Target Residence Time-Guided Optimization on TTK Kinase Results in Inhibitors with Potent Anti-Proliferative Activity

Article

Target Residence Time-Guided Optimization on TTK Kinase Results in Inhibitors with Potent Anti-Proliferative Activity

Joost C.M. Uitdehaag, Jos de Man, Nicole Willemsen-Seegers, Martine B.W. Prinsen, Marion A.A. Libouban, Jan Gerard Sterrenburg, Joeri J.P. de Wit, Judith R.F. de Vetter, Jeroen A.D.M. de Roos, Rogier C. Buijsman and Guido J.R. Zaman Netherlands Translational Research Center B.V., Kloosterstraat 9, 5349AB Oss, The Netherlands

Correspondence to Guido J.R. Zaman: [email protected] http://dx.doi.org/10.1016/j.jmb.2017.05.014 Edited by Georg Schulz

Abstract The protein kinase threonine tyrosine kinase (TTK; also known as Mps1) is a critical component of the spindle assembly checkpoint and a promising drug target for the treatment of aggressive cancers, such as triple negative breast cancer. While the first TTK inhibitors have entered clinical trials, little is known about how the inhibition of TTK with small-molecule compounds affects cellular activity. We studied the selective TTK inhibitor NTRC 0066-0, which was developed in our own laboratory, together with 11 TTK inhibitors developed by other companies, including Mps-BAY2b, BAY 1161909, BAY 1217389 (Bayer), TC-Mps1-12 (Shionogi), and MPI-0479605 (Myrexis). Parallel testing shows that the cellular activity of these TTK inhibitors correlates with their binding affinity to TTK and, more strongly, with target residence time. TTK inhibitors are therefore an example where target residence time determines activity in in vitro cellular assays. X-ray structures and thermal stability experiments reveal that the most potent compounds induce a shift of the glycine-rich loop as a result of binding to the catalytic lysine at position 553. This “lysine trap” disrupts the catalytic machinery. Based on these insights, we developed TTK inhibitors, based on a (5,6-dihydro)pyrimido[4,5-e]indolizine scaffold, with longer target residence times, which further exploit an allosteric pocket surrounding Lys553. Their binding mode is new for kinase inhibitors and can be classified as hybrid Type I/Type III. These inhibitors have very potent anti-proliferative activity that rivals classic cytotoxic therapy. Our findings will open up new avenues for more applications for TTK inhibitors in cancer treatment. © 2017 Elsevier Ltd. All rights reserved.

Introduction Kinases are the target of more than 30 registered small-molecule drugs, while more than 300 kinase inhibitors are currently investigated in clinical studies [1,2]. Well-known examples of successful kinase inhibitors are epidermal growth factor receptor (EGFR), ABL, and RAF inhibitors, which are applied for targeted therapy in lung cancer, chronic myeloid leukemia, and metastatic melanoma, respectively. However, given the fact that the human genome encodes more than 500 kinases, there are still many kinases that remain to be explored [1,2]. Early drug discovery projects on protein kinases commonly focus on improving the potency (IC50) of inhibitors in an enzyme activity assay, which is related 0022-2836/© 2017 Elsevier Ltd. All rights reserved.

to the binding affinity (KD) of the compound to the target. Affinity data of drug candidates are supplemented with selectivity profiling data on large panels of biochemical assays of other kinases [1]. The selectivity of a kinase inhibitor in such a panel can be expressed as a theoretical “selectivity entropy” (Ssel,) [3]. Ssel is a single-value parameter, similar to KD. Use of Ssel allows the ordering of compounds on selectivity and therefore can be used to give priority to selective hits at an early stage in the project [4]. A recent development in kinase drug discovery is the increased focus on the “target residence time” (τ) of kinase inhibitors. Copeland et al. [5] suggested that the time a compound resides on its target is a more important determinant of its biological activity J Mol Biol (2017) 429, 2211–2230

2212 than KD or IC50, because the concentration of a compound in living systems continuously changes due to drug metabolism and excretion. Indeed, the biological action of drugs with long target residency can endure even after they have been cleared from the systemic circulation [5–7]. This may limit off-target activities and was one of the reasons to develop irreversible binding kinase inhibitors [8], such as the EGFR inhibitors afatinib and osimertinib, and the BTK inhibitor ibrutinib. Indeed, in a Phase I trial, ibrutinib showed a plasma half-life of 7–8 h, whereas full target occupancy was maintained over 24 h, owing to ibrutinib's long residence time [9]. Also, non-covalently binding kinase inhibitors can have long residence times. For instance, lapatinib, an allosteric EGFR/HER2 inhibitor drug [10], has a substantially prolonged residence time (21,000 s) on EGFR compared to other EGFR inhibitor drugs such as gefitinib (131 s) and erlotinib (300 s) [10,11]). In the cases of BTK and FGFR, tunable residence times have even been achieved [12]. In general, the residence times of drug–target interactions range from minutes to hours [5–7,11]. Moreover, in cells, the availability of a compound at its target is thought to be primarily limited by its diffusion through the lipid bilayer [5]. Therefore, inhibitors with slow off-rates often have better cellular potency. This was demonstrated for lapatinib and nilotinib [10,13]. Because kinase domains are soluble, they are amenable to protein crystallography and other biophysical techniques that can be used to relate activity to binding mode. These studies can facilitate the design of novel compounds with improved properties. For instance, at a molecular level, slow off-rates of kinase inhibitors have been associated with alternative binding modes that require substantial structural rearrangements [14], as illustrated by lapatinib [10] and by the p38α inhibitor BIRB-796 [15]. Three types of kinase inhibition have been distinguished: inhibitors that bind primarily to the structurally conserved ATP binding site are called Type I. Inhibitors that induce a conformational shift in a segment containing Asp (D), Phenylalanine (F), and glycine (G), referred to as the DFG loop, are called Type II. Inhibitors that bind near the catalytic lysine in combination with ATP itself are called type III [2,14,16]. Other groups have added further subclasses, such as Type 1½ and Type IV inhibitors [2,14,16]. A good example of a relatively unexplored kinase is threonine tyrosine kinase (TTK; also known as Mps1). TTK is not a member of a thoroughly studied protein kinase family such as TK, CGMC, or MAP kinases. Nevertheless, it is a promising drug target [17–20]. TTK is a critical component of the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures the fidelity of chromosome segregation. Inhibition of TTK activity with small-molecule kinase inhibitors leads to chromosome segregation errors by allowing mitotic exit in the presence of unattached

TTK inhibitors with long target residence time

kinetochores [21,22]. After several rounds of cell division, the accumulation of chromosome segregation errors results in cancer cell death by apoptosis [17,20,23,24]. Initially, TTK drug discovery focused on tool inhibitors such as SP-600125, a dual TTK/JNK inhibitor [25], and reversine, which also inhibits Aurora B [26]. Follow-up compounds aimed at improving selectivity were based on the reversine scaffold, for instance, MPI-0479605 [27], which is selective over Aurora B, and Mps1-IN-1 and -3 [22,28]. Alternative scaffolds were progressed from screening, such as AZ3146 [29], the related Mps1-IN-2 [22], NMS-P715 [21], Mps-BAY2b [23], and CCT-251455 [30]. TC-Mps1-12 has been derived from a JNK inhibitor scaffold [31]. More recently, TTK inhibitors were described with increased cellular potency, such as PF-7006 and PF-3837, which were, however, discontinued because of a poor therapeutic window [32]. The most advanced compounds are NTRC 0066-0, which is in preclinical development [24], CFI-402257 [20], BAY 1161909 [19], and BAY 1217389 [19], which are in clinical phase I (see www.clinicaltrials.gov under NCT02792465, NCT02138812, and NCT02366949, respectively). Recently, we described the biological characterization of NTRC 0066-0, a (5,6-dihydro)pyrimido[4,5-e] indolizine (Fig. 1a–b) [24]. NTRC 0066-0 has a low selectivity entropy and a relatively long target residence time on TTK and inhibits the proliferation of diverse cancer cell lines with a potency in the same range as that of classic chemotherapeutic agents such as doxorubicin [24]. NTRC 0066-0 inhibited tumor growth in a mouse xenograft model of the human triple-negative breast cancer (TNBC) cell line MDA-MB-231. In a genetic mouse model of TNBC, combination of NTRC 0066-0 with a therapeutic dose of docetaxel resulted in increased mouse survival and extended tumor remission, without toxicity [24]. These data position NTRC 0066-0 as a candidate drug for TNBC, which is characterized by high expression levels of TTK [18]. Despite this progress, little is known about how binding to TTK at a molecular level leads to cellular potency. Therefore, we engaged in a study of the relationship among binding mode, residence time, and anti-proliferative activity of a collection of 12 TTK inhibitors with different chemical scaffolds (Fig. 1c). In addition, we characterized their activity in a panel of 66 cancer cell lines [33] and resolved the X-ray protein crystal structure of the kinase domain of TTK in complex with NTRC 0066-0 and also with MPI0479605, Mps- BAY2b, TC-Mps1-12, BAY 1161909, and BAY 1217389. This shows that the TTK inhibitors with the best cellular potency have the longest residence times. They bind in a unique way that is a hybrid of type I and type III inhibitors. To demonstrate the value of these insights, we developed new TTK inhibitors with long residence times and potent anti-proliferative activities.

