Characterization of Covalent PyrazolopyrimidineâMKK7 Complexes

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Characterization of Covalent Pyrazolopyrimidine–MKK7 Complexes and Report on a Unique DFG-in/Leu-in Conformation of the Mitogen Activated Protein Kinase Kinase 7 (MKK7) Patrik Wolle, Julian Engel, Steven Smith, Lisa Goebel, Elisabeth Hennes, Jonas Lategahn, and Daniel Rauh J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00472 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

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Journal of Medicinal Chemistry

Characterization of Covalent Pyrazolopyrimidine– MKK7 Complexes and Report on a Unique DFGin/Leu-in Conformation of the Mitogen Activated Protein Kinase Kinase 7 (MKK7) Patrik Wolle,1,2 Julian Engel,1,† Steven Smith,1,† Lisa Goebel,1,2 Elisabeth Hennes,1,† Jonas Lategahn,1,2,†,* and Daniel Rauh1,2,* 1TU

Dortmund University, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Strasse 4a, 44227

Dortmund (Germany). 2Drug

Discovery Hub Dortmund (DDHD) am Zentrum für Integrierte Wirkstoffforschung (ZIW), 44227

Dortmund (Germany). KEYWORDS: Mitogen Activated Protein Kinase Kinase 7 (MKK7), JNK-Signaling, Drug Discovery, Covalent Inhibitors.

ABSTRACT

Pyrazolopyrimidines are well established as covalent inhibitors of protein kinases such as the epidermal growth factor receptor (EGFR) or Bruton’s tyrosine kinase (BTK) and we recently described their potential

in

targeting

MKK7.

Herein,

we

report

the

structure–activity

relationship

of

pyrazolopyrimidine-based MKK7 inhibitors and solved several complex crystal structures to gain ACS Paragon Plus Environment

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insights into their binding mode. In addition, we present two structures of apo-MKK7, exhibiting a DFG-out and an unprecedented DFG-in/Leu-in conformation.

INTRODUCTION Mitogen activated protein kinase kinase 7 (MKK7) is a member of the c-JUN N-terminal kinase (JNK) signaling pathway which is involved in several cellular regulation processes including the response to stress and cellular development.1 It is associated with a variety of diseases, such as multiple myeloma, or with various neurological disorder diseases like Alzheimer’s disease.2, 3 However, previous attempts to directly inhibit JNK led to numerous side effects and therefore the inhibition of upstream modulators was pursued.4 Therefore, research interests have focused on MKK7, because it takes a central role in JNK regulation and is one of only two known activators which phosphorylate JNK at a TXY motif.5 MKK4 is known to phosphorylate JNK as well as the p38 MAPK, in contrast with MKK7 which only phosphorylates the different isoforms of JNK.6 This renders it an interesting specific target for the development of selective JNK pathway modulators which can then be used to unravel currently unknown functions of the pathway, to investigate the exact role of MKK7 in different cellular processes, or for the development of selective drugs. We, therefore, set out to identify modulators of this kinase. In the present work, a screening endeavor revealed compounds based on the 4-amino-pyrazolopyrimidine scaffold, e.g., ibrutinib,7, 8 to exhibit a pronounced inhibitory effect toward MKK7 (Figure 1A). This type of inhibitors binds to the ATP binding pocket and shares the same interactions as the adenosine of the ATP-molecule. This class of inhibitory agents allows for the introduction of a piperidine linker equipped with an acrylamide that forms a covalent bond with a reactive cysteine which is similarly positioned in EGFR, BTK and MKK7 (Figure 1B). The identified inhibitors were further characterized in complex with MKK7 by means of MS analysis as well as protein X-ray crystallography. We present insights into their binding mode and

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also show the crystal structures of wild-type MKK7 in its apo-form exhibiting a DFG-out and a unique DFG-in/Leu-in conformation.

Figure 1. A) Focused screen of EGFR-targeted inhibitors against MKK7 at a concentration of 50 µM, results grouped by scaffold. B) Binding pose of an AMPPNP (Adenosine 5′-(β,γ-imido)triphosphate) molecule in the ATP-binding site of EGFR (yellow, PDB: 3VJN) aligned with ibrutinib-bound EGFR (green, PDB: 5YU9) which exhibits a similar binding site as compared to MKK7.

