Universal and Quantitative Method To Evaluate Inhibitor Potency for

Publication Date (Web): May 18, 2017 ... that the potency of compounds can be quantitatively evaluated by performing an activity-based protein profili...
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Universal and Quantitative Method To Evaluate Inhibitor Potency for Cysteinome Proteins Using a Nonspecific Activity-Based Protein Profiling Probe Tomoya Sameshima,* Yukiya Tanaka, and Ikuo Miyahisa Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2 chome, Fujisawa, Kanagawa, Japan S Supporting Information *

ABSTRACT: Recently, there have been a limited number of new, validated targets for small-molecule drug discovery in the pharmaceutical industry. Although there are approximately 30 000 genes in the human genome, only 2% are targeted by currently approved smallmolecule drugs. One reason that many targets remain neglected by drug discovery programs is the absence of biochemical assays enabling evaluation of the potency of inhibitors in a quantitative and highthroughput manner. To overcome this issue, we developed a biochemical assay to evaluate the potency of both reversible and irreversible inhibitors using a nonspecific thiol-labeling fluorescent probe. The assay can be applied to any targets with a cysteine residue in a pocket that can accommodate small-molecule ligands. By constructing a mathematical model, we showed that the potency of compounds can be quantitatively evaluated by performing an activity-based protein profiling assay. In addition, the validity of the theory was confirmed experimentally using epidermal growth factor receptor kinase as a model target. This approach provides an assay system for targets for which biochemical assays cannot be developed. Our approach can potentially not only expand the number of exploitable targets but also accelerate the lead optimization process by providing quantitative structure−activity relationship information. compound is necessary to introduce a fluorophore to the probe compound. Recently, fluorescence polarization-activity-based protein profiling (fluopol-ABPP) was reported as an approach to address targets for which biochemical assays cannot be developed.5−7 Fluopol-ABPP is a fluorescence polarization (FP)-based assay that measures the ability of compounds to block labeling of enzymes by an ABPP probe, a compound that contains a chemically reactive warhead and a traceable tag moiety (i.e., fluorophore or biotin). The assay can be performed in a homogeneous and high-throughput screening (HTS) format and does not require highly optimized fluorescent probes because covalent bond formation will always capture the target given sufficient incubation time. Despite this desirable feature, identification of a general ABPP probe for all targets is challenging because its covalent bond formation is achieved by a catalytic mechanism within the target. Another study introduced a more general technique using a nonspecific covalent probe, such as fluorescence-labeled maleimide,8 that enabled universal application to all “cysteinome” targets, proteins that have a cysteine residue in the cavity of the protein.

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ver the past few decades, there have been a limited number of new, validated targets for small-molecule drug discovery in the pharmaceutical industry. Among approximately 30 000 genes in the human genome, approximately 3000 have been suggested as being disease related.1 However, only 549 of them are targeted by the current FDA-approved small-molecule drugs.2 One reason that many targets are neglected by drug discovery research is the absence of high-throughput biochemical assays. Enzyme inhibitors are typically discovered and evaluated by their ability to modulate enzymatic activity.3 To develop an enzymatic assay, however, a substrate for the target should be identified, and adequate enzymatic activity is required to produce a sufficient signal for robust reproducibility and sensitivity. For targets for which enzymatic assays cannot be applied, a binding assay using a traceable (i.e., radioactive, fluorescent) probe is an alternative approach.4 In this type of assay, the potency of inhibitors is evaluated by displacement of a probe from the target protein. Homogeneous fluorescencebased displacement assays have been widely applied because of lower costs and higher throughput. As a tracer ligand for a displacement assay, appropriate affinity (usually ≤1 μM) to a target protein is required to obtain a sufficient signal-tobackground ratio (S/B). To design a fluorescent probe, information on the X-ray structure of the protein−compound complex or structure−activity relationships (SAR) of a parent © 2017 American Chemical Society

