Discovery of a Class of Novel Tankyrase Inhibitors that Bind to Both

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Brief Article

Discovery of a Class of Novel Tankyrase Inhibitors that Bind to Both the Nicotinamide Pocket and the Induced Pocket Howard Bregman, Hakan Gunaydin, Yan Gu, Steve Schneider, Cindy Wilson, Erin F DiMauro, and Xin Huang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301607v • Publication Date (Web): 14 Jan 2013 Downloaded from http://pubs.acs.org on January 16, 2013

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Journal of Medicinal Chemistry

Discovery of a Class of Novel Tankyrase Inhibitors that Bind to Both the Nicotinamide Pocket and the Induced Pocket Howard Bregman#1, Hakan Gunaydin#2, Yan Gu2, Steve Schneider2, Cindy Wilson3, Erin F. DiMauro1, Xin Huang*2 1

Department of Medicinal Chemistry, 2Department of Molecular Structure and Characterization, 3Therapeutic Innovation Unit, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, U.S.A. ABSTRACT: Potent and selective inhibitors of tankyrases have recently been characterized to bind to an induced pocket.

Here we report the identification of a novel potent and selective tankyrase inhibitor that binds to both the nicotinamide pocket and the induced pocket. The crystal structure of human TNKS1 in complex with this “dual-binder” provides a molecular basis for their strong and specific interactions and suggests clues for the further development of tankyrase inhibitors.

Two different classes of potent and selective small molecule tankyrase inhibitors, inhibitors of Wnt response The two highly homologous human tankyrase isoforms, (IWRs) with IWR1 (1) and IWR2 (2) as examples and TNKS1 and TNKS2, are members of the poly ADP-ribose XAV939 (3), have been identified.6-7 1 inhibits TNKS1 and polymerase (PARP) family of 17 proteins that share a cataTNKS2 with IC50 of 131 nM and 56 nM, respectively, but lytic PARP domain.1 These PARP proteins cleave NAD+ does not inhibit PARP1 or PARP2 up to a concentration of into ADP-ribose and nicotinamide and transfer the ADP18.75 µM;6 2 is equipotent to 1 in a cellular luciferaseribose units onto their substrates, resulting in a postbased reporter assay. 3 was originally developed as a translational modification referred to as poly-ADP riboPARP1/2 inhibitor, albeit a weak one with IC50 of 2.2 µM sylation (PARsylation). The cellular functions of many and 0.11 µM for PARP1 and PARP2, respectively, and it PARP proteins remain unknown. was recently reported to be a more potent inhibitor of PARP1 and PARP2, the two best characterized family TNKS1 and TNKS2 with IC50 of 11 nM and 4 nM, respecmembers, are key players in homologous recombination tively.6 As expected, 3 binds to the nicotinamide pocket of DNA damage response and have been pursued as cancer TNKS1 and TNKS2 through interactions similar to those targets for over a decade.2 A few PARP1/2 inhibitors such observed in other PARP inhibitor complexes (Figure 1B),8 as ABT-888 and AZD2281 (Figure 1) have shown promismaintaining the three aforementioned, conserved hydrogen ing results in clinical trials.3 They contain functional bonds with a serine hydroxyl, as well as the oxygen and groups that resemble nicotinamide. Structural studies of NH from a glycine main chain. In these crystal structures, PARP inhibitor complexes reveal that these compounds are the cyclic amide of 3 mimics the binding motif of ABTanchored in the nicotinamide pocket in a very similar man888’s primary amide. There is also a stacking interaction ner.4 Using ABT-888 as a representative example, the carbetween the pyrimidinone of 3 and the Tyr1224 side chain boxamide oxygen forms hydrogen bonds with both the side of TNKS1 (or the Tyr1071 side chain of TNKS2). IWR chain hydroxyl of Ser470 and the main chain NH of compounds, however, do not share these features for anGly429 in PARP2, while one of the hydrogens on the prichoring in the nicotinamide pocket. The crystal structures mary amide forms a hydrogen bond with the main chain of the TNKS1/2 complex9 and the TNKS2/1 complex10 oxygen of Gly429 in PARP2. In addition, the imidazole of reveal that these IWR compounds do not bind to the nicoABT-888 stacks with the side chain of Tyr472 of PARP2. tinamide pocket but instead occupy a different pocket, Recently, tankyrases have gained increased attention as which is not present in either apo or 3 bound tankyrase potential drug targets. They were first discovered as factors structures. This induced pocket only becomes available that regulate telomere homeostasis by modifying the negaupon the binding of IWR compounds and it was created by tive regulator of telomere length, TRF1.5 Recently axin, the the movement of Phe1188 of the α3 helix and the D-loop away from one another. Another class of tankyrase inhibiconcentration-limiting component of the β-catenin destructors that bind to the same induced pocket in a very similar tion complex, was identified as a substrate for the tankyway have also been recently identified.11 rases. Tankyrase catalyzed PARsylation marks axin for Although ligand binding to the induced pocket appears to degradation, supporting Wnt pathway dysregulation. close down the nicotinamide pocket,9-11 we hypothesized Tankyrase inhibition antagonizes the Wnt signal transducthat one could extend the induced pocket binding tankyrase tion pathway by stabilizing axin and promoting β-catenin inhibitors into the nicotinamide pocket.9 Herein, we predegradation.6 Therefore, inhibition of tankyrase activity sent the discovery of such a class of novel tankyrase inhibimay be a promising strategy for the treatment of cancer. tors that bind to both the nicotinamide pocket and the ACS Paragon Plus Environment Introduction