RESULTS Our lab features a Robotics-Assisted Screening Platform for Efficient Ligand Discovery, RASPELD, which allows for screening campaigns in an academic environment.9 We performed a focused ADP-Glo biochemical screen against MKK7 at a screening concentration of 50 µM. Due to the structural similarity between EGFR and MKK7, we selected 101 EGFR-targeted and structurally related compounds that were developed in our lab. Fifty-five compounds have been already published,10-18 and their results are discussed below. The selection parameters comprised covalent, reversible, and covalentACS Paragon Plus Environment


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reversible inhibitors, among which the latter can bind covalently to the kinase, but when it comes to degradation of the protein, the compound is released again.12 As positive controls, we included ibrutinib as well as an inhibitor for MKK7 that was recently reported by Wolle and Hardick and colleagues with a low nanomolar IC50.18 DMSO served as the negative control and we observed a Z′ value of 0.76 which indicates a favorable assay robustness. We observed 19 compounds that possessed ≤35% residual activity and were termed hits (hit rate 35%), which is reasonable for a focused screen. Interestingly, the hit rate was considerably higher within compounds based on the pyrazolopyrimidine scaffold, while none of the compounds based on other scaffolds showed a reasonable inhibitory effect toward MKK7 (Figure 1A, Table 1 and Table S1). Therefore, we next recorded dose–response curves for an expanded set of pyrazolopyrimidines against MKK7 and determined the IC50 values in a biochemical assay (Table 1). The results showed a wide range of activities matching the results of the screen. The compounds 1b, 1h and 1m revealed high inhibitory potency, with the iodine-substituted molecule (1b) having an IC50 value of 35 nM. The highest activity was observed with compounds that exhibited a hydrogen-bond donor in a distinct position, that is, the para-phenol substituted compound 1h, with an IC50 of 8.6 nM and the indazole-bearing inhibitor 1m with an IC50 of 19 nM. Interestingly, compounds that did not bind covalently lost their activity against MKK7. This result is best exemplified with compounds 1d–1e and 1g, which possessed an acrylamide warhead in close proximity to the reactive Cys218 in MKK7, while the corresponding compounds 2a–2c exhibited propionamides instead, which could not form the covalent bond to the reactive cysteine. This outcome was also observed with compounds 4a and 4b which carried a 2-cyano-3-cyclopropylacrylamide group for covalent-reversible targeting of Cys218; these compounds did not inhibit the kinase activity. The reactivity or the increased steric demand of the group did not seem to support the covalent bond formation.


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Table 1. Hit validation and expanded analysis of pyrazolopyrimidine-based inhibitors against MKK7

MKK7 residual activity [%] @ 50 µM

MKK7 residual activity [%] @ 50 µM

MKK7 IC50 [nM]

MKK7 IC50 [nM]

1a

38 ± 20

631 ± 87

1k

17 ± 1

149 ± 30

1b

28 ± 18

35 ± 3

1l

28 ± 10

113 ± 25

1c

37 ± 12

307 ± 9

1m

9a

19 ± 9

1d

38 ± 15

413 ± 127

2a

n.d.

2611 ± 1503

1e

32 ± 18

154 ± 23

3a

n.d.

>10000

1f

41 ± 29

666 ± 195

3b

n.d.

>10000

1g

35 ± 8

159 ± 117

3c

n.d.

>10000

1h

n.d.

8.6 ± 2.9

4a

n.d.

>10000

1i

42 ± 31

1572 ± 105

4b

n.d.

>10000

1j

32 ± 23

302 ± 104

ibrutinib

35 ± 15

78 ± 21

Cpd

R

asingle

Cpd

R

measurement; n.d. = not determined.


-


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To correlate these results with the formation of a covalent bond, we incubated these compounds with MKK7 and performed subsequent mass spectrometry to identify a new, larger molecular ion species (Figure S1). For the compounds 1a–1j, 1l–1m, we were able to obtain a complete labeling of the kinase with the corresponding inhibitor. For the inhibitors 1f– and 2a, we observed a partial labeling, corresponding to the reduced inhibitory potency of the compounds. As expected, compounds 3a–3c showed no binding due to the absence of the reactive warhead. The compounds 4a and 4b showed partial labelling. Additionally, we tested the influence of incubation time and of competition with ATP on covalent bond formation (Figure 2). The analysis showed a pronounced time dependency of covalent bond formation for compound 1b and full labelling of the kinase was achieved after 20 min of incubation. We performed the identical analysis in the presence of 500 µM ATP which revealed, as expected, a marked impact on the covalent bond formation. The high concentration of ATP in the solution blocks the ATP-binding pocket and hampers inhibitor binding, so that a complete labeling was only achieved after 60 min. This result offers a possible explanation for the observed poor activity of the reversible and the covalent-reversible compounds 3–4. For the covalent compounds, activity could be detected in the ADP-Glo assay system because of the pre-incubation time of 30 min before adding ATP. The preincubation time gave enough time to form the covalent bond, preventing any competition between the inhibitor and the co-factor ATP for the binding site.


