Evaluation of different glutathione S-transferase-tagged protein captures for screening E6/E6AP interaction inhibitors using AlphaScreen.
Human papillomavirus (HPV) infection is responsible for the development of cervical cancer and its premalignant lesions in women. The virus-encoded oncogene E6 is a promising target for an anti-HPV drug therapy. The authors describe the development of a homogenous screening assay for inhibitors of the E6 interaction with its cellular target, the E6-associated protein (E6AP), based on AlphaScreen technology. The E6 protein was expressed and purified as glutathione S-transferase (GST) fusion protein, and the binding to a biotinylated E6AP peptide was monitored using GST-detecting Acceptor beads coated either with anti-GST antibody or glutathione. After optimization of the assay conditions, a commercial library of 3000 compounds was screened for inhibitors. Active compounds were retested and counterscreened for E6/E6AP specificity using biotinylated GST as a control protein. The results obtained with both types of GST-detecting reagents correlated very well and demonstrated the great potential of the newly developed glutathione-coated Acceptor beads as a detection reagent for GST fusion proteins.
Sehr
Peter
P
Chemical Biology Core Facility EMBL Meyerhofstrasse 1 D-69117 Heidelberg, Germany. sehr@embl.de
Pawlita
Michael
M
Lewis
Joe
J
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004353:Drug Evaluation, Preclinical; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010865:Pilot Projects; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase