RESUMEN
We introduce a novel strategy to sample bioactive chemical space, which follows-up on hits from fragment campaigns without the need for a crystal structure. Our results strongly suggest that screening a few hundred or thousand fragments can substantially improve the selection of small-molecule screening subsets. By combining fragment-based screening with virtual fragment linking and HTS fingerprints, we have developed an effective strategy not only to expand from low-affinity hits to potent compounds but also to hop in chemical space to substantially novel chemotypes. In benchmark calculations, our approach accessed subsets of compounds that were substantially enriched in chemically diverse hit compounds for various activity classes. Overall, half of the hits in the screening collection were found by screening only 10% of the library. Furthermore, a prospective application led to the discovery of two structurally novel histone deacetylase 4 inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Bibliotecas de Moléculas Pequeñas/química , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Enzimas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Modelos Moleculares , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
Differentiation is accompanied by extensive epigenomic reprogramming, leading to the repression of stemness factors and the transcriptional maintenance of activated lineage-specific genes. Here we use the mammalian Hoxa cluster of developmental genes as a model system to follow changes in DNA modification patterns during retinoic acid-induced differentiation. We find the inactive cluster to be marked by defined patterns of 5-methylcytosine (5mC). Upon the induction of differentiation, the active anterior part of the cluster becomes increasingly enriched in 5-hydroxymethylcytosine (5hmC), following closely the colinear activation pattern of the gene array, which is paralleled by the reduction of 5mC. Depletion of the 5hmC generating dioxygenase Tet2 impairs the maintenance of Hoxa activity and partially restores 5mC levels. Our results indicate that gene-specific 5mC-5hmC conversion by Tet2 is crucial for the maintenance of active chromatin states at lineage-specific loci.
Asunto(s)
5-Metilcitosina/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Diferenciación Celular , Línea Celular , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Unión al ADN/genética , Dioxigenasas , Células Madre Embrionarias/citología , Proteínas de Homeodominio/genética , Humanos , Hidroxilación , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/genéticaRESUMEN
DNA methylation plays an important role in the self-renewal of hematopoietic stem cells and in the commitment to the lymphoid or myeloid lineages. Using purified CD34⺠hematopoietic progenitor cells and differentiated myeloid cell populations from the same human samples, we obtained detailed methylation profiles at distinct stages of hematopoiesis. We identified a defined set of differentiation-related genes that are methylated in CD34⺠hematopoietic progenitor cells but show pronounced DNA hypomethylation in monocytes and in granulocytes. In addition, by comparing hematopoietic progenitor cells from umbilical cord blood to hematopoietic progenitor cells from peripheral blood of adult donors we were also able to analyze age-related methylation changes in CD34⺠cells. Interestingly, the methylation changes observed in older progenitor cells showed a bimodal pattern with hypomethylation of differentiation-associated genes and de novo methylation events resembling epigenetic mutations. Our results thus provide detailed insight into the methylation dynamics during differentiation and suggest that epigenetic changes contribute to hematopoietic progenitor cell aging.