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O NH 2 O O P O

O

O

HN

NH N

O

N

AZD2281 O

O NH 2

HO

HN

OH

N

N

O

NH

N

F

ABT-888

O

NH 2

O

N

P O

GLY429 (PARP2)

SER470 (PARP2) OH

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N

O O

N

N

H O

SER1221 (TNKS1) OH

O

N HO

OH

H O

GLY1185 (TNKS1)

HN O

HN

O N

R

NH

S N

NAD+ R = H IWR1/ 1 R = Me IWR2/2

XAV939/ 3

CF 3

Figure 1. Chemical structures of NAD+, ABT-888, AZD2281, XAV939, IWR1, and IWR2 and the binding modes of ABT888 and XAV939 to PARP2 and TNKS1 respectively. The nicotinamide in NAD+ and the nicotinamide-mimetics in ABT888, AZD2281, and XAV939 are highlighted in red. induced pocket. Results and Discussion As shown in Figure 2, induced pocket binding tankyrase inhibitors such as 2 bind to tankyrases through three hydrogen bonds. One of the two carbonyl oxygens of the pyrrolidine dione group is hydrogen bonded to the main chain NH of Tyr1213 and the carbonyl oxygen of the amide group is hydrogen bonded to the main chain NH of Asp1198. The

CH at the 6-position of the quinoline also forms a CH…O=C hydrogen bond with the main chain carbonyl oxygen of Gly1196. Moreover, the quinoline group in 2 engages in a hydrophobic interaction with the side chain of Phe1188 and a stacking interaction with the side chain of His1201 of the D-loop. These hydrogen bond interactions and the hydrophobic interactions are crucial for 2 to bind to the induced pocket of tankyrase and have also been observed for other induced pocket binders.11

Figure 2. Identification of 4 from substructure search using the binding motif derived from the crystal structure of the TNKS1/2 complex (PDB entry 4DVI).

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This induced pocket binding motif derived from the TNKS1/2 co-crystal structure was used in a substructure search against a compound library and about 1000 diverse compounds were identified (Figure 2). These compounds were further characterized in the enzymatic TNKS1 autoparsylation assay and compound 4 was found to be the most potent among them with an IC50 of 8 nM (Table 1). Interestingly, 4 appeared to possess not only the binding motif for the induced pocket but also the binding motif for the nicotinamide pocket. Table 1 Compound activity in TNKS1/2 and PARP2 biochemical assays IC50 (µM) TNKS1 TNKS2 PARP2 Wnt Pathway 0.151 0.038 > 25 136 1 0.192 0.036 n.d. 163 2 0.068 0.017 n.d. 272 3 0.008 0.002 0.931 36 4 In order to determine the binding mode of 4 to tankyrase, we co-crystallized the TNKS1/4 complex. These crystals diffracted to 2.4 Å with synchrotron radiation. There are four crystallographically independent TNKS1/4 complexes in the crystal structure, highly similar to each other (with a backbone rmsd of 0.6 Å). The TNKS1/4 complex structure reveals that 4 does indeed bind to the nicotinamide pocket in addition to the induced pocket (Figure 3). A.

