ABSTRACT
High-throughput screening (HTS) efforts to discover "hits" typically rely on the large-scale parallel screening of individual compounds with attempts to screen mixtures of compounds typically and, unfortunately, giving rise to false positives and false negatives due to the nature of the HTS readout (% inhibition/activation above a defined threshold) that makes deconvolution virtually intractable. Bioaffinity screening methods have emerged as an alternative or orthogonal method to classic HTS. One of these methods, frontal affinity chromatography coupled to mass spectrometry detection (FAC-MS), although still a relatively new technique, is turning out to be a viable screening tool. However, to push FAC-MS more to the forefront as a moderate primary HTS system (or a secondary screening assay), automation needs to be addressed. An automated FAC-MS system is described using 2 columns containing immobilized hERbeta, whereby while 1 column is being regenerated, the other is being used. The authors are extrapolating that in a continuous 24-h operation, the number of ligands screened could potentially approach 10,000. In addition, preliminary structure-activity relationship binding information (typically not seen in early primary HTS) can be obtained by observing the rank order of the library members in the various mixtures.
Subject(s)
Chromatography, Affinity/methods , Estrogen Receptor beta/metabolism , Mass Spectrometry/methods , Amino Acid Sequence , Amino Acid Substitution , Automation , Chromatography, Affinity/instrumentation , Estrogen Receptor beta/chemistry , Estrogen Receptor beta/genetics , Histidine/chemistry , Humans , Ligands , Mass Spectrometry/instrumentation , Molecular Sequence Data , Protein Structure, Tertiary , Serine/metabolismABSTRACT
The emergence of a relatively new technique resulting from a combination of frontal affinity chromatography coupled with MS detection (FAC-MS) has extended the capabilities of MS in drug discovery and development. Its application in a broad range of biological systems, together with its label-free operation, relatively high throughput, ability to rank ligands and determine Kd, makes FAC-MS a universal tool enabling convenient and efficient screening in the identification of new potential drug leads. Here we will highlight FAC-MS screening studies and discuss where it can be applied in evaluating multiple protein-binding sites, protein-protein interactions and inactive proteins, and also in determining selectivity.
Subject(s)
Chromatography, Affinity , Drug Design , Drug Evaluation, Preclinical/methods , Mass Spectrometry , Proteins/chemistry , Binding Sites , Ligands , Models, Molecular , Molecular ConformationABSTRACT
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.
