Discovery of Inactive Conformation-Selective Kinase Inhibitors by

Jun 2, 2017 - Achieving selectivity across the human kinome is a major hurdle in kinase inhibitor drug discovery. Targeting inactive (vs active) kinas...
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Discovery of inactive conformation-selective kinase inhibitors by utilizing cascade assays Weixue Wang, Daniel Krosky, and Kay Ahn Biochemistry, Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017 Downloaded from on June 5, 2017

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Discovery of inactive conformation-selective kinase inhibitors by utilizing cascade assays Weixue Wang*, Daniel Krosky, and Kay Ahn Molecular & Cellular Pharmacology, Lead Discovery, Janssen Research and Development, Spring House, PA 19477, U.S.A. ABSTRACT: Achieving selectivity across the human kinome is a major hurdle in kinase inhibitor drug discovery. Targeting inactive (versus active) kinase conformations offers advantages in achieving selectivity due to their more diversified structures. Discovery of inactive conformation-selective inhibitors, however, has been hampered partly by the lack of general assay methods. Herein, we present that such inhibitors can be discovered by utilizing kinase cascade assays. This type of assays is initiated with the target kinase in its unphosphorylated, inactive conformation, which is activated during the assay. Inactive conformation-selective inhibitors stabilize the inactive kinase, block activation and yield reduced kinase activity. We investigate properties of the assay by mathematical modeling, as well as by proof-of-concept experiments using the BRAF-MEK1 cascade. The present study demonstrates effective identification of inactive conformation-selective inhibitors by cascade assays, reveals key factors that impact results, and provides guidelines for successful cascade assay development.

INTRODUCTION Dysregulation of protein kinases are implicated in numerous diseases including cancer,1,2 diabetes, and inflammation.3 Kinases are of great interest in the context of drug discovery,4 as they constitute one of the largest protein families in the druggable genome.5 However, despite FDA approval of 28 kinase inhibitors between 2001 and 2015,6 drug discovery programs targeting protein kinases still face high failure rates. One of the major hurdles is achieving selectivity across the human kinome, which stems from the high structural conservation at the ATP binding pocket of activated kinases.7 Furthermore, the wide use of activated kinases in high-throughput screens and lead optimization assays bias inhibitor discovery towards so-called Type I kinase inhibitors that bind in the ATP binding pocket.8 Targeting inactive protein kinases can offer better opportunities for selective inhibitor discovery due to their higher structural diversity.7,9,10 This strategy was demonstrated serendipitously by the discovery of imatinib, a selective inhibitor of the tyrosine kinase BCR-Abl.11 Imatinib is a prototypical Type II kinase inhibitor,8 which occupies the ATP binding pocket as well as an additional site formed in the inactive “DFG-out” conformation of the activation loop.10,11 Analyses of Type II inhibitors showed that they appear to be more selective than Type I inhibitors,13,14 although they are not a general solution to the problem of kinase inhibitor selectivity.12-14 Achieving selectivity by stabilization of other inactive conformations and binding to other pockets have also been explored, as exemplified by allosteric inhibitors of MEK18,15 and TrkA16. Regardless of the preferred inactive conformation, these inhibitors reduce the target kinase activity by stabilization of the inactive form and inhibition of activation, as demonstrated in several examples including MEK1 inhibitors PD098059,17 PD184352 (CI-1040) and U0126,18,19 p38α inhibitor BIRB796,20 EGFR/Erb-B inhibitor lapatinib (Tykerb),21 and c-MET inhibitor ARQ197.22