Received: March 3, 2017 Revised: May 17, 2017 Published: May 18, 2017 2921

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plate. Subsequently, 3.5 μL of the buffer solution containing 30 μM of CI-1033, an irreversible EGFR kinase inhibitor,10,11 or DMSO was added to each well of the assay plate. After 4 h of incubation at room temperature, 3.5 μL of buffer solution containing different concentrations of BODIPY-MA was added to the assay plate. The final concentrations of Tb-SA, EGFR kinase, and CI-1033 were 0.17 nM, 0.1 nM, and 10 μM, respectively. The plates were sealed using MicroAmp Optical Adhesive Film (Thermo Fisher Scientific, Waltham, MA) to avoid evaporation, and the signal was measured using an EnVision plate reader every 5 min. During the time course measurement, the temperature of the assay plate was maintained at 25 °C, and the top seal was heated to 2 °C higher than the assay plate to avoid condensation on the seal. To obtain a specific EGFR kinase binding signal, the TR-FRET signal in the absence of CI-1033 was subtracted from that in the presence of 10 μm CI-1033. The progress curves were globally fitted with a single exponential equation, and the observed association rate constants were plotted against the BODIPYMA concentration. A linear equation was fit to the plot data, and the slope was used to determine the association rate constant of BODIPY-MA to EGFR kinase. The data analysis was performed using Prism 5 software (GraphPad, La Jolla, CA). Determination of kinact/KI Values of Irreversible Inhibitors by Performing the ABPP Assay. To determine kinact/KI values of irreversible inhibitors, the Tb-SA/EGFR kinase premix was added to the assay buffer containing BODIPY-MA and test compounds. The final concentrations of BODIPY-MA, Tb-SA, and biotinylated EGFR kinase were 200, 0.17, and 0.1 nM, respectively. The plate was incubated for 180 min to reach the reaction equilibrium, and the TR-FRET signal was measured to monitor enzyme occupancy by the probe. The percentage of inhibition was calculated according to eq 1:

Although fluopol-ABPP is an excellent widely applicable approach, there is no theoretical background that relates IC50 values measured in the assay to the true potencies of test compounds. To optimize the inhibitory activities of compounds using a cell-free assay, the SARs of the compounds should be described in terms of quantitative metrics that can be extrapolated to results of cell-based assays and in vivo phenomena. As quantitative metrics, affinity (Ki) and specificity constants (kinact/KI) are determined for reversible and irreversible inhibitors, respectively.3 Association of IC50 values measured by ABPP assays with these metrics will provide quantitative SAR information and contribute to rational lead optimization. To address this issue, we constructed a theoretical model that enables quantitative evaluation of test compounds by performing an ABPP assay. In this model, the potencies of reversible and irreversible inhibitors can be calculated quantitatively in the same assay format by selecting optimal incubation times. By use of a nonspecific thiol-labeling probe, the ABPP assay can be applied to any targets with a cysteine residue in a suitable binding pocket. In this study, we experimentally verified our theory using epidermal growth factor receptor (EGFR) kinase as a model target. Our study demonstrates the quantitative evaluation of reversible and irreversible inhibitors using a nonspecific covalent ABPP probe, o-maleimide BODIPY (BODIPY-MA).9

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MATERIALS AND METHODS Materials. Recombinant avi-tag fused EGFR kinase protein (variant1, 696−1022) was expressed in Sf9 cells and purified by NiNTA-column and subsequent gel-filtration chromatography. Biotinylation of avi-tag was achieved by coexpression of biotin ligase (BirA) in the expression cell. The proteins thus obtained were stored at −80 °C until use. BODIPY-MA was synthesized as described previously.9 Terbium-conjugated streptavidin (TbSA) was purchased from Cisbio (Codolet, France). HEPES solution and Kinase Tracer 199 were purchased from Thermo Fisher Scientific (Waltham, MA). Tween-20 was obtained from Bio-Rad (Hercules, CA). CI-103310,11 was from Haoyuan Chemexpress (Shanghai, China). Other reagents were obtained from Wako (Osaka, Japan). Biochemical assays were performed using an assay buffer composed of 50 mM HEPES (pH, 7.2− 7.5), 10 mM MgCl2, 1 mM EGTA, and 0.01% (w/v) Tween20. Time-Resolved FRET (TR-FRET) Assay. TR-FRET assays were performed using 384-well, white, flat-bottomed plates (ref no. 784075, Greiner Bio-One, Frickenhausen, Germany), and the signal was measured using an EnVision plate reader (PerkinElmer, Waltham, MA). The solution in each well was excited using a laser (λ = 337 nm) reflected by a dichroic mirror (D400/D505 (PerkinElmer), and fluorescence signals from terbium (Tb) and BODIPY were detected using two emission filters (CFP 486; PerkinElmer for Tb and Emission 515; PerkinElmer for BODIPY). To measure the TR-FRET signal between Tb and Kinase Tracer 199, the following filter set was used: dichroic mirror (D400/D630; PerkinElmer), emission filter APC 665 for Kinase Tracer 199 (PerkinElmer), and Cy5 620 (PerkinElmer) for Tb. Throughout this study, Tb-SA and the biotinylated EGFR kinase complex were incubated for >1 h at room temperature before initiation of the reaction. Determination of the Association Rate Constant of BODIPY-MA to EGFR Kinase. First, 3.5 μL of the Tb-SA/ EGFR kinase premix was dispensed into each well of the assay

