Biophysical Analysis

Biophysical analyses give insight into the compound-target-interaction on a molecular level. The way a compound acts on the target shapes the drug’s PK/PD profile and efficacy in vivo. Therefore, in a targeted drug discovery approach, knowledge of the molecular behavior of your lead candidates early in the drug discovery process aids modeling the structure-activity relationship for optimal modifications of the drug design.

Our Reaction Oncology Platform enables the investigation of various aspects of the mechanism of action of a new drug, including:

Binding affinity
Binding kinetics and residence time
Target Occupancy
Binding Reversibility
Competition with Substrates or Co-factors
Stoichiometry
Thermodynamics of the compound-target interaction

The knowledge of the compound-target interaction can be expanded by examination of the structure of the compound-target complex via NMR and X-ray crystallography via an external partner.

Biophysical Analysis - Example 1

Question

Determination of substrate-specific competitive binding of a methyltransferase inhibitor

Challenge

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.

Achievement

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.

PRMTS

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.

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Surface Plasmon Resonance Binding Studies of Epigenetic Targets

Biophysical Analysis - Example 2

Question

Mutant-specific binding affinity of KRAS inhibitors

Challenge

The determination of the selectivity of a small molecule compound on a selection of KRAS mutant proteins

Achievement

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.

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Tools for the discovery of KRAS Pathway Inhibitors

Biophysical Analysis - Example 3

Question

Stoichiometry and thermodynamic parameter determination of BET protein inhibitor JQ1

Challenge

The thermodynamic parameters and the stoichiometry of JQ1 binding to bromodomain BRD4 needed to be determined.

Achievement

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.

BRD4

Biophysical Analysis - Example 4

Question

Determination of association and dissociation constants of two methyltransferase inhibitors

Challenge

Two inhibitors of methyltransferase PRMT5 were compared regarding their potency and binding kinetics.

Achievement

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.

PRMT5

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Surface Plasmon Resonance Binding Studies on Epigenetic Targets with PRMT5 and BRD4

Surface Plasmon Resonance: A key technology for high-quality leads

How does SPR aid in drug discovery?

Surface Plasmon Resonance (SPR) is a highly sensitive method detecting changes in the molecular weight of biomolecules, for example, when binding to a compound. SPR provides information on specificity, affinity, kinetics, thermodynamics for a wide range of biomolecules. The data generated by SPR gives insight into molecular recognition processes to advance rational drug design.

Compounds with the same binding affinity can have different rates of drug-target formation and breakdown, which can result in very different biological activity profiles. Integrating kinetic information with affinity and potency data early in the drug discovery process helps to ensure that superior compounds are not being discarded at the early stages of a project. A compound with lower potency may actually be a superior drug candidate, something that SPR analysis is particularly good at teasing out.

SPR data can also be used to eliminate promiscuous binders that achieve enzyme inhibition using a non-specific aggregation-type binding mechanism, which is non-optimizable for medicinal chemistry, and appear as false positives in biochemical inhibition assays.

Why optimize for kinetics instead of just potency or binding affinity?

The behavior of a molecule as a drug depends on the binding kinetics, which cannot be obtained from equilibrium parameters like IC50 and KD alone.

Also, increasing the potency will not necessarily result in decreased off-rates for molecules with affinities in the nanomolar to micromolar range.

Slow off-rates can lead to kinetic selectivity even if there is no thermodynamic selectivity (i.e., two drugs bind one target with the same affinity or the same drug binds two targets with the same affinity). Differences in binding kinetics can result in drug-target complexes with lifetimes that differ by orders of magnitude, even if the equilibrium affinity or potency is the same. As such, a drug with a slow off-rate to the desired target and a faster off-rate to unintended targets may still be viable even if potency data alone suggests off-target effects.

The kinetics of drug binding and unbinding, especially the residence time, play a crucial role in a drug’s in vivo efficacy. SPR can rank the kinetic selectivity of drug analogs for the selection of the best drug candidates.