While the advantage of targeting inactive kinases became clear years ago, it has not been widely practiced, largely due to limitations of current assay methods. In functional assays utilizing activated kinases, inactive form-selective inhibitors can be missed, since they typically bind weakly to the activated form. Functional assays measuring the basal activity of inactive kinases have led to discovery of several inactive formselective inhibitors.17,23 However, this method requires preparation of inactive kinases free of the activated form (discussed later in this manuscript), a condition not always guaranteed in practice. In contrast to enzymatic methods, screening inactive kinases using binding assays, such as mass spectrometry,16 fluorescent labels in kinases (FLiK)24,25, and probe displacement using ATP-competitive probes have been reported and had successes. However, these assays have substantial limitations, including having dynamic conformations accessible only during enzymatic turnover unavailable for compound interactions. Moreover, in the case of FLiK assays, mutagenesis and fluorescent labeling not only require steps that are not guaranteed to work for other kinases, but also can perturb protein structure and dynamics; in the case of probe displacement assays, ATP noncompetitive inhibitors may be overlooked. In the present study, we demonstrate how kinase cascade assays can be used as a general strategy for the discovery of inactive conformation-selective inhibitors. This type of assay takes advantage of kinase activation by phosphorylation, a common regulatory mechanism of most protein kinases.26,27 The term “cascade assay” has referred to any coupled assay involving more than one kinase, but here we use it to refer to the specific type of assays described below (Fig. 1). In a kinase cascade assay, the target kinase is present in an unphosphorylated, inactive form, and is pre-incubated with test compounds. During the assay, the target kinase becomes activated by either an upstream kinase or autophosphorylation, and reaction progress is monitored by measuring the catalytic activity of the activated kinase (Fig. 1A). Inactive form-selective inhibitors would bind to and stabilize the inactive kinase, pre-

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venting its activation (Fig. 1B). Compounds that inhibit only the activated kinase (Fig. 1C), or bind to both forms but do not block activation (Fig. 1D) are also detected. These compounds can be confirmed in a triage assay using the activated kinase. If the activation is achieved by an upstream kinase, a separate triage assay needs to be implemented to exclude compounds that inhibit the upstream kinase.

Figure 1. Kinase cascade assay. (A) Inactive kinase is activated during the assay, typically by phosphorylation on the activation loop. The activated kinase then catalyzes substrate phosphorylation. (B) Inactive conformation-selective inhibitors stabilize the target in its inactive state and inhibit its activation, resulting in reduced product formation, thus being detected by the assay. (C, D) Compounds that inhibit the activated form, but not kinase activation would also be detected. Such compounds may bind preferentially to the activated form (C), or bind to both forms with similar affinities (D). The kinase domain is represented as an Nterminal lobe, a C-terminal lobe, and an activation loop. Inhibitors are represented in magenta, and phosphorylation on the activation loop in yellow. The cartoon representation of a kinase is adapted from reference 28.

Although the kinetics of kinase activation by either an upstream kinase 29 or by autophosphorylation28,30 has been studied thoroughly, the effects of inhibitors of different modes of action on kinase activation kinetics have not been reported. Cascade assays have been used for potency measurements of inactive form-selective inhibitors in a few cases,31,32 but establishing general parameters of cascade assays have not been investigated, and critical factors that affect the sensitivity of cascade assays to detect inactive conformation-selective inhibitors are not well understood. In contrast to active kinase assays, which can be formatted according to general principles of assay development,33 cascade kinase assays are more complex (e.g. non-linear progress curves) and generally have more parameters that require optimization. To provide a comprehensive theoretical framework to understand cascade assays, and to predict how these parameters affect assay results, we performed mathematical modeling. We further validated key conclusions of the mathematical modeling by proof-of-concept experiments using the BRAF-MEK1 cascade.

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Mathematical Modeling of Cascade Assays. We considered four common activation mechanisms, including activation by an upstream kinase (Fig. 2A), and activation by three possible autophosphorylation scenarios: intramolecular (cis) autophosphorylation (Fig. 2B), intermolecular (trans) autophosphorylation by the inactive kinase itself (Fig. 2C), or by the activated kinase (Fig. 2D).30 Mechanisms that are more complicated than these four may exist.28 In the present study, we have focused on cascade assays involving activation by an upstream kinase (Fig. 2A); modeling of other scenarios are presented in Supporting Information.

