RESUMO
We present the synthesis and application of a molecule containing both the powerful influenza neuraminidase (NA) inhibitor phospha-oseltamivir and d-biotin, connected via an undecaethylene glycol spacer. It inhibits influenza virus neuraminidase (from the H3N2 X31 virus) in the same range as oseltamivir, with a slow off-rate, and produces a stable NA-coated surface when loaded onto streptavidin-coated biosensors. Purified X31 virus binds to these loaded biosensors with an apparent dissociation constant in the low picomolar range and binding of antibodies to the immobilized virus could be readily detected. The compound is thus a potential candidate for the selective immobilization of influenza virus in influenza diagnosis, vaccine choice, development or testing.
Assuntos
Antivirais/farmacologia , Biotina/farmacologia , Inibidores Enzimáticos/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/metabolismo , Oseltamivir/farmacologia , Antivirais/síntese química , Antivirais/química , Biotina/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Conformação Molecular , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Orthomyxoviridae/enzimologia , Oseltamivir/química , Relação Estrutura-AtividadeRESUMO
The synthesis of conjugates of phospha-oseltamivir to the well established reporter groups fluorescein and biotin and an approach to multimeric inhibitors is described. We report powerful inhibition of the influenza neuraminidase by these probes and quantify fluorescence quenching during binding of the fluorescein conjugate through titration with the neuraminidase. Thus, we show that they could be useful tools to efficiently inhibit, detect and quantify the virus and the neuraminidase in biological systems.
Assuntos
Antivirais/química , Biotina/química , Fluoresceína/química , Neuraminidase/antagonistas & inibidores , Orthomyxoviridae/enzimologia , Orthomyxoviridae/isolamento & purificação , Oseltamivir/química , Antivirais/síntese química , Antivirais/farmacologia , Biotina/síntese química , Biotina/farmacologia , Química Click , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fluoresceína/síntese química , Fluoresceína/farmacologia , Humanos , Influenza Humana/tratamento farmacológico , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológico , Oseltamivir/síntese química , Oseltamivir/farmacologia , Ligação ProteicaRESUMO
Cyclin-dependent kinase 1 (Cdk1) is thought to trigger centrosome separation in late G2 phase by phosphorylating the motor protein Eg5 at Thr927. However, the precise control mechanism of centrosome separation remains to be understood. Here, we report that in G2 phase polo-like kinase 1 (Plk1) can trigger centrosome separation independently of Cdk1. We find that Plk1 is required for both C-Nap1 displacement and for Eg5 localization on the centrosome. Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927. Nevertheless, Plk1-driven centrosome separation is slow and staggering, while Cdk1 triggers fast movement of the centrosomes. We find that actin-dependent Eg5-opposing forces slow down separation in G2 phase. Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation. Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement. Our findings implicate the modulation of MT stability in G2 and M phase as a regulatory element in the control of centrosome separation.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Centrossomo/metabolismo , Cinesinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Linhagem Celular , Humanos , Quinase 1 Polo-LikeRESUMO
A series of sialosides modified at the 4- and 9-hydroxy group were synthesised and tested for inhibition of the viral haemagglutinin-esterase activity from various Orthomyxoviruses and Coronaviruses. While no inhibition of the sialate-4-O-acetylesterases from mouse hepatitis virus strain S or sialodacryoadenitis virus was found, a 9-O-methyl derivative displayed inhibitory activity against recombinant sialate-9-O-acetylesterase from influenza C virus.
