ATLAS--a high-throughput affinity-based screening technology for soluble proteins: technology application using p38 MAP kinase.

Abstract: A general affinity-based screening assay for discovery of lead compounds binding to potential protein drug targets that is based upon protein thermal unfolding and aggregation is described. ATLAS (Any Target Ligand Affinity Screen) (Anadys Pharmaceuticals, Inc., San Diego, CA) is a simple, homogeneous, and high-throughput affinity-based screening technology that can identify compounds that bind and protect the target protein from thermal unfolding, denaturation, and subsequent aggregation. ATLAS detection of thermally unfolded and aggregated hexahistidine [(His)6]-tagged proteins uses time-resolved fluorescence resonance energy transfer between two anti-(His)6 antibodies, labeled with either a donor or acceptor fluorophore, that are simultaneously bound to the aggregated protein. The ATLAS assay is simple to perform and easily automated for screening large compound libraries. The technology is applicable to lead discovery for soluble proteins of known and unknown functions, and particularly for proteins that are difficult to assay functionally. The ATLAS technology has been evaluated using p38 mitogen-activated protein (MAP) kinase as the target protein. Known inhibitors of p38 MAP kinase were examined by ATLAS and a functional assay; the results showed good correlation between the two methods.

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.