RESUMO
The activity of pleconaril in cell culture against prototypic enterovirus strains and 215 clinical isolates of the most commonly isolated enterovirus serotypes was examined. The latter viruses were isolated by the Centers for Disease Control and Prevention during the 1970s and 1980s from clinically ill subjects. Pleconaril at a concentration of =0.03 microM inhibited the replication of 50% of all clinical isolates tested. Ninety percent of the isolates were inhibited at a drug concentration of =0.18 microM. The most sensitive serotype, echovirus serotype 11, was also the most prevalent enterovirus in the United States from 1970 to 1983. Pleconaril was further tested for oral activity in three animal models of lethal enterovirus infection: coxsackievirus serotype A9 infection in suckling mice, coxsackievirus serotype A21 strain Kenny infection in weanling mice, and coxsackievirus serotype B3 strain M infection in adult mice. Treatment with pleconaril increased the survival rate in all three models for both prophylactic and therapeutic dosing regimens. Moreover, pleconaril dramatically reduced virus levels in target tissues of coxsackievirus serotype B3 strain M-infected animals. Pleconaril represents a promising new drug candidate for potential use in the treatment of human enteroviral infections.
Assuntos
Antivirais/uso terapêutico , Infecções por Coxsackievirus/tratamento farmacológico , Enterovirus/efeitos dos fármacos , Oxidiazóis/uso terapêutico , Animais , Animais Recém-Nascidos , Animais Lactentes , Antivirais/farmacocinética , Antivirais/farmacologia , Células Cultivadas , Infecções por Coxsackievirus/virologia , Enterovirus/classificação , Enterovirus/isolamento & purificação , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Oxidiazóis/farmacocinética , Oxidiazóis/farmacologia , Oxazóis , Sorotipagem , Replicação Viral/efeitos dos fármacosRESUMO
Pleconaril (VP 63843) is a novel orally bioavailable small molecule with broad antipicornavirus (enterovirus and rhinovirus) activity. Ten independently derived pleconaril-resistant variants of coxsackievirus B3 were isolated from cell culture. The molecular basis of drug resistance and the biologic properties of the drug-resistant viruses were investigated. RNA sequence analysis revealed amino acid changes in the drug-binding pocket of the resistant variants. Thermal stability studies showed the drug-resistant viruses to be significantly less stable than wild type virus. When evaluated in a murine model in which wild type virus infection is 100% lethal, the drug-resistant viruses showed attenuated virulence with both reduced mortality and delayed time to death. Virus titers in heart and spleen were dramatically lower in drug-resistant virus-infected mice than in wild type virus-infected animals. The study results indicate that pleconaril-resistant virus variants are attenuated and significantly less virulent than drug-sensitive wild type virus.
Assuntos
Antivirais/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/genética , Enterovirus Humano B/patogenicidade , Oxidiazóis/farmacologia , Animais , Sítios de Ligação/genética , Capsídeo/genética , Proteínas do Capsídeo , Resistência Microbiana a Medicamentos , Variação Genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxazóis , RNA Viral/genética , Análise de Sequência de RNARESUMO
The nucleotide sequences of rbsD, rbsA, and rbsC have been determined. These genes encode components of the high affinity ribose transport system in Escherichia coli, and together with the sequences of rbsB (Groarke, J.M., Mahoney, W.C., Hope, J.N., Furlong, C.E., Robb, F.T., Zalkin, H., and Hermodson, M.A. (1983) J. Biol. Chem. 258, 12952-12956) and rbsK (Hope, J.N., Bell, A.W., Hermodson, M.A., and Groarke, J.M. (1986) J. Biol. Chem. 261, 7663-7668), they complete the nucleotide sequence of the first five genes of the rbs operon. Nuclease S1 mapping places the transcriptional start site for the operon 29 base pairs upstream from the most likely translational start site for rbsD. The open reading frames of rbsD, rbsA, and rbsC encode proteins of 139, 501, and 321 amino acid residues, respectively. The character of the proteins varies widely, from very hydrophilic for the rbsA product to exceedingly hydrophobic for the rbsC product. The intercistronic spaces between the three genes are very short, with the stop codons of the upstream genes overlapping the ribosome-binding sites of the downstream genes. This may imply translational control of expression of these genes, the products of which presumably form a membrane-bound transport complex.
Assuntos
Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Genes , Proteínas Periplásmicas de Ligação , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/isolamento & purificação , Membrana Celular , Enzimas de Restrição do DNA , Endonucleases , Escherichia coli/metabolismo , Peso Molecular , Óperon , Ribose/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
The nucleotide sequence of a 1455-base pair TaqI-HinfI fragment of the rbs operon of Escherichia coli K12 has been determined. It includes the 3' terminus of rbsB (the gene for ribose-binding protein) and the entire rbsK gene, encoding ribokinase. Potential consensus promoter sequences and a stable stem-loop structure are present in the rbsB-rbsK intercistronic region. The regulatory significance of these sequence features is discussed with respect to the rbs operon. rbsK has been cloned downstream from the Serratia marcescens trp promoter on a multicopy plasmid. Cells harboring this plasmid, when grown on minimal ribose plus ampicillin, express ribokinase at the level of 2% of the soluble protein, and induction with indoleacrylic acid raises ribokinase levels another 8-fold. Ribokinase has been purified to homogeneity (216 mumol/min/mg) from a strain harboring this plasmid. Protein sequence analyses of peptides generated by cyanogen bromide cleavage and o-iodosobenzoic acid cleavage confirmed the translation initiation site and the reading frame of the DNA sequence. Amino acid compositions of native ribokinase and the C-terminal dodecapeptide agree with the predicted amino acid compositions, confirming the accuracy of the DNA sequence and the translation termination site.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Genes , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/genética , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , Escherichia coli/enzimologia , Óperon , Fosfotransferases/isolamento & purificação , PlasmídeosRESUMO
Ribose-binding protein is exported to the periplasmic compartment of Escherichia coli by a process that involves proteolytic cleavage of an amino-terminal extension of amino acids from the precursor form of the protein. In a collection of mutants isolated as defective in the Rbs transport system, a strain was identified that contained only precursor ribose-binding protein, none of which was exported to its normal location in the periplasm. The mutated rbsB contained a base substitution that results in a change of leucine to a proline at position-17 in the signal sequence. A pseudorevertant of the mutant contained proteolytically processed, active ribose-binding protein in the periplasm. The pseudorevertant rbsB carried a second mutation: serine at position-15 in the signal sequence was changed to phenylalanine. Isolation of a signal sequence mutant and a corresponding pseudorevertant without specific selection or site-directed mutagenesis emphasizes the possibility of obtaining export mutants without the use of procedures that could bias or limit the range of mutations found. Explanation of the extreme phenotype of the mutant and the effective correction of that phenotype in the pseudorevertant requires extension of current notions of critical features of signal sequences.
Assuntos
Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Mutação , Proteínas Periplásmicas de Ligação , Ribose/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/isolamento & purificação , Colífagos/genética , Fragmentos de Peptídeos/análise , Plasmídeos , Seleção Genética , Transdução GenéticaRESUMO
The amino acid sequence of the D-ribose-binding protein from Escherichia coli K12 was determined from the DNA sequence of the gene. Protein sequence analyses covering 80% of the protein were consistent with the sequence deduced from the DNA. The mature binding protein has 271 amino acid residues and shows substantial homology to D-galactose-binding protein. A signal peptide sequence of 23 or 25 residues was also deduced from the DNA sequence. It shows the characteristic features of prokaryotic signal peptides.