Targeting the MKK7âJNK (Mitogen-Activated Protein Kinase Kinase 7

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Brief Article

Targeting the MKK7–JNK (Mitogen-Activated Protein Kinase Kinase 7–c Jun N-Terminal Kinase) Pathway with Covalent Inhibitors Patrik Wolle, Julia Hardick, Shane J.F. Cronin, Julian Engel, Matthias Baumann, Jonas Lategahn, Josef Penninger, and Daniel Rauh J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00102 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

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Journal of Medicinal Chemistry

Targeting the MKK7–JNK (Mitogen-Activated Protein Kinase Kinase 7– c-Jun N-Terminal Kinase) Pathway with Covalent Inhibitors Patrik Wolle,1,2,‡ Julia Hardick,1,2,‡ Shane J.F. Cronin,3 Julian Engel,1,† Matthias Baumann,4 Jonas Lategahn,1,2 Josef M. Penninger,3 and Daniel Rauh1,2,* 1Faculty

of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund (Germany). Discovery Hub Dortmund (DDHD) am Zentrum für integrierte Wirkstoffforschung (ZIW), 44227 Dortmund (Germany). 3Institute of Molecular Biotechnology, Austrian Academy of Sciences, Dr. Bohr Gasse 3, AT-1030 Vienna (Austria). 4Lead Discovery Center GmbH, Otto-Hahn-Strasse 15, 44227 Dortmund (Germany). 2Drug

KEYWORDS: mitogen-activated protein kinase kinase 7 (MKK7), click chemistry, drug discovery, covalent inhibitor. ABSTRACT: The protein kinase MKK7 is linked to neuronal development and the onset of cancer. The field, however, lacks highquality functional probes that would allow for the dissection of its detailed functions. Against this background, we describe an effective covalent inhibitor of MKK7 based on the pyrazolopyrimidine scaffold.

INTRODUCTION The development of highly potent and selective chemical probes to determine the function of proteins in cells is absolutely necessary to study their role in cellular signaling. Many other ways to explore the function of a protein are possible, but most of them are less efficient or not specific.1 Therefore small molecule modulators, with a specific target and high potency can be used to look deeper into the function of single proteins, for example protein kinases, which are highly involved in cellular signaling.2 The human kinome comprises about 500 protein kinases,3 which renders the design of selective kinase inhibitors a major challenge due to the highly conserved ATP pocket.4 One promising approach is to target cysteines with reactive groups that form a covalent linkage.5 The covalent bond formation leads to higher residence time and therefore an increase in potency and selectivity.5, 6 Although covalent inhibitors have been avoided in medicinal chemistry in the past, given concerns regarding their potential for off-target reactivity and immune responses,7 this approach was recently successfully exemplified in covalently targeting protein kinases such as EGFR (epidermal growth factor receptor) and BTK (Bruton’s tyrosine kinase) with clinically approved drugs, osimertinib,810 afatinib,11, 12 and ibrutinib (1)13, 14. These address a cysteine at the end of the hinge region connecting the N-terminal and C-terminal part of the kinase domain. In the entire kinome only 11 kinases bear a cysteine at this position (Figure S1). One of these kinases is MKK7 (Figure 1a), a member of the mitogen-activated protein kinase family.15 It is part of the c-Jun N-terminal kinase (JNK) pathway, which is involved in stress signaling, for example. JNK’s were identified in numerous human diseases, like Parkinson’s and Alzheimer’s