Figure 2. Four possible mechanisms of kinase activation. (A), activation by an upstream kinase; (B), activation by cisautophosphorylation; (C), activation by transautophosphorylation, by the inactive kinase; (D), activation by trans-autophosphorylation, by the activated kinase. The cartoon representation of a kinase is adapted from reference 28.

To model cascade assays involving activation by an upstream kinase, we adopted an approach similar to those used in modeling of kinase activation,28,30 and built our model based on the following assumptions and approximations: (1) Reactions and equilibria presented in Scheme 1 were considered. The upstream kinase (E′) can potentially phosphorylate four species of the inactive kinase E, each with a different rate of activation (Scheme 1A). (2) Michaelis-Menten kinetics is not considered for activation reactions shown in (A), assuming [E] is much smaller than the KM of the upstream kinase. (3) No ligand depletion (i.e. [S] and [I] are equal to the total concentration of substrate and inhibitor, respectively). (4) Peptide or protein substrates are not modeled explicitly. For kinases that exhibit interdependent KM(ATP) and KM(peptide/ protein) values, this treatment is valid if the consumption of substrates is negligible in comparison with the total amount of substrates. In addition, kinase inhibitors are often non-competitive with respect to these substrates. The amount of protein/peptide phosphorylation can be inferred from the amount of ADP produced. (5) Partial inhibition is not considered. Tertiary complexes of enzyme-inhibitor-substrate are catalytically inactive. (6) The concentration of ADP produced by autophosphorylation is negligible.

Materials and Methods ACS Paragon Plus Environment

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Biochemistry ܻ = ‫ ݉݋ݐݐ݋ܤ‬+

Scheme 1. Activation reactions (A) and chemical equilibria (B) considered in the mathematical modeling for cascade assays involving activation by an upstream kinase. E represents the inactive kinase; E* the activated kinase; E′ the upstream kinase; S the substrate (ATP); P the product (ADP); I the inhibitor. The factors α0 and α reflect how the binding of one ligand affects the binding of the other to the inactive and active kinases, respectively. According to Scheme 1A, the rate of kinase activation is

ௗሾா೅∗ ሿ ௗ௧

= ሺ݇௔ ሾ‫ܧ‬ሿ + ݇௕ ሾ‫ܵܧ‬ሿ + ݇௖ ሾ‫ܫܧ‬ሿ + ݇ௗ ሾ‫ܫܵܧ‬ሿሻሾ‫ ܧ‬ᇱ ሿ


= ݇௢௕௦ ሺሾܶሿ − ሾ‫ ∗்ܧ‬ሿሻ



where [E T] is the total concentration of activated kinase. Expressing concentrations of each species in terms of [I], [S], [E*T], the total target kinase concentration [T], as well as equilibrium and rate constants shown in Scheme 1, Eq. 1 can be rewritten as ௗሾா೅∗ ሿ ௗ௧


݇௢௕௦ = ሾ‫ ܧ‬ᇱ ሿ ൭


௞ೌ ሾೄሿ ሾ಺ሿ ሾ಺ሿሾೄሿ ା ା ಼ೄబ ಼ವబ ഀబ ಼ವబ ಼ೄబ

௞೎ ሾೄሿ಼ವబ ಼ವబ ሾೄሿ ଵା ሾ಺ሿ ା ା ഀబ ಼ೄబ ሾ಺ሿ಼ೄబ




ሾ಺ሿ಼ೄబ ሾ಺ሿ ା ഀబ ಼ವబ ሾೄሿ಼ವబ

಼ೄబ ଵା ሾೄሿ ା


௞೏ ഀ ಼ ഀ ಼ ഀ ಼ೄబ ಼ವబ ൱. ଵା బሾ಺ሿವబ ା బሾೄሿೄబ ା బ ሾೄሿሾ಺ሿ


By integration, we can calculate the total concentration of activated kinase at time t: ሾ‫ ∗்ܧ‬ሿ = ሾܶሿሺ1 − ݂݁ ି௞೚್ೞ௧ ሻ, (4) where ݂ =

