Ligand-dependent structural dynamics assays

National Center For Advancing Translational Sciences

Investigators

Abstract

The structural dynamics response (SDR) assay has been demonstrated using quantitative high throughput screening (qHTS) in 1536-well microtiter plate format with enzymes from the following enzyme classes: ATP cofactor-dependent monooxygenase using firefly luciferase (FLuc); oxidoreductase using dihydrofolate reductase (DHFR); tyrosine kinase using Abelson tyrosine (ABL1) kinase domain; serine/threonine protein kinase using protein kinase A (PKA); isomerase using co-factor independent phosphoglycerate mutase (iPGM); NAD+- and ATP-dependent DNA ligases, using E. coli DNA ligase and bacteriophage T7 DNA ligase, respectively. We have demonstrated that the SDR assay is capable of discriminating ligand binding affinities allowing the determination of structural-activity relationships (SAR) among chemotype classes. As a proof-of-concept this has been demonstrated experimentally using the monooxygenase FLuc to establish SAR series for 29 chemotype series composed of between 3 and 47 compounds. We have demonstrated that the SDR assay can reliably distinguish protein kinase subtype selectivity. Using the tyrosine kinase ABL1 and serine/threonine kinase PKA we demonstrated the accurate identification of ABL1 kinase vs. PKA inhibitors from a library of kinase inhibitors enriched for tyrosine kinases. A major advantage of the SDR assay may be the method’s potential to detect allosteric-site ligands often silent, for example, in biochemical assays measuring active-site catalytic activity. This feature was demonstrated using the ABL1 kinase domain myristate binding-site, an auxiliary pocket distinct from the ATP-binding site. Here, while inactive in the functional kinase assay, the FDA-approved myristate-binding pocket drug, asciminib gave a measurable gain-of-signal SDR. The exquisite sensitivity of the SDR assay has proven useful for the detection of ligand binding in cellular lysates. In this scenario the target-sensor fusion protein is expressed either from a transfected plasmid or endogenously edited gene, the latter readily accomplished using the small 11-amino acid alpha-peptide. For example, DHFR-C-alpha peptide in HEK293 cell lysate from a CRISPR/Cas9-mediated edit of the DHFR gene, was used to establish an SDR assay. Using methotrexate (MTX), a potent DHFR ligand, both low- and NADPH-dependent high-affinity binding states could be measured. The SDR assay accurately rank ordered a collection of DHFR-binding antifolates and their NADPH-cofactor synergistic binding.