Use of proximity ligation to screen for inhibitors of interactions between vascular endothelial growth factor A and its receptors.
BACKGROUND
Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs.


METHODS
We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule.


RESULTS
The PLAs were successful for monitoring the formation and inhibition of VEGF-A-receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC(50))] from a dose-response curve.


CONCLUSIONS
The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique provides dose-response curves, allowing IC(50) values to be calculated.
Gustafsdottir
Sigrun M
SM
Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, SE-75185 Uppsala, Sweden.
Wennström
Stefan
S
Fredriksson
Simon
S
Schallmeiner
Edith
E
Hamilton
Andrew D
AD
Sebti
Said M
SM
Landegren
Ulf
U
D000345:Affinity Labels; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001011:Aorta / Q000166:cytology; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000494:pharmacology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D008024:Ligands; D010766:Phosphorylation; D000072483:Placenta Growth Factor; D011257:Pregnancy Proteins / Q000494:pharmacology; D040262:Receptors, Vascular Endothelial Growth Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D013552:Swine; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D052157:Aptamers, Nucleotide; D008024:Ligands; C000608036:PGF protein, human; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D000072483:Placenta Growth Factor; D040262:Receptors, Vascular Endothelial Growth Factor; D040301:Vascular Endothelial Growth Factor Receptor-2