B

Figure 3. (A) Crystal Structure of 4 (in purple) bound to TNKS1 superimposed with 2 (in green; PDB entry 4DVI) and 3 (in yellow; PDB entry 3UH4) and the binding mode of 4 to TNKS1. (B) 4 binds to the nicotinamide pocket through three hydrogen bonds and to the induced pocket through another three hydrogen bonds. There are also hydrophobic interactions between 4 and TNKS1 (His1201 and Phe1188 for example). The crystal structure of TNKS1/4 complex is very similar to that of TNKS1/2 complex (with a backbone rmsd of 0.8 Å) except for two major differences (Figure 2 and 3). The side chain of Tyr1224 in TNKS1, conserved in all PARP family members and critical for stacking with nicotinamide and nicotinamide mimicking PARP inhibitors, rotates ~50⁰ to interact with 2 bound in the induced pocket in the crystal structure of TNKS1/2 complex.9 This rotation closes the nicotinamide binding site and could prevent binding of the nicotinamide to the now smaller cavity (Figure 2). In the crystal structure of the TNKS1/4 complex, the Tyr1224 side chain rotates back and opens up, thereby enabling the interaction of the nicotinamide pocket with the quinazolinone of 4 (Figure 3). The other major difference is the orientation of the central phenyl of the inhibitors. 2 adopts an energically less favorable conformation in the crystal structure of TNKS1/2 complex in which the central phenyl is almost perpendicular to the norbornyl group but rotated by about 60⁰ away from the plane of the amide group.9 In the crystal structure of TNKS1/4, the central phenyl in 4, on the other hand, is co-planar to the two amides, which is a much more stable conformation. Structure comparison reveals that 4 overlays very well with both 3 and 2 with the quinazolinone of 4 occupying the nicotinamide pocket and the rest of the molecule occupying the induced pocket (Figure 3). Reminiscent of 3, the carbonyl oxygen of the quinazolinone in 4 is hydrogen bonded to the side chain hydroxyl of Ser1221 and the main chain NH of Gly1185 in TNKS1 while the NH of the quinazoline is hydrogen bonded to the main chain oxygen of Gly1185. The quinazolinone ring is also parallel to the Tyr1224 side chain forming a π–π stacking interaction with Tyr1224. Similar to 2, there are three more hydrogen bonds between