ሾ்ሿିሾாబ∗ ሿ ሾ்ሿ

, the fraction of inactive kinase at the begin-

ning of the assay. We then derive the rate of product formation: ௗሾ௉ሿ ௗ௧

= ݇௖௔௧ ሾ‫ܵ ∗ ܧ‬ሿ + ݇௖௔௧,଴ ሾ‫ܵܧ‬ሿ = ‫ݒ‬௦ − ሺ‫ݒ‬௦ − ‫ݒ‬௦ᇱ ሻ݂݁ ି௞೚್ೞ௧ (5)

where ‫ݒ‬௦ =

௞೎ೌ೟ ሾ்ሿ

ሾ಺ሿ಼ೄ ሾ಺ሿ ା ഀ಼ವ ሾೄሿ಼ವ

ೄା ଵା ሾೄሿ

, and ‫ݒ‬௦ᇱ =

௞೎ೌ೟,బ ሾ்ሿ ሾ಺ሿ಼ . ಼ೄబ ሾ಺ሿ ଵା ሾೄሿ ା ାሾೄሿ಼ೄబ ഀబ ಼ವబ


Integrating Eq. 5, we obtain Eq. 6 for calculation of [P]: ሾܲሿ =

൫௩ೞ ି௩ೞᇲ ൯௙ ௞೚್ೞ

ሺ݁ ି௞೚್ೞ௧ − 1ሻ + ‫ݒ‬௦ ‫ݐ‬


Using Eq. 6 and equations derived for other activation mechanisms (Supporting Information), reaction progress curves can be simulated. Dose-response curves can be extracted by plotting [P] as a function of [I] at a given time point. IC50 values can then be determined by fitting dose-response curves to the four-parameter logistic equation:


ଵାଵ଴ሺ೗೚೒಺಴ఱబ ష೉ሻ∗ಹ೔೗೗ ೞ೗೚೛೐


where X is the compound concentration in log units, Y the measured signal proportional to product concentration, Bottom the signal at maximal inhibition, and Top the signal at no inhibition. Chemicals and Reagents. Unphosphorylated, inactive MEK1 and constitutively active V600E BRAF were purchased from ThermoFisher Scientific. Pyruvate kinase/lactic dehydrogenase enzyme mix from rabbit muscle was obtained from Sigma-Aldrich. ADP Glo kinase kit34 was ordered from Promega. MEK1 inhibitors PD098059, U-0126, CI-1040, Trametinib, Cobimetinib were purchased from Cayman Chemical; Selumetinib from Selleckchem. Tween-20 was obtained from Enzo; all other reagents were purchased from SigmaAldrich. BRAF – MEK1 Cascade Assay. Unphosphorylated, inactive MEK1 was activated by constitutively active V600E BRAF in the assay.29 ADP production was detected either continuously by coupling to NADH oxidation using pyruvate kinase/lactic dehydrogenase (PK/LDH assay),29 or at the endpoint by ADP Glo kinase kit. Dose-response curves were fit to the four-parameter logistic equation (Equation 7) to determine IC50 values. For continuous ADP detection by PK/LDH assay, 0.4 µL inhibitor (DMSO solution) was dispensed to each well of UVStar 384-well microplates (Greiner Bio-One) using an Echo 550 acoustic dispenser (Labcyte Inc.), followed by addition of 20 µL solution containing 533 µM NADH, 2 mM phosphoenolpyruvate, 20 µM ATP, and 10-fold dilution of PK/LDH enzyme mix. Cascade reactions were initiated by addition of 20 µL of a solution containing 40 nM MEK1 and 0.4 nM V600E BRAF to each well. Reaction progress was monitored by recording NADH absorbance at 340 nm for up to 10 hours at room temperature, using a Tecan Infinite M1000 plate reader. Assay mixture without MEK1 and V600E BRAF was used as background, and was subtracted to correct absorption change caused by photo-degradation of NADH. For end-point detection of ADP by ADP Glo kinase assay kit, 0.04 µL inhibitor (DMSO solution) was dispensed to each well of a white 384-well ProxiPlate (PerkinElmer) using an Echo 550 (Labcyte Inc.). 2 µL 10 nM MEK1 and 0.8 or 4 nM V600E BRAF was added to each well. After a 15 min incubation, 2 µL 20 µM ATP or 400 µM ATP was added to each well to initiate the cascade reaction. The plate was incubated at room temperature. At the assay end-point, 4 µL ADP-Glo Reagent was added to each well to quench the reaction. 5 µL ADP-Glo Detection Reagent was added to each well 40 min later. The plate was incubated for at least 60 min before reading luminescence from each well. All assays were performed in buffer containing 40 mM Tris·HCl, pH 7.4, 10 mM MgCl2, 0.001% Tween-20, 0.2 mg/mL BSA, and 2 mM DTT.