TTK inhibitors with long target residence time

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Fig. 1. Chemical structure of TTK inhibitors used in this study. (a) Development of NTRC 0066-0 from predecessors 1 and 2. IC50, TTK refers to the biochemical assay. IC50, MOLT4 refers to a proliferation assay using the leukemic cell line MOLT4. (b) Structure–activity relationships in the (5,6-dihydro)pyrimido[4,5-e]indolizine series; res. time refers to residence time. (c) Structures of reference inhibitors. Compounds are displayed so that the hinge binding element is top left. Hinge binding atoms are colored red. The oxygen in the amide group that binds Lys553 is colored green.

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Results Discovery of NTRC 0066-0 We initiated a search for new TTK inhibitors using our EntropySelect™ drug discovery platform [3] that simultaneously tracks the potency and selectivity of scaffolds. First, we identified compound 1, a (5,6-dihydro)pyrimido[4,5-e]indolizine, which had good activity in a biochemical enzyme assay of TTK (IC50 = 8.7 nM; Fig. 1a). In order to characterize its selectivity, compound 1 was tested in a so-called QuickScout™ kinase panel † , consisting of 20 tyrosine kinases, 30 cell cycle kinase, and 22 other serine/threonine kinases. At 1 μM, compound 1 inhibited the activity of 48 out of 72 tested kinases by more than 90% (Supplementary Table S1). This corresponds to an estimated Ssel of 3.2, classifying compound 1 as a general kinase inhibitor [4]. Therefore, we focused the optimization of compound 1 on improving its kinase selectivity profile. By making use of available structural information and known pharmacophores of published TTK inhibitors, we subjected compound 1 to several iterative optimization rounds. Introducing a methoxy group at the 2-position of the aniline ring (compound 2a) significantly improved the biochemical potency, target residence time, and selectivity of the series (Fig. 1b). At a concentration of 1 μM, compound 2a inhibited 19 out of 72 kinases in the QuickScout™ panel, which corresponds to an estimated Ssel of 1.9 (Supplementary Table S1). Meanwhile, resolution of the crystal structure of compound 1 in complex with TTK showed room for substitutions at the amide functionality of the indolizine ring. Introduction of an aniline group (compound 2b) resulted in a significant increase of biochemical selectivity. Furthermore, 3 out of 72 kinases were inhibited, corresponding to an estimated Ssel of 0.7 (Supplementary Table S1). Biochemical potency and residence time were retained (Fig. 1b). However, the improvement in selectivity was accompanied by a significant drop in cellular potency in a cancer cell line proliferation assay (Fig. 1a). This finding prompted us to focus on optimizing the potency of the inhibitor series on TTK while retaining the excellent selectivity profile. As a 3D structure of compound 2b in complex with TTK showed that the aryl group was engaged in cation–π interactions (see below), we synthesized and tested a variety of substituents around this group. A di-fluoro substituent (2c) significantly increased potency, probably by fixing the compound's binding conformation (Fig. 1b). Moreover, more electron-donating substituents such as fluoroethyl- (2d), dimethyl- (2e), and diethyl- (NTRC 0066-0) were more effective, consistent with the presence of a cation–π interaction. We also found that the di-ethyl-phenyl could be replaced by bio-isosteric

TTK inhibitors with long target residence time

aromatic rings, such as di-ethyl-pyrrazole (2f; Fig. 1b). The optimized lead, NTRC 0066-0, has subnanomolar potency in a TTK enzyme assay and inhibits the proliferation of cancer cell lines from diverse tumor tissue origin with IC50 from 11 to 290 nM at 5-day incubation time (Table 1) [24]. In a panel of 276 kinases, it only inhibits TTK by more than 90%, when tested at 100 times the IC50 for TTK, that is, at 100 nM [24]. This corresponds to Ssel of 0.26, ranking NTRC 0066-0 among the 8% most selective kinase inhibitors known [4]. Comparing the cellular potency of TTK inhibitors To compare the anti-proliferative activity of NTRC 0066-0 with other TTK inhibitors, we extended the reference collection used in an earlier study [24] with other well-characterized TTK inhibitors, such as TC-Mps1-12, NMS-P715, Mps1-IN-1 to -IN-3, BAY 1161909, and BAY 1217389 (Fig. 1c). The selectivity of these inhibitors has been demonstrated by profiling experiments on large kinase assay panels [19,21,22,27,31]. Therefore, we focused on comparing their effects on TTK in our biochemical and cell-based assay panel (see Table 1 for data and Supplementary Table S2 for standard deviations). Initially, a proliferation assay with the acute lymphoblastic leukemia cell line MOLT4 was used, in which TTK inhibitors nearly always inhibit proliferation by 100% after 5 days of incubation, with potent IC50 values (Table 1). The inhibitor IC50s from this panel range from 3.8 μM for Mps1-IN-1 to 30 nM for NTRC 0066-0. To get data on more cell lines, we tested all 12 inhibitors on a panel of 66 cancer cell line proliferation assays (Oncolines™ panel; Fig. 2a) [33]. The geometrically averaged IC50 values from this panel range from 6.0 μM (Mps1-IN-1) to 96 nM for NTRC 0066-0. Also, BAY 1217389, BAY 1161909, and reversine have relatively potent cellular activities (Table 1 and Fig. 2a). Cellular potency correlates with biochemical IC50 Next, we studied how the cellular activities of TTK inhibitors relate to their biochemical characteristics. We analyzed the 11 reference inhibitors, together with NTRC 0066-0 and its derivatives generated during lead optimization (Fig. 1). There is a clear correlation between biochemical activity and potency in the cell proliferation assay [Spearman rank correlation (ρSpearman) = 0.71; Fig. 2b], confirming, as shown before [24] that the anti-proliferative effects are due to TTK inhibition. However, since the nominal enzyme concentration in the kinase assay was 3.9 nM, ligand depletion effects prevented reliable IC50 determination below this value.

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TTK inhibitors with long target residence time Table 1. In vitro characterization of TTK inhibitors a–i Inhibitor name

IC50 (nM)a

kd (1000/s)b

KD (nM)c

res. time (s)d

ΔTm (°C)e

IC50,MOLT4 (nM)f

IC50, panel ave (nM)g

IC50, panel min (nM)h

IC50, 66-panel max (nM)i

NTRC 0066-0 BAY 1217389 reversine BAY 1161909 MPI-0479605 TC-Mps1-12 Mps1-IN-3 Mps1-IN-2 AZ3146 Mps-BAY2b NMS-P715 Mps1-IN-1

0.90 1.0 2.0 2.4 1.9 7.7 15 48 5.2 6.7 1.3 39

0.27 0.41 0.10 0.54 3.4 47 8.7 49 30 28 0.83 8.2

0.56 1.1 0.050 2.4 1.7 2.2 3.5 43 5.5 11 1.3 22

3700 2400 9600 1900 300 21 120 20 33 35 1200 120

17 12 14 19 14 12 10 9.3 11 10 13 9.3

30 55 200 200 170 580 2300 940 790 1200 1900 3800

96 110 240 380 440 870 1100 1300 1700 1800 1800 6100

11 16 42 110 64 230 25 410 310 560 520 420

290 280 3300 820 22,000 2600 3100 3200 6700 6200 2900 9900

Data sorted on IC50,panel (ave). For standard deviations of the data, see Supplementary Table S2. a IC50 from TTK biochemical assay. b Dissocation constant from SPR experiments. c Binding constant from SPR experiments. d Residence time from SPR experiments. e Midpoint temperature of unfolding, difference with DMSO control. f IC50 from MOLT4 proliferation assay. g Geometrical average IC50 from a 66-cell line panel. h Lowest IC50 in cell line panel. i Highest IC50 in cell line panel.

Cellular potency correlates best with target residence time As a next step, we set up a surface plasmon resonance (SPR) binding assay for TTK. SPR does not suffer from ligand depletion and directly measures target residence time (Fig. 3 and Supplementary Fig. S1a). The KD was calculated from the apparent association rate constant (ka) and dissociation rate constant (kd) through KD = kd/ka (Fig. 4a–d). The KDs from SPR predict cellular potency with broadly the same accuracy as the IC50s from the activity assay (ρSpearman = 0.72; Fig. 4a), even though the SPR data were obtained using the kinase domain, whereas full-length enzyme was used in the activity assay. The correlation between the KD values from SPR and the IC50s from the enzyme activity assay was relatively low (ρSpearman = 0.59), which may be related to ligand depletion (Supplementary Fig. S1b). We also studied if the apparent ka or kd separately relates to cellular potency (IC50, MOLT4). There is a poor correlation between IC50,MOLT4 and ka (Fig. 4b, ρSpearman = 0.30). In contrast, there is a strong correlation between the dissociation rate constant (kd, the inverse of residence time) and IC50, MOLT4 (Fig. 4c, ρSpearman = 0.84). This correlation is significantly higher than that between IC50, MOLT4 and KD (0.72, p = 0.01) in Williams' test for paired correlations. To study the correlations when taking measurement errors into account, we recalculated these correlations using all SPR replicates (285 experiments on 67 inhibitors). This resulted in Spearman correlations of

0.71 and 0.79 between cellular activity and KD and kd, respectively. Again, the correlation is significantly higher for kd (p = 0.005, Williams' test), indicating that target residence time (τ) better predicts the cellular activity of TTK inhibitors than KD. Potent TTK inhibitors stabilize the TTK kinase domain Because shifts in the temperature of protein unfolding can provide information about compound-induced conformational changes [34], we studied the inhibitors in a thermal stability assay. Without inhibitor bound, the TTK kinase domain unfolds at 50 °C, but this can be increased by more than 19 °C in the presence of 10 μM inhibitor (Table 1 and Fig. 4e). This is considerable, since in a study of 156 inhibitors on 60 kinases, only two inhibitors shifted the unfolding Tm by more than 15 °C [34]. Unfortunately, the protein concentration (2.4 μM) required in the thermal shift assay precludes the determination of affinity constants for potent inhibitors. Nevertheless, there is a clear relation between the extent of thermal stabilization and kd (Fig. 4f, ρSpearman = − 0.85). Relatively fast dissociating compounds, such as Mps-BAY2b, AZ3146, MPI-0479605, and Mps1-IN-1, shift unfolding by 8 to 11 °C, whereas more slowly dissociating compounds, such as BAY 1161909, BAY 1217389, and NTRC 0066-0, shift unfolding by 14 to 19 °C (Table 1). This indicates that the binding of long residence TTK inhibitors induces a conformational shift that stabilizes the kinase domain.

