RESUMEN
Neovascular age-related macular degeneration (NVAMD) is a prevalent cause of vision loss. Intraocular injections of VEGF-neutralizing proteins provide benefit, but many patients require frequent injections for a prolonged period. Benefits are often lost over time due to lapses in treatment. New treatments that sustain anti-angiogenic activity are needed. This study tested the safety and expression profile of a lentiviral Equine Infectious Anemia Virus (EIAV) vector expressing endostatin and angiostatin (RetinoStat®). Patients with advanced NVAMD were enrolled at three centers in the United States, and the study eye received a subretinal injection of 2.4 × 104 (n = 3), 2.4 × 105 (n = 3), or 8.0 × 105 transduction units (TU; n = 15). Each of the doses was well-tolerated with no dose-limiting toxicities. There was little or no ocular inflammation. There was one procedure-related serious adverse event (AE), a macular hole, which was managed without difficulty and resolved. There was a vector dose-related increase in aqueous humor levels of endostatin and angiostatin with high reproducibility among subjects within cohorts. Mean levels of endostatin and angiostatin peaked between 12 and 24 weeks after injection of 2.4 × 105 TU or 8.0 × 105 TU at 57-81 ng/mL for endostatin and 15-27 ng/mL for angiostatin, and remained stable through the last measurement at week 48. Long-term follow-up demonstrated expression was maintained at last measurement (2.5 years in eight subjects and >4 years in two subjects). Despite an apparent reduction in fluorescein angiographic leakage that broadly correlated with the expression levels in the majority of patients, only one subject showed convincing evidence of anti-permeability activity in these late-stage patients. There was no significant change in mean lesion size in subjects injected with 8.0 × 105 TU. These data demonstrate that EIAV vectors provide a safe platform with robust and sustained transgene expression for ocular gene therapy.
Asunto(s)
Endostatinas/genética , Terapia Genética , Vectores Genéticos/administración & dosificación , Lentivirus/genética , Degeneración Macular/terapia , Anciano , Anciano de 80 o más Años , Angiostatinas/genética , Estudios de Cohortes , Femenino , Humanos , Inyecciones Intraoculares , Degeneración Macular/genética , Masculino , Dosis Máxima ToleradaRESUMEN
The synthesis and inhibitory potencies of a novel series of ß-amino alcohols, based on the hit-compound 3-[3'-(4''-cyclopent-2'''-en-1'''-ylphenoxy)-2'-hydroxypropyl]-5,5 dimethylimidazolidine-2,4-dione as specific inhibitors of mycobacterial N-acetyltransferase (NAT) enzymes are reported. Effects of synthesised compounds on growth of Mycobacterium tuberculosis have been determined.
Asunto(s)
Amino Alcoholes/química , Antituberculosos/química , Arilamina N-Acetiltransferasa/antagonistas & inhibidores , Amino Alcoholes/síntesis química , Amino Alcoholes/farmacología , Antituberculosos/síntesis química , Antituberculosos/farmacología , Arilamina N-Acetiltransferasa/metabolismo , Sitios de Unión , Dominio Catalítico , Simulación por Computador , Mycobacterium/efectos de los fármacos , Mycobacterium/enzimología , Relación Estructura-ActividadRESUMEN
New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules-triazoles-in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.
Asunto(s)
Antituberculosos/química , Antituberculosos/farmacología , Arilamina N-Acetiltransferasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Triazoles/química , Triazoles/farmacología , Antituberculosos/aislamiento & purificación , Arilamina N-Acetiltransferasa/química , Inhibidores Enzimáticos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Humanos , Mycobacterium bovis/efectos de los fármacos , Mycobacterium bovis/enzimología , Mycobacterium bovis/genética , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Conformación Proteica , Relación Estructura-Actividad , Triazoles/aislamiento & purificaciónRESUMEN
Arylamine N-acetyltransferase (NAT) enzymes are widespread in nature. They serve to acetylate xenobiotics and/or endogenous substrates using acetyl coenzyme A (CoA) as a cofactor. Conservation of the architecture of the NAT enzyme family from mammals to bacteria has been demonstrated by a series of prokaryotic NAT structures, together with the recently reported structure of human NAT1. We report here the cloning, purification, kinetic characterisation and crystallographic structure determination of NAT from Mycobacterium marinum, a close relative of the pathogenic Mycobacterium tuberculosis. We have also determined the structure of M. marinum NAT in complex with CoA, shedding the first light on cofactor recognition in prokaryotic NATs. Surprisingly, the principal CoA recognition site in M. marinum NAT is located some 30 A from the site of CoA recognition in the recently deposited structure of human NAT2 bound to CoA. The structure explains the Ping-Pong Bi-Bi reaction mechanism of NAT enzymes and suggests mechanisms by which the acetylated enzyme intermediate may be protected. Recognition of CoA in a much wider groove in prokaryotic NATs suggests that this subfamily may accommodate larger substrates than is the case for human NATs and may assist in the identification of potential endogenous substrates. It also suggests the cofactor-binding site as a unique subsite to target in drug design directed against NAT in mycobacteria.