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Figure 2. Time dependent mass spectrometry analysis of compound 1b incubated with MKK7. Spectra colored in black were recorded in the absence of ATP and spectra in red in the presence of 500 µM ATP.

To gain further insight into the binding mode of these compounds, we obtained complex crystal structures and were able to solve the structures of MKK7 in complex with the compounds 1a, 1b, 1h, 1k and 1m by means of X-ray crystallography (Figure 3). These compounds showed a similar binding pose where the amine at the 4-position interacts with the carbonyl oxygen of Glu213 and the cyclic nitrogen at the 3-position interacts via a hydrogen bond with the amine of the backbone amide of Met215. Furthermore, clear electron density revealed covalent bond formation with Cys218 and also showed a hydrogen bond interaction between the carbonyl of the acrylamide and the sidechain of the Lys221. For compound 1b, we observed an interaction between a water molecule and the N7 of the scaffold (Figure 3B). This interaction was only found in this structure and may be caused by the iodide which led to a different configuration of the crystal water in near proximity to the ligand. Furthermore, compound 1k revealed an additional water-mediated hinge contact to the Gly216 backbone carbonyl (Figure 3D). Despite this result, the compound’s binding mode resembled one found previously in EGFR, however, ACS Paragon Plus Environment


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the flexible methionine gatekeeper side chain adopted a slightly different orientation (Figure 3F). For the two most potent compounds 1h and 1m, we found additional interactions, which were formed between Asp182 and the phenol group of compound 1h and with the N1 of the pyrrolopyridine moiety of compound 1m (Figure 3C and 3E). These interactions appear to be important for the increased inhibitory activity and should be incorporated in future MKK7 inhibitors.

Figure 3. Crystal structures of MKK7 covalently bound to the compounds 1a, 1b, 1h, 1k and 1m. The hinge region is displayed in white, the αC helix in blue and the DFG-triad (comprised of Asp277, Phe278 and Gly279) is displayed in green; 2Fo-Fc maps were contoured at an r.m.s.d. of 1. A) ACS Paragon Plus Environment

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compound 1a at a resolution of 2.7 Å (PDB: 6QHO), B) compound 1b at a resolution of 2.5 Å (PDB: 6QFT), C) compound 1h at a resolution of 2.3 Å (PDB: 6QG4), D) compound 1k at a resolution of 2.1 Å (PDB: 6QG7), E) compound 1m at a resolution of 2.5 Å (PDB: 6QHR), and F) alignment of MKK7 and EGFR-T790M bound with compound 1k (PDB: 6QG7, 5J9Y).

In addition to the generation of six complex crystal structures of MKK7, we were also able to obtain two apo structures (Figure 4), and interestingly, one of them showed an inactive-like kinase conformation. An inactive conformation of MKK7 was recently described by Shraga et al.,19 who observed a DFG-out conformation of mutated MKK7-C218S. However, here we present wild-type MKK7 in the conformation defined by the DFG-motif being rotated outwards so that Asp277 pointed towards the glycine-rich loop and the Phe278 pointed toward the binding site of the triphosphate (Figure 4A). This conformation represents a semi-inactive state of the kinase; catalytic activity is not possible in this conformation. Also, it would not allow binding of sterically demanding compounds because Asp277 blocks the pocket before Asp182. Interestingly, this conformation was provoked by the addition of a bulky type II kinase inhibitor AD8020, 21 during crystallization which did not, however, bind to the kinase (IC50 >20 µM; Figure S2). In the DFG-in conformation we observed that the hinge region formed a small turn that was termed the ‘Leu-in’ conformation and did not allow ligand binding to the hinge region (Figure 4B). This turn, provoked by a peptide flip of the backbone between Met215 and Leu214, is not seen in the DFG-out conformation and could not be detected once a compound became bound to the kinase.