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Fig. 2. Relation between cellular activity and biochemical properties of TTK inhibitors. (a) IC50s of all reference inhibitors in the proliferation assays of the 66 cell line panel. Cell line names on the x-axis. (b) Correlation between anti-proliferative IC50 (on MOLT4 cells) and biochemical IC50. Reference inhibitors of Fig. 1c are displayed in red, NTRC 0066-0 analogs in gray. ρSpearman indicates the Spearman rank correlation.

TTK inhibitors with long target residence time

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Fig. 3. SPR profiles of TTK inhibitors. Sensorgrams of single-cycle kinetics experiments of TTK inhibitor binding to the TTK kinase domain. The actual response is shown by the colored line. The fitted response, using a 1:1 binding model, is displayed as a black line. x-axis: time from 0 to 3000 s, y-axis: relative response. The NTRC 0066-0 curve [24] is included for reference. Sensorgrams of the other reference inhibitors are available in Supplementary Fig. S1a.

X-ray structure of NTRC 0066-0 bound to TTK To better characterize compound-induced conformational changes, we determined the X-ray structure of a number of TTK inhibitor complexes. First, we resolved the structure of TTK bound to NTRC 0066-0 at 2.3-Å resolution [Fig. 5; Protein Data Bank (PDB) id: 5N87]. The central pyrimido-indolizine ring of NTRC 0066-0 fits in the hydrophobic cleft of the ATP binding pocket (Fig. 5a), where the aminopyrimidine ring interacts with the hinge region of TTK (Glu603–Asn606). The methoxy substitution occupies a small pocket near Cys604 (Fig. 5b), which is specific for TTK [30]. The diethyl-phenyl engages in a cation–π interaction with the amino group of the catalytic residue Lys553 (Fig. 5c). This interaction is strengthened by the two ethyl substitutions on the phenyl ring that optimally orient the ring and by the contact between the amide oxygen of NTRC 0066-0 and the Nζ atom of Lys553 (Fig. 5c). When the complex of NTRC 0066-0 and TTK is compared to a X-ray structure of apo-TTK (PDB id: 3CEK) [35], crystallized under similar conditions, it appears that the region of residues Gly532 to Phe540 (known as the glycine-rich loop) has shifted downward ~ 1 Å, narrowing the ATP binding cleft (Fig. 5d). Gly534 and Gly535 have undergone a peptide bond reorientation (peptide flip). In the NTRC 0066-0-bound complex, Asp664 in the so-called DFG loop has assumed a different rotameric isoform and forms a strong hydrogen bond with Glu571 (Fig. 5c). This is the glutamate that, in catalytically competent TTK, forms an essential salt bridge to Lys553 [30,35]. Thus, NTRC 0066-0 monopolizes binding interactions with Lys553 and provides an alternative binding

partner for Glu571, thereby locking TTK into an inactive conformation. The shift of the glycine-rich loop is related to compound potency To get a better idea of the possibilities for binding in the TTK active site, we determined X-ray structures of TTK in complex with MPI-0479605 (PDB id: 5N7V), Mps-BAY2b (5N84), TC-Mps1-12 (5N93), BAY 1161909 (5N9S), and BAY 1217389 (5NAD; Fig. 6 and Supplementary Table S3). In addition, we downloaded X-ray structures of complexes with reversine (5LJJ), NMS-P715 (2X9E), Mps1-IN-1 (3GFW), and Mps1-IN-2 (3H9F). We were therefore able to compare experimental 3D structures of most of the compounds characterized in the experiments summarized in Table 1. The set was completed with various other available TTK inhibitor complexes, for instance, that with CCT251455 (4C4J) and different CFI scaffolds (4ZEG, 4O6L; Supplementary Fig. S2). To investigate which molecular event is connected to the increase in thermal stability of TTK after inhibitor binding, we superimposed all X-ray structures. It appears that the glycine-rich loop adopts the most variable positions of the regions resolved in electron density (Fig. 7a), consistent with the variation described for the binding of NTRC 0066-0 (Fig. 4d). When we quantify the shift by the position of the Ser533 Cα backbone oxygen, there is a clear correlation with IC50 in the cellular assay (Fig. 7b). Thus, the shift of the glycine-rich loop is most likely the molecular event leading to thermal stabilization of the kinase domain and the long residence time of the most potent inhibitors.

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Fig. 4. Relation between cellular activity and biochemical properties of TTK inhibitors. (a–c) Relation between anti-proliferative IC50 (on MOLT4 cells) and SPR data. (a) Binding constant (KD). (b) Association rate (ka). (c) Dissociation rate (kd). Note that the residence time is defined as the inverse of kd. (d) Comparison of association and dissociation rates for the reference inhibitors. The reversine-like and ATP-pocket binding inhibitors (green, triangles) and lysine-targeting inhibitors (yellow, lozenge) can be distinguished as separate groups. Diagonals represent equipotency lines with pertinent KDs indicated within the frame. (e) Representative thermal unfolding curves of the TTK kinase domain in the presence of TTK inhibitors. For clarity, all fluorescence signals at 40 °C were set to 0. The arrow indicates an example midpoint of unfolding (Tm). (f) Correlation between Tm and inhibitor dissociation rate (kd). ΔTm is relative to the control without inhibitor. ρSpearman indicates the Spearman rank correlation.

TTK inhibitors with long target residence time

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Fig. 5. Binding mode of NTRC 0066-0. (a) Overview of the TTK kinase domain with NTRC 0066-0 (gray) bound (PDB id: 5N87). ATP refers to the ATP binding cleft. DFG refers to the DFG loop. (b) Interactions of NTRC 0066-0 near the hinge region in the ATP pocket. (c) Interactions of NTRC 0066-0 near the catalytic site. Dotted lines indicate important interactions. The dark gray moiety is a PEG molecule from the crystallization buffer, which is also present in apo-TTK [35]. (d) Overlay of the NTRC 0066-0/TTK complex with apo-TTK (yellow; PDB id: 3CEK) [35], with focus on the glycine-rich loop where the largest conformational differences are observed.

X-ray structures distinguish several inhibitor classes As pointed out earlier [36], nearly all studied TTK inhibitors (including NTRC 0066-0) contain a common pyrimidin-2-amine motif, which interacts with the backbone of the hinge region (red in Fig. 1c). On this basis, the X-ray structures show two basic inhibitor binding modes. The first (“reversine-like”) is used by reversine and reversine-like compounds such as MPI-0479605, Mps1-IN-1, and -IN-3 (Fig. 6a). These all provide three nitrogen atoms for interaction with

the hinge, two from the pyrimidin-2-amine motif, and a third from the pyrrole ring of the scaffold. A hydrophobic moiety on the pyrimidine ring, embodied by a cyclohexyl in MPI-0479605 and reversine, and a propyl-sulfonyl in Mps1-IN-1 (Fig. 1c), occupies a pocket that is lined by Met671 and Pro673. These amino acids are part of a string of residues from amino acids 667 to 675 that is called the activation segment [30,37] and that is resolved in the electron density map of the MPI-0479605 complex (Fig. 6a) and the reversine complex (Supplementary Fig. S2) [38].

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TTK inhibitors with long target residence time

Fig. 6. Binding modes of TTK inhibitors. Active site detail of TTK X-ray structures in complex with reference inhibitors. (a–e) PDB ids: 5N7V, 5N93, 5N84, 5N9S, 5NAD, respectively, this work. (f) PDB id: 3X9E [21].