RESULTS AND DISCUSSION Key Properties of Cascade Assays Revealed by Mathematical Modeling. Mathematical modeling shows that the cascade assay can detect inactive form-selective inhibitors, which would be otherwise missed in assays of activated kinases. For example, consider an allosteric and inactive conformation-selective inhibitor that is non-competitive with respect to ATP (α0 = α = 1) with a KD0 value (dissociation constant of

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the inhibitor-inactive kinase complex) of 2 µM and a KD value (dissociation constant of the inhibitor-activated kinase complex) of 200 µM. Reaction progress curves of 20 nM inactive kinase activated by 0.2 nM upstream kinase in the presence of the inhibitor were calculated using Eq. 6 (Fig. 3A). A lag phase is apparent at the beginning of the time course, which is a common characteristic of enzymes undergoing activation.28,30 Dose-response curves at seven time points are extracted and fit to Eq. 7 to determine their IC50 values (Fig. 3B). The IC50 values range between 2 to 6 µM, depending on the end-point of the cascade assay (Fig. 3C, blue trace). If this compound were assayed against the activated form, the IC50 value would be 200 µM, and would be missed under standard screening conditions that are usually set up with compound concentrations at 10 – 20 µM. Similar results were obtained with cascade assays following other activation mechanisms (Figs. S1-3).

Figure 3. Mathematical modeling of a cascade assay involving activation by an upstream kinase in the presence of an inactive conformation-selective inhibitor. (A) Simulated reaction progress curves of 20 nM inactive kinase and 0.2 nM upstream kinase, at twelve inhibitor concentrations. (B) Simulated dose-response curves at seven time points, extracted from (A). (C) IC50 values at fourteen time-points, at two upstream kinase concentrations, 0.2 or 2 nM. (D) the same set of IC50 data as in (C), but plotted as a function of cascade reaction time normalized by half-lives of activation. The KD0 value was set to be 2 µM and KD value 200 µM. Other simulation parameters are: ka = kb = 35 µM-1min-1 and kc = kd = 0 µM-1min-1 (i.e. inhibitor-bound forms do not get activated); [T] = 20 nM; [E'] = 0.2 or 2 nM; [S] = 10 µM; KM = 10 µM; kcat = 5 min-1; kcat,0 (kcat of the inactive kinase) = 0 min-1; f (fraction of the inactive form in the kinase preparation used for the assay) = 1; α0 = α = 1. ATP concentration (10 µM) does not change in the simulation, as is the case when ADP is detected continuously by pyruvate/lactic dehydrogenase (PK/ LDH) assay, and ADP is recycled back to ATP.35

The simulation also demonstrates that kinase cascade assays tolerate small amounts of pre-existing activated kinase, which is often unavoidable in inactive kinase preparations. This can be demonstrated with the following example. Let us again consider the same inhibitor and cascade reaction discussed in Fig. 3, and assume that the inactive kinase preparation con-