disease, rheumatoid arthritis and inflammatory diseases.16 MKK7 is activated by several MAP3 kinases and in turn phosphorylates JNK together with MKK4.17 In contrast to MKK4, which can also phosphorylate p38 MAPK, MKK7 phosphorylates only the different isoforms of JNK.18 The exact role of MKK7 in different cellular processes is still a topic of current research. For example, it seems to be an important part of neuronal development and is involved in the generation and growth of dendrites.19 It was also found to be involved in some special cases of cancer development.20 So far only few inhibitors are published to target MKK7, and most of them inhibit MKK7 as an off-target. Sogabe et al. showed a derivative of hypothemycin – a known natural product addressing a cysteine right before the DFG-motif in several kinases,21 among them MEK1 (Cys276) – to bind covalently to Cys218 in MKK7 and provokes an IC50 of 1.3 µM.22 More recently, Shraga et al. identified small fragments through docking endeavors, that covalently bind to MKK7 and inhibit cellular JNK activity with EC50 values in the micromolar range.23 Therefore, further research requires the development of highly potent and selective probes to gain further insight in to MKK7 biology. We recently reported pyrazolopyrimidine-based inhibitors that target gatekeeper-mutant (T790M) drug resistance in EGFR,24, 25 and were able to solve their crystal structure.24 The structure of apo MKK7 is known, and the alignment of MKK7 apo with the ligand bound EGFR structure shows high similarity regarding the ATP binding pocket. Both kinases carry a methionine gatekeeper, a reactive cysteine at the end of the hinge region as well as the conserved catalytic lysine (Figure 1 and Figure S1). Furthermore, the pyrazolopyrimidine-based inhibitor of BTK, ibrutinib (1), was reported to exhibit MKK7 off-target activity.26 We therefore

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considered pyrazolopyrimidines well-suited as ligands of MKK7 that could alkylate the analogous cysteine with a Michael acceptor system. Here, we present the structure-based design, synthesis and evaluation of a pyrazolopyrimidine-based covalent inhibitor to specifically target MKK7. Mass spectrometry demonstrated the covalent bond formation and subsequent X-ray structures yielded insight into the binding mode, as well as the final evidence for targeting Cys218 in MKK7. Biochemical and cellular characterization revealed the inhibitory potency and kinome profiling as well as pharmacokinetic analysis substantiate our approach.

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(Figure 2a) and therefore, we synthesized the derivatives 4–6 that were believed to occupy the observed pocket. This focused series of triazole-containing compounds showed mixed results when they were tested in the biochemical assay (Table 1). The para-keto benzene derivative 4a set the lead with an IC50 value of 10 nM against MKK7, provoking a 4times increased potency as compared to the initial compound 3a. It is worth noting that 4a markedly improved the selectivity towards EGFR and its mutant variant. The derivatives 5 and 6, which showed moderate inhibition (788 and 669 nM, respectively), seemed not to fit properly into the backpocket. As a reference, we tested ibrutinib, an approved inhibitor of BTK, which inhibited MKK7 with an IC50 of 78 nM. We synthesized the reversible counterpart 4b of the most potent inhibitor 4a. Subsequent testing revealed a complete loss of activity against MKK7 which led to the conclusion that these compounds form a covalent bond to Cys218, that is inevitable for inhibitory activity. Table 1. Half-maximal inhibitory concentrations (IC50) determined for 4-aminopyrazolopyrimidine-based inhibitors 1–6 against MKK7 and EGFR.a

Figure 1. Similarity between the binding site of MKK7 and EGFR-T790M. (A) Sequence overlay of MKK7-wt and EGFRT790M which share the same cysteine in the ATP binding site. (B) Deeper insight into the ATP binding pocket to illustrate the high similarity between the EGFR structure bound pyrazolopyrimidine based compound 2 (PDB ID: 5J9Z; left) and MKK7 apo protein (PDB ID: 2DYL; right). Important amino acid sidechains are highlighted – catalytic lysine (purple), methionine gatekeeper (cyan) and the reactive cysteine (red). IC50 [nM] Compound

RESULTS Rational Design and Biochemical Characterization of Pyrazolopyrimidine-Based MKK7-Inhibitors. We took advantage of the previously described 4-aminopyrazolopyrimidine EGFR inhibitor 2,24 which showed a high inhibitory effect on EGFR and its mutants and moderate potency on MKK7 (Table 1). Structural alignment of 2 in complex with EGFR and MKK7 showed the already mentioned high similarity of the two binding sites as well as some space in the backpocket for further compound design and optimization (Figure 1). Therefore, compound 3a served as a suitable starting point since it provides an alkyne as the derivatization point for copper-catalyzed click-chemistry.25 Furthermore, compound 3a showed a significant inhibitory effect against MKK7 in biochemical assays. We were able to gain a co-crystal structure of the kinase in complex with 3a and verified the additional space in the backpocket of MKK7 between the Met212-gatekeeper and the catalytic Lys165