Compounds that are not related to reversine can also bind in a similar mode. Mps1-IN-2 and AZ3146 contain a cyclopentyl that is not attached to the pyrimidine but to a neighboring ring. In its complex with TTK, Mps1-IN-2 is oriented in such a way that also the cyclopentyl interacts with the activation segment (Supplementary Fig. S2) [22], which is resolved in electron density. Also, CCT-251455, which has a hydrophobic BOC group protruding from a second ring, stabilizes the activation segment in a similar way (Supplementary Fig. S2) [30]. An interesting variation is provided by TC-Mps1-12 (Fig. 6b), which induces a peptide flip in the hinge at amino acid 604, as was described earlier for an analog (PDB id: 3VQU) [31]. As with the other compounds, TC-Mps1-12 contains a hydrophobic group (tert-butyl)

that interacts with the activation segment, which is resolved in the TC-Mps1-12/TTK complex (Fig. 6b). The common theme for all these inhibitors is the stabilization of the activation segment across the active site, which forms an antiparallel β-sheet with the glycine-rich loop (Fig. 6a and b) and occupies the area where a protein substrate would bind [37]. Thus, the reversine-like binding mode involves blocking the ATP pocket by binding to the hinge and blocking substrate binding by stabilizing the activation segment. Inhibitor binding modes that target Lys553 A different binding orientation is observed in the X-ray structures of inhibitors that contain an amide

TTK inhibitors with long target residence time

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Fig. 7. Shift of the glycine-rich loop after inhibitor binding. (a) The overlay of five representative X-ray structures is shown. For apo-TTK, PDB id 3CEK was used; for other PDB ids, see Fig. 6. (b) Glycine-rich loop shift correlates with IC50 in the proliferation assay of MOLT4 cells. The shift was quantified by monitoring the position of the indicated atom, relative to apo-TTK (PDB id: 3CEK). Every single label represents one X-ray structure. Red: reference inhibitors. Green: NTRC 0066-0. Black: NTRC 0066-0 analogs. The label “5” indicates compound 5.

pharmacophore (green in Fig. 1c), such as NTRC 0066-0, Mps-BAY2b, BAY 1161909, BAY 1217389, NMS-P715, and also the CFI scaffolds. These inhibitors bind to the hinge in the ATP pocket and, in addition, employ their amide to bind the catalytic Lys553 (Figs. 6c–f). A first subset of these inhibitors approaches Lys553 from a back pocket. Under the crystallization conditions applied, this pocket is usually occupied by a polyethylene glycol (PEG) molecule that wraps around Lys553 (e.g., Fig. 5c) [35]. In the complexes of TTK with Mps-BAY2b, BAY 1161909, and BAY 1217389, it is (partly) filled by inhibitor substituents (Figs. 6c–e, respectively). For instance, in the BAY 1161909 complex, the pocket is filled by a fluorobenzene, which engages in cation–π interactions with Lys553, shielding it from Glu571 (Fig. 6d). In the complexes of Mps-BAY2b (Fig. 6c), BAY 1217389 (Fig. 6e), and a CFI-402257 analog (Supplementary Fig. S2) [39], the pocket is filled by a cyclopropyl that engages, in the latter two complexes, in stacking interactions with Glu571, potentially reducing its catalytic proficiency (Fig. 6e). In some of these complexes, the activation segment is resolved in electron density. In the complex with BAY 1161909, it assumes a different position compared to the reversine-like inhibitor complexes (Fig. 6d). In the complexes with BAY 1217389 (Fig. 6e) and the CFI-402257 analog (Supplementary Fig. S2F), it binds at the same location, because the loop is stabilized by properly positioned hydrophobic substituents (a difluoro-methoxy-benzyl for BAY 1217389, a pyridine for the CFI-402257 analog). A second subset of amide-containing inhibitors approaches Lys553 from the front, blocking it from interaction with Glu571. This is seen in the complex

with NTRC 0066-0 (Fig. 5c), as discussed above, with NMS-P715 (Fig. 6f) and a CFI-401870 analog (Supplementary Fig. S2e) [29]. In all X-ray structures, Glu571 now binds to Asp664. When these complexes are superimposed on structures with a resolved activation segment, steric clashes appear, for example, between Met671 and the diethylphenyl moieties of NTRC 0066-0 and between the activation segment backbone and the glycine-rich loop in its position after NTRC 0066-0 binding (Supplementary Fig. S3a). Therefore, this second subclass appears to actively destabilize the binding of the activation segment. A common theme of all the amide-containing inhibitors is that they disrupt the Lys553/Glu571 pair by monopolizing binding to Lys553 and providing an alternative binding partner to Glu571. Therefore, we propose the name “lysine traps” for this class. Binding interactions near Ile531 stabilize shift of the glycine-rich loop To further investigate the relationship between TTK inhibitor binding and cellular potency, we studied the differences between NTRC 0066-0 and NMS-P715, which share a common binding mode (Figs. 5c and 6f). Nevertheless, NTRC 0066-0 has about 60-fold higher cellular potency than NMS-P715 and a more dramatic glycine-rich loop shift (Fig. 7a). Superimposition of X-ray structures suggested that the CF 3 -ether substituent of NMS-P715 blocks a full shift of the glycine-rich loop since the trifluoro substituent of NMS-P715 is positioned 0.83 Å higher in the ATP pocket than the methoxy substituent of NTRC 0066-0 (Fig. 8a), leading to putative steric clashes with the side

2222 chains of Ile531 and Gln541 in the NTRC 0066-0-bound position. Since these residues are at the start and at the end of the glycine-rich loop, these interactions might underlie the potency differences between the two inhibitors. To better understand this, we synthesized a hybrid molecule (3a) containing the NMS-P715 substituents on the NTRC 0066-0 scaffold (Fig. 8b). Inhibitor 3a had a four times better cellular potency than NMS-P715 (Fig. 8c), indicating that the pyrimido-indolizine scaffold contributes to cellular potency. Next, we replaced the trifluoromethoxy group of 3a by hydrogen (3b) and methoxy (3c). Especially shifting from CF3O- to CH3Oresulted in a 10-fold increase in cellular potency and a 3.6-fold increase in residence time (Fig. 8c and Supplementary Table S2). Although this structure– activity relationship contrasts with other data [40], it proves that not only the interactions with Lys553 but also those near the attachment points of the glycine-rich loop contribute to the effectivity of NTRC 0066-0.

TTK inhibitors with long target residence time

Different inhibitor types show different kinetic behavior To study if the different subclasses of TTK inhibitors could also be distinguished from their kinetic profiles, we looked at the plot of apparent ka and kds from the SPR experiments (Fig. 4d). This shows that the lysine-trap inhibitors form a separate group with relatively slow on- and off-rates compared to the inhibitors with reversine-like binding modes (Fig. 4d and Supplementary Table S2). The only exception is Mps-BAY2b (relatively fast ka and kd). In contrast, stabilization of the activation segment in the X-ray structures does not predict slow dissociation kinetics, as illustrated by the very different binding kinetics of activation segment-stabilizing compounds; for instance, reversine and BAY 1217389 on the one hand, and Mps1-IN-2 and TC-Mps1-12 on the other hand all stabilize the segment but have different binding kinetics (Fig. 4d). Thus, most likely, the slow kinetics of the lysine trap inhibitors are not

Fig. 8. Analysis of activity differences between NTRC 0066-0 and NMS-P715. (a) Overlay of 3D structures of TTK/ NMS-P715 complex (PDB id: 2X9E, green) and the TTK/NTRC 0066-0 complex (PDB id: 5N87, red) at the hinge region. (b) Chemical structures of hybrid TTK inhibitors containing the NTRC 0066-0 tricyclic core and NMS-P715-like substituents. (c) Activities of 3a to 3c in biochemical, biophysical, and cellular assays for TTK. Apparently, a methoxy substituent results in improved cellular potency.

TTK inhibitors with long target residence time

caused by activation segment stabilization but rather by the rearrangements in the glycine-rich loop, which are required for inhibitor binding and dissociation. New TTK inhibitors with increased cellular potency To test our hypothesis that binding to Lys553 can effectively improve the inhibitor dissociation rate, we generated compounds designed to fit better in the pocket surrounding Lys553. The NTRC 0066-0 variant containing diethyl-pyrrazole (2f) was used to attach a PEG linker (Fig. 9a, compound 4), which was predicted to fit at the position of the bound PEG molecule (Fig. 5c). Furthermore, an analog of 4 was designed with the aim of picking up additional interactions in the front pocket (compound 5). The affinities and residence times of 4 and 5 are substantially longer than NTRC 0066-0 (17,000 s and 14,000 s, respectively; Fig 9b and c). The estimated selectivity entropies of 4 and 5 in the QuickScout™ panel are 0.0033 and 0.0036, respectively, indicating very good selectivity (Supplementary Table S1). Both 4 and 5 increase the stability of TTK better than any other inhibitor, with ΔTm of 5 going up to 24 °C. Concurrently, the cellular activity of 4 and 5

2223 improved, respectively, 5- to 10-fold compared to NTRC 0066-0, both in the MOLT4 assay and in the full cell panel average (Fig. 9c). The crystal structure of compound 5 bound to TTK (Fig. 9d) shows a binding mode similar to NTRC 0066-0 with the PEG linker wrapping around Lys553 and the ether oxygens forming a cryptate with the Nζ.-atom (Fig. 9d). The front pocket substituent of compound 5 picks up interactions with Lys615, which can explain its superior affinity compared to 4, which probably cannot interact in this way. Superimposition of structures shows that the glycine-rich loop in the compound 5 complex is displaced further downward compared to NTRC 0066-0, confirming this event as a key indicator of compound potency (Fig. 9e). TTK inhibitors 4 and 5 have strong effects on chromosome segregation We have previously shown that NTRC 0066-0 reduces time in mitosis and induces chromosome mis-segregation in human cervical cancer HeLa cells [24,41]. To show that the potent anti-proliferative potency of 4 and 5 is due to the inhibition of TTK, we studied their effects, alongside those of NTRC

Fig. 9. Structure-guided design of TTK inhibitors with increased cellular potency. (a) Chemical structures of the new compounds 4 and 5 based on the NTRC 0066-0 scaffold. (b) Overlay of single-cycle kinetic binding experiments, showing that 4 and 5 have longer residence time on TTK than NTRC 0066-0. Response was normalized. (c) In vitro characterization of 4 and 5. res. time: residence time. IC50,66-panel: geometrical average of IC50s in the 66 cell line panel. (d) X-ray structure of 5 bound to the TTK kinase domain (PDB id: 5NA0), active site detail. (e) Overlay showing conformational changes in the glycine-rich loop after binding of 5 (see also Fig. 7a).

