Custom-tailored Epigenetic Drug Discovery

Custom-tailored epigenetic screening and mechanism of action studies can be performed with either our portfolio of assays or with an epigenetic protein or substrate provided by the customer. Customers are welcome to consult with our scientists to identify the best approach for a mechanism of action analysis based on the nature of the compound and research goals.

Use a suite of biophysical assays for determination of target-compound interaction on the molecular level
Determine whether your compound needs the presence of a cofactor or competes with the substrate
Custom-tailor our portfolio of epigenetic assays to suit your individual screening needs

Reaction Biology is a partner for integrated drug discovery, offering services to support every step of the drug discovery process.

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Kinetics of target-analyte binding

Surface Plasmon Resonance Assay (SPR). SPR is commonly used to determine the kinetics of target-analyte binding. The assay detects changes in the molecular mass of a target after binding of the analyte. The target is immobilized to a sensor chip, and the analyte flows to the target. Target binding is monitored in real-time for both, association and dissociation.

Substrate competition

Example of a cofactor analysis by SPR. EPZ015666 is a substrate-competitive inhibitor that binds to its target enzyme, PRMT5/ MEP50, only in the presence of SAM or SAM analogs such as MTA and SAH.
No dose-dependent responses were observed for analyte binding to apoprotein (left figure). The binding to the MTA-bound target is relatively weak (KD~16μM) with fast kinetics (on/off). The binding affinity increased by ~10-fold for the SAH-bound target (KD~1μM). While the on-rates are similar for the MTA- and SAH-bound conditions, the off-rates are approximately 100X slower. The highest affinity (KD~3nM) and slowest off-rate (100x less than SAH-bound) was observed for analyte binding to the SAM-bound target. Single-cycle kinetics, that do not require a return to the baseline in between doses, was used due to the slow off-rate observed for this condition. A slower off-rate indicates longer occupancy of the analyte on the target.

The analyte was tested with 7 concentrations depicted in different colors.

Mechanism of Action

EPZ015666 = a substrate-competitive inhibitor that binds only in the presence of SAM or SAM analog.

LLY-283 = an inhibitor that binds the SAM-binding pocket but appears to be non-competitive for both SAM and substrate.

JNJ-64619178 = a pseudo-irreversible inhibitor that binds the SAM-binding pocket and reaches into the substrate pocket.

PRMT5:MEP50 complex was immobilized to a sensor chip and the binding of tool molecules was measured. In the absence of co-factor (SAM or SAM analogs SAH or MTA), EPZ015666 exhibited a relatively weak affinity for PRMT5:MEP50 with minimal signal changes observed. LLY-283 and JNJ-64619178, however, exhibited tight binding. While the on-rates are similar for the two compounds, the off-rates differ by ~10-fold with JNJ-64619178 exhibiting longer target engagement resulting in tighter binding. A kinetic titration was used to inject the molecules from low to a high concentration in a single cycle due to the slow off-rates.

Custom-tailored Epigenetic Drug Discovery

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