⎛μ −T⎞ ⎟⎟ percentage of inhibition = 100 × ⎜⎜ H ⎝ μH − μL ⎠

(1)

where T is the value of the wells containing test compounds, and μH and μL are the mean values of the 0% and 100% inhibition control wells, respectively. The values of the 100% and 0% inhibition controls were the signals obtained in the presence and absence of 10 μM CI-1033, respectively. The inhibitory activities against the concentrations of the inhibitors were determined using the IC50 equation. Data analysis was performed using GraphPad Prism 5 software. IC50 values obtained from the ABPP assay were converted to kinact/KI using eq 2.11 (k inact /KI)inhibitor =

k probe[P] IC50

(2)

where kprobe and [P] indicate the reaction rate constant to the target protein and concentration of ABPP probe, respectively. Determination of the Ki Values of Reversible EGFR Kinase Inhibitors Using Kinase Tracer 199 or BODIPY-MA. An Access Echo555 (Labcyte, Sunnyvale, CA) was used to dispense 25 nL of test inhibitors in DMSO into an assay plate. Subsequently, 2.5 μL of assay buffer containing Kinase Tracer 19912 or BODIPY-MA was added to each well of the assay-ready plate. The reaction was initiated by addition of 2.5 μL of the Tb-SA/ EGFR kinase premix. The final concentrations of Kinase Tracer 199, BODIPY-MA, Tb-SA, and EGFR kinase were 20, 50, 0.17, 2922

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reactive cysteine residue (Cys797) in the catalytic domain that is the binding site for small-molecule inhibitors10,14 and (2) various reversible and irreversible inhibitors with a wide range of potencies have been reported.10,11 For an assay system, we used TR-FRET because of its high sensitivity and simple assay procedure. As a nonspecific thiol-reactive fluorescent probe, omaleimide BODIPY (BODIPY-MA) (Figure 2A) was used because it emits strong fluorescence only when it reacts to a cysteine residue on a protein,9 which decreases the background signal (Figure S1). Because streptavidin has no cysteine residue (Table S1) and is thus unreactive to the BODIPY-MA probe (Figure S2), EGFR kinase was labeled with terbium-conjugated streptavidin via biotin on the avi-tag of EGFR kinase. To determine the association rate constant of BODIPY-MA to EGFR, the change in the TR-FRET signal was measured at various concentrations of BODIPY-MA in the presence or absence of CI-1033, an EGFR kinase inhibitor that binds covalently and irreversibly binds to Cys797 in the ATP-binding region.10,11 As shown in Figure 2B, an increase in the TR-FRET signal was obtained only in the absence of CI-1033, which suggested that Cys797 in the ATP-binding site is preferentially modified by BODIPY-MA among six cysteine residues of EGFR kinase. From the progress curve analysis (Figure 2C), kprobe, the reaction rate constant of BODIPY-MA, was calculated as (2.9 ± 0.07) × 103 M−1 s−1 (Figure 2D, the error is the SEM of the curve fitting). The half-time of the reaction was 21 min in the presence of 200 nM BODIPY-MA (Figure 2D). Because a longer than 5× half-time incubation is required to occupy >97% of the receptor,11 we selected 180 min as the incubation time for compound evaluation. Next, we tested whether the potencies of irreversible inhibitors could be quantitatively measured using the ABPP assay. The kinact/KI values of EGFR kinase inhibitors (Table S2) were determined from the probe displacement assay against the BODIPY-MA probe. In the presence of a 200-nM probe and a gradient of inhibitor concentrations, enzyme occupancy by the probe was monitored using TR-FRET. From IC50 values in this assay, the kinact/KI for each inhibitor was determined using eq 2. These values showed excellent agreement with the kinact/KI values previously determined by kinetic experiment.11 When plotting the results in log−log space, a linear relationship with a high correlation coefficient was observed (r = 0.99) (Figure 3). This result indicated that the kinact/KI of an irreversible inhibitor could be quantitatively determined using a nonspecific cysteinelabeling probe. Determination of Ki Values of Reversible Inhibitors Using the ABPP Assay. Inhibition of reversible inhibitors can also be monitored using the ABPP assay because reversible inhibitors block access of the ABPP probe to the target protein and delay the reaction rate. As stated in previous reports, the assay should be performed before the reaction reaches completion because inhibition by a reversible inhibitor in the ABPP assay decreases in a time-dependent manner.5,8,15 Specifically, the ABPP assay should be performed at the initiation of the reaction to evaluate the potencies of reversible inhibitors. To apply the ABPP assay to quantitative SAR studies of reversible inhibitors, we investigated the relationship between IC50 values in the initial phase of the ABPP assay and the affinity (Ki) values of inhibitors. The reaction scheme of the ABPP assay is described in Figure 1B for evaluation of reversible inhibitors. If the interaction between an inhibitor and