Asunto(s)
Arilamina N-Acetiltransferasa/metabolismo , Coenzima A/metabolismo , Evolución Molecular , Variación Genética , Células Procariotas/enzimología , Secuencia de Aminoácidos , Arilamina N-Acetiltransferasa/química , Arilamina N-Acetiltransferasa/genética , Arilamina N-Acetiltransferasa/aislamiento & purificación , Clonación Molecular , Coenzima A/análisis , Secuencia Conservada , Cristalografía por Rayos X , Dimerización , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Espectrometría de Masas , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium/enzimología , Sistemas de Lectura Abierta , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Rhodococcus sp. strain RHA1, a soil bacterium related to Mycobacterium tuberculosis, degrades an exceptionally broad range of organic compounds. Transcriptomic analysis of cholesterol-grown RHA1 revealed a catabolic pathway predicted to proceed via 4-androstene-3,17-dione and 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA). Inactivation of each of the hsaC, supAB, and mce4 genes in RHA1 substantiated their roles in cholesterol catabolism. Moreover, the hsaC(-) mutant accumulated 3,4-DHSA, indicating that HsaC(RHA1), formerly annotated as a biphenyl-degrading dioxygenase, catalyzes the oxygenolytic cleavage of steroid ring A. Bioinformatic analyses revealed that 51 rhodococcal genes specifically expressed during growth on cholesterol, including all predicted to specify the catabolism of rings A and B, are conserved within an 82-gene cluster in M. tuberculosis H37Rv and Mycobacterium bovis bacillus Calmette-Guérin. M. bovis bacillus Calmette-Guérin grew on cholesterol, and hsaC and kshA were up-regulated under these conditions. Heterologously produced HsaC(H37Rv) and HsaD(H37Rv) transformed 3,4-DHSA and its ring-cleaved product, respectively, with apparent specificities approximately 40-fold higher than for the corresponding biphenyl metabolites. Overall, we annotated 28 RHA1 genes and proposed physiological roles for a similar number of mycobacterial genes. During survival of M. tuberculosis in the macrophage, these genes are specifically expressed, and many appear to be essential. We have delineated a complete suite of genes necessary for microbial steroid degradation, and pathogenic mycobacteria have been shown to catabolize cholesterol. The results suggest that cholesterol metabolism is central to M. tuberculosis's unusual ability to survive in macrophages and provide insights into potential targets for novel therapeutics.
Asunto(s)
Colesterol/genética , Colesterol/metabolismo , Hidrolasas/genética , Macrófagos/microbiología , Familia de Multigenes , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/genética , Rhodococcus/genética , Hidrolasas/metabolismo , Mycobacterium tuberculosis/metabolismo , ARN Mensajero/metabolismo , Rhodococcus/metabolismoRESUMEN
Mycobacterium bovis BCG and Mycobacterium tuberculosis possess a single arylamine N-acetyltransferase whose gene is predicted to occur within a six-gene operon. Deletion of the nat gene caused an extended lag phase in M. bovis BCG and a cell morphology associated with an altered pattern of cell wall mycolates. Analysis of cDNA from M. bovis BCG shows that during in vitro growth all the genes in the putative nat operon are expressed and the open reading frames are contiguous, supporting the existence of an operon. Two genes in the operon, Mb3599c and Mb3600c, are predicted to encode homologues of enzymes annotated as a 2,3-dihydroxybiphenyl 1,2-dioxygenase (bphC5) and a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD2), respectively, in Rhodococcus RHA1. As predicted, M. bovis BCG cell lysates metabolized the BphC substrate 2,3-dihydroxybiphenyl (2,3-DHB) to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), a BphD substrate, which was subsequently hydrolysed. Immunoprecipitation of the BphD homologue from these lysates led to an accumulation of HOPDA. M. bovis BCG growth on both solid and liquid media was inhibited with either 2,3-DHB or an inhibitor of BphC, 3-chlorocatechol (3-CC). In addition, incubation with 2,3-DHB affects the lipid composition of the cell wall resulting in a diminished level of mycolates and an altered cell morphology similar to the Deltanat strain. We propose the enzymes encoded by the putative operon have a similar endogenous role to that of the NAT enzyme and are part of a pathway important for cell wall synthesis.
Asunto(s)
Arilamina N-Acetiltransferasa/genética , Proteínas Bacterianas/metabolismo , Mycobacterium bovis/genética , Operón , Arilamina N-Acetiltransferasa/metabolismo , Proteínas Bacterianas/genética , Compuestos de Bifenilo/química , Compuestos de Bifenilo/metabolismo , Forma de la Célula , Pared Celular/química , Biología Computacional , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Hidrolasas/genética , Hidrolasas/metabolismo , Lípidos/química , Estructura Molecular , Mycobacterium bovis/enzimología , Mycobacterium bovis/ultraestructura , Ácidos Micólicos/química , Ácidos Micólicos/metabolismo , Sistemas de Lectura Abierta , Regiones Promotoras GenéticasRESUMEN
Mycolic acids represent a major component of the unique cell wall of mycobacteria. Mycolic acid biosynthesis is inhibited by isoniazid, a key frontline antitubercular drug that is inactivated by mycobacterial and human arylamine N-acetyltransferase (NAT). We show that an in-frame deletion of Mycobacterium bovis BCG nat results in delayed entry into log phase, altered morphology, altered cell wall lipid composition, and increased intracellular killing by macrophages. In particular, deletion of nat perturbs biosynthesis of mycolic acids and their derivatives and increases susceptibility of M. bovis BCG to antibiotics that permeate the cell wall. Phenotypic traits are fully complemented by introduction of Mycobacterium tuberculosis nat. We infer from our findings that NAT is critical to normal mycolic acid synthesis and hence other derivative cell wall components and represents a novel target for antituberculosis therapy. In addition, this is the first report of an endogenous role for NAT in mycobacteria.