scanKINETIC℠ - Association/Dissociation Kinetics
Reversibility and Dissociation Kinetics Studies
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Classify inhibitors as irreversible, reversible-slow dissociation or reversible-rapid dissociation
Irreversible, covalent inhibitors and reversible inhibitors that dissociate slowly from a kinase target can have unusual properties in both cellular and in vivo pharmacology models. For example, target inhibition can be maintained for several hours or more, even after the inhibitor has been “washed out” or cleared. In the absence of target dissociation data, these pharmacology results can be difficult to interpret, particularly when multiple inhibitors are being compared. Furthermore, while irreversible/slowly dissociating inhibitors may be desirable for some drug discovery programs, these properties may not be ideal in all cases. KINOMEscan offers a dissociation kinetics service that classifies inhibitors as irreversible, reversible-slow dissociation, or reversible-rapid dissociation.
Experimental Design for Reversibility and Dissociation Kinetics Studies
For each inhibitor (Samples A & B) two parallel dose response curves are prepared and equilibrated with the kinase of interest either continuously (Sample A) or with an intervening reaction dilution step (Sample B). Samples are then read out and the data are fit to the Hill binding equation to calculate Kd values.
Model Data for Reversibility and Dissociation Kinetics Studies
For reversible, rapidly dissociating inhibitors (left panel), the apparent Kd value for Sample B is higher than the Kd value for Sample A by a multiple equal to the Sample B reaction dilution factor (10-fold in this example). For reversible, slowly dissociating inhibitors (center panel), the apparent Kd value for Sample B is higher than the Kd value for Sample A by a multiple less than the Sample B reaction dilution factor, since for Sample B, the inhibitor only partially dissociates after the reaction dilution step. For irreversible inhibitors (right panel), the Kd values for Samples A&B are equivalent, since for Sample B, the inhibitor fails to dissociate after the reaction dilution step [click graph to enlarge].
Association Kinetics Studies
Identify slowly associating inhibitors early in the drug discovery process
The association rates for kinase-inhibitor complexes are variable and can be both inhibitor and kinase-dependent. While many kinase-inhibitor complexes form rapidly (< minutes), others can have extraordinarily slow association rates, requiring several hours to reach equilibrium. Slowly associating inhibitors can yield conflicting potency data in various in vitro, cellular, and in vivo experiments, where the inhibitor-target equilibration time is often assay specific. High affinity, slowly associating inhibitors also have extremely slow dissociation kinetics; the desirability of these kinetic properties ultimately depends on other inhibitor characteristics (e.g. pharmacokinetics) and upon the goals of a specific drug discovery program. In addition, slow association kinetics can provide insight into inhibitor binding mode, as it is well documented that Type II inhibitors, which bind a catalytically inactive “DFG-out” enzyme conformation, often bind more slowly than Type I inhibitors, which are less conformation selective. The KINOMEscan
association kinetics service enables the identification of slowly associating inhibitors early in the drug discovery process.
Slow Association Kinetics: p38-alpha/BIRB-796
Association kinetics analysis for the Type II p38-alpha inhibitor BIRB-796. BIRB-796 is a highly potent Type II inhibitor reported to have ultra-slow association kinetics for its target, p38-alpha. In this KINOMEscan association kinetics study, Kd measurements for this interaction were made as a function of co-incubation time (1 or 24 hr). The data reveal a dramatic reduction (30-fold) in the apparent Kd value for the 24 hr time point relative to the 1 hr time point, which is consistent with published studies demonstrating ultra-slow association kinetics for this interaction [click graph to enlarge].
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