Identification of ligand binding by protein stabilization: comparison of ATLAS with biophysical and enzymatic methods.
ATLAS (Any Target Ligand Affinity Screen) (Anadys Pharmaceuticals, Inc., San Diego, CA) is a homogeneous, affinity-based high-throughput screening technology based on protein thermal denaturation and the ability of ligands to bind and stabilize the target protein from unfolding. To further understand the assay sensitivity for the identification of ligands that bind to soluble protein targets, firefly luciferase was chosen to characterize the technology. Luciferase is a multidomain protein with a complex unfolding pathway. Binding of ATP results in a stabilizing conformational rearrangement of the domains. Using luciferase to characterize the ATLAS technology allowed us to evaluate the generality of the screening method for the identification of ligand binding to any target. Luciferase inhibitors identified from functional screens were used to assess the capability of ATLAS to rank order inhibitors. Comparison of the ATLAS 50% effective concentration with other biophysical and biochemical methods offered insight into optimizing ATLAS assay conditions to maximize sensitivity to compound binding and protein stabilization. The results show the importance of characterizing the thermal unfolding and aggregation behavior of the protein to allow the ATLAS screen to be optimally designed.
Thompson
Peggy A
PA
Department of Biology, Anadys Pharmaceuticals, Inc., San Diego, California, USA. pthompson@anadyspharma.com
Wang
Shaohui
S
Howett
Lindsay J
LJ
Wang
Mei-Mei
MM
Patel
Rupal
R
Averill
April
A
Showalter
Richard E
RE
Li
Bin
B
Appleman
James R
JR
D000255:Adenosine Triphosphate / Q000494:pharmacology; D055592:Biophysical Phenomena; D001703:Biophysics; D002138:Calibration; D002151:Calorimetry; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics; D006639:Histidine; D006358:Hot Temperature; D007202:Indicators and Reagents; D007700:Kinetics; D008024:Ligands; D008027:Light; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics; D009842:Oligopeptides; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D012542:Scattering, Radiation
D004791:Enzyme Inhibitors; C471213:His-His-His-His-His-His; D007202:Indicators and Reagents; D008024:Ligands; D009842:Oligopeptides; D011506:Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D008156:Luciferases