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tains activated kinase. In the presence of 5% activated form, an IC50 value of 4 µM is obtained by the cascade assay at t1/2 (Fig. 4, orange trace), only 2-fold higher than the KD0 value of 2 µM for the inactive form. In contrast, if the potency is assayed by measuring basal activities of the inactive kinase (no kinase activation in the assay), trace amounts of activated kinase increase the IC50 value significantly. For the same inactive conformation-selective inhibitor discussed above, the IC50 value would increase to 7.9 µM when assayed in the presence of as low as 1% activated form, nearly 4-fold higher than the KD0 value; or 79 µM in the presence of 5% activated form, nearly 40-fold higher than the KD0 value (Fig. S4; assuming the activated kinase is 10-fold more active than the inactive kinase). As apparent in Fig. 3C, the inactive conformation-selective inhibitor exhibits increased IC50 values with increasing reaction time in cascade assays. This is due to the generation of increasing concentrations of the activated kinase, which is inhibited weakly by the inactive conformation-selective inhibitor. The simulation further shows that the faster the kinase is activated, the faster the IC50 value increases. For example, when the rate of kinase activation is increased 10-fold by adding 10-fold more upstream kinase, the IC50 value also increases 10-fold faster (Fig. 3C, yellow trace). If the time scale is normalized by the half-life of kinase activation in the absence of the inhibitor (t1/2 = 0.693/kobs, at [I] = 0), the two curves in Fig. 3C overlap as shown in Fig. 3D. Therefore, balancing the extent of target kinase activation with the amount of product generated for robust assay readout is critical for establishing cascade assays that are sensitive to inactive conformationselective inhibitors; faster target kinase activation requires shorter reaction times. In the case shown in Fig. 3, at reaction time equals to t1/2 (99 min with 0.2 nM upstream kinase, or 9.9 min with 2 nM upstream kinase), the IC50 value is only 1.2fold higher than the KD0 value. When the reaction time is extended to ten half-lives, the IC50 value increases to over 8-fold higher than the KD0 value, which is unfavorable for the discovery of inactive conformation-selective inhibitors. In practice, the earliest assay end-point is limited by the handling time for initiating and quenching the reaction, and/or the minimum amount of product required for reliable detection.

Figure 4. Effect of pre-existing activated kinase on the IC50 value of inactive conformation-selective inhibitors measured by cascade assay. Reaction time is normalized to the half-life of kinase activation (t1/2). Simulation parameters are the same as those described in Fig. 3 legend, except that the fraction of pre-existing activated kinase is set to be 0, 5, 25, 50, and 100%. Upstream kinase concentration [E'] is set to be 0.2 nM.

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Although tolerable when present at low levels, the preexisting activated kinase changes the behavior of cascade assays. It causes higher IC50 values when the reaction time is short (Fig. 4), because at early time points most of the product is derived from the activated form, which is inhibited weakly by inactive conformation-selective inhibitors. At later reaction time points, as most of the product comes from target kinase that was activated during the cascade assay, the IC50 value drops, but not to the level where there is no pre-existing activated kinase. High levels of pre-existing activated kinase are detrimental, as the minimal IC50 value of a given inactive conformation-selective inhibitor increases as the percentage of the pre-existing activated kinase increases (Fig. 4). Therefore, the amount of the pre-existing activated form should be minimized in the kinase preparation; if there are such impurities, the reaction time should be long enough to reach the minimal IC50 value. In the case shown in Fig. 4, with 5% pre-existing activated kinase, the cascade reaction should proceed for about one half-life, to reach the lowest IC50 value. Experimental Validation of Cascade Assays. To validate some of the key implications from mathematical modeling, and to demonstrate experimentally that cascade assays can indeed identify inactive conformation-selective inhibitors, we experimentally investigated the inhibition of MEK1 using a cascade assay with its upstream kinase BRAF. The BRAFMEK1 cascade is part of the MAPK/ERK signaling pathway, which is often hyper-activated in cancers, and is a target of antiproliferative drugs.36,37 Multiple selective allosteric inhibitors targeting inactive MEK1 have been developed.37,38 Here, we used the oncogenic BRAF mutant V600E to activate MEK1.39 Reaction kinetics of this cascade has been established, and has been used to measure BRAF activity.29 We first briefly revisit the basic kinetic behavior of this cascade in the absence of inhibitors. Reaction progress of MEK1 activation was followed by monitoring the ATPase activity of the activated MEK1, which correlates with its kinase activity.29 To study reaction kinetics conveniently, ADP production was coupled to NADH oxidation for detection by absorbance at 340 nm, using a continuous pyruvate kinase/lactic dehydrogenase (PK/LDH) assay.29 At a fixed V600E BRAF concentration, the reaction is pseudo-first order with respect to MEK1, as evidenced by the overlapping progress curves when normalized by MEK1 concentration (Fig. S5).

