ABSTRACT
We have integrated two complementary methods, high-throughput virtual screening with a "high-content" wet screening technique based on frontal affinity chromatography with mass spectrometry detection (FAC-MS), for identification of hits against the erythropoietin-producing hepatocellular B2 (EphB2) receptor tyrosine kinase domain. Both an EphB2-directed virtual screen combining docking and scoring and a kinase-directed pharmacophore search strategy were used to identify a compound set enriched in bioactive compounds against EphB2. The coupling of virtual screening methodologies with FAC-MS is a unique hybrid approach that can be used to increase the efficacy of both hit discovery and optimization efforts in drug discovery and has successfully identified hits, in particular 19a (36% shift, IC(50) = 5.2 microM, K(d) = 3.3 microM), as inhibitors for EphB2, a potential cancer target.
Subject(s)
Antineoplastic Agents/chemistry , Receptor, EphB2/antagonists & inhibitors , Receptor, EphB2/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Cell Line, Tumor , Chromatography, Affinity , Databases, Factual , Enzyme-Linked Immunosorbent Assay , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Humans , Mass Spectrometry , Models, Molecular , Molecular Weight , Naphthoquinones/chemistry , Naphthoquinones/pharmacology , Phosphorylation , Protein Structure, Tertiary , Quantitative Structure-Activity Relationship , Receptor, EphB2/metabolism , Sulfides/chemistry , Sulfides/pharmacologyABSTRACT
Utilizing frontal affinity chromatography with mass spectrometry detection (FAC-MS), we have identified novel applications in the discovery of small-molecule hits to protein targets that are difficult if not impossible to accomplish using traditional assays. We demonstrate for the first time an ability to distinguish between competitive ligands for the ATP and substrate sites of protein kinase C independently in the same experiment and show that ATP competitive ligands using a functionally inactive receptor tyrosine kinase can be identified. This ability of FAC-MS to simultaneously monitor binding at the ATP and substrate binding sites, as well as measure ligand binding to both active and inactive kinases, suggests that FAC-MS can be used as a "global kinase binding assay".
Subject(s)
Chromatography, Affinity/methods , Drug Evaluation, Preclinical/methods , Mass Spectrometry/methods , Protein Kinase Inhibitors/chemistry , Protein Kinases/chemistry , Alkaloids/chemistry , Alkaloids/metabolism , Animals , Benzophenanthridines/chemistry , Benzophenanthridines/metabolism , Binding Sites , Binding, Competitive , Catalytic Domain , Humans , Imidazoles/chemistry , Imidazoles/metabolism , Mice , Molecular Structure , Peptides/chemistry , Peptides/metabolism , Phosphorylation , Protein Binding , Protein Kinase C-alpha/antagonists & inhibitors , Protein Kinase C-alpha/chemistry , Protein Kinase C-alpha/metabolism , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Pyridines/chemistry , Pyridines/metabolism , Quinazolines/chemistry , Quinazolines/metabolism , Receptor, EphB2/antagonists & inhibitors , Receptor, EphB2/chemistry , Receptor, EphB2/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
FAC-MS offers a convenient method for measuring the relative binding strengths of ligands in a mixture and enables a rapid ranking and identification of ligands in the mixture as potential hits against immobilized targets. Using immobilized EphB2 receptor tyrosine kinase as the target and known kinase inhibitors, the results of FAC-MS screening (% shift) have been shown to correlate with the binding constant, K(d), and with IC(50) results from the more traditional ELISA assay. Therefore, since FAC-MS can accommodate a wide variety of target proteins, its applications could play a broad role in drug discovery not only at the hit discovery stage but also during the subsequent more rigorous screening at the hit-to-lead and lead optimization stages.
Subject(s)
Enzyme Inhibitors/chemistry , Receptor, EphB2/chemistry , Chromatography, Affinity/methods , Enzyme-Linked Immunosorbent Assay , Ligands , Mass Spectrometry , Models, Molecular , Receptor, EphB2/antagonists & inhibitorsABSTRACT
A new knowledge-based scoring function was developed in this work to facilitate the rapid ranking of ligands in databases. The acronym of the method is BHB based on the descriptors it utilizes: buriedness, hydrogen bonding, and binding energy. Receptor buriedness is a measure of how well molecules occupy the binding pocket in comparison to known high-affinity ligands or, alternatively, whether they have contact with identified residues in the pocket. The possibility of hydrogen bond formation is checked for selected residues that are recognized as being important in the binding of known ligands. The approximate binding energy is calculated from the thermodynamic cycle using the optimized bound and free solvent conformations of the ligand-receptor system. The information necessary for the scoring function can ideally be gleaned from the 3D structure of the receptor-ligand complex. Alternatively, the descriptors can be derived from the 3D structure of the unbound receptor, provided this receptor has a known ligand that binds to the given site with nanomolar activity. We show that the new scoring functions provide up to 12 times improvement in enrichment compared to the popular commercial docking program GOLD.
