sodíko-draslíková ATPasa chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