gress curves shown in (A). Concentration of inactive MEK1 was 20 nM; V600E BRAF was 0.2 nM; ATP was 10 µM.

We then studied MEK1 inhibition by the inactive conformation-selective inhibitor PD09805917,40 using the BRAF – MEK1 cascade assay. By fitting reaction progress curves to Eq. 6 (Fig. 5A), a KD0 value of 2.3 µM was obtained; by fitting dose-response curves at seven time points to the fourparameter logistic equation (Eq. 7), IC50 values of 2-5 µM were determined (Fig. 5B). In good agreement with the prediction of mathematical modeling shown in Fig. 3, the IC50 value increased over time (Fig. 5B). The results are also in consistent with the published IC50 value range of 2-7 µM, which were determined by measuring the basal activity of inactive MEK1 using the MEK1-ERK2-myelin basic protein (MBP) coupled assay.17,40 We next studied the effect of activation kinetics on IC50 value, using the BRAF – MEK1 cascade by varying the concentration of BRAF. In this experiment, cascade assay reactions were carried out at two V600E BRAF concentrations (0.4 and 2 nM). Reactions were quenched at several time points, and ADP production was determined using the ADP Glo kinase kit.34 With a 5-fold higher V600E BRAF concentration, the IC50 value increased 3.3-fold faster over time (Fig. 6), reminiscent of the mathematical modeling result shown in Fig. 3C.

Figure 6. IC50 values of PD098059 as a function of cascade reaction time. In this assay, 5 nM MEK1 was activated by V600E BRAF at 0.4 nM (closed circle) or 2 nM (open circle). ADP produced at each time-point was measured using an ADP-Glo kinase kit.34 ATP concentration was 200 µM to ensure less than 10% ATP consumption even with the longest reaction time, since the ADP-Glo assay does not recycle ADP back to ATP.

Table 1. MEK1 inhibitors tested by cascade assay in an end-point format using ADP Glo kinase kita

Figure 5. Inhibition of inactive MEK1 by PD098059 as measured by the BRAF – MEK1 cascade assay. (A) cascade reaction progress curves at twelve PD098059 concentrations. Dashed lines are fitted progress curves using Eq. 6. Details of data fitting are presented in Supporting Information. (B) IC50 values of PD098059 as a function of reaction time (bottom x-axis) or reaction time normalized by t1/2 (top x-axis). Closed circles represent experimental values, and open circles are results derived from the fitted pro-

Compound name

IC50 values, this study

IC50 values, literature


2.2±0.6 µM

2-7 µM b


45±3 nM

72 nM c

CI-1040 (PD184352)

6.7±1.0 nM

2.3 nM b


1.7±0.1 nM

0.7 nM d


13±2 nM

14 nM e

Cobimetinib (GDC-0973)

