RESUMO
The DNA cis-syn cyclobutane photoproduct formed between two adjacent cytosine residues is highly mutagenic and responsible for the tandem CC to TT transition. However, its instability has prevented its in vitro study, so far. With a view to prepare oligodeoxynucleotides containing the CC cyclobutane lesion, we have synthesized in good yield a ditetrazolouracil cyclobutane dinucleotide photoproduct as a stable precursor of this photoproduct. Our approach also overcomes the low photochemical reactivity of the cytosine-cytosine deoxydinucleoside monophosphate.
Assuntos
Citosina/análogos & derivados , Fosfatos de Dinucleosídeos/química , Dímeros de Pirimidina/química , Uracila/análogos & derivados , Acetona/química , Raios UltravioletaRESUMO
The [2 + 2] photocycloaddition of natural pyrimidine nucleobases is devoid of regioselectivity. Although modified pyrimidines have been developed to selectively obtain syn-cyclobutane isomers, the targeted formation of anti-cyclobutane isomers has not been addressed yet. Herein, using NMR analyses and DFT calculations, we demonstrate that the acetone photosensitized excitation of the 4-tetrazolouracil motif in the nucleoside series specifically provides anti-cyclobutane photoproducts in 51% yield. In addition, the cis stereomer formation is preferred over the trans-cyclobutane formation (71:29).
Assuntos
Ciclobutanos , Dímeros de Pirimidina , Reação de Cicloadição , Isomerismo , Nucleosídeos , Dímeros de Pirimidina/química , Raios UltravioletaRESUMO
Antimicrobial susceptibility tests (AST) are based on the minimal inhibitory concentration (MIC), the method used worldwide to guide antimicrobial therapy. Despite its relevance in correctly predicting clinical outcome for most acute infections, this approach is misleading for multiple clinical cases in which pathogens do not grow rapidly, uniformly or with physical protection. This behaviour, named 'metabolic evasion' (ME), enables bacteria to survive antimicrobials. ME can result from different, and sometimes combined, bacterial mechanisms such as biofilms, intracellular growth, persisters or dormancy. We discuss how ME can influence the MIC-based probability of target attainment. We identify clinical cases in which this approach is undermined by ME and propose a new approach that takes ME into account in order to improve patient management and the evaluation of innovative drugs.
Assuntos
Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Bactérias/metabolismo , Árvores de Decisões , Humanos , Resultado do TratamentoRESUMO
An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.
RESUMO
An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.
RESUMO
The flavivirus RNA genome contains a conserved cap-1 structure, (7Me)GpppA(2'OMe)G, at the 5' end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)- and the (nucleoside-2'O)-MTases (2'O-MTase), reside in a single domain of non-structural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 2'O-MTase activity of NS5MTase of dengue virus (NS5MTase(DV)) using purified, short, capped RNA substrates ((7Me)GpppAC(n) or GpppAC(n)). NS5MTase(DV) methylated both types of substrate exclusively at the 2'O position. The efficiency of 2'O-methylation did not depend on the methylation of the N7 position. Using (7Me)GpppAC(n) and GpppAC(n) substrates of increasing chain lengths, it was found that both NS5MTase(DV) 2'O activity and substrate binding increased before reaching a plateau at n=5. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K(m) values for (7Me)GpppAC(5) and the co-substrate S-adenosyl-L-methionine (AdoMet) were determined (0.39 and 3.26 microM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 2'O-MTase activity of NS5MTase(DV) showed a low turnover of 3.25x10(-4) s(-1). Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTase(DV), which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC(50) 0.34 microM) and sinefungin (IC(50) 0.63 microM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays.
Assuntos
Vírus da Dengue/enzimologia , Metiltransferases/metabolismo , Oligonucleotídeos/metabolismo , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Inibidores Enzimáticos/farmacologia , Cinética , Metilação , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Estrutura Terciária de Proteína , S-Adenosilmetionina/metabolismoRESUMO
Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR). We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. The NS3:186-619 construct occurs as a dimer in solution and in the crystals. We show that NS3:186-619 displays both ATPase and RTPase activities, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus.
Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Vírus do Nilo Ocidental/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Viral methyltransferases are involved in the mRNA capping process, resulting in the transfer of a methyl group from S-adenosyl-L-methionine to capped RNA. Two groups of methyltransferases (MTases) are known: (guanine-N7)-methyltransferases (N7MTases), adding a methyl group onto the N7 atom of guanine, and (nucleoside-2'-O-)-methyltransferases (2'OMTases), adding a methyl group to a ribose hydroxyl. We have expressed and purified two constructs of Meaban virus (MV; genus Flavivirus) NS5 protein MTase domain (residues 1-265 and 1-293, respectively). We report here the three-dimensional structure of the shorter MTase construct in complex with the cofactor S-adenosyl-L-methionine, at 2.9 angstroms resolution. Inspection of the refined crystal structure, which highlights structural conservation of specific active site residues, together with sequence analysis and structural comparison with Dengue virus 2'OMTase, suggests that the crystallized enzyme belongs to the 2'OMTase subgroup. Enzymatic assays show that the short MV MTase construct is inactive, but the longer construct expressed can transfer a methyl group to the ribose 2'O atom of a short GpppAC(5) substrate. West Nile virus MTase domain has been recently shown to display both N7 and 2'O MTase activity on a capped RNA substrate comprising the 5'-terminal 190 nt of the West Nile virus genome. The lack of N7 MTase activity here reported for MV MTase may be related either to the small size of the capped RNA substrate, to its sequence, or to different structural properties of the C-terminal regions of West Nile virus and MV MTase-domains.