2224 0066-0, on mitosis in the TNBC cell line MDA-MB231 by time lapse imaging (Fig. 10a). Under the vehicle condition, these cells show normal mitosis, starting from nuclear envelope breakdown (0 min) and finishing with anaphase (35 min). One of the daughter cells is represented at 175 min with no major defects (Fig. 10a, top). Treatment with 100 nM NTRC 0066-0 reduced mitotic timing to 18 min (Fig. 10b). It also induced mis-segregation and mitotic slippage (Fig. 10a, second line). Images show the lack of proper chromosome alignment and precipitated DNA decondensation. At 225 min, the sole daughter cell has a double DNA content and an irregular nuclear shape (Fig. 10a). Since the anti-proliferative IC50s in MDA-MB-231 (33, 13, and 3.5 nM for NTRC 0066-0, 4, and 5, respectively) suggested that 20 nM would be a good dose to discriminate between the compounds, we next reduced the NTRC 0066-0 concentration to 20 nM. This restored normal mitosis and mitotic

TTK inhibitors with long target residence time

timing was similar to vehicle-treated cells (Fig. 10b), however, with a slight induction of mis-segregation (Fig. 10c). In the example scenario, the cell eventually proceeded to anaphase with a chromosome bridge, and the daughter cell harbors a micronucleus (Fig. 10a, third line), indicating that the SAC remained active. At 20 nM, compounds 4 and 5 effectively reduced time in mitosis to 22 min and 18 min, respectively (Fig. 10b). Representative images after treatment with 4 show a cell decondensing its DNA before metaphase. This results in a polyploid cell with micronuclei (Fig. 10a, fourth line). In the scenario of compound 5, the cell divides rapidly with chromosome bridges, and the one daughter cell is binucleated with multiple micronuclei (Fig. 10a, fifth line). Similar to NTRC 0066-0, compounds 4 and 5 also induce chromosome mis-segregation errors, such as chromosome bridges, lagging chromosomes, and mitotic slippage, but at lower concentrations (Fig. 10b and c). Such phenotypes are typical of an inhibited SAC, which is a

Fig. 10. Effects on the cell cycle of novel TTK inhibitors. (a) Time-lapse imaging study of mitosis in MDA-MB-231 cells in the presence and absence of TTK inhibitors. Representative images describe the course (in minutes) of mitosis in the presence of TTK inhibitors or treated with vehicle. Images show a normal mitosis, chromosome bridges, mitotic slippages, and micronuclei. (b) Quantification of mitotic timing and (c) chromosome mis-segregation. Averages of three independent experiments on 30 cells each are shown. The bar graphs represent means and standard deviation.

TTK inhibitors with long target residence time

consequence of TTK inhibition. Inhibitors 4 and 5 therefore are the evidence that cellular potency can be increased by prolonging residence time and thermal stabilization of TTK.

Discussion TTK is a promising drug target for treatment of aggressive cancers with a high unmet medical need, such as TNBC [24,30], pancreatic cancer [42], and hepatocellular carcinoma [43]. The first TTK inhibitors have recently entered clinical trials [19,20]. With the aim of understanding the structure–activity relationship of current TTK inhibitors, and to support further drug discovery, we investigated the relationship between the biochemical properties of TTK inhibitors, their binding modes, and cellular potencies. We used this information to design TTK inhibitors with exceptionally potent cellular activity. To illustrate how extraordinary the potencies of our inhibitors are, we compared our data to that of other anti-proliferative agents. In a recent profiling of 122 compounds in the same cell panel used in Fig. 2a, the log-averaged IC50 of the compounds is 1300 nM [33]. The log-average IC50 of compound 5 (11 nM) would rank eighth in that list, only surpassed by widely used cytotoxic agents such vincristine (6.4 nM) and docetaxel (6.3 nM). The most potent IC50 that our TTK inhibitors achieve, 720 pM (for compound 5 in the A427 cell line; Table S2), is only matched by 4 out of 6118 entries, that is, epothilone B, dasatinib, actinomycin-D, and gemcitabine. In the NCI60 [44] and GDSC [45] datasets, which contain more than 1.1 million and more than 224,000 dose– response curves, the IC50 of 720 pM of compound 5 ranks among the 0.06% and 0.11% most potent, respectively. Concomitantly, the residence time of compound 5 on TTK of 4.7 h is among the longest measured for reversible kinase inhibitors, rivaling that of lapatinib [10]. In a recent kinetic binding study of 35 different inhibitors on 46 different kinases, compound 5 would rank sixth with regard to target residence time [11]. To explain these effects, we determined seven new X-ray structures of TTK–inhibitor complexes. Analysis of these structures shows that TTK inhibitors can assume two basic binding modes, of which the first stabilizes the activation segment (Figs. 6a and b). The resolution of this segment is not an experimental artifact, because it has been observed in X-ray structures determined under various crystallization conditions [22,30,38,39]. The structure might represent an auto-inhibited state of TTK, in analogy with the auto-inhibited states of cyclin-dependent kinases (CDKs), Aurora, JNK, and other kinases [37,46]. In these examples, a phosphorylation or co-factor binding event induces the dissociation of the activation segment, allowing the protein substrate to bind.

2225 Also TTK needs multiple phosphorylation events for full activation [47]. In comparison with these well-described auto-inhibited kinases [37,46], the TTK activation segment has a unique conformation in covering the ATP binding site and in its antiparallel β-sheet interactions with the glycine-rich loop, suggesting a tight regulation of TTK activation. After the activation of TTK, returning to the activation-segmentbound state upon inhibitor binding would cost conformational entropy. This might explain why interactions with the activation segment generally do not give inhibitors a better KD or better anti-proliferative activity (Table 1 and Fig. 4d). A superior approach to improve the potency of TTK inhibitors is to contact Lys553. The lysine trap class of TTK inhibitors generally has slow binding kinetics (Fig. 4d) and stabilizes the TTK kinase domain against thermal unfolding (Fig. 4e). On a molecular level, binding to Lys553 is accompanied by a compound-induced shift of the glycine-rich loop, which closes the ATP binding pocket (Fig. 7a). Most likely, this prevents the rapid diffusion of the inhibitor out of the pocket, resulting in a long residence time. The inhibitors characteristically exploit an allosteric hydrophobic cavity around the Lys553/Glu571 catalytic pair (Fig. 6). This pair is disrupted through the binding of Lys553 and a simultaneous neutralizing of Glu571, through binding with either Asp664 or the inhibitor itself (Fig. 5c and 6f). Some lysine trap inhibitors, such as NTRC 0066-0, also actively block the binding of the activation segment, which facilitates more extended glycine-rich loop shifts, such as that observed in the complex with the most potent inhibitor 5. Although NTRC 0066-0 and compound 5 were designed with a long residence time in mind, it is surprising that the residence time of TTK inhibitors correlates significantly better with cellular IC50 than the binding constant KD does (Fig. 4c). Usually, the advantages of residence time only become apparent in an “open” system, where compound can freely diffuse away from its target, and not in the “closed” equilibrium system of a cellular assay. At first sight, the separate correlations are counterintuitive, since KD = kd/ka, and if the association rate ka is solely governed by diffusion, KD and kd will always differ by a constant factor, resulting in their correlations with IC50 being identical [48]. However, as indicated by our 3D structures, association most likely consists of a series of steps, including the glycine-rich loop movement and disruption of the catalytic pair. In such cases, ka is a composite of diffusion and intramolecular compounddependent steps [5,49]. As a result, KD and apparent kd differ in a compound-dependent way. This is confirmed by the far-from-perfect correlation (ρSpearman = 0.69) between KD and kd in our data. The association rates of the lysine trap inhibitors are ~ 10 5 M − 1 s − 1 (Supplementary Table S2), in the same range as other kinase inhibitors that bind after induced fit rearrangements, such as BIRB-796 to

2226 p38α and lapatinib to EGFR (ka = 10 4–10 5 M −1 s −1) [10,11,15]. This is substantially slower than diffusioncontrolled rates (10 6–10 7 M −1 s −1) [48], indicating that conformational changes indeed limit the rate of binding. Interestingly, lapatinib is also an example in which residence time correlates with cellular efficacy. In the case of TTK, consistent and prolonged inhibition of SAC activity might be more efficacious than intermittent inhibition by inhibitors with more rapid on- and off-rates, but this needs to be further investigated. Our findings are therefore a good illustration that residence time can also impact cellular IC50s in a closed system. The lysine trap binding mode can also be applied to other kinases. Comparing our 3D structures with other inhibitor binding modes, via the KLIFS database [9], indeed reveals three kinase inhibitor complexes where an aromatic group captures the lysine at the active site (PDB ids: 2R3Q, 3MJ2, and 3TTJ, involving, respectively, CDK2, ITK, and JNK3 kinases). These complexes can also be classified as lysine traps. The allosteric pocket around Lys553 resembles the cavity filled by type III binders, of which MEK inhibitors are the best known example (Supplementary Fig. S3b) [50]. As type III binders do not bind into the ATP pocket, our inhibitors assume a hybrid binding mode between type I and type III. This could be exploited to inhibit other kinases such as the BMP2-inducible kinase 2, GAK, ULK3, MAP2K4, and CaMKK, which have structural homology with TTK [51]. It would be interesting to see if lysine trap inhibitors for such other targets would also show a strong relation between residence time and cellular potency.

