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Investigative Tools & MoA Services

Introduction

Learn more about your kinase inhibitor’s biochemical mechanism of action. KINOMEscan® offers a suite of investigative tools that provides a detailed biochemical characterization of the interaction between inhibitors and their targets. The thermodynamic, kinetic, and structural information provided by these tools enables a detailed comparison of inhibitors from common or distinct lead series and facilitates the interpretation of data from downstream cellular and pharmacology models.

KdELECT

Quantitative interaction affinities measured for any kinase/inhibitor combination
Inhibitor binding constants (Kd values) are calculated from duplicate 11-point dose-response curves (plus DMSO control). Measurements are made under optimized conditions that generate true thermodynamic Kd values, as opposed to IC50 values, which can depend on the individual assay conditions (e.g. the ATP concentration). For this reason, KdELECT data facilitate a direct comparison of inhibitor affinity across kinases.

Binding constant (Kd) determinationfor the interaction between Compound F and Aurora Kinase B (AURKB). Kd values were calculated by fitting dose-response curves to the Hill binding equation using the Levenberg–Marquardt algorithm. Duplicate Kd values calculated for this interaction were 36.5 nM and 24.3 nM.
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scanMODE

Structural Insights from Biochemistry

Classifies inhibitors as Type I or Type II in the absence of a cocrystal structure.

A panel of ABL assay pairs differentially phosphorylated on the activation loop (A-loop), scanMODE classifies inhibitor binding mode by measuring the phosphorylation state-dependence of inhibitor affinity. scanMODE capitalizes on four key observations: 1) Type II inhibitors, which recognize a catalytically inactive “DGF-out” enzyme conformation, bind preferentially to the non-phosphorylated, unactivated state of ABL, whereas Type I inhibitor binding is phosphorylation state-independent; 2) an inhibitor’s binding mode is most often maintained across kinases (e.g. imatinib is a Type II ABL inhibitor and a Type II LCK inhibitor); 3) inhibitors that primarily target kinases other than ABL are correctly classified as Type I or Type II when tested against the differentially phosphorylated ABL assay pairs; and 4) a significant fraction of kinase inhibitors have sufficient off-target affinity for ABL and/or clinically relevant ABL mutants to qualify for a scanMODE screen. These observations support the use of scanMODE assays as a general tool to classify the binding mode of inhibitors targeting a wide range of kinases. Key reference: Wodicka et al., Chemistry & Biology (2010) 17(11):1241-9.

Binding constant (Kd) determinations were measured for interactions between imatinib, a known Type II inhibitor, and ABL preparations differentially phosphorylated on the A-loop. Imatinib exhibited a 30-fold affinity preference for the non-phosphorylated state (Kd = 1.4 nM) relative to the phosphorylated state (Kd = 56 nM) [click graph to enlarge].
Binding constant (Kd) determinations were measured for interactions between the Type I inhibitor dasatinib and ABL preparations differentially phosphorylated on the A-loop. The Kd values for dasatinib were nearly identical for the phosphorylated and non-phosphorylated states (Kd = 0.019 and 0.027 nM, respectively) and consistent with an activation state insensitive interaction [click graph to enlarge].
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Reversibility and Dissociation Kinetics Studies

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].
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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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