Assuntos
Flavivirus/enzimologia , Metiltransferases/química , Metiltransferases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Flavivirus/genética , Metiltransferases/genética , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
We report high-throughput structure-based virtual screening of putative Flavivirus 2'-O-methyltransferase inhibitors together with results from subsequent bioassay tests of selected compounds. Potential inhibitors for the S-adenosylmethionine binding site were explored using 2D similarity searching, pharmacophore filtering and docking. The inhibitory activities of 15 top-ranking compounds from the docking calculations were tested on a recombinant methyltransferase with the RNA substrate (7Me)GpppAC(5). Local and global docking simulations were combined to estimate the ligand selectivity for the target site. The results of the combined computational and experimental screening identified a novel inhibitor, with a previously unknown scaffold, that has an IC(50) value of 60 microM.
Assuntos
Vírus da Dengue/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Metiltransferases/biossíntese , RNA Mensageiro/biossíntese , Sítios de Ligação , Bioensaio , Vírus da Dengue/enzimologia , Vírus da Dengue/genética , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Concentração Inibidora 50 , Ligantes , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Estrutura Molecular , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Montagem de Vírus/efeitos dos fármacosRESUMO
Treatment of 2'-deoxy-3',5'-dithexyldimethylsilyl-5,6-dihydrouridine with Lawesson's reagent led to the expected C4-thiolated derivative together with a number of oxathiaphosphepane isomers which resulted from the heat reversible incorporation of an AnPS2 unit within the 2'-deoxyribose moiety explaining the subsequent anomerisation of the 5,6-dihydropyrimidine nucleosides.
Assuntos
Compostos Organotiofosforados/química , Nucleosídeos de Pirimidina/química , Tiouridina/análogos & derivados , Tiouridina/síntese química , Uridina/análogos & derivados , Uridina/química , Desoxirribose/química , Temperatura Alta , EstereoisomerismoRESUMO
Under aqueous conditions, 4-azidouracil/tetrazolo[1,5-c]pyrimidin-5(6H)-one nucleosides undergo a very efficient photochemical nitrogen elimination and ring expansion to 1,3,5-triazepin-2,4-dione nucleosides whose structure has been confirmed by X-ray crystallography. In contrast, when the photolysis was attempted under anhydrous conditions in the presence of a nucleophile, a ring contraction reaction occurred, affording 2-oxoimidazolone nucleosides. A mechanism to account for the formation of ring expansion and contraction reactions and involving a carbodiimide intermediate is proposed which is reminiscent of the known photochemical behavior of 2-azidopyridines/tetrazolo[1,5-a]pyridines.
Assuntos
Azepinas/síntese química , Nucleosídeos/síntese química , Fotoquímica/métodos , Azidas/química , Carbodi-Imidas/química , Nucleosídeos/química , Piridinas/química , Solventes , Uracila/química , ÁguaRESUMO
4-Thio oxathiaphosphepane nucleosides - undergo a rearrangement in pyridine that leads selectively to the beta anomer of the 2'-deoxy-5,6-dihydro-4-thiouridine derivative . This diastereoselective reaction proceeds through a multistep mechanism initiated by the addition of pyridine at the C1'position of - and concomitant opening of the oxathiaphosphepane. This was confirmed by the trapping of the corresponding intermediate in the closely related DMAP series. In contrast, LR thiation of in pyridine leads to a new class of modified nucleosides containing an oxathiaphospholane moiety. The quantitative conversion of into the corresponding 5,6-dihydrocytosine derivative with NH3-MeOH is also reported.
Assuntos
Desoxicitidina/análogos & derivados , Desoxiuridina/análogos & derivados , Radiação Ionizante , Tiouridina/análogos & derivados , Desoxicitidina/química , Desoxiuridina/síntese química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Tiouridina/síntese químicaRESUMO
Treatment of 4-azido-2-pyrimidinone nucleoside 1 with MeOH/K2CO3 or ammonium hydroxide led to derivatives 2 and 3, respectively. Both compounds derived from a nucleophilic addition at the modified base 2-position. These results contrast with the previously reported sensitivity of the 4-azido-2-pyrimidinone nucleoside 6-position to nucleophiles.