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1.
Biochem J ; 477(4): 801-814, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32011652

RESUMO

Autophagy is a critical cellular homeostatic mechanism, the dysfunction of which has been linked to a wide variety of disease states. It is regulated through the activity of specific kinases, in particular Unc-51 like autophagy activating kinase 1 (ULK1) and Phosphatidylinositol 3-kinase vacuolar protein sorting 34 (VPS34), which have both been suggested as potential targets for drug development. To identify new chemical compounds that might provide useful chemical tools or act as starting points for drug development, we screened each protein against the Published Kinase Inhibitor Set (PKIS), a library of known kinase inhibitors. In vitro screening and analysis of the published selectivity profiles of the hits informed the selection of three relatively potent ATP-competitive inhibitors against each target that presented the least number of off-target kinases in common. Cellular assays confirmed potent inhibition of autophagy in response to two of the ULK1 inhibitors and all three of the VPS34 inhibitors. These compounds represent not only a new resource for the study of autophagy but also potential chemical starting points for the validation or invalidation of these two centrally important autophagy kinases in disease models.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Autofagia , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Descoberta de Drogas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Osteossarcoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Fosforilação , Células Tumorais Cultivadas
2.
Nucleic Acids Res ; 45(14): 8474-8483, 2017 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-28582530

RESUMO

Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing.


Assuntos
Proteínas de Helminto/genética , RNA de Helmintos/genética , RNA Líder para Processamento/genética , Ribonucleoproteínas/genética , Trans-Splicing , Animais , Animais Geneticamente Modificados , Sequência de Bases , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Helminto/metabolismo , Microscopia de Fluorescência , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA de Helmintos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Líder para Processamento/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo
3.
SLAS Discov ; 23(3): 225-241, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29460707

RESUMO

High-throughput screening (HTS) is a proven method for discovering new lead matter for drug discovery and chemical biology. To maximize the likelihood of identifying genuine binders to a molecular target, and avoid wasting resources following up compounds with unproductive/nonspecific mechanisms of action, it is important to employ a range of assays during an HTS campaign that build confidence of target engagement for hit compounds. Biophysical methods that measure direct target/compound engagement have established themselves as key techniques in generating this confidence, and they are now integral to the latter stages of HTS triage at the European Lead Factory (ELF). One relatively new technique that the ELF is using is microscale thermophoresis (MST), which measures the differences in rate of movement through a temperature gradient that are caused when single molecular species form complexes. Here we provide an overview of the MST assay development workflow that the ELF employs and a perspective of our experience to date of using MST to triage the output of HTS campaigns and how it compares and contrasts with the use of other biophysical techniques.


Assuntos
Bioensaio/métodos , Ensaios de Triagem em Larga Escala/métodos , Biofísica/métodos , Desenho de Fármacos , Descoberta de Drogas/métodos , Europa (Continente) , Bibliotecas de Moléculas Pequenas/química , Temperatura
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