Materials and Methods TTK inhibitors All inhibitors were synthesized in our laboratories according to patent or literature procedures (see Supplementary Methods), except compounds TC-Mps1-12 and Mps1-IN-1, which were purchased from Tocris (Bristol, UK), Mps1-IN-2 and reversine from Axon MedChem (Reston, USA), Mps1-IN-3 from Sigma-Aldrich (St. Louis, USA), and AZ3146 from Selleck (Houston, USA). Compounds were stored as solids at room temperature and dissolved in dimethylsulfoxide (DMSO) before all experiments. For analysis methods and spectra, see Supplementary Methods. Biochemical assays The inhibition of the kinase activity of biochemically purified full-length TTK (Life Technologies, Madison, USA) was determined in a IMAP® fluorescence polarization-based assay (Molecular Devices, Sunnyvale), as previously described [24]. Briefly,

TTK inhibitors with long target residence time

kinase inhibitors and TTK were diluted in IMAP reaction buffer, which consists of 10 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.01% Tween-20, 0.1% NaN3, and 1 mM dithiotreitol (DTT). After preincubation of 1 h in the dark at room temperature, fluorescein-labeled myelin basic protein MBP-derived substrate peptide (Molecular Devices, Sunnyvale, USA) was added, followed by ATP to start the reaction. Final enzyme concentration was 3.9 nM, final substrate concentration was 50 nM, and final ATP concentration was 5 μM. The reaction was allowed to proceed for 2 h at room temperature in the dark. Fluorescein polarization was measured on an Envision multimode reader (Perkin Elmer, Waltham, USA). Data were fit using a four parameter logistics curve in XLfit™5. Experiments were performed at least three times in duplicate (Supplementary Table S2). Broad kinase selectivity profiling was carried out at Carna Biosciences (Kobe, Japan). Purification of TTK kinase domain TTK was expressed and purified according to a protocol developed at the Structural Genomics Consortium [22]. Plasmid TTKA-c013/SGC2-B08 was obtained from Source Bioscience (Nottingham, U.K.). TTK was expressed in the Escherichia coli Rosetta™ strain (Invitrogen, Carlsbad, USA) in LB medium with 50 μg/L kanamycin and 35 μg/L chloramphenicol, to OD600 of 0.6–0.8 at 37 °C in a shaker incubator. After this, 1 mM IPTG was added, followed by further incubation for 4 h at room temperature. Bacterial pellets were frozen at − 20 °C. For purification, cells were resuspended in binding buffer [50 mM Hepes (pH 7.5), 500 mM NaCl, and 5% (vol/vol) glycerol]. We added 10 mM imidazole and EDTA-free protease inhibitors (Roche, 1 tablet per 50 mL), and the cells were lysed using an Avastin liquid homogenizer, followed by centrifugation. The supernatant was incubated for 1 h at 4 °C with NiNTA Superflowsepharose beads (Qiagen, Venlo, the Netherlands), after which the beads were isolated and washed with binding buffer with 50 mM imidazole. Protein was eluted with binding buffer and 250 mM imidazole. The next day, the protein-containing fractions were further purified by size-exclusion chromatography on a Superdex 75 column running in 50 mM Hepes (pH 7.5), 150 mM NaCl, and 5 mM DTT. Peak fractions were pooled and subsequently flash-frozen in dry ice/ethanol in 50 μl aliquots at − 80 °C. Specifically for protein crystallography, peak fractions were concentrated at 4 °C on a Spin-X UF 30-kDa filter (Corning, USA) to 8–10 mg/ml, before flashfreezing and storage. Thermal shift assays An aliquot of purified TTK kinase domain was thawed and diluted to 4.8 μM in IMAP buffer [10 mM

2227

TTK inhibitors with long target residence time

Tris (pH 7.5), 10 mM MgCl2, 0.01% Tween-20, and 0.1% NaN3]. Subsequently, 10 μl of the protein was mixed with 5 μl of 40 μM compound of interest in IMAP buffer, in a Greiner 96-well PCR plate (cat. no. 652260). After 30-min incubation on ice, 5 μl of 1250 times diluted SyproOrange (Invitrogen, Carlsbad, USA) in IMAP buffer was added. Final concentrations were 2.4 μM TTK and 10 μM compound. SyproOrange was 5000 times diluted. Immediately after incubation, the plate was sealed with TopSeal A-plus (PerkinElmer, Groningen, the Netherlands) and transferred to a Biorad CFX96, where the temperature was increased from 20 °C to 90 °C in 0.5 °C increment per 5 s. Reported values were measured in quadruplicate, in three independent experiments (Supplementary Table S2). Melting temperatures were determined by taking the minimum of the first derivative of the melting curve. SPR Binding kinetics were determined by SPR using a Biacore T200 (GE Healthcare). Because initial tests with full-length TTK did not yield stable surfaces, we used the His-tagged kinase domain of TTK, as described previously [24]. Briefly, TTK kinase domain was immobilized on a Ni-NTA sensor chip with His-tag capturing and amine-coupling to a level of 4000–6000 RU. Inhibitors were diluted in binding buffer to a final concentration of DMSO of 1% (vol/vol). Compound binding was measured in binding buffer (10 mM Tris, 10 mM MgCl2, 0.01% Tween-20, and 1 mM DTT at pH 6.8) with 1% (vol/vol) DMSO using single cycle kinetics by injecting an increasing concentration range of 1 – 3.16 – 10 – 31.6 – 100 nM. Flow rate was 30 μl/min, and association time per injection was 100 s. Dissociation following the last injection was monitored for at least 30 min. No regeneration was carried out. From the compound signal, we subtracted the buffer injection and the reference channel signals (double referencing). Resulting data were fit using the Biacore Evaluation software to the simple 1:1 Langmuir binding model. All kinetic constants were within the working range of Biacore T200. To determine the reliability of the curve fit, we applied standard Biacore checks. The uniqueness of a fit (U value), the standard error of the ka and kd, and the mass transfer constant were monitored as outlined before [11].

normal distribution of residuals. Therefore, all regression figures were constructed using log-transformed data. To quantify correlations, we used the Spearman rank correlation, which is independent of log transformation and therefore gives similar values if “normal” or log values are used. To test if a correlation between log(kd) and log(IC50) is better than correlation between log(KD) and log(IC50), we used a one-sided Williams' test for paired correlations, as implemented by the function paired.r in the package psych in R [52,53]. We took into account the confounding correlations between log(kd) and log(KD). To incorporate measurement errors in the SPR data, we repeated the correlation analysis on a dataset that included all SPR replicates separately (Supplementary Fig. S4C). TTK crystallography Clear prism-shaped crystals were grown 1 to 3 weeks before a soaking experiment by hanging drop vapor diffusion at room temperature above a reservoir of 1000 μl containing 32–37% PEG400 (Acros, Geel, Belgium), 0.1 M Na/K phosphate (pH 6.3), and 250 mM NaCl. Drops were composed of 1 μl protein sample and 1 μl mother liquor. For soaking, crystals were harvested and transferred to a sitting drop containing 10 μl mother liquor with 100 μM inhibitor and 1% (vol/vol) DMSO. After 48 h, crystals were flash-frozen by dipping them into liquid nitrogen, without the use of additional cryoprotectant. For compound 5, this soaking procedure led to problems with crystal cracking, presumably because of conformational rearrangements in the protein. In this case, crystals were exposed to an increasing concentration of compound 5 in mother liquor from 10 nM to 10 μM in the course of 5 days, after which crystals were flashfrozen. All data were collected in a semi-automated fashion at the European Synchrotron Radiation Facility at the ID29 and ID30A-1 beamlines. Diffraction patterns were interpreted by sequential use of Mosflm, Pointless, Aimless, cTruncate, Amore, and Refmac5 within the CCP4 suite [54] and further manually fitted in Wincoot [55]. Ideal ligand stereochemistry was defined with Accelrys DS and imported in CCP4 with ProDrg. Superpositions were performed in Wincoot. Pictures were generated using Pymol [56]. Data and refinement details can be found in Supplementary Table S3. Cell proliferation assays

Correlation analysis To study the relations between IC50s and kinetic parameters, we used linear regression and correlation analysis. The linear relationship between parameters was assessed from the distribution of residuals, quantile–quantile plots, and Shapiro–Wilk statistics in the statistical program R [52] (see Supplementary Figs. S4A and S4B). Only regressions of log-transformed data showed a sufficiently

All cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, U.S.A.) and cultured in the media as recommended by ATCC, supplemented with 10% bovine calf serum. All cells used were within nine passages of the original ATCC vial. The cell lines were authenticated by short tandem repeat analysis at ATCC and by targeted exome sequencing of 25 cancer genes [33]. Cell proliferation assays with the MOLT4 cell line and

2228 on a panel of 66 cancer cell lines (Oncolines™) were performed as described previously [33]. MOLT4 experiments were performed at least three times in duplicate. Oncolines™ cell panel profiling was performed once in duplicate. Briefly, proliferation assays were carried out in 384-well plates with incubation with compound for 120 h (5 days). Precise starting cell number was optimized for each cell line (for MOLT4, this was 1600 cells/well). Compounds were serially diluted in 3.16-fold steps in 100% DMSO to obtain a nine-point dilution series, followed by further dilution in aqueous buffer. A volume of 5 μl was transferred to the cells to generate a test concentration range from 3.16 μM to 3.16 nM in duplicate. The final DMSO concentration during incubation was 0.4% in all wells. After 5 days, 25 μl ATPlite™ 1 Step solution was added (Perkin Elmer, Groningen, The Netherlands) to each well. Luminescence was recorded on an Envision multimode reader. Percentage growth compared to uninhibited control was used for curve fitting. IC50s were derived from a four-parameter logistics curve fit using XLfit™5. Live-cell imaging MDA-MB-231 cells were cultured in Leibovitz L15 CO2-independent cell culture medium in a 6-well glass bottom chamber (LabTek Corp., Australia). Then, 2 h before imaging, cells were preincubated with SiR-DNA (Spirochrome, Switzerland) to visualize DNA. Cells treated with compound or DMSO were imaged every 5 min in a heated chamber at 37 °C, using a 40 × NA 0.95 air objective on an IX71 microscope (Olympus) controlled by SoftWoRx 6.0 software (Applied Precision). Image Z-stacks were acquired with 3-μm intervals using a sCMOS camera (DeltaVision RT; GE Healthcare, Issaquah, USA) and processed using ImageJ software (NIH, Bethesda, USA). Statistical analysis of chromosome segregation was performed with GraphPad Prism v6.0 f.