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Figure 4. Crystal structures of apo-MKK7. The hinge region is displayed in white, the αC helix in blue and the DFG-motif is displayed in green; 2Fo-Fc maps were contoured at an r.m.s.d. of 1. A) MKK7 with a DFG-out conformation at a resolution of 2.3 Å (PDB: 6QFR) and B) MKK7 with a DFG-in/Leuin conformation at a resolution of 2.2 Å (PDB: 6QFL). The QR-codes provide an augmented reality view of 3D models of the binding sites.22

DISCUSSION AND CONCLUSIONS We were able to identify a set of potent covalent inhibitors of MKK7 that are based on the pyrazolopyrimidine scaffold. A potent inhibitor of this kinase must bind covalently due to high competition with the natural co-factor ATP. X-ray crystallography revealed a crucial interaction of the inhibitors with Asp182 within the αC helix to gain additional potency. Corresponding compounds provoked an inhibition of the kinase activity with low nanomolar IC50 values. Furthermore, we presented a DFG-out conformation of wild-type MKK7 and we observed a unique DFG-in conformation with a Leu-in state at the hinge region. The herein described molecules can be further developed to act as chemical probes and the identified protein–ligand


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interactions may guide the development of novel potent and selective inhibitors to investigate the functions of MKK7.

EXPERIMENTAL SECTION Protein Purification For the crystallization, a construct of MKK7 was used containing amino acids 117-423 with a non-cleavable N-terminal His6-Tag. For the assay, the construct contained amino acids 1-462 with a TEV-cleavable N-Terminal His6-Tag and 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 a French press and centrifuged. The supernatant was then loaded on a Ni-affinity chromatography (Qiagen NiNTA Superflow, 5 mL). The column was washed with buffer A (50 mM Tris, 500 mM NaCl, 25 mM imidazole, 5% glycerol, pH 8) and eluted with a linear 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 column (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 pyrazolopyrimidine-based inhibitors 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%


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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 XDS and scaled using XSCALE. Structure determination and refinement of the complex crystal structures were solved by molecular replacement with PHASER using structure 2DYL as template.23, 24 The MKK7 molecule in the asymmetric unit was manually adjusted using the program COOT.25 The refinement was performed with REFMAC5.23 Inhibitor topology files were generated using the Dundee PRODRG2 server. Refined structures were validated with PROCHECK and the PDB validation server. Data collection, structure refinement statistics, PDB ID codes, and further details for data collection are provided in Tables S2 and S3. PyMOL was used for generating the figures.26

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 series of the compound was generated with the Echo 520 (LabCyte) acoustic pipetting robot and the liquid handling was performed with a Multidrop dispenser (Thermo Scientific). The readout was performed with a Tecan M1000 Infinite plate reader.

Mass Spectrometry 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 used for the measurements. The MS spectra were recorded on a VelosPro IonTrap (Thermo Scientific) with an EC 50/3 Nucleodur C18 1.8 µm column (Macherey and


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Nagel) and a gradient of the mobile phase A (0.1% formic acid in water) to B (0.1% formic acid in acetonitrile). For visualization of the spectra, XCalibur and MagTran were used.

Synthesis All final compounds were purified to ≥95% purity confirmed by LC/MS analysis using a gradient of A (0.1% formic acid in water) to B (0.1% formic acid in acetonitrile). The synthesis of the compounds used was previously reported.10-18

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Supplementary Figures, MS-based analysis of covalent bond formation and crystal structure of apo-MKK7 with a DFG-out conformation with 2Fo-Fc map contoured at an r.m.s.d. of 0.1; Supplementary Tables, X-ray data collection and refinement statistics (PDF). Molecular formula strings, MFS (CSV). Accession Codes. PDB codes are 6QHO (MKK7/1a), 6QFT (MKK7/1b), 6QG4 (MKK7/1h), 6QG7 (MKK7/1k), 6QHR (MKK7/1m), 6QFR (Apo-MKK7, DFG-out), 6QFL (Apo-MKK7, DFG-in/Leu-in). Authors will release the atomic coordinates and experimental data upon article publication.


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AUTHOR INFORMATION Corresponding Author *Phone: +49 (0)231/755-7080, Twitter: @DDHDortmund, Web: DDHDortmund.de. eMail: [email protected] (J.L.), [email protected] (D.R.). Present Addresses †J.E.: Lead Discovery Center GmbH, Otto-Hahn-Strasse 15, 44227 Dortmund (Germany); S.S.: agap2 Frankfurt, Bockenheimer Landstraße 72, 60323 Frankfurt am Main (Germany); E.H.: Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany); J.L.: PearlRiver Bio GmbH, Otto-Hahn-Strasse 15, 44227 Dortmund (Germany). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources 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


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AMPPNP, Adenosine 5′-(β,γ-imido)triphosphate; BTK, Bruton’s tyrosine kinase; EGFR, epidermal growth factor receptor; JNK, c-JUN N-terminal Kinase; MKK7, mitogen activated protein kinase kinase 7; RASPELD, Robotics-Assisted Screening Platform for Efficient Ligand Discovery. REFERENCES 1.

