RESUMEN
Intercellular adhesion molecule-1 (ICAM-1) is up-regulated on numerous cell types in response to inflammatory cytokines. Tumor necrosis factor-alpha (TNF-alpha) activates the ICAM-1 promoter through a variant nuclear factor-kappaB (NF-kappaB) site at -187/-178 bp upstream of the transcription start site. In this investigation, we provide biochemical and functional evidence that an adjacent CCAAT/enhancer binding protein (C/EBP) site and this variant NF-kappaB site define a composite element for activation of the ICAM-1 promoter in certain cell lines. We detected an endogenous TNF-alpha-inducible DNA-protein complex in nuclear extracts from A549, HeLa, and EVC304 cells that contained both RelA and C/EBPbeta but not other family members. Complex formation required intact C/EBP and NF-kappaB sites and was absolutely dependent on translocation of RelA into the nucleus. Complex formation and cooperative binding were also demonstrated using recombinant proteins, and as above, both binding sites were necessary. Interestingly, the RelA/C/EBPbeta complex was not detected in either Jurkat or Raji cells, indicating cell type specificity. Functional studies with various reporter gene constructs revealed that both binding sites were required for maximal activation of the ICAM-1 promoter in response to TNF-alpha and for synergistic activation by RelA and C/EBPbeta. This is the first detailed analysis of how RelA and C/EBPbeta function to regulate ICAM-1 expression, and this study has important implications for how this gene is activated in specific cell types.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Molécula 1 de Adhesión Intercelular/genética , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Sitios de Unión , Transporte Biológico , Proteínas Potenciadoras de Unión a CCAAT , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Sinergismo Farmacológico , Células HeLa , Humanos , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-rel , Factor de Transcripción ReIA , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Like other nonnucleoside inhibitors of HIV-1 reverse transcriptase, the dipyridodiazepinone nevirapine (Viramune, 1) selects for drug resistant variants of HIV-1, both in cell culture and in patients. In particular, the mutation of residue 181 from tyrosine to cysteine (Y181C) is associated with resistance to most reported nonnucleoside inhibitors. Introduction of an arylethyl substituent at the 8-position of the tricyclic dipyridodiazepinone skeleton confers enhanced potency against Y181C RT. Several analogues of this series display good broad spectrum potency against a panel of mutant enzymes.
Asunto(s)
Antivirales/síntesis química , Azepinas/síntesis química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Mutación , Piridinas/síntesis química , Inhibidores de la Transcriptasa Inversa/síntesis química , Sustitución de Aminoácidos , Antivirales/química , Antivirales/farmacología , Azepinas/química , Azepinas/farmacología , Línea Celular Transformada , Farmacorresistencia Microbiana , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , VIH-1/genética , Humanos , Técnicas In Vitro , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Nevirapina/química , Nevirapina/farmacología , Piridinas/química , Piridinas/farmacología , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-ActividadRESUMEN
Nevirapine (I) is the first human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor to reach regulatory approval. As a result of a second generation program around the tricyclic core system of nevirapine, 2-chloro-5, 11-dihydro-11-ethyl-5-methyl-8-(2-(pyridin-4-yl)ethyl)-6H-dipyrido[3, 2-b:2',3'-e][1,4]diazepin-6-one (II)1a and 2-chloro-5, 11-dihydro-11-ethyl-5-methyl-8-phenylethyl-6H-dipyrido[3,2-b:2', 3'-e][1,4]diazepin-6-one (III)1a were identified as broad spectrum HIV-1 RT inhibitors. A detailed examination of replacing either of the methylenes of the 8-ethyl linker of II or III is presented. It was found that 8-aryloxymethyl and 8-arylthiomethyl are the preferred pattern of substitution for potency against RT. The most potent compounds were further evaluated against a panel of clinically significant mutant RT enzymes (K103N, V106A, G190A, P236L) and in cytotoxicity and in vitro metabolism assays. The most potent compound was 2-chloro-8-phenylthiomethyl analogue 37 which displayed sub-100 nM activity against all HIV-1 RT enzymes tested.
Asunto(s)
Antivirales/síntesis química , Azepinas/síntesis química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Nevirapina/análogos & derivados , Piridinas/síntesis química , Inhibidores de la Transcriptasa Inversa/síntesis química , Animales , Antivirales/química , Antivirales/farmacología , Azepinas/química , Azepinas/farmacología , Disponibilidad Biológica , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Farmacorresistencia Microbiana , Estabilidad de Medicamentos , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , VIH-1/fisiología , Humanos , Técnicas In Vitro , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Mutación , Nevirapina/síntesis química , Nevirapina/química , Nevirapina/farmacocinética , Nevirapina/farmacología , Piridinas/química , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
The syntheses of 11-ethyl and 11-cyclopropyldipyrido[2,3-b:3',2'-f]azepines, analogs of the HIV-1 reverse transcriptase inhibitor nevirapine 1, are described. These compounds exhibit potency equivalent to nevirapine in the inhibition of wild-type and some mutant RT enzymes.