RESUMEN
Studies indicate that MAO-B is induced in peripheral inflammatory diseases. To target peripheral tissues using MAO-B inhibitors that do not permeate the blood-brain barrier (BBB) the MAO-B-selective inhibitor deprenyl was remodeled by replacing the terminal acetylene with a CO2H function, and incorporating a para-OCH2Ar motif (compounds 10a-s). Further, in compound 32 the C-2 side chain corresponded to CH2CN. In vitro, 10c, 10j, 10k, and 32 were identified as potent reversible MAO-B inhibitors, and all four compounds were more stable than deprenyl in plasma, liver microsomal, and hepatocyte stability assays. In vivo, they demonstrated greater plasma bioavailability. Assessment of in vitro BBB permeability showed that compound 10k is a P-glycoprotein (P-gp) substrate and 10j displayed mild interaction. Importantly, compounds 10c, 10j, 10k, and 32 displayed significantly reduced BBB permeability after intravenous, subcutaneous, and oral administration. These polar MAO-B inhibitors are pertinent leads for evaluation of efficacy in noncentral nervous system (CNS) inflammatory disease models.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/metabolismo , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Perros , Desarrollo de Medicamentos , Humanos , Células de Riñón Canino Madin Darby , Estructura Molecular , Inhibidores de la Monoaminooxidasa/síntesis química , Inhibidores de la Monoaminooxidasa/química , Enfermedades del Sistema Nervioso/metabolismoRESUMEN
Four potential binding sites for LexA were identified upstream of the Mycobacterium tuberculosis lexA gene. A mutational analysis of these sites in a lexA-lacZ reporter construct revealed that only one of these SOS boxes was required for DNA-damage-mediated regulation of lexA expression. A novel DNA-damage-inducible gene, Rv2719c, was identified that was divergently transcribed relative to lexA; the other three SOS boxes were found to be involved in regulating expression of this novel mycobacterial-specific gene. The SOS boxes lay in the respective promoter regions of the genes that they regulated.
Asunto(s)
Proteínas Bacterianas/genética , Daño del ADN/fisiología , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Respuesta SOS en Genética/fisiología , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano/análisis , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/fisiología , Respuesta SOS en Genética/genética , Homología de Secuencia de AminoácidoRESUMEN
Annually, Mycobacterium tuberculosis is the cause of approximately three million deaths worldwide. It would appear that currently available therapies for this disease are inadequate. The identification of genes involved in mycobacterial virulence will facilitate the design of new prophylactic and therapeutic interventions. A method for high-resolution comparison of bacterial genomes has been developed to facilitate the identification of genes possibly involved in the virulence of clinically relevant mycobacteria. This 'two-dimensional bacterial genome display' (2DBGD) method utilizes two-dimensional DNA electrophoresis to separate, on the basis of size and G+C content, genomic fragments generated with different restriction endonucleases. The use of this method to identify genomic differences between species, strains and, most importantly, isogenic mutants of mycobacteria is reported. That 2DBGD can be used to identify differences resulting from either insertional mutagenesis using a gentamicin-resistance gene or from a frameshift mutation is demonstrated.
Asunto(s)
Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , Genoma Bacteriano , Mutación , Mycobacterium/clasificación , Proteínas Bacterianas/genética , Composición de Base , Electroforesis en Gel Bidimensional/métodos , Humanos , Mycobacterium/genética , Infecciones por Mycobacterium/microbiología , Complejo Mycobacterium avium/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
High-resolution comparison of bacterial genomes facilitates the identification of the genetic changes responsible for clinically relevant phenotypes. For this purpose we have established a method for the display and comparison of high G+C bacterial genomes in two dimensions. Here we describe the application of two-dimensional bacterial genomic display to resolve the genomes of Bordetella pertussis, Mycobacterium avium and Mycobacterium tuberculosis, and its utility in strain comparison and detection of insertion and substitution mutations.
Asunto(s)
Bacterias/genética , ADN Bacteriano/genética , Electroforesis en Gel Bidimensional/métodos , Genoma Bacteriano , Bacterias/patogenicidad , Composición de Base , Bordetella pertussis/genética , Análisis Mutacional de ADN , ADN Bacteriano/química , Mutación , Mycobacterium avium/genética , Mycobacterium tuberculosis/genética , Reproducibilidad de los Resultados , Especificidad de la Especie , Virulencia/genéticaRESUMEN
The bases of the mycobacterial SOS box important for LexA binding were determined by replacing each base with every other and examining the effect on the induction of a reporter gene following DNA damage. This analysis revealed that the SOS box was longer than originally thought by 2 bp in each half of the palindromic site. A search of the Mycobacterium tuberculosis genome sequence with the new consensus, TCGAAC(N)(4)GTTCGA, identified 4 sites which were perfect matches and 12 sites with a single mismatch which were predicted to bind LexA. Genes which could potentially be regulated by these SOS boxes were ascertained from their positions relative to the sites. Examination of expression data for these genes following DNA damage identified 12 new genes which are most likely regulated by LexA as well as the known M. tuberculosis DNA damage-inducible genes recA, lexA, and ruvC. Of these 12 genes, only 2 have a predicted function: dnaE2, a component of DNA polymerase III, and linB, which is similar to 1,3,4,6-tetrachloro-1,4-cylcohexadiene hydrolase. Curiously, of the remaining 10 genes predicted to be LexA regulated, 7 are members of the M. tuberculosis 13E12 repeat family, which has some of the characteristics of mobile elements.