High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation.
The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990 s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor beta signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z' factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery.
Molecular Medicine, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Dag Hammarskjölds Väg 20, S-751 85 Uppsala, Sweden. karl-johan.leuchowius@genpat.uu.se
Karl-Johan
KJ
Leuchowius
Malin
M
Jarvius
Malin
M
Wickström
Linda
L
Rickardson
Ulf
U
Landegren
Rolf
R
Larsson
Ola
O
Söderberg
Mårten
M
Fryknäs
Jonas
J
Jarvius
D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013678:Technology, Pharmaceutical / Q000379:methods*; D014443:Tyrosine / Q000378:metabolism; D015262:Xenobiotics / Q000302:isolation & purification / Q000494:pharmacology*
D000906:Antibodies; D047428:Protein Kinase Inhibitors; D015262:Xenobiotics; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D020797:Receptor, Platelet-Derived Growth Factor beta