MKK7b

EGFR-WTc

EGFRL858R/T790Mc

2

302  104

35  14d

3  1d

3a

40  8

1489  659d

693  336d

4a

10  3

8006  3453

>10000

4b

>10000

649  129

677  52

5

788  172

61

28 ± 9

6

669  161

85  6

104  16

78  21

70%) and



also a high plasma protein binding (>93%), features that are frequently observed for covalent inhibitors. The potent inhibitor 4a displayed a moderate microsomal stability in liver microsomes (154 and 124 µL/min/mg) but obviously improved as compared to the reference ibrutinib, which revealed a clearance of 234 and >1000 µL/min/mg in human and mouse liver microsomes, respectively (Figure S8). Despite the high clearance in microsomes, the overall favorable profile of 4a encourages us to investigate its in vivo pharmacokinetic properties in the future. Table 2. Physicochemical and in vitro pharmacokinetic parameters for 4a and 1 as a reference compound. 4a

ibrutinib (1)

cLogPa

1.78

3.63

LLEb

6.22

3.48

8

36

PAMPA [% Flux]

n.d.

38

Caco-2 Papp (A–B) [10-6 cm/s]

20

40

Caco-2 Papp (B–A) [10-6 cm/s]

13

0.9

Caco-2 ratio Papp (B–A)/(A–B)

0.6

0.02

MDCKII-MDR1 Papp (A–B) [10-6 cm/s]

9

22

MDCKII-MDR1 Papp (B–A) [10-6 cm/s]

4

3

MDCKII-MDR1 ratio Papp (B–A)/(A–B)

0.40

0.13

Plasma Stability human / mouse [% Remain]

>99 / 70.4

79.7 / 97.5

Plasma Protein Binding human / mouse [% Bound]

96.0 / 92.7

97.6 / 98.9

Microsomal Stability (Phase I) CLint human / mouse [µL/min/mg]

154 / 124

234 / >1000

SolRank [µM]

acLogP

was calculated with Seurat (consensus model using ChemAxon and Klopman’s models and the PhysProp database). bLigand lipophilicity efficiency (LLE) was calculated from pIC50(MKK7) – cLogP. n.d. = not detectable.

DISCUSSION AND CONCLUSIONS In summary, we have developed a potent, selective, and covalent inhibitor of MKK7, which shows an excellent biochemical inhibitory effect and selectivity over EGFR and its mutant variant. A kinase profiling study displayed high selectivity for only seven off-target enzymes because of the isostructural cysteine residues in six of these kinases. Mass experiments revealed the covalent bond formation and further MS/MS studies confirmed the intended cysteine had been targeted. X-ray analysis confirmed the predicted binding mode and provided insight into the binding site for further compound optimization. Additionally, western blot analysis showed the high cellular inhibitory effect of 4a on the phosphorylation of JNK as well as the downstream signaling

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in the JNK-pathway. Besides the high clearance in human and mouse liver microsomes, inhibitor 4a demonstrated an overall good pharmacokinetic profile, thus rendering it a valuable probe for further investigation to elucidate the function of MKK7 in cellular studies and in vivo.