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the remaining molecule and TNKS1: one between one amide oxygen and the main chain NH of Tyr1213, one between the other amide oxygen and the main chain NH of Asp1198, and one CH…O=C hydrogen bond between the methoxy phenyl and the main chain oxygen of Gly1196. The methoxy phenyl group in 4, as the quinoline group in 2, also engages in a hydrophobic interaction with the side chain of Phe1188 and a stacking interaction with the side chain of His1201 of the D-loop. Inhibitors that bind to both the nicotinamide pocket and the induced pocket of tankyrases may possess advantages in terms of chemical space, potency and selectivity. Since the nicotinamide pocket has been well explored for designing PARP inhibitors, it may be challenging to identify novel chemotypes that bind solely to the nicotinamide pocket for the inhibition of tankyrases. While a new class of tankyrase inhibitors that bind to the previously unknown induced pocket such as IWRs have been reported recently, compound 4, identified here in a simple substructure search, represents a unique class of novel tankyrase inhibitors that bind to both the nicotinamide pocket and the induced pocket and it is likely that other dual binding chemotypes may also be discovered with more comprehensive approaches such as high throughput screening.12 These tankyrase inhibitors interact with both the nicotinamide pocket and the induced pocket and thus should have greater inhibitory activity than those that bind to either the nicotinamide or the induced pocket alone. Consistent with this analysis, 4 is about 18 fold more potent than 1 and about 8 fold more potent than 3 in the TNKS1 enzyme assay. Furthermore, 4 is found to be a much more potent Wnt pathway inhibitor than 1 and 3 in a cell-based assay (Table 1). Despite the high conservation of the nicotinamide pocket among all PARP family members, these dual-pocket binding tankyrase inhibitors should also maintain the same good selectivity over other PARP proteins as the induced pocket binding tankyrase inhibitors since the residues forming the induced pocket of tankyrase are much more variable among other PARP family members. Indeed, the selectivity of 1 and 4 for TNKS1 over PARP2 is greater than 100 fold (Table 1), whereas the selectivity of 3 is only 10 fold.6 The TNKS1/4 complex structure and molecular modeling analysis suggest a number of distinct routes to further optimize compound 4. While the sulfur atom in the thiopyrano group of 3 interacts with Phe1061 of TNKS2,8b the equivalent phenyl of 4 is not involved in any interaction with TNKS1 and could be replaced with a thiopyrano moiety to potentially improve potency. Replacement of the central phenyl in 1 with a trans-cyclohexyl moiety improves the wnt inhibition potency by 6 fold13 and similar modifications of the central phenyl group in 4 may also generate compounds with more favorable binding geometries and enhanced interaction with tankyrase. The methoxy phenyl of 4 also does not appear to be optimal for stacking with the His1201 side chain and could be modified as well. Preliminary metabolic stability studies indicated enzymatic cleavage of the amide bond in 1 to be the primary clearance mechanism for IWRs14 and it is likely that the two amides in 4 would present similar metabolic liability. The TNKS1/4 crystal structure suggests that the two amides can

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be replaced by other more stable moieties that maintain the same hydrogen bonding interactions. In summary, we have identified and characterized the first of a new class of novel tankyrase inhibitors that bind to both the nicotinamide pocket and the induced pocket. The crystal structure of the inhibitor bound to TNKS1 provides insights into the molecular basis for the key interactions between these dual binding inhibitors and tankyrases and will be an important tool towards improving the activity and pharmacokinetic properties of dual pocket binding tankyrase inhibitors. Materials and Methods Human TNKS1 (1091-1325) with an N-terminal His6 tag, TNKS1 (1104-1314) with a C-terminal His6 tag, and TNKS2 (946-1162) with an N-terminal His6 tag were expressed and purified as previously described.9 Compounds 1, 2, 3, and 4 were purchased commercially. Their purity was reported to be > 98% by vendors and confirmed by us using Agilent 1100 Series HPLC system (Agilent Zorbax Eclipse XDB-C8 4.6 x 150 mm, 5 micron, 5-100% CH3CN in H2O with 0.1% TFA for 15 min at 1.5 mL/min). 1030 compounds were identified from a substructure search against a compound library with ISIS/BASE using the induced pocket binding motif derived from the TNKS1/2 cocrystal structure (Figure 2). These compounds were then characterized in the enzymatic TNKS1 autoparsylation assay. TNKS1 biochemical activity was assayed using an assay buffer (50 mM MOPS pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.01% Tween-20, 0.05% BSA, 1 mM DTT) as follows: 0.25 nM of His6TNKS1(1091-1325) is incubated in the presence of compound (DMSO 1.85% final) in a Perkin Elmer white 384 well Proxiplate PlusTM with 400 nM of NAD for 60 minutes at room temperature. The assay was stopped in the dark with 0.6 µM inhibitor and the following detection components: 0.05 µg/mL monoclonal anti-PAR antibody (Trevigen) pre-bound for 60 minutes with 0.63 µg/mL protein G AlphaLISA® acceptor bead and 5 µg/mL AlphaLISA® nickel chelate donor bead (Perkin Elmer). The microplate was covered with ThermowellTM Sealing Tape (Costar) and incubated 16 hours at room temperature and read on a Perkin Elmer Envision® multi label reader using the default program set with laser excitation at 680 nm and emission at 615 nm. IC50 values were calculated from Percent of Control (POC) values according to the following formula: ((experimental-LO)/(HI-LO))*100 with the absence of inhibitor as high control (HI) and the presence of 3uM compound 3 as the low (LO) control. The most potent compound from the TNKS1 biochemical assay, along with compounds 1, 2 and 3, was further characterized in enzymatic TNKS2 and PARP2 assays as well as a cellular Wnt3a-induced Super-Top Flush (STF) transcriptional assay. The TNKS2 biochemical assay is as described above except 4 nM His6TNKS2 (946-1162) and 250 nM NAD was used. The PARP2 biochemical assay was purchased as a kit from BPS. The Wnt3a-induced STF assay in HEK293 cells was previously described.7 The TNKS1/4 complex was obtained by incubating TNKS1(1104-1314)His6 at 10 mg/ml with 4 in 2-fold mo-


