Specialized assays help to narrow an existing hit list and guide lead finding and optimization. They also improve understanding of the compound’s in vitro and, ultimately, in vivo behaviors.
Hit Characterization
Understanding the mode of inhibition is crucial for knowledge-based hit identification and efficient lead optimization.
- High-substrate assays (orthosteric vs. allosteric/competitive vs. uncompetitive):
(Un)competitive orthosteric inhibitors can be identified in high-substrate assays. If an increasing substrate concentration (assay at substrate KM versus multiple of KM) leads to increased or decreased IC50, this is a hint there is competitive or uncompetitive binding to the active site, respectively.
Comparison of IC50 determinations with high versus low substrate assay
- Variable (pre-)incubation time, jump dilution, and variable enzyme concentration
It is critical to know when mathematical assumptions fail, such as for tight binding and time-dependent inhibition (observed for potent and slow or covalent inhibitors, respectively). Variable (pre)incubation times of inhibitor and target or jump dilution assays provide information about the compound’s binding kinetic behaviors in high throughput. Variable enzyme concentration assays enable identification of tight binders.
- Biochemical association and dissociation rate (kon, koff/residence time) determination
Knowledge about the kinetics of formation and the lifetime of the inhibitor–target protein interaction adds an important layer of information to affinity, per se. In some cases, it is a better predictor of in vivo efficacy and selectivity. In many cases, it influences the outcome of cell-based assays—especially for slow binders. The finding of structure–kinetic relationships can help in lead optimization.In our lab, we developed a high-throughput method for determination of binding kinetics, namely, the kinetic probe competition assay (kPCA).We can also perform surface plasmon resonance (SPR) assays.
- Covalent binders
Irreversible mechanisms of drug action allow for overcoming otherwise intractable challenges, such as competition with high-abundance and/or high-affinity substrates. Moreover, the ultimately prolonged residence time can provide benefits regarding kinetic selectivity, potency, and longer duration of action. We have developed a high-throughput platform for identification and characterization of covalent compounds.
If reversibility experiments and/or MS analysis hint at covalent binding, characterization of covalent compounds is possible with binding assays in high throughput.
- Thermodynamic analyses
Enthalpic and entropic components reflect different types of interactions (e.g., favorable binding enthalpy hints at the presence of strong hydrogen bonds and van der Waals interactions). Therefore, enthalpic efficiency has been proposed as a critical determinant during drug discovery.
Isothermal titration calorimetry (ITC), SPR, and equilibrium and kinetic probe competition assays (see above) at various temperatures can be utilized for thermodynamic analyses of compounds of interest.
- Activators
Binding of, for example, activator peptides can influence binding affinity and kinetics of compound binding. Thus, the addition of activators in assays can serve as a better predictor for the cellular and in vivo situations.
These and further tailored assays can be developed to characterize your compounds in depth and explore their modes of inhibition.
If you are interested in more details about our readout technologies and previous targets or about specific steps of the development of screening assays, refer to the following:
Publications from our NUVISAN team:
- Georgi, V., et al., Considerations for improved performance of competition association assays analysed with the Motulsky–Mahan’s “kinetics of competitive binding” model. Br J Pharmacol (2019)
- Georgi, V., et al., Binding Kinetics Survey of the Drugged Kinome. JACS (2018)
- Heroven, C., et al., Halogen-Aromatic π Interactions Modulate Inhibitor Residence Times. Angew Chem Int Ed Engl (2018)
- Witte, W., et al., In vitro and in silico analysis of the effects of D2 receptor antagonist target binding kinetics on the cellular response to fluctuating dopamine concentrations. Br J Pharmacol (2018)
- Schuetz, D., et al., Kinetics for Drug Discovery: An industry-driven effort to target drug residence time. Drug Discov Today (2017)
Further examples can be found in our HTS publication list.
