Determination of substrate-specific competitive binding of a methyltransferase inhibitor
The inhibitor EPZ015666 is a substrate competitive inhibitor that binds only in the presence of SAM or SAM analogues. We have determined how the binding affinity and kinetics of the inhibitor change with different co-factors.
Surface plasmon resonance (SPR) measures the direct binding of an inhibitor to its target. SPR is a flow based assay with the target immobilized on a surface and the inhibitor flowing over that surface which can be performed in the presence of co-factors or substrates.
Methyltransferase PRMT5 uses S-adenosylmethionine (SAM) as methionine donor for it’s enzymatic reaction. During it’s usual enzymatic activity the substrate and SAM both reach into PRMT5 which catalyses the transfer from SAM to the substrate. Inhibitor EPZ015666 occupies the substrate binding pocket to inhibit PRMT5 activity. However, the inhibitor was found to bind with different affinities when the co-factor SAM or its analogues were bound to PRMT5.
Left: PRMT5 interacts with a substrate and methionine donor SAM for methionine transfer.
Right: Inhibitor EPZ05666 occupies the substrate binding pocket. The interaction with methionine increases the binding affinity of the inhibitor.
Surface Plasmon Resonance Binding Studies of Epigenetic Targets
Mutant-specific binding affinity of KRAS inhibitors
The determination of the selectivity of a small molecule compound on a selection of KRAS mutant proteins
Using Thermal Shift Assays, KRAS wild type and mutant proteins were used for affinity binding analysis with several KRAS inhibitors. ARS-1620 and AMG510 were confirmed to specifically bind to KRAS G12C, which is a relevant mutant for tumor progression.
Tools for the discovery of KRAS Pathway Inhibitors
Stoichiometry and thermodynamic parameter determination of BET protein inhibitor JQ1
The thermodynamic parameters and the stoichiometry of JQ1 binding to bromodomain BRD4 needed to be determined.
The isothermal titration method was used to characterize the interaction of bromodomain 1 of BRD4 protein and the small molecule compound JQ1. JQ1 was titrated to the bromodomain 1, and the change in the temperature of the solution was measured. The change in temperature represents the binding energy which is the sum of all molecular interactions that contribute to the binding of the inhibitor to the target. The results indicate the forces that drive binding, providing guidance for structure-activity-relationship studies.
Stoichiometry measurements below show that one molecule of inhibitor binds to one molecule of the target. This knowledge, too, is valuable for drug optimization.
Determination of association and dissociation constants of two methyltransferase inhibitors
Two inhibitors of methyltransferase PRMT5 were compared regarding their potency and binding kinetics.
Surface plasmon resonance measures in real-time the association and dissociation of inhibitors to their target. We compared two methyltransferase inhibitors regarding their binding kinetics and found differences in the dissociation constant kd. JNJ-64619178 did not dissociate off its target as fast as LLY-183 did, resulting in a 300x higher binding affinity proposing JNJ-64619178’s higher potency and possibly lower off-target effects.
Surface Plasmon Resonance Binding Studies on Epigenetic Targets with PRMT5 and BRD4