Assuntos
Acetilesterase/antagonistas & inibidores , Antivirais/química , Gammainfluenzavirus/química , Ácido N-Acetilneuramínico/análogos & derivados , Proteínas Virais de Fusão/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Acetilesterase/química , Animais , Antivirais/síntese química , Coronavirus/química , Coronavirus/enzimologia , Desenho de Fármacos , Hemaglutininas Virais/química , Gammainfluenzavirus/enzimologia , Camundongos , Ácido N-Acetilneuramínico/síntese química , Orthomyxoviridae/química , Orthomyxoviridae/enzimologia , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Especificidade por Substrato , Torovirus/química , Torovirus/enzimologia , Proteínas Virais de Fusão/química , Proteínas Virais/químicaRESUMO
We describe the synthesis of mimetics of the alpha2-3 and alpha2-6 sialogalactoside substrates of influenza neuraminidase which include the oseltamivir pharmacophore, and report the sub-nanomolar affinities for these novel neuraminidase inhibitors. The challenge of synthesizing a Phospha-Oseltamivir/Tamiphosphor monoester involving the secondary 3-hydroxy group of galactose required to mimic the alpha2-3 sialogalactoside has been overcome by palladium-promoted coupling of the oseltamivir-derived vinyl iodide with a protected galactose-3-phosphonate. The difference in binding of these two inhibitors to a given influenza neuraminidase should be a function of its alpha2-3/alpha2-6-selectivity, an important, but not yet fully understood factor in the adaptation of highly pathogenic avian influenza viruses to human hosts.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Galactose/química , Vírus da Influenza A Subtipo H1N1/enzimologia , Neuraminidase/metabolismo , Oseltamivir/química , Oseltamivir/metabolismo , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Oseltamivir/farmacologia , Ligação ProteicaRESUMO
With a Hunsdiecker-Barton iododecarboxylation strategy, we converted the carboxylate group of the oseltamivir precursor into exemplary phosphonate monoesters. In all cases, K(i) values towards influenza virus sialidase remained in the sub-nanomolar range. We have thus made valuable structural space available for the design of novel oseltamivir-based tools for influenza virus research.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Neuraminidase/metabolismo , Oseltamivir/síntese química , Oseltamivir/farmacologia , Humanos , Neuraminidase/antagonistas & inibidores , Organofosfonatos/síntese química , Organofosfonatos/química , Oseltamivir/análogos & derivadosRESUMO
A variable synthesis of a new class of sialylmimetics which provides access to pseudo-sialosides containing the successful cyclohexene motif in the sialic acid mimicking part has been developed. The D- and L-xylo cyclohexenephosphonate scaffolds allow attachment of selected aglycons or aglycon mimetics via mixed phosphonate diester strategies and some target compounds thus synthesized displayed promising inhibitory properties when tested with parasitic or bacterial sialidases.
Assuntos
Cicloexanos/química , Ésteres/química , Ésteres/farmacologia , Ácido N-Acetilneuramínico/química , Neuraminidase/antagonistas & inibidores , Ácidos Fosfóricos/química , Animais , Benzeno/química , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/enzimologia , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ésteres/síntese química , Metilação , Estrutura Molecular , Neuraminidase/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/enzimologia , Relação Estrutura-Atividade , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologiaRESUMO
Based on a strategy previously reported by us, we have synthesized D-xylo configured cyclohexenephosphonates designed to mimic the transition state of the sialidase reaction. The double bond orientation corresponds to the benchmark inhibitor Neu5Ac2en and we could selectively introduce hydroxyalkyl substituents in order to simulate the glycerol side-chain of neuraminic acid. The inhibitory activity of a set of compounds towards bacterial sialidases was tested and interesting differences in activity were found.
Assuntos
Cicloexanos/síntese química , Inibidores Enzimáticos/síntese química , Glicerol/síntese química , Neuraminidase/antagonistas & inibidores , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/enzimologia , Clostridium perfringens/crescimento & desenvolvimento , Cicloexanos/farmacologia , Cicloexenos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Glicerol/farmacologia , Neuraminidase/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/enzimologia , Salmonella typhimurium/crescimento & desenvolvimento , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/enzimologia , Vibrio cholerae/crescimento & desenvolvimentoAssuntos
Lectinas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA de Plantas/genética , Escherichia coli/genética , Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutação , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Reaction of gluconolactone 2 with allylmagnesium bromide at low temperatures afforded ketopyranose 3, which could easily be converted into open-chain ketoses (R)-6 and (S)-6. Their reaction with lithioacetylide 9 afforded propargylic alcohol derivatives (R)-10 and (S)-10, which could not be cyclized directly to the desired C-ketosides. They were converted by standard procedures into (R)-14 and (S)-14 and then into dicobalthexacarbonyl complexes (R)-16 and (S)-16. A facile acid-catalyzed ring closure gave the desired C-ketosides (R)-18 α/ß and (S)-18α/ß, respectively, in different ratios. In order to demonstrate that removal of the protective groups and hydrogenation of the CC triple bond proceed smoothly, (R)-18 α was transformed into the deprotected target molecule (R)-1 α. For the assignment of the new chiral centers at C-2/2' and at C-8, (S)-18α was transformed into azido derivative (S)-22α, which underwent intramolecular cycloaddition to afford the spiro derivative (S)-25α. Because of the conformational constraints in this molecule, unequivocal configurational assignment was possible with the help of NMR data.