RESUMO
High-content phenotypic screening has become the approach of choice for drug discovery due to its ability to extract drug-specific multi-layered data. In the field of epigenetics, such screening methods have suffered from a lack of tools sensitive to selective epigenetic perturbations. Here we describe a novel approach, Microscopic Imaging of Epigenetic Landscapes (MIEL), which captures the nuclear staining patterns of epigenetic marks and employs machine learning to accurately distinguish between such patterns. We validated the MIEL platform across multiple cells lines and using dose-response curves, to insure the fidelity and robustness of this approach for high content high throughput drug discovery. Focusing on noncytotoxic glioblastoma treatments, we demonstrated that MIEL can identify and classify epigenetically active drugs. Furthermore, we show MIEL was able to accurately rank candidate drugs by their ability to produce desired epigenetic alterations consistent with increased sensitivity to chemotherapeutic agents or with induction of glioblastoma differentiation.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Descoberta de Drogas/métodos , Epigênese Genética/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Histonas/genética , Proteínas de Neoplasias/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Relação Dose-Resposta a Droga , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Histonas/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Aprendizado de Máquina , Microscopia de Fluorescência , Proteínas de Neoplasias/metabolismoRESUMO
The recent re-emergence of Zika virus (ZIKV)1, a member of the Flaviviridae family, has become a global emergency. Currently, there are no effective methods of preventing or treating ZIKV infection, which causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women. However, the pathology induced by ZIKV is unique among flaviviruses, and knowledge of the biology of other family members cannot easily be extrapolated to ZIKV. Thus, structure-function studies of ZIKV proteins are urgently needed to facilitate the development of effective preventative and therapeutic agents. Like other flaviviruses, ZIKV expresses an NS2B-NS3 protease, which consists of the NS2B cofactor and the NS3 protease domain and is essential for cleavage of the ZIKV polyprotein precursor and generation of fully functional viral proteins. Here, we report the enzymatic characterization of ZIKV protease, and we identify structural scaffolds for allosteric small-molecule inhibitors of this protease. Molecular modeling of the protease-inhibitor complexes suggests that these compounds bind to the druggable cavity in the NS2B-NS3 protease interface and affect productive interactions of the protease domain with its cofactor. The most potent compound demonstrated efficient inhibition of ZIKV propagation in vitro in human fetal neural progenitor cells and in vivo in SJL mice. The inhibitory scaffolds could be further developed into valuable research reagents and, ultimately, provide a roadmap for the selection of efficient inhibitors of ZIKV infection.
Assuntos
Sítio Alostérico , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/química , Zika virus/enzimologia , Sequência de Aminoácidos , Animais , Antivirais/antagonistas & inibidores , Antivirais/química , Sequência de Bases , Ativação Enzimática , Feminino , Flavivirus/química , Expressão Gênica , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/química , RNA Helicases/efeitos dos fármacos , Fatores de Transcrição SOXB1/genética , Alinhamento de Sequência , Serina Endopeptidases/química , Serina Endopeptidases/efeitos dos fármacos , Células-Tronco , Proteínas não Estruturais Virais/efeitos dos fármacos , Proteínas Virais/química , Proteínas Virais/genética , Zika virus/química , Zika virus/genética , Zika virus/crescimento & desenvolvimento , Infecção por Zika virus/virologiaRESUMO
Alternative splicing plays a major role in transcriptome diversity and plasticity, but it is largely unknown how tissue-specific and embryogenesis-specific alternative splicing is regulated. The highly conserved splicing factor Slu7 is involved in 3' splice site selection and also regulates alternative splicing. We show that Slu7 has a unique spatial pattern of expression among human and mouse embryonic and adult tissues. We identified several functional Ets binding sites and GC-boxes in the human Slu7 (hSlu7) promoter region. The Ets and GC-box binding transcription factors, Elk-1 and Sp1, respectively, exerted opposite effects on hSlu7 transcription: Sp1 protein enhances and Elk-1 protein represses transcription in a dose-dependent manner. Sp1 protein bound to the hSlu7 promoter in vivo, and depletion of Sp1 by RNA interference (RNAi) repressed hSlu7 expression. Elk-1 protein bound to the hSlu7 promoter in vivo, and depletion of Elk-1 by RNAi caused an increase in the endogenous level of hSlu7 mRNA. Further, depletion of either Sp1 or Elk-1 affected alternative splicing. Our results provide indications of a complex transcription regulation mechanism that controls the spatial and temporal expression of Slu7, presumably allowing regulation of tissue-specific alternative splicing events.