and 0.1 nM, respectively. After 30 min incubation at room temperature, the TR-FRET signal was measured using an EnVision plate reader. The values of 100% and 0% inhibition control were set to the FRET ratio in the presence and absence of 10 μM CI-1033. The percentage of inhibition was calculated using eq 1, and IC50 values were determined using GraphPad Prism 5 software to fit a logistic equation curve. IC50 values obtained using the Kinase Tracer 199 was converted to Ki using the Cheng−Prusoff equation (eq 3).13 ⎛ [P] ⎞ IC50 = K i⎜1 + ⎟ Kd ⎠ ⎝

(3)

where [P] and Kd indicate the concentration and dissociation constant of Kinase Tracer 199 to EGFR kinase, respectively.

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RESULTS Evaluation of the kinact/KI Values of Irreversible Inhibitors Using a Nonspecific Thiol-Reactive Probe. At first, we experimentally verified whether the kinact/KI of irreversible inhibitors could be quantitatively determined using a nonspecific thiol-reactive probe. In the previous study, the kinact/KI values of the irreversible inhibitors were measured using a target-specific irreversible probe compound.11 Although this is a convenient high-throughput assay, it cannot be applied to all targets because preparation of a target-specific irreversible probe is usually challenging unless its ligand is identified. For a more universal application of this approach, we used a nonspecific thiol-labeling fluorescent probe as a tracer molecule because it reacts to the target if a reactive and accessible cysteine residue is present in the pocket. In the ABPP assay using an irreversible probe, the reaction scheme is described as shown in Figure 1A. In this assay, the

Figure 1. Reaction model of the TR-FRET ABPP assay for irreversible (A) and reversible (B) inhibitors. R, I, RI, RI*, and P represent target protein, inhibitor, reversible protein−inhibitor complex, irreversible protein−inhibitor complex, and ABPP probe, respectively.

probe (P) is irreversible and competitive with the test inhibitors (I). The signal is observable only when it forms a complex with the target protein (RP). At t = 0, assays are initiated by addition of protein (referred to as R) to solutions containing a fixed concentration of the probe and various concentration of a test inhibitor. In this model, we considered the following assumptions: (1) the probe and test inhibitors bind mutually and exclusively to R, (2) they show 1:1 binding to R, and (3) no receptor-mediated ligand depletion occurs. According to a previous report,11 the relationship between the kinact/KI values of the test inhibitors and IC50 values at infinite time can be expressed as eq 2 (see Materials and Methods). As a model target for the ABPP assay, we selected EGFR kinase for the following two reasons: (1) the protein contains a 2923

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Figure 2. Time course of the TR-FRET signal derived from BODIPY-MA binding to EGFR kinase. (A) Chemical structure of BODIPY-MA. (B) Time course of the TR-FRET signal in the absence (black) or presence (red) of 10 μM CI-1033. The concentration of BODIPY-MA was 200 nM. Data are given as the mean ± SEM (n = 4). (C) Time course of the corrected TR-FRET signal at varied concentrations of BODIPY-MA. The TRFRET signal of target-specific binding was obtained by subtracting the signal from a sample containing 10 μM CI-1033 from that without the inhibitor. Data are given as the mean ± SEM (n = 4). The solid lines are the single exponential curves, C[1 − exp(−kobs)t], fit to the data by leastsquares fitting. R2 value of global fitting was 0.99. (D) Replot of the kobs values versus probe concentrations. Data are presented with an SEM of curve fitting as in panel C. The replot data was fitted with the following function, kprobe[BODIPY-MA] (R2 = 0.99). kprobe was determined to be (2.9 ± 0.07) × 103 (M−1 s−1) (mean ± SEM of curve fitting).