TTK inhibitors with long target residence time

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jmb.2017.05.014.

Acknowledgments The authors thank Linda Brouwers for purifying TTK; Antoon van Doornmalen and Jelle Dylus for help with cellular experiments; Stephanie Monaco, Matthew Bowler, and Salyha Ali at the European Synchrotron Radiation Facility for data collection; Anastassis Perrakis and Eleanor von Castelmur for diffraction tests at the Netherlands Cancer Institute; René Medema for facilitating imaging experiments at the Netherlands Cancer Institute; and Ross McGuire and Mario van der Stelt for critical reading of the manuscript. Author contributions: Concept and design: J.C.M.U., J.M., R.C.B., G.J.R.Z; execution of experiments: N.W.-S., M.B.W.P., M.A.A.L., J.C.M.U., J.G.S., J.J.P.W., J.R.F.V., J.A.D.M.R.; analysis of results: J.C.M.U., J.M., N.W.-S., M.B.W.P., M.A.A.L., J.G.S., J.J.P.W., J.R.F.V., J.A.D.M.R., R.C.B., G.J.R.Z.; writing of manuscript: J.C.M.U., G.J.R.Z. Competing financial interests: R.C.B. and G.J.R.Z. are founders and shareholders of Netherlands Translational Research Center B.V. Received 25 March 2017; Received in revised form 10 May 2017; Accepted 16 May 2017 Available online 21 May 2017 Keywords: target residence time; cellular activity; kinase; spindle assembly checkpoint; NTRC 0066-0 †www.carnabio.com

Data reproducibility Multiplicity and variation of all assay data can be found in Supplementary Table S2, where N means the number of experiments on separate occasions, and n means the number of replicates in a single experiment. For IC50s, ka, kd, and KD, averages and standard deviations were based on 10log values and reported in Supplementary Table S2.

Abbreviations used: TTK, threonine tyrosine kinase; SAC, spindle assembly checkpoint; TNBC, triple-negative breast cancer; SPR, surface plasmon resonance; PDB, Protein Data Bank; PEG, polyethylene glycol; DMSO, dimethylsulfoxide; ATCC, American Type Culture Collection.

Accession numbers The X-ray structures of the complexes have been submitted to the Protein Data Bank under ids 5N87 (NTRC 0066-0), 5N7V (MPI-0479605), 5N84 (MpsBAY2b), 5N93 (TC-Mps1-12), 5N9S (BAY 1161909), 5NAD (BAY 1217389), and 5NA0 (compound 5).

References [1] P. Cohen, D.R. Alessi, Kinase drug discovery—what's next in the field? ACS Chem. Biol. 8 (2013) 96–104.

TTK inhibitors with long target residence time

[2] D. Fabbro, 25 years of small molecular weight kinase inhibitors: potentials and limitations, Mol. Pharmacol. 87 (2015) 766–775. [3] J.C.M. Uitdehaag, G.J.R. Zaman, A theoretical entropy score as a single value to express inhibitor selectivity, BMC Bioinf. 12 (2011) 94. [4] J.C.M. Uitdehaag, F. Verkaar, H. Alwan, J. de Man, R.C. Buijsman, G.J.R. Zaman, A guide to picking the most selective kinase inhibitor tool compounds for pharmacological validation of drug targets, Br. J. Pharmacol. 166 (2012) 858–876. [5] R.A. Copeland, D.L. Pompliano, T.D. Meek, Drug target residence time and its implications for lead optimization, Nat. Rev. Drug Discov. 5 (2006) 730–739. [6] D.C. Swinney, Biochemical mechanisms of drug action: what does it take for success? Nat. Rev. Drug Discov. 3 (2004) 804–808. [7] G. Dahl, T. Åkerud, Pharmacokinetics and the drug-target residence time concept, Drug Discov. Today 18 (2013) 697–707. [8] T. Barf, A. Kaptein, Irreversible protein kinase inhibitors: balancing the benefits and risks, J. Med. Chem. 55 (2012) 6243–6262. [9] J.C. Byrd, R.R. Furman, S.E.C. Coutre, I.W. Flinn, J.A. Burger, K.A. Blum, B. Grant, J.P. Sharman, M. Coleman, W.G. Wierda, J.A. Jones, et al., Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukema, New Engl. J. Med. 369 (2013) 32–42. [10] E.R. Wood, A.T. Truesdale, O.B. McDonald, D. Yuan, A. Hassell, S.H. Dickerson, B. Ellis, C. Pennisi, E. Horne, K. Lackey, K.J. Alligood, et al., A unique structure for epidermal growth factor receptor bound to GW572016 (lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells, Cancer Res. 64 (2004) 6652–6659. [11] N. Willemsen-Seegers, J.C.M. Uitdehaag, M.B.W. Prinsen, J.R.F. de Vetter, J. de Man, M. Sawa, Y. Kawase, R.C. Buijsman, G.J.R. Zaman, Compound selectivity and target residence time of kinase inhibitors studies with surface plasmon resonance, J. Mol. Biol. 429 (2016) 574–586. [12] J.M. Bradshaw, J.M. McFarland, V.O. Paavilainen, A. Bisconte, D. Tam, V.T. Phan, S. Romanov, D. Finkle, J. Shu, V. Patel, et al., Prolonged and tunable residence time using reversible covalent kinase inhibitors, Nat. Chem. Biol. 11 (2015) 525–531. [13] M. Puttini, S. Redaelli, L. Moretti, S. Brussolo, R.H. Gunby, L. Mologni, E. Marchesi, L. Cleris, A. Donella-Deana, P. Drueckes, et al., Characterization of compound 584, an Abl kinase inhibitor with lasting effects, Haematologica 93 (2008) 653–661. [14] Z. Fang, C. Grütter, D. Rauh, Strategies for the selective regulation of kinases with allosteric modulators: exploiting exclusive structural features, ACS Chem. Biol. 8 (2012) 58–70. [15] C. Pargellis, L. Tong, L. Churchill, P.F. Chirillo, T. Gilmore, A.G. Graham, P.M. Grob, E.R. Hickey, N. Moss, S. Pav, et al., Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site, Nat. Struct. Biol. 9 (2002) 268–272. [16] A.J. Kooistra, G.K. Kanev, O.P.J. van Linden, R. Leurs, I.J.P. de Esch, C. de Graaf, KLIFS: a structural kinase-ligand interaction database, Nucleic Acids Res. (2016) D365–D371. [17] A. Janssen, G.J.P.L. Kops, R.H. Medema, Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 19,108–19,113.