EXPERIMENTAL SECTION Protein Purification. For the crystallization a construct of MKK7 was used containing amino acids 117-423 with a non-cleavable Nterminal His6-Tag. For the Assay the construct contained amino acids 1-462 with a TEV-cleavable N-Terminal His6-Tag, containing the activation mutations S273D and T275D. The protein for crystallization was expressed in BL21 DE3 E. coli at 18 °C for 20 h, while the protein for the assay was expressed in SF9 insect cells. The cells were lysed using French press and centrifuged. The supernatant was then loaded on a Ni-Affinity Chromatography (Qiagen Ni-NTA Superflow 5mL). Washed with buffer A (50 mM Tris, 500 mM NaCl, 25 mM Imidazole, 5% glycerol, pH 8) and eluted with a gradient of buffer B (50 mM Tris, 500 mM NaCl, 500 mM Imidazole, 5% glycerol, pH 8). The protein was concentrated, and then applied to a gel filtration chromatography (GE HiLoad 16/600 75pg) using buffer C (25 mM Tris, 100 mM NaCl, 10% glycerol, pH 7.4). The eluted protein fractions were combined and concentrated to 10 mg/mL and then used for crystallization. Protein crystallization and Data collection. Crystallization of MKK7 with Inhibitor 3a and 4a was performed by incubating 10 mg/mL MKK7 with a threefold molar excess of inhibitor (10 mM DMSO stock) for 1 h at 4 °C. Crystals were grown using the hanging drop method at 20 °C after mixing 1 μL protein-inhibitor solution with 1 μL reservoir solution (180-220 mM Sodium Citrate, 15-25% PEG3350). All crystals were frozen with further addition of 20% (v/v) glycerol. The data sets were collected at the PX10SA beamline of the Swiss Light Source (PSI,Villingen, Switzerland). All data sets were processed with XDS29 and scaled using XSCALE29. Structure Determination and Refinement of the complex crystal structures were solved by molecular replacement with PHASER30 using PDB ID: 2DYL as template. The MKK7 molecule in the asymmetric unit was manually adjusted using the program COOT31. The refinement was performed with REFMAC532. Inhibitor topology files were generated using the Dundee PRODRG2 server33. Refined structures were validated with PROCHECK33 and the PDB validation server. Data collection, structure refinement statistics, PDB ID codes, further details for data collection are provided in Table S1. PyMOL34 was used for generating the figures. ADP-Glo Assay. The ADP-Glo Assay was performed according to the instructions provided by the supplier with 20 nM Kinase (MKK7 beta 1, S273D T275D) and 5 µM Substrate (JNK1 K55M). The dilution of the compound was done with the Echo 520 (LabCyte) acoustic pipetting robot and the liquid handling was performed with a multidrop (Thermo Scientific). The readout was performed with a Tecan M1000 infinite plate reader. Mass spectrometry. The MS experiments were performed on a VelosPro IonTrap (Thermo Scientific). The protein was diluted to a concentration of 1 mg/mL and combined with 2-fold molar excess of the compound and incubated for 30 min on ice. Then the solution was centrifuged, and the supernatant was then used for the measurements. The measurement was performed with an EC 50/3 Nucleodur C18 1.8µm column (Macherey and Nagel) and gradient of the mobile phase A (0.1% formic acid in water) to B (0.1% formic acid in acetonitrile). For the spectra’s visualization, XCalibur and MagTran were used. For the MS/MS experiments the protein was incubated with the compound as described before. Then denaturized using SDS-sample buffer (50 mM Tris-HCl) pH 6.8, 2% SDS, 10% glycerol, 1% betamercaptoethanol, 12.5 mM EDTA and 0.02% bromphenol blue) and then separated via SDS-Gel electrophoresis with a 15% SDS-gel. The corresponding band was cut out and washed with 200 µL solution A (3:1 25 mM ammonium hydrogen carbonate and