k probeR 0[P] d[RP] (t = 0) = [I] dt 1+ K i

That is, if the probe and test compounds are mixed with the target protein simultaneously, IC50 values of the compounds correspond to the Ki value (detailed derivation is expressed in the Supporting Information, Appendix I). Because the amount of [RP] is proportional to the velocity of [RP] formation in a linear region of the reaction, IC50 values of [RP] are also equal to Ki values of a reversible inhibitor. Next, we determined the Ki values of EGFR kinase inhibitors from their IC50 values using our ABPP assay and a conventional binding assay to investigate the validity of our theory for reversible inhibitors. Because our theory of the ABPP assay is based on a rapid equilibrium assumption, we evaluated the time dependency of EGFR kinase inhibitors by performing the TRFRET binding assay using Kinase Tracer 199,12 a commercially available reversible EGFR kinase ligand, to select inhibitors for further analysis. Among them, 18 compounds with no time dependency were selected (Table S3). Ki was determined from the IC50 value in the presence of 20 nM Kinase Tracer 199 using the Cheng−Prusoff equation (the Kd of Kinase Tracer 199 to EGFR kinase was 150 ± 14 nM (mean ± SEM of curve fitting); Figure S3). Next, we determined the Ki values of EGFR kinase inhibitors using a TR-FRET-based ABPP assay. To determine the assay condition, we examined the linear region of the BODIPY-MA binding reaction to EGFR kinase. From the progress curve in the presence of 50 nM BODIPY-MA (Figure 4A), we selected 30 min as an incubation time for evaluation of the reversible inhibitors. Under this condition, the S/B ratio and Z′ factor of the assay were calculated to be 2.3 and 0.57 (Figure S4), respectively, which suggested that the assay can be performed with high quality, exhibiting a wide separation

Figure 3. Correlation of kinact/KI measured by the kinetic assay11 and TR-FRET ABPP assay (r = 0.99). Data are presented as the mean ± SEM of log-transformed kinact/KI values of three independent experiments.

protein is in rapid equilibrium, the amount of [RP] is described as a function of [I] (eq 4): ⎧ ⎛ ⎞⎫ ⎪ ⎜ k probe[P] ⎟⎪ ⎨ [RP] = R 0 1 − exp⎜ − [I] t ⎟⎬ ⎜ ⎟⎪ ⎪ 1 + ⎝ Ki ⎠⎭ ⎩

(5)

(4)

where Ro, [RP], and [I] represent the concentrations of the total target protein, protein−probe adduct, and inhibitor, respectively. By differentiating eq 4 with respect to t, the initial velocity of probe adduct formation is expressed as eq 5: 2924

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Figure 4. Evaluation of Ki values of reversible EGFR kinase inhibitors using the TR-FRET ABPP assay. (A) Time course of target-specific TR-FRET signal in the presence of 50 nM BODIPY-MA. Data are given as the mean ± SEM (n = 4). The solid line represents a linear regression curve (R2 = 0.96). The intercept was fixed at 0, and the slope was calculated as 0.0081 ± 0.0002 (mean ± SEM of curve fitting). (B) Correlation between Ki values obtained from the reversible Kinase Tracer 199 and TR-FRET ABPP approach (r = 0.97). Each point represents the mean ± SEM of pKi values of three independent experiments.