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lar excess for 30 minutes at 4 ⁰C. Crystals of TNKS1/4 were obtained at 4 ⁰C in hanging drops by mixing 0.5 µL of TNKS1/4 complex with 0.5 µL of well solution containing 100 mM sodium acetate pH 4.6, 0.2 M ammonium acetate, 30% PEG4000. Crystals appeared overnight and grew to maximum size in a few days. These crystals belong to the spacegroup P21212 with unit cell parameters of a=158.6, b=77.1, c=84.5 Å. Paratone-N mineral oil was used as cryo protectant and diffraction data were collected on beamline 5.0.1 at the Advanced Light Source (ALS), Berkeley, CA and processed with HKL2000. The TNKS1/2 complex structure was solved by molecular replacement with AMoRe using the TNKS1/2 structure (4DVI) as the template. Model building was carried out with QUANTA and refinement was done using CNX.

ASSOCIATED CONTENT Supporting Information

Details on data processing and refinement statistics. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

Coordinates for the TNKS1&4 structure have been deposited in the Protein Data Bank with access codes 4I9I.

AUTHOR INFORMATION Corresponding Author

*Corresponding Author (Tel: 617-444-5045; Fax: 617-5779511; Email: [email protected]) Author Contributions #

These authors contributed equally to this work

ACKNOWLEDGMENT We are grateful to Drs. Paul Rose, Doug Whittington, and Nigel Walker for critical review of the manuscript.

ABBREVIATIONS TNKS, tankyrase; PARP, poly ADP-ribose polymerase; melting temperature; IWRs, inhibitors of Wnt response;

REFERENCES 1. Schreiber, V.; Dantzer, F.; Ame, J. C.; de Murcia, G., Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol 2006, 7 (7), 517-528. 2. Martin, S. A.; Lord, C. J.; Ashworth, A., DNA repair deficiency as a therapeutic target in cancer. Curr Opin Genet Dev 2008, 18 (1), 80-86. 3. (a) Fong, P. C.; Boss, D. S.; Yap, T. A.; Tutt, A.; Wu, P.; Mergui-Roelvink, M.; Mortimer, P.; Swaisland, H.; Lau, A.; O'Connor, M. J.; Ashworth, A.; Carmichael, J.; Kaye, S. B.; Schellens, J. H.; de Bono, J. S., Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 2009, 361 (2), 123-134; (b) Rouleau, M.; Patel, A.; Hendzel, M. J.; Kaufmann, S. H.; Poirier, G. G., PARP inhibition: PARP1 and beyond. Nat Rev Cancer 2010, 10 (4), 293-301.