2.7±0.2 nM

0.9 nM f


Inhibitor potencies were evaluated by the BRAF – MEK1 cascade assay, using 5 nM inactive MEK1, 0.4 nM V600E BRAF, and 10 µM ATP. ADP production was determined using ADP-

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Glo34 after 120 min reaction. Cited IC50 values were determined by the following methods: b measuring basal activity of inactive MEK1, coupled to ERK2 activation and subsequent MBP phosphorylation;17 c measuring ERK2 phosphorylation by immunoprecipitated MEK1 or constitutively active MEK1 mutant;18 d measuring activity of MEK1 after activation by BRAF in the presence of the inhibitor;31 e measuring ERK2 phosphorylation by constitutively active MEK1 mutant;41 f measuring MBP peptide phosphorylation by ERK2 in the CRAF-MEK1-ERK2 cascade.32

Finally, we extended our study to include a panel of selective MEK1 inhibitors of diverse modes of action. These inhibitors include U-0126,18,19 CI-1040,15,19 and trametinib,31 all of which are ATP-noncompetitive, inactive conformationselective, and inhibit MEK1 activation; selumetinib, which is ATP-noncompetitive and inhibits the activated form but not MEK1 activation;41 and cobimetinib, which is ATP uncompetitive, and binds to both inactive and activated MEK1.42 IC50 values determined by the cascade assay are comparable with published values from using several different assay methods (Table 1). These results show that the cascade assay is sensitive to inhibitors with a wide range of mechanisms of action, including inactive-conformation inhibitors.

CONCLUSIONS In summary, utilization of cascade assays is an effective method for discovering inhibitors that target inactive kinases, and provides significant advantages over previous approaches. For example, compared with assays that measure basal activity of inactive kinases, cascade assays are more tolerant of small amounts of pre-existing activated kinase, which is typically difficult to avoid in practice. Accessibility to both inactive and active conformations as well as other dynamic conformations in a functional kinase assay are other advantages over binding assays or basal activity assays. In addition, cascade assays do not require mutations or fluorescent labeling on the target kinase, avoiding perturbations of kinase structure and dynamics. The present study provides a theoretical framework to understand cascade assays. Although mathematical models were built upon multiple assumptions and simplifications, they capture main features of the assays. Most notably, the potency of a given inactive form-selective inhibitor measured by a cascade assay depends on the reaction time, the rate of kinase activation, and the amount of pre-existing activated form. These features are confirmed by the proof-of-concept study of known MEK1 inhibitors using the BRAF-MEK1 cascade. Our work establishes general guidelines for assay development. In the case of activation by an upstream kinase, in the absence of pre-existing activated kinase, the IC50 value of an inactive conformation-selective inhibitor increases over time. To achieve high sensitivity for identifying inactive formselective inhibitors, reaction times that are shorter than the activation half-life (t1/2) are preferable, if enough product can be generated for robust assay readout. In addition, faster kinase activation requires shorter reaction times, as t1/2 is shorter. In the presence of pre-existing activated kinase, longer reaction times are necessary to reach the highest potency (i.e. lowest IC50 values). Additional triage assays are required to determine if inhibitors found by cascade assays are inactive conformation-selective, as cascade assays can identify inhibitors of different modes of action. This guidance allows for the development of optimized cascade assays that maximize the chances for the discovery of inactive conformation-selective inhibitors. These types of inhibitors have a better chance of

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being kinome selective, and is critical to the success of kinase drug discovery programs.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ Details for mathematical modeling of cascade assay, experimental methods, and supplementary figures (file type, PDF document).

AUTHOR INFORMATION Corresponding Author * [email protected]

Funding Sources This work was supported by Janssen Research and Development, LLC.

ACKNOWLEDGMENT We thank Dr. Gaochao Tian for helpful discussions on mathematical modeling.

ABBREVIATIONS ATP, adenosine triphosphate. ADP, adenosine diphosphate. NADH, nicotinamide adenine dinucleotide, reduced form. PK/LDH, pyruvate kinase/lactic dehydrogenase.

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