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acetonitrile) for 15 min at 37 °C. The solution was replaced with 200 µL solution B (1:1 25 mM ammonium hydrogen carbonate and acetonitrile) and incubated again for 15 min at 37 °C. After removing the solution, the samples were treated with 100 µL reductive solution C (50 mM DTT, 25 mM ammonium hydrogen carbonate) for 1 hour at 37 °C. For the following alkylation the solution was replaced with 100 µL solution D (55 mM Iodacetamide, 25 mM ammonium hydrogen carbonate) and incubated for one hour at 20 °C. The alkylation solution was replaced by solution B and washed two times for 15 min at 37 °C. The solution was removed, and the samples were dehydrated with 10 µL acetonitrile for 10 min. The acetonitrile was removed and 40 µL of the digest solution (0.1 mg/mL trypsin, 25 mM ammonium hydrogen carbonate) was added and incubated for 20 hours at 30 °C. After incubation, 3.5 µL of a 10% TFA solution was added and incubated for 30 min at 0 °C. The solution was then used for further experiments. Cell culture, western blotting and antibodies. DRGs were collected and dissociated as previously described.35 DRGs were cultured in laminin-coated 12-well plates for three hours. Media was replaced, and compounds were then added for a further 2 hours unless indicated otherwise. The neurons were lysed, and proteins extracted using RIPA buffer supplemented with phosphatase inhibitors and Western blots were performed for phosphor-cJun (cell signaling, 3270), cJun (cell signaling, 9165), phospho-JNK (cell signaling, 4668) and JNK (cell signaling, 9252). SolRank. For determining the kinetic solubility, the compound was diluted from a 10 mM stock in DMSO to a final concentration of 500 μM in 50 mM Hepes buffer, pH 7.4. Following an incubation of 90 min at room temperature on a shaker, the aqueous dilution was filtered through a 0.2 μm PVDF filter, and the optical density between 250 and 500 nm was measured at intervals of 10 nm. The kinetic solubility was calculated from the area under the curve (AUC) between 250 and 500 nm and normalized to absorption of a dilution of the compound in acetonitrile. PAMPA (Parallel artificial membrane permeability assay). The compound was diluted from a 10 mM stock in DMSO to a final concentration of 500 µM in 50 mM Hepes buffer pH 7.4 and transferred onto a transwell membrane covered with a membraneforming solution of 10% 1,2-Dioleyl-sn-glycero-3-phosphocholine (Sigma Aldrich) and 0.5% (w/v) cholesterol (Sigma Aldrich) in dodecane. Following an incubation of 16 hours at room temperature in a wet chamber, the optical density of the solution in the receiver well was measured between 250 and 500 nm in intervals of 10 nm. The percent flux was calculated from the AUC between 250 and 500 nm and normalized to the absorption of the compound following a 16 hours incubation in a parallel transwell containing a membrane covered with 50% MeOH in 50 mM Hepes buffer pH 7.4.36 Caco-2 assay. For Caco-2 cell assay, a 10 mM DMSO stock of the compound was diluted to a final concentration of 5 μM in HBSS buffer pH 7.4 and incubated for 2 hours at 37 °C and 5% CO2 on a monolayer of Caco-2 cells (ATCC) that had been grown on a transwell membrane (Millipore, Schwalbach, Germany) for 21 days. The compound concentration was measured in the receiver as well as the donor well. Apparent permeability (Papp) from either the apical to basolateral direction or vice versa was calculated by the equation: Papp = 1/AC0 (dQ/dt), where A is the membrane surface area, C0 is the donor drug concentration at t = 0, and dQ/dt is the amount of drug transported within the given time period of 2 hours. MDCKII-MDR1. To measure cellular permeability, compounds were applied at a concentration of 10 µM in HBSS to either the apical (A) or basolateral (B) side of a MDCKII-MDR cell monolayer and incubated for 2 hours at 37 °C. Compound concentrations on each side of the monolayer were determined by LC-MS/MS and the apparent permeability (Papp) was calculated in the apical to basolateral (A–B) and basolateral to apical (B–A) directions according to the following equation: Papp (A–B) = (CB  VB  0.001) / (t  A  Ct0,A). Plasma stability. Plasma stability was measured by LC/MS-based determination of the percentage of remaining compounds at a

concentration of 5 µM after incubation in plasma obtained from different species for 1 h at 37 °C. Plasma protein binding. Assessment of plasma protein binding was measured by equilibrium dialysis by incubating plasma with the compound of interest at a concentration of 5 µM for 6 h at 37 °C followed by LC/MS-based determination of final compound concentrations. Microsomal stability assay (phase I). Metabolic stability under oxidative conditions was measured in liver microsomes from different species by LC/MS-based measuring of depletion of compound at a concentration of 3 µM over time up to 60 minutes for HLM and MLM at 37 °C. Based on the compound half-life t1/2, the in vitro intrinsic clearance CLint was calculated. Chemistry. All final compounds were purified to ≥95% purity confirmed by NMR analysis as well as LC/MS analysis. Detailed synthetic procedures are described in the supporting information.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supplementary Tables and Figures, and Experimental Section – Chemistry (PDF) Molecular formula strings, MFS (CSV)

Accession Codes PDB codes for MKK7 bound with 3a and 4a are 6IB0 and 6IB2, respectively.

AUTHOR INFORMATION Corresponding Author *Prof. Dr. Daniel Rauh, Phone: +49 (0)231/755-7080, eMail: [email protected], Twitter: @DDHDortmund, Web: DDHDortmund.de.