2229 [18] V. Maire, C. Baldeyron, M. Richardson, B. Tesson, A. Vincent-Salomon, E. Gravier, B. Marty-Prouvost, L. De Koning, G. Rigaill, A. Dumont, et al., TTK/hMps1 is an attractive therapeutic target for triple-negative breast cancer, PLoS One 8 (2013) e63712. [19] A.M. Wengner, G. Siemeister, M. Koppitz, V. Schulze, D. Kosemund, U. Klar, D. Stoeckigt, R. Neuhaus, P. Lienau, B. Bader, et al., Novel Mps1 kinase inhibitors with potent antitumor activity, Mol. Cancer Ther. 15 (2016) 583–592. [20] J.M. Mason, X. Wei, G.C. Fletcher, R. Kiarash, R. Brokx, R. Hodgson, I. Beletskaya, M.R. Bray, T.W. Mak, Functional characterization of CFI-402257, a potent and selective Mps1/ TTK kinase inhibitor, for the treatment of cancer, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) 3,127–3,132, http://dx.doi.org/ 10.1073/pnas.1700234114. [21] R. Colombo, M. Caldarelli, M. Mennecozzi, M.L. Giorgini, F. Sola, P. Cappella, C. Perrera, S.R. Depaolini, L. Rusconi, U. Cucchi, et al., Targeting the mitotic checkpoint for cancer therapy with NMS-P715, an inhibitor of MPS1 kinase, Cancer Res. 70 (2010) 10,255–10,264. [22] N. Kwiatkowski, N. Jelluma, P. Filippakopoulos, M. Soundararajan, M.S. Manak, M. Kwon, H.G. Choi, T. Sim, Q.L. Deveraux, S. Rottmann, et al., Small molecule kinase inhibitors provide insight into Mps1 cell cycle function, Nat. Chem. Biol. 6 (2010) 359–368. [23] M. Jemaà, L. Galluzzi, O. Kepp, L. Senovilla, M. Brands, U. Boemer, M. Koppitz, P. Lienau, S. Prechtl, V. Schulze, et al., Characterization of novel Mps1 inhibitors with preclinical anticancer activity, Cell Death Differ. 20 (2013) 1532–1545. [24] A.R.R. Maia, J. de Man, A. Janssen, J.Y. Song, M. Omerzu, J.G. Sterrenburg, M.B.W. Prinsen, N. Willemsen-Seegers, J.A.D.M. de Roos, et al., Inhibition of the spindle assembly checkpoint kinase TTK enhances the efficacy of docetaxel in a triple-negative breast cancer model, Ann. Oncol. 26 (2015) 2180–2192. [25] M. Schmidt, Y. Budirahardja, R. Klompmaker, R.H. Medema, Ablation of the spindle assembly checkpoint by a compound targeting Mps1, EMBO Rep. 6 (2005) 866–872. [26] S. Santaguida, A. Tigher, A.M. D'Alise, S.S. Taylor, A. Musacchio, Dissecting the role of Mps1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine, J. Cell Biol. 190 (2010) 73–87. [27] K.D. Tardif, A. Rogers, J. Cassiano, B.L. Roth, D.M. Cimbora, R. McKinnon, A. Peterson, T.B. Douce, R. Robinson, I. Dorweiler, et al., Characterisation of the cellular and antitumor effects of MPI-0479605, a small molecule inhibitor of the mitotic kinase Mps1, Mol. Cancer Ther. 10 (2011) 2267–2275. [28] B.A. Tannous, M. Kerami, P.M. van der Stoop, N. Kwiatkowski, J. Wang, W. Zhou, A.F. Kessler, G. Lewandrowski, L. Hiddingh, N. Sol, et al., Effects of the selective Mps1 inhibitor Mps1-IN-3 on glioblastoma sensitivity to antimitotic drugs, J. Natl. Cancer Inst. 105 (2013) 1322–1331. [29] L. Hewitt, A. Tighe, S. Santaguida, A.M. White, C.D. Jones, A. Musacchio, S. Green, S.S. Taylor, Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1-C-Mad2 core complex, J. Cell Biol. 190 (2010) 25–34. [30] S. Naud, I.M. Westwood, A. Faisal, P. Sheldrake, V. Bavetsias, B. Atrash, K.J. Cheung, M. Liu, A. Hayes, J. Schmitt, et al., Structure-based design of orally bioavailable 1H-pyrrolo[3,2c]pyridine inhibitors of mitotic kinase monopolar spindle 1 (Mps1), J. Med. Chem. 56 (2013) 10,045–10,065. [31] K. Kusakabe, N. Ide, Y. Daigo, T. Itoh, K. Higashino, Y. Okano, G. Tadano, Y. Tachibana, Y. Sato, M. Inoue, et al.,

2230

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

Diaminopyridine-based potent and selective Mps1 kinase inhibitors binding to an unusual flipped peptide conformation, ACS Med. Chem. Lett. 3 (2012) 560–564. R. Martinez, A. Blasina, J.F. Hallin, W. Hu, I. Rymer, J. Fan, R.L. Hoffman, S. Murphy, M. Marx, G. Yanochko, et al., Mitotic checkpoint kinase Mps1 has a role in normal physiology which impacts clinical utility, PLoS One 10 (2015) e0138616. J.C.M. Uitdehaag, J.A.D.M. de Roos, M.B.W. Prinsen, N. Willemsen-Seegers, J.R.F. de Vetter, J. Dylus, A.M. van Doornmalen, J. Kooijman, M. Sawa, S.J.C. van Gerwen, et al., Cell panel profiling reveals conserved therapeutic clusters and differentiates the mechanism of action of different PI3K/mTOR, aurora kinase and EZH2 inhibitors, Mol. Cancer Ther. 15 (2016) 3097–3109. O. Fedorov, B. Marsden, V. Pogacic, P. Rellos, S. Müller, A.N. Bullock, J. Schwaller, M. Sundström, S. Knapp, A systematic interaction map ot validated kinase inhibitors with Ser/Thr kinases, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 20,523–20,528. M.L.H. Chu, L.M.G. Chavas, K.T. Douglas, P.A. Eyers, L. Tabernero, Crystal structure of the catalytic domain of the mitotic checkpoint kinase Mps1 in complex with SP600125, J. Biol. Chem. 283 (2008) 21,495–21,500. Y. Liu, Y. Lang, N. Kumar Patel, G. Ng, R. Laufer, S.-W. Li, L. Edwards, B. Forrest, P.B. Sampson, M. Feher, et al., The discovery of orally bioavailable tyrosine threonine kinase (TTK) inhibitors: 3-(4-(heterocyclyl)phenyl)-1H-indazole-5carboxamides as anticancer agents, J. Med. Chem. 58 (2015) 3366–3392. R. Bayliss, A. Fry, T. Haq, S. Yeoh, On the molecular mechanism of mitotic kinase activation, Open Biol. 2 (2012) 120,136. Y. Hiruma, A. Koch, S. Dharadhar, R.P. Joosten, A. Perrakis, Structural basis of reversine selectivity in inhibiting Mps1 more potently than aurora B kinase, Proteins 84 (2016) 1761–1766. Y. Liu, R. Laufer, N. Kumar Patel, G. Ng, P.B. Sampson, S.-W. Li, Y. Lang, M. Feher, R. Brokx, I. Beletskaya, et al., Discovery of pyrazolo[1,5-a]pyrimidine TTK inhibitors: CFI-402257 is a potent, selective, bioavailable anticancer agent, ACS Med. Chem. Lett. 7 (2016) 671–675. M. Caldarelli, M. Angiolini, T. Disingrini, D. Donati, M. Guanci, S. Nuvolini, H. Posteri, F. Quartieri, M. Silvagni, R. Colombo, Synthesis and SAR of new pyrazolo[4,3-h]quinazoline-3carboxamide derivatives as potent and selective Mps1 kinase inhibitors, Bioorg. Med. Chem. Lett. 21 (2011) 4507–4511. M.A.A. Libouban, J.A.D.M. de Roos, J.C.M. Uitdehaag, N. Willemsen-Seeger, S. Mainardi, J. Dylus, J. de Man, B. Tops, J.P.P. Meijerink, Z. Storchova, et al., Stable aneuploid tumor cells are more sensitive to TTK inhibition than chromosomally unstable cell lines, Oncotarget (2017), http://dx.doi.org/10. 18632/oncotarget.16213.

TTK inhibitors with long target residence time

[42] R.B. Slee, B.R. Grimes, R. Bansal, J. Gore, C. Blackburn, L. Brown, R. Gasaway, J. Jeon, J. Victorino, K.L. March, et al., Selective inhibition of pancreatic ductal adenocarcinoma cell growth by the mitotic MPS1 kinase inhibitor, NMS-P715, Mol. Cancer Ther. 13 (2013) 307–315. [43] X. Liu, W. Liao, Q. Yuan, Y. Ou, J. Huang, TTK activates Akt and promotes proliferation and migration of hepatocellular carcinoma cells, Oncotarget 6 (2015) 34,309–34,320. [44] R.H. Shoemaker, The NCI60 human tumour cell line anticancer drug screen, Nat. Rev. Cancer 6 (2006) 813–823. [45] F. Iorio, T.A. Knijnenburg, D.J. Vis, G.R. Bignell, M.P. Menden, M. Schubert, N. Aben, E. Gonçalves, S. Barthorpe, H. Lightfoot, et al., A landscape of pharmacogenomic interactions in cancer, Cell 166 (2016) 740–754. [46] B. Nolen, S. Taylor, G. Ghosh, Regulation of protein kinases: controlling activity through activation segment conformation, Mol. Cell 15 (2004) 661–675. [47] W. Wang, Y. Yang, Y. Gao, Q. Xu, F. Wang, S. Zhu, W. Old, K. Resing, N. Ahn, M. Lei, et al., Structural and mechanistic insights into Mps1 kinase activation, J. Cell. Mol. Med. 13 (2008) 1679–1694. [48] T.D. Pollard, A guide to simple and informative binding assays, Mol. Biol. Cell 21 (2010) 4061–4067. [49] A. Fersht, The basic equations of enzyme kinetics. In: Structure and Mechanism in Protein Science, a Guide to Enzyme Catalysis and Protein Folding, in: M.R. Julet (Ed.) W.H. Freeman, New York, USA, 1999 page 122–123. [50] J.F. Ohren, H. Chen, A. Pavlovsky, C. Whitehead, E. Zhang, P. Kuffa, C. Yan, P. McConnell, C. Spessard, C. Banotai, et al., Structures of human MAP kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition, Nat. Struct. Mol. Biol. 11 (2004) 1192–1197. [51] A. Prlić, S. Bliven, P.W. Rose, W.F. Bluhm, C. Bizon, A. Godzik, P.E. Bourne, Pre-calculated protein structure alignments at the RCSB PDB website, Bioinformatics 26 (2010) 2983–2985. [52] R. Core Team, R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, 2014 Vienna, Austria. http://www.R-project.org/. [53] E.J. Williams, The treatment of heterogenous data, in: W.A. Shewhart, S.S. Wilks (Eds.), Regression Analysis, Wiley, New York, USA 1959, pp. 129–162. [54] M.D. Winn, C.C. Ballard, K.D. Cowtan, E.J. Dodson, P. Emsley, P.R. Evans, R.M. Keegan, E.B. Krissinel, A.G.W. Leslie, A. McCoy, et al., Overview of the CCP4 suite and current developments, Acta Cryst D67 (2011) 235–242. [55] P. Elmsley, B. Lohkamp, W.G. Scott, K. Cowtan, Features and development of Coot, Acta Cryst D66 (2010) 486–501. [56] W.L. DeLano, The Pymol Molecular Graphics System. DeLano Scientific, 2002 San Carlos, USA, http://www. pymol.org.