RESUMEN
Development of new antimycobacterial agents for Mycobacterium avium complex (MAC) infections is important particularly for persons coinfected with human immunodeficiency virus. The objectives of this study were to evaluate the in vitro activity of 2, 4-diamino-5-methyl-5-deazapteridines (DMDPs) against MAC and to assess their activities against MAC dihydrofolate reductase recombinant enzyme (rDHFR). Seventy-seven DMDP derivatives were evaluated initially for in vitro activity against one to three strains of MAC (NJ168, NJ211, and/or NJ3404). MICs were determined with 10-fold dilutions of drug and a colorimetric (Alamar Blue) microdilution broth assay. MAC rDHFR 50% inhibitory concentrations versus those of human rDHFR were also determined. Substitutions at position 5 of the pteridine moiety included -CH(3), -CH(2)CH(3), and -CH(2)OCH(3) groups. Additionally, different substituted and unsubstituted aryl groups were linked at position 6 through a two-atom bridge of either -CH(2)NH, -CH(2)N(CH(3)), -CH(2)CH(2), or -CH(2)S. All but 4 of the 77 derivatives were active against MAC NJ168 at concentrations of < or =13 microg/ml. Depending on the MAC strain used, 81 to 87% had MICs of < or =1.3 microg/ml. Twenty-one derivatives were >100-fold more active against MAC rDHFR than against human rDHFR. In general, selectivity was dependent on the composition of the two-atom bridge at position 6 and the attached aryl group with substitutions at the 2' and 5' positions on the phenyl ring. Using this assessment, a rational synthetic approach was implemented that resulted in a DMDP derivative that had significant intracellular activity against a MAC-infected Mono Mac 6 monocytic cell line. These results demonstrate that it is possible to synthesize pteridine derivatives that have selective activity against MAC.
Asunto(s)
Antiinfecciosos/síntesis química , Antagonistas del Ácido Fólico/síntesis química , Mycobacterium/efectos de los fármacos , Mycobacterium/enzimología , Pteridinas/síntesis química , Pirimidinas/síntesis química , Tetrahidrofolato Deshidrogenasa/metabolismo , Antibacterianos , Antiinfecciosos/farmacología , Línea Celular , Supervivencia Celular , Recuento de Colonia Microbiana , Antagonistas del Ácido Fólico/farmacología , Humanos , Macrófagos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Monocitos/efectos de los fármacos , Monocitos/microbiología , Pteridinas/farmacología , Pirimidinas/farmacología , Proteínas Recombinantes/farmacología , Relación Estructura-ActividadRESUMEN
Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing. Western blotting of total T. gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed. Both form enzymatically active homotetramers when overexpressed in Escherichia coli. The specific activity of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-expressed in E. coli, HGPRT-I.HGPRT-II heterotetramers form. The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT-II. Mass spectrometric analysis of cross-linked homo- and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I.HGPRT-II and suggests that the predominant heterotetramer consists of one HGPRT-I subunit and three HGPRT-II subunits. The implications of this finding are discussed.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/química , Isoenzimas/química , Toxoplasma/enzimología , Animales , Biopolímeros , Western Blotting , Cromatografía en Gel , Escherichia coli/genética , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/aislamiento & purificación , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Dihydrofolate reductase is an essential bacterial enzyme necessary for the maintenance of intracellular folate pools in a biochemically active reduced state. In this report, the Mycobacterium avium folA gene was identified by functional genetic complementation, sequenced, and expressed for the first time. It has an open reading frame of 543 bp with a G + C content of 73%. The translated polypeptide sequence shows 58% identity to the consensus sequence of the conserved regions from eight other bacterial dihydrofolate reductases. Recombinant M. avium dihydrofolate reductase was expressed actively in Escherichia coli, and SDS-PAGE analysis revealed a 20 kDa species, agreeable with that predicted from the polypeptide sequence:
Asunto(s)
Genes Bacterianos , Complejo Mycobacterium avium/enzimología , Complejo Mycobacterium avium/genética , Tetrahidrofolato Deshidrogenasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Cartilla de ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Recent reports indicate that polyclonal infections may play an important role in multiple drug resistance in Mycobacterium avium infections. We report here on the isolation of a single M. avium strain that appeared to have smooth colony morphology upon initial isolation on a Lowenstein-Jensen slant. Primary subculture onto Middlebrook 7H10, however, revealed three distinct morphotypes representing smooth opaque (SmO), smooth transparent (SmT), and rough (Rg) colony morphologies. All three morphotypes were identified as M. avium by standard biochemical procedures, Genprobe analysis, and mycolic acid patterns. Subsequent restriction fragment length polymorphism analysis, using SalI- and PvuII-digested genomic DNA, revealed identical patterns for hybridization with the IS1245 probe. Thin-layer chromatographic analysis of lipids from the three morphotypes revealed that only the SmT morphotype possessed what appeared to be lipid components similar to, but unlike, previously described serovar-specific glycopeptidolipid antigens. Further analysis of internally radiolabeled deacylated lipids from the SmT morphotype, by high-performance liquid chromatography and thin-layer chromatography, disclosed that some of these components can be internally radiolabeled with [14C] phenylalanine and [14C]mannose. These results suggest that these components are structurally similar to previously described glycopeptidolipid antigens. This is apparently the first report of a monoclonal infection involving a single strain of M. avium presenting with all three colony morphotypes, SmO, SmT, and Rg.