ORCID Patrik Wolle: 0000-0001-6309-6773 Julia Hardick: 0000-0002-0062-1849 Jonas Lategahn: 0000-0001-5993-7082 Daniel Rauh: 0000-0002-1970-7642

Present Addresses †Lead Discovery Center GmbH, Otto-Hahn-Strasse 15, 44227 Dortmund (Germany).

Author Contributions ‡P.W. and J.H. contributed equally to this work.

ACKNOWLEDGMENT This work was co-funded by the German Federal Ministry for Education and Research (NGFNPlus and e:Med) (Grant No. BMBF 01GS08104, 01ZX1303C), the Deutsche Forschungsgemeinschaft (DFG), the German federal state North Rhine-Westphalia (NRW) and the European Union (European Regional Development Fund: Investing In Your Future) (EFRE800400), NEGECA (PerMed NRW), EMODI and Drug Discovery Hub Dortmund (DDHD).

ABBREVIATIONS ADME, absorption, distribution, metabolism, and excretion; BTK, Bruton’s tyrosine kinase; CNS, central nervous system; EGFR, epidermal growth factor receptor; JNK, c-Jun N-terminal kinase;



LLE, ligand-lipophilicity efficiency; MKK7, mitogen-activated protein kinase kinase 7; PAMPA, parallel artificial membrane permeability assay.

