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1.
J Med Chem ; 45(1): 1-18, 2002 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-11754575

RESUMEN

The stereospecific synthesis of a series of alpha-methylpyrrolidine-5,5-trans-lactam inhibitors of human cytomegalovirus (HCMV) protease is described. Examination of the SAR in this series has defined the size and chirality of the alpha-substituent, optimized the acyl substituent on the lactam nitrogen, and defined the steric constraint of this functionality. The SAR of the functionality on the pyrrolidine nitrogen of the trans-lactam has been investigated, and this has led to the discovery of potent serine protease inhibitors that are highly selective for the viral enzyme over the mammalian enzymes elastase, thrombin, and acetylcholine esterase. The mechanism of action of our lead compounds has been established by mass spectrometry, and enzymatic degradation of HCMV deltaAla protease acylated with these inhibitors showed that Ser 132 is the active site nucleophile. The crystal structure of HCMV protease was obtained and used to model the conformationally restricted, chiral (S)-proline-alpha-methyl-5,5-trans-lactams into the active site groove of the enzyme, enabling us to direct and rationalize the SAR in this series. The activity against HCMV deltaAla protease is the greatest with inhibitors based on the dansyl-(S)-proline alpha-methyl-5,5-trans-lactam template, which have low nanomolar activity against the viral enzyme.


Asunto(s)
Antivirales/síntesis química , Citomegalovirus/química , Lactamas/síntesis química , Inhibidores de Proteasas/síntesis química , Pirroles/síntesis química , Serina Endopeptidasas/química , Antivirales/química , Sitios de Unión , Cristalografía por Rayos X , Lactamas/química , Espectrometría de Masas , Modelos Moleculares , Inhibidores de Proteasas/química , Pirroles/química , Estereoisomerismo , Relación Estructura-Actividad
2.
J Med Chem ; 46(21): 4428-49, 2003 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-14521407

RESUMEN

A series of chiral, (S)-proline-alpha-methylpyrrolidine-5,5-trans-lactam serine protease inhibitors has been developed as antivirals of human cytomegalovirus (HCMV). The SAR of the functionality on the proline nitrogen has shown that derivatives of para-substituted phenyl ureas > para-substituted phenyl sulfonamides > para-substituted phenyl carboxamide for activity against HCMV deltaAla protease, producing para-substituted phenyl ureas with single figure nM potency (K(i)) against the viral enzyme. The SAR of the functionality on the lactam nitrogen has defined the steric and electronic requirements for high human plasma stability while retaining good activity against HCMV protease. The combination of high potency against HCMV deltaAla protease and high human plasma stability has produced compounds with significant in vitro antiviral activity against human cytomegalovirus with the 6-hydroxymethyl benzothiazole derivative 72 being equivalent in potency to ganciclovir. The parent benzothiazole 56 had good pharmacokinetics in dogs with 29% bioavailability and good brain and ocular penetration in guinea pigs.


Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/enzimología , Lactamas/síntesis química , Lactamas/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Pirroles/síntesis química , Pirroles/farmacología , Serina Endopeptidasas/metabolismo , Animales , Antivirales/sangre , Disponibilidad Biológica , Encéfalo/metabolismo , Células Cultivadas , Perros , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática , Ojo/metabolismo , Ganciclovir/farmacología , Cobayas , Semivida , Humanos , Indicadores y Reactivos , Cinética , Espectrometría de Masas , Modelos Moleculares , Inhibidores de Proteasas/sangre , Relación Estructura-Actividad , Especificidad por Sustrato
3.
Methods ; 31(3): 199-206, 2003 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-14511952

RESUMEN

Delivery of antigen-encoding genes by nucleic acid vaccine vectors offers tremendous advantages in power and flexibility over conventional antigen delivery systems. Of the many forms of nucleic acid vaccine that can be constructed, circular DNA plasmids are the simplest. In this article, we consider the components that make up a generic DNA plasmid vaccine. We discuss some of the issues encountered when optimizing these vectors to exploit their potential for in vivo expression and presentation of antigens and thereby maximizing the immune response.


Asunto(s)
Vacunas de ADN/genética , Secuencia de Aminoácidos , Antígenos/genética , Secuencia de Bases , Expresión Génica , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Plásmidos/metabolismo , Vacunas de ADN/inmunología
4.
J Med Virol ; 69(2): 240-50, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12683414

RESUMEN

Infection of Balb/c mice with murine cytomegalovirus (MCMV) has been used extensively as a model system with which to study host mechanisms of immunity to cytomegaloviruses. In this model, the cytotoxic T-lymphocyte (CTL) response crucial for clearing infected cells is dominated by CTLs specific for the MCMV nonapeptide 168YPHFMPTNL176 encoded by the immediate-early 1 (IE-1) gene. The intradermal injection of plasmid pcDNA89 encoding IE-1 has been shown to offer some protection from viral challenge. In the present studies, the protective efficacy of immunisation with pcDNA89 given by intradermal injection was compared with particle-mediated DNA delivery (PMDD) and contrasted with that induced by injection with the K181 MCMV strain and with temperature-sensitive mutants (tsm) derived from the K181 strain. Modest protection was afforded by pcDNA89 immunisation given by PMDD, but none was observed after intradermal injection. PMDD immunisation induced a frequency of 168YPHFMPTNL176-specific interferon-gamma (IFN-gamma)-secreting splenocytes, which was equivalent to that after K181 infection and significantly higher than tsm immunisation. Whereas tsm-immunised mice were completely protected from MCMV challenge, PMDD-immunised mice were only weakly protected. Tsm immunisation protected mice completely against challenge with natural isolates having sequence variation in the IE-1 nonapeptide, while PMDD-immunised mice were weakly protected from isolates encoding 168YPHFMPTNL176 and were not protected against isolates encoding 168YPHFMPPSL176 or 168YLDFMPPNL176. Thus, while IE-1-specific IFN-gamma-secreting splenocytes do contribute to immunity from MCMV challenge, their presence in isolation is insufficient to provide complete protection and they may not be involved in the protection observed against MCMV isolates having IE-1 sequence variation.


Asunto(s)
Infecciones por Herpesviridae/prevención & control , Proteínas Inmediatas-Precoces/inmunología , Interferón gamma/metabolismo , Muromegalovirus/inmunología , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Transactivadores/inmunología , Células 3T3 , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales , Vacunas contra Citomegalovirus/administración & dosificación , Vacunas contra Citomegalovirus/genética , Vacunas contra Citomegalovirus/inmunología , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/genética , Inmunización , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Muromegalovirus/genética , Muromegalovirus/patogenicidad , Péptidos/química , Bazo/citología , Bazo/inmunología , Transactivadores/química , Transactivadores/genética , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología
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