4. Karlberg, T.; Hammarstrom, M.; Schutz, P.; Svensson, L.; Schuler, H., Crystal structure of the catalytic domain of human PARP2 in complex with PARP inhibitor ABT-888. Biochemistry 2010, 49 (6), 1056-1058. 5. Smith, S.; Giriat, I.; Schmitt, A.; de Lange, T., Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Science 1998, 282 (5393), 1484-1487. 6. Huang, S. M.; Mishina, Y. M.; Liu, S.; Cheung, A.; Stegmeier, F.; Michaud, G. A.; Charlat, O.; Wiellette, E.; Zhang, Y.; Wiessner, S.; Hild, M.; Shi, X.; Wilson, C. J.; Mickanin, C.; Myer, V.; Fazal, A.; Tomlinson, R.; Serluca, F.; Shao, W.; Cheng, H.; Shultz, M.; Rau, C.; Schirle, M.; Schlegl, J.; Ghidelli, S.; Fawell, S.; Lu, C.; Curtis, D.; Kirschner, M. W.; Lengauer, C.; Finan, P. M.; Tallarico, J. A.; Bouwmeester, T.; Porter, J. A.; Bauer, A.; Cong, F., Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 2009, 461 (7264), 614-620. 7. Chen, B.; Dodge, M. E.; Tang, W.; Lu, J.; Ma, Z.; Fan, C. W.; Wei, S.; Hao, W.; Kilgore, J.; Williams, N. S.; Roth, M. G.; Amatruda, J. F.; Chen, C.; Lum, L., Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat Chem Biol 2009, 5 (2), 100-107. 8. (a) Kirby, C. A.; Cheung, A.; Fazal, A.; Shultz, M. D.; Stams, T., Structure of human tankyrase 1 in complex with small-molecule inhibitors PJ34 and XAV939. Acta Crystallogr Sect F Struct Biol Cryst Commun 2012, 68 (Pt 2), 115-118; (b) Karlberg, T.; Markova, N.; Johansson, I.; Hammarstrom, M.; Schutz, P.; Weigelt, J.; Schuler, H., Structural basis for the interaction between tankyrase-2 and a potent Wnt-signaling inhibitor. J Med Chem 2010, 53 (14), 5352-5355. 9. Gunaydin, H.; Gu, Y.; Huang, X., Novel Binding Mode of a Potent and Selective Tankyrase Inhibitor. PLoS One 2012, e33740 10. Narwal, M.; Venkannagari, H.; Lehtio, L., Structural basis of selective inhibition of human tankyrases. J Med Chem 2012, 55 (3), 1360-1367. 11. Shultz, M. D.; Kirby, C. A.; Stams, T.; Chin, D. N.; Blank, J.; Charlat, O.; Cheng, H.; Cheung, A.; Cong, F.; Feng, Y.; Fortin, P. D.; Hood, T.; Tyagi, V.; Xu, M.; Zhang, B.; Shao, W., [1,2,4]Triazol-3-ylsulfanylmethyl)-3phenyl-[1,2,4]oxadiazoles: Antagonists of the Wnt Pathway That Inhibit Tankyrases 1 and 2 via Novel Adenosine Pocket Binding. J Med Chem 2012, 55 (3), 1127-1136. 12. Narwal, M.; Fallarero, A.; Vuorela, P.; Lehtio, L., Homogeneous Screening Assay for Human Tankyrase. J Biomol Screen 2012. 13. Lanier, M.; Schade, D.; Willems, E.; Tsuda, M.; Spiering, S.; Kalisiak, J.; Mercola, M.; Cashman, J. R., Wnt inhibition correlates with human embryonic stem cell cardiomyogenesis: a structure-activity relationship study based on inhibitors for the wnt response. J Med Chem 2012, 55 (2), 697-708. 14. Lu, J.; Ma, Z.; Hsieh, J. C.; Fan, C. W.; Chen, B.; Longgood, J. C.; Williams, N. S.; Amatruda, J. F.; Lum, L.; Chen, C., Structure-activity relationship studies of smallmolecule inhibitors of Wnt response. Bioorg Med Chem Lett 2009, 19 (14), 3825-3827.



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Table S1. Data Collection and refinement statistics for TNKS1/4 structure TNKS1/4 Space group Unit cell a, b, c (Å) Mol/ASU

P21212 158.62, 77.12, 84.51 4

Wavelength (Å)

1.0000

Resolution (Å)

2.40

Rmerge (%)

6.6 (36.0)

I/σI

18.0 (3.6)

Reflections (total/unique) Completeness (%)

166776/39973 96.6 (90.6)

Protein atoms

6711

Inhibitor atoms

172

Water molecules

175

Rwork/Rfree (%)

25.0/28.6

Rms deviations Bond length (Å)

0.013

Bond Angle (⁰)

1.94

Values in parentheses are for the highest resolution shell.


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Table of Contents Graphic


Discovery of a Class of Novel Tankyrase Inhibitors that Bind to Both

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