5-fold of half-time, and longer incubation. As reported in a previous report,11 irreversibility of test compounds can be evaluated by comparing IC50 values at two different incubation times after the reaction reaches equilibrium. If IC50 values at 5fold of half-time and longer incubation are equivalent, the test compounds are considered to be irreversible inhibitors, and the potency is evaluated by kinact/KI. On the other hand, if IC50 values after overnight incubation are larger than those of 5-fold of half-time incubation, the test compounds are expected to be reversible inhibitors, and the potency should be evaluated by Ki obtained from IC50 values of the reaction at the initial phase. Some compounds are known to show slow-binding behaviors.3 The typical slow-binding reaction scheme (induced fit model) is shown in Figure S5. In this scheme, the inhibitor (I) forms the initial complex (RI) with target protein (R) under rapid equilibrium conditions followed by isomerization into a tighter binding complex (RI*). Potency of such compounds can be evaluated using the ABPP assay in analogy to an enzymatic assay; namely, when the decrease in IC50 values is observed after preincubation, the test compound is considered to be a slow-binding inhibitor. As shown in Supporting Information (Appendix II), IC50 values after a sufficiently long incubation correspond to the true affinity of the inhibitors (Ki* = (Ki /(1 + k5/k6)), whereas those measured without preincubation correspond to the affinity of the initial encounter complex (Ki). There are some points that must be kept in mind when evaluating slow-binding inhibitor using ABPP assay. First, the assay should be conducted in a linear phase of the reaction of ABPP probe as is the case with rapid reversible inhibitors. Second, the reaction time of the ABPP probe should be much shorter than the time scale of slow-binding behaviors because long incubation will shift the equilibrium to the target−probe complex. As stated above, the assay should be performed in a linear phase of the reaction to evaluate the potencies of reversible inhibitors. Under such conditions, a highly sensitive assay format is required because only 10−20% of the maximum signal can be obtained. To meet the needed criteria, we applied TR-FRET as a detection system for the ABPP assay. TR-FRET is based on the ratiometric measurement of donor and acceptor

between signal and background and low data variability. Using this assay, we evaluated the Ki values of EGFR kinase inhibitors from their IC50 values. The Ki values obtained from the ABPP assay were well correlated with those obtained from the conventional TR-FRET binding assay (r = 0.97, Figure 4B), which indicated that the affinity of reversible inhibitors can be quantitatively determined from their IC50 values in the initial phase of the ABPP assay.

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DISCUSSION In this study, we applied the theoretical background that supports the association between IC50 values obtained from the ABPP assay with the true potencies of test compounds (Ki or kinact/KI). Using a nonspecific thiol-reactive probe, we found that the assay can potentially be applied to any target that has a cysteine residue in a suitable binding pocket. It should be noted that IC50 values of ABPP assay do not always reflect IC50 values of the biological response. For example, inhibitory activity of allosteric inhibitors cannot be evaluated unless they alter the structure of the pocket containing target cysteine residue because our assay is based on the assumption that ABPP probe and test inhibitors share the same binding site. To interpret the assay result using the ABPP assay, the inhibitory mechanism of test compounds should be carefully examined. Our assay enabled quantitative evaluation of the potencies of reversible and irreversible inhibitors in the same assay format by selecting optimal incubation times. After a long incubation time, the kinact/KI values of irreversible inhibitors can be determined quantitatively, but the potency of reversible inhibitors is underestimated because the occupancy of covalent ABPP increases in a time-dependent manner. In contrast, IC50 values of the initial velocity of the reaction correspond to the Ki values of reversible inhibitors and do not reflect the kinact/KI values of irreversible inhibitors (see mathematical derivation in the Supporting Information, Appendix I). For these reasons, it is important to optimize the incubation times for the ABPP assay depending on the properties of the test compounds. To evaluate a compound collection containing both reversible and irreversible inhibitors, we recommend the measurement of binding signal at three different time points; reaction initiation, 2925