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18. Wolle, P.; Hardick, J.; Cronin, S. J. F.; Engel, J.; Baumann, M.; Lategahn, J.; Penninger, J. M.; Rauh, D. Targeting the MKK7-JNK (mitogen-activated protein kinase kinase 7-c-Jun Nterminal kinase) pathway with covalent inhibitors. J. Med. Chem. 2019, 62, 2843-2848. 19. 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. 20. Dar, A. C.; Das, T. K.; Shokat, K. M.; Cagan, R. L. Chemical genetic discovery of targets and anti-targets for cancer polypharmacology. Nature 2012, 486, 80-84. 21. Plenker, D.; Riedel, M.; Brägelmann, J.; Dammert, M. A.; Chauhan, R.; Knowles, P. P.; Lorenz, C.; Keul, M.; Bührmann, M.; Pagel, O.; Tischler, V.; Scheel, A. H.; Schutte, D.; Song, Y.; Stark, J.; Mrugalla, F.; Alber, Y.; Richters, A.; Engel, J.; Leenders, F.; Heuckmann, J. M.; Wolf, J.; Diebold, J.; Pall, G.; Peifer, M.; Aerts, M.; Gevaert, K.; Zahedi, R. P.; Buettner, R.; Shokat, K. M.; McDonald, N. Q.; Kast, S. M.; Gautschi, O.; Thomas, R. K.; Sos, M. L. Drugging the catalytically inactive state of RET kinase in RET-rearranged tumors. Sci. Transl. Med. 2017, 9(394), eaah6144. 22. 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. 23. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 1997, 53, 240-255.


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24. Kabsch, W. Automatic indexing of rotation diffraction patterns. J. Appl. Cryst. 1988, 21, 67-71. 25. Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 2004, 60, 2126-2132. 26. DeLano, W. L. The PyMOL Molecular Graphics System.


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Figure 1. A) Focused screen of EGFR-targeted inhibitors against MKK7 at a concentration of 50 µM, results grouped by scaffold. B) Binding pose of an AMPPNP (Adenosine 5′-(β,γ-imido)triphosphate) molecule in the ATP-binding site of EGFR (yellow, PDB: 3VJN) aligned with ibrutinib-bound EGFR (green, PDB: 5YU9) which exhibits a similar binding site as compared to MKK7. 177x77mm (300 x 300 DPI)



Figure 2. Time dependent mass spectrometry analysis of compound 1b incubated with MKK7. Spectra colored in black were recorded in the absence of ATP and spectra in red in the presence of 500 µM ATP. 83x79mm (300 x 300 DPI)


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Figure 3. Crystal structures of MKK7 covalently bound to the compounds 1a, 1b, 1h, 1k and 1m. The hinge region is displayed in white, the αC helix in blue and the DFG-triad (comprised of Asp277, Phe278 and Gly279) is displayed in green; 2Fo-Fc maps were contoured at an r.m.s.d. of 1. A) compound 1a at a resolution of 2.7 Å (PDB: 6QHO), B) compound 1b at a resolution of 2.5 Å (PDB: 6QFT), C) compound 1h at a resolution of 2.3 Å (PDB: 6QG4), D) compound 1k at a resolution of 2.1 Å (PDB: 6QG7), E) compound 1m at a resolution of 2.5 Å (PDB: 6QHR), and F) alignment of MKK7 and EGFR-T790M bound with compound 1k (PDB: 6QG7, 5J9Y). 177x152mm (300 x 300 DPI)



Figure 4. Crystal structures of apo-MKK7. The hinge region is displayed in white, the αC helix in blue and the DFG-motif is dis-played in green; 2Fo-Fc maps were contoured at an r.m.s.d. of 1. A) MKK7 with a DFG-out conformation at a resolution of 2.3 Å (PDB: 6QFR) and B) MKK7 with a DFG-in/Leu-in conformation at a resolution of 2.2 Å (PDB: 6QFL). The QR-codes provide an augmented reality view of 3D models of the binding sites.22 162x77mm (300 x 300 DPI)


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Table of Contents Figure 53x54mm (300 x 300 DPI)


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