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(14) Byrd, J. C.; Furman, R. R.; Coutre, S. E.; Flinn, I. W.; Burger, J. A.; Blum, K. A.; Grant, B.; Sharman, J. P.; Coleman, M.; Wierda, W. G.; Jones, J. A.; Zhao, W.; Heerema, N. A.; Johnson, A. J.; Sukbuntherng, J.; Chang, B. Y.; Clow, F.; Hedrick, E.; Buggy, J. J.; James, D. F.; O'Brien, S. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N. Engl. J. Med. 2013, 369, 32-42. (15) Tournier, C.; Whitmarsh, A. J.; Cavanagh, J.; Barrett, T.; Davis, R. J. The MKK7 gene encodes a group of c-Jun NH2-terminal kinase kinases. Mol. Cell. Biol. 1999, 19, 1569-1581. (16) Sabapathy, K. Role of the JNK pathway in human diseases. Prog. Mol. Biol. Transl. Sci. 2012, 106, 145-169. (17) Yamauchi, J.; Kaziro, Y.; Itoh, H. Differential regulation of mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7) by signaling from G protein beta gamma subunit in human embryonal kidney 293 cells. J. Biol. Chem. 1999, 274, 1957-1965. (18) Wang, X.; Destrument, A.; Tournier, C. Physiological roles of MKK4 and MKK7: insights from animal models. Biochim. Biophys. Acta 2007, 1773, 1349-1357. (19) Yamasaki, T.; Kawasaki, H.; Arakawa, S.; Shimizu, K.; Shimizu, S.; Reiner, O.; Okano, H.; Nishina, S.; Azuma, N.; Penninger, J. M.; Katada, T.; Nishina, H. Stress-activated protein kinase MKK7 regulates axon elongation in the developing cerebral cortex. J. Neurosci. 2011, 31, 16872-16883. (20) Tornatore, L.; Sandomenico, A.; Raimondo, D.; Low, C.; Rocci, A.; Tralau-Stewart, C.; Capece, D.; D'Andrea, D.; Bua, M.; Boyle, E.; van Duin, M.; Zoppoli, P.; Jaxa-Chamiec, A.; Thotakura, A. K.; Dyson, J.; Walker, B. A.; Leonardi, A.; Chambery, A.; Driessen, C.; Sonneveld, P.; Morgan, G.; Palumbo, A.; Tramontano, A.; Rahemtulla, A.; Ruvo, M.; Franzoso, G. Cancer-selective targeting of the NF-kappaB survival pathway with GADD45beta/MKK7 inhibitors. Cancer Cell 2014, 26, 938. (21) Chaikuad, A.; Koch, P.; Laufer, S. A.; Knapp, S. The cysteinome of protein kinases as a target in drug development. Angew. Chem. Int. Ed. 2018, 57, 4372-4385. (22) Sogabe, Y.; Matsumoto, T.; Hashimoto, T.; Kirii, Y.; Sawa, M.; Kinoshita, T. 5Z-7-Oxozeaenol covalently binds to MAP2K7 at Cys218 in an unprecedented manner. Bioorg. Med. Chem. Lett. 2015, 25, 593-596. (23) Shraga, A.; Olshvang, E.; Davidzohn, N.; Khoshkenar, P.; Germain, N.; Shurrush, K.; Carvalho, S.; Avram, L.; Albeck, S.; Unger, T.; Lefker, B.; Subramanyam, C.; Hudkins, R. L.; Mitchell, A.; Shulman, Z.; Kinoshita, T.; London, N. Covalent docking identifies a potent and selective MKK7 inhibitor. Cell Chem. Biol. 2019, 26, 98-108 e5. (24) Engel, J.; Becker, C.; Lategahn, J.; Keul, M.; Ketzer, J.; Mühlenberg, T.; Kollipara, L.; Schultz-Fademrecht, C.; Zahedi, R. P.; Bauer, S.; Rauh, D. Insight into the inhibition of drug-resistant mutants of the receptor tyrosine kinase EGFR. Angew. Chem. Int. Ed. 2016, 55, 10909-10912. (25) Engel, J.; Smith, S.; Lategahn, J.; Tumbrink, H. L.; Goebel, L.; Becker, C.; Hennes, E.; Keul, M.; Unger, A.; Müller, H.; Baumann, M.; Schultz-Fademrecht, C.; Günther, G.; Hengstler, J. G.; Rauh, D. Structure-guided development of covalent and mutantselective pyrazolopyrimidines to target T790M drug resistance in epidermal growth factor receptor. J. Med. Chem. 2017, 60, 77257744. (26) Lanning, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert, A. M.; Hett, E. C.; Johnson, T. O.; Joslyn, C.; Kath, J. C.; Niessen, S.; Roberts, L. R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B.; Whiteley, L. O.; Hayward, M. M.; Cravatt, B. F. A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors. Nat. Chem. Biol. 2014, 10, 760-767. (27) Wolle, P.; Müller, M. P.; Rauh, D. Augmented reality in scientific publications-taking the visualization of 3D structures to the next level. ACS Chem. Biol. 2018, 13, 496-499. (28) Schultes, S.; de Graaf, C.; Haaksma, E. E. J.; de Esch, I. J. P.; Leurs, R.; Krämer, O. Ligand efficiency as a guide in fragment hit selection and optimization. Drug Discov. Today: Technol. 2010, 7, e157-e162. (29) Kabsch, W. Automatic processing of rotation diffraction


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Table of Contents artwork


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Figure 1. Similarity between the binding site of MKK7 and EGFR-T790M. (A) Sequence overlay of MKK7-wt and EGFR-T790M which share the same cysteine in the ATP binding site. (B) Deeper insight into the ATP binding pocket to illustrate the high similarity between the EGFR structure bound pyra-zolopyrimidine based compound 2 (PDB ID: 5J9Z; left) and MKK7 apo protein (PDB ID: 2DYL; right). Important amino acid sidechains are highlighted – catalytic lysine (purple), methionine gatekeeper (cyan) and the reactive cysteine (red). 83x77mm (300 x 300 DPI)



Figure 2. Crystal structure of MKK7 in complex with (A) compound 3a and (B) compound 4a. Shown is the electron density (2FO-FC map contoured at an RMSD of 1) of MKK7/3a (PDB ID: 6IB0) at a resolution of 2.6 Å and of MKK7/4a (PDB ID: 6IB2) at a resolution of 2.1 Å. Shown are also the amino acid sidechains of the hinge region (white) and the α helix C (green). The QR-codes provide an augmented reality view of 3D models of the binding sites.24 83x59mm (300 x 300 DPI)


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Figure 3. Western blot analysis of the inhibitory effect of compound 4a and 4b against the JNK pathway. 83x68mm (300 x 300 DPI)



Table of Contents artwork 71x55mm (300 x 300 DPI)


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