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Biochemistry fluorescence and is less susceptible to background noise, scattered light, and autofluorescence from test compounds than is FP; thus, it provides stable assay results.16 As a fluorescent probe, we used BODIPY-MA, which emits a high fluorescence signal only when it reacts with a thiol residue9 and thus gives a low background signal (Figure S1). Combining the properties of TR-FRET and BODIPY-MA, we constructed a highly robust assay system (S/B = 2.3, Z′ = 0.57, Figure S4). On the basis of the reaction rate of BODIPY-MA to EGFR (2.9 × 103 M−1 s−1, Figure 2C,D), robust ABPP assay system is likely to develop as long as the target cysteine residue has approximately 10-fold higher reactivity than GSH (3.9 × 102 M−1 s−1).17 In addition, the assay can be performed in a homogeneous format. Because of its high sensitivity and simple procedure, the assay can easily be applied to HTS of a large-scale compound library. To develop an ABPP assay based on TR-FRET, the target must be labeled with a tag-affinity protein conjugated with a donor fluorophore.16 The tag-affinity antibody usually used in the TR-FRET assay has cysteine resides and is susceptible to modification by a nonspecific thiol-labeling probe, thus leading to a nonspecific fluorescence signal (Figure S2). On the other hand, streptavidin has no cysteine residue (Table S1) and is unreactive to nonspecific cysteine-labeling reagents (Figure S2). For this reason, biotinylated proteins via avi-tag or chemical labeling are desirable for development of TR-FRET-based ABPP assays. Some proteins have cysteine residues on the protein surface as well as in the catalytic site, so nonspecific labeling probes may react with nontargeted residues. In fact, the kinase domain of EGFR kinase has six cysteine residues, and two of them (Cys781 and Cys797) are exposed on the surface (PDB code: 2ITY). The reason for the preferential labeling of Cys797 is considered to be that the side chain of the thiol residue is the most solvent-exposed compared with those of the other cysteine residues. In fact, a previous report showed that Cys797 is preferentially oxidized by H2O2,10 which suggested that Cys797 is considered to be the most susceptible to attack from electrophilic small-molecule compounds. Our ABPP assay can be easily applied for targets, such as cysteine proteases, for which cysteine residues in their active site are activated.18 If nucleophilicity of the targeted cysteine residue is not sufficient and other cysteine residues are modified by the ABPP probe, the ABPP assay may not be developed due to the high background signal. In this case, nontargeted cysteine residues should be replaced by other residues, such as serine or alanine, to avoid nonspecific labeling. Theoretically, our approach can also be applied to targets with no cysteine in the catalytic site by introducing a cysteine residue. In the case of the protein kinase family, sequence alignment indicates that there are nine kinases that also possess a cysteine at the same position as Cys797 of EGFR kinase.19 It has been reported that some EGFR kinase covalent inhibitors cross-react with these kinases,20 which indicates that all kinases possessing a cysteine residue at the position corresponding to Cys797 in EGFR kinase are expected to be covalently modified by electrophilic compounds. For example, a c-Src mutant that genetically introduced a cysteine residue at the position corresponding to Cys797 in EGFR kinase was modified by EGFR kinase covalent inhibitors.21 On the basis of these findings, it should be possible to apply the ABPP approach by introducing a cysteine mutant. Achieving selective inhibition of specific protein kinases is challenging because of the similarity of the ATP-binding sites among the kinase family.22 The ABPP

approach will enable development of a common assay format and evaluation of inhibitory activity against a large amount of kinase. The ABPP assay is expected to be applicable beyond the protein kinase target class. For example, bromodomains, epigenetic acetyl lysine-recognizing reader proteins, are known to contain a deep cavity the size of which is sufficient to accommodate small-molecule ligands.23,24 Despite the high druggability, inhibitors of some bromodomains have not been identified, partly because of the absence of an HTS-applicable biochemical assay. A previous report showed that 55 out of 61 bromodomains contained one or more cysteine residues distributed across three areas in the vicinity of the substrate binding site.25 By exploiting the cysteine residues using nonspecific thiol-reactive probes, the ABPP assay can be developed for HTS of intractable bromodomains analogous to those in the kinase family. In conclusion, in this study, we quantitatively evaluated reversible and irreversible inhibitors using a nonspecific thiollabeling ABPP probe. Our approach has the potential to not only increase the number of drug targets that can be screened but also accelerate the lead optimization process by providing quantitative SAR information.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00190. Supplemental methods and results, summary of kinact/KI and Ki values of test compounds evaluated by conventional and our ABPP approach methods, derivation of ordinary equation model of ABPP assay (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Tomoya Sameshima: 0000-0002-3580-3095 Author Contributions

T.S. and I.M. designed the experiments; T.S. and Y.T. performed the experiments; T.S. analyzed the experimental data; T.S. and I.M. wrote the paper. All authors read and approved the final manuscript. Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Shoichi Okubo, Mika Gotou, and Yoshihiko Hirozane for their technical assistance. We also thank Satoshi Sogabe and Phillip A. Schwartz for their critical advice on the EGFR kinase structure. We appreciate Junichi Sakamoto and Junji Matsui for their assistance in this research and for their critical reading of the manuscript. The authors would like to thank Enago (www.enago.jp) for the English language review. 2926

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ABBREVIATION Fluopol-ABPP, fluorescence polarization-activity-based protein profiling; FP, fluorescence polarization; TR-FRET, timeresolved fluorescence resonance energy transfer; EGFR, epidermal growth factor receptor; BODIPY-MA, o-maleimide BODIPY; SAR, structure−activity relationship; Tb, terbium; SA, streptavidin; HTS, high-throughput screening

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DOI: 10.1021/acs.biochem.7b00190 Biochemistry 2017, 56, 2921−2927