Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway
| Autor: | Claudia Rutz, Ralf Schülein, Patrick Scheerer, Jamina Eckhard, Martin Neuenschwander, Kurt Vermeire, Marc Nazaré, Gunnar Kleinau, Becky Provinciael, Jens-Peter von Kries, Wolfgang Klein, Edgar Specker |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Cell Membranes Endoplasmic Reticulum Biochemistry SUBSTRATE CHANNEL Post-Translational Modification Lipid bilayer Multidisciplinary Secretory Pathway Chemistry ENDOTHELIN B RECEPTOR Translocon Small molecule Cell biology Multidisciplinary Sciences Cell Processes Science & Technology - Other Topics Medicine Technology Platforms Cellular Structures and Organelles Signal Peptides Research Article SIGNAL-PEPTIDE Sec61 High-throughput screening Science Green Fluorescent Proteins Biosynthesis Research and Analysis Methods 03 medical and health sciences Inhibitory Concentration 50 Microsomes Humans Integral Membrane Proteins PROTEIN-TRANSLOCATION Molecular Biology Techniques Molecular Biology Molecular Biology Assays and Analysis Techniques Science & Technology Cell-Free System Endoplasmic reticulum Biology and Life Sciences Proteins Membrane Proteins Cell Biology High Throughput Screening High-Throughput Screening Assays DRUG DISCOVERY 030104 developmental biology Secretory protein HEK293 Cells Pyrimidines Membrane protein Pyrazoles APRATOXIN Ribosomes SEC Translocation Channels |
| Zdroj: | PLoS ONE PLoS ONE, Vol 13, Iss 12, p e0208641 (2018) |
| ISSN: | 1932-6203 |
| Popis: | The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs. ispartof: PLOS ONE vol:13 issue:12 ispartof: location:United States status: published |
| Databáze: | OpenAIRE |
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