Screening and Structural Analysis of Flavones Inhibiting Tankyrases

Article pubs.acs.org/jmc

Screening and Structural Analysis of Flavones Inhibiting Tankyrases Mohit Narwal,†,‡ Teemu Haikarainen,† Adyary Fallarero,‡ Pia M. Vuorela,‡ and Lari Lehtiö*,† †

Biocenter Oulu and Department of Biochemistry, University of Oulu, Oulu, Finland Pharmaceutical Sciences, Department of Biosciences, Abo Akademi University, Turku, Finland

‡

S Supporting Information *

ABSTRACT: Flavonoids are known for their beneficial effects on human health, and therefore the therapeutic potential of these compounds have been extensively studied. Flavone has been previously identified as a tankyrase inhibitor, and to further elucidate whether tankyrases would be inhibited by other flavonoids, we performed a systematic screening of tankyrase 2 inhibitory activity using 500 natural and naturally derived flavonoids covering nine different flavonoid classes. All identified tankyrase inhibitors were flavones. We report crystal structures of all the hit compounds in complex with the catalytic domain of human tankyrase 2. Flavone derivatives in all 10 crystal structures bind to the nicotinamide binding site of tankyrase 2. Potencies of the active flavones toward tankyrases vary between 50 nM and 1.1 μM, and flavones show up to 200-fold selectivity for tankyrases over ARTD1. The molecular details of the interactions revealed by cocrystal structures efficiently describe the properties of potent flavone derivatives inhibiting tankyrases.

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INTRODUCTION Tankyrases are members of the poly(ADP-ribose) polymerase (PARP) family (EC 2.4.2.30), recently renamed as diphtheria toxin-like ADP-ribosyltransferase (ARTD) family.1 ARTDs covalently modify acceptor proteins by transferring an ADPribose moiety to glutamate or lysine residues of acceptor proteins (Figure 1). Tankyrases 1 and 2 (TNKS1/PARP5a/ ARTD5 and TNKS2/PARP5b/ARTD6, respectively) are multidomain proteins consisting of four domains: histidine proline serine rich domain (only present in TNKS1), ankyrin repeats, sterile α motif (SAM), and a catalytic ART domain. The overall sequence identity between TNKS1 (1327 amino acids) and TNKS2 (1166 amino acids) is 82%, and sequence identity of the catalytic domain is 89%. The catalytic domain cleaves NAD+ to nicotinamide and ADP-ribose, which is covalently attached to an acceptor protein or to a growing ADP-ribose polymer (Figure 1). Tankyrases are present at mitotic centrosomes, golgi apparatus, cytoplasm, and nuclear pore complexes.2−7 Tankyrases have functional redundancies as the knockout of both genes in mice is embryonically lethal, but inactivation of either one alone does not lead to obvious changes in the phenotype.8 Tankyrases are attractive drug targets for cancer. Their anticancer therapeutic potential is related to the functions of tankyrases in telomere homeostasis, in mitosis, and in Wnt signaling.9 In normal cells, telomeres shorten during each cell division. This shortening signals cells to cease division and regulates cellular senescence. In cancer cells, stable telomere length is maintained through the up-regulation of telomerase, which adds TTAGGG repeats to the chromosome ends. Tankyrases regulate telomere lengths indirectly through TRF1 © 2013 American Chemical Society

(telomere repeat binding factor 1), which protects telomeres from telomerase by binding to telomeric DNA. ADPribosylation of TRF1 by TNKS1 inhibits the binding of TRF1 to telomeres, allowing access to telomerase. Accordingly, partial knockdown of TNKS1 leads to telomere shortening.10 Thus, tankyrase inhibition provides a viable strategy for cancer therapy, and accordingly the treatment of cancer cells in combination with telomerase and ARTD inhibitors leads to telomere shortening and cell death.11 In addition to telomere homeostasis, tankyrases appear to be crucial for mitosis. TNKS1 binds to NuMA, an essential protein for mitotic spindle assembly. TNKS1 localizes to spindle poles in complex with NuMA and PARsylates NuMA in vitro and in vivo.12 TNKS1 knockdown leads to defects in the assembly of bipolar spindles, and RNA interference of TNKS1 causes cancer cells to arrest in mitosis with multipolar spindles. This suggests that tankyrase inhibition could also be used to arrest the growth of cancer cells.13 Wnt signaling pathway is crucial for embryonic development and tissue homeostasis, and it is often overactivated in cancers. The pathway controls the proteolysis of transcriptional coactivator, β-catenin. In the absence of Wnt ligands, β-catenin is bound to the multiprotein destruction complex formed by adenomatous polyposis coli (APC), glycogen synthase kinase 3 β (GSK3β), and axis inhibition protein (Axin). Tankyrases regulate the destruction complex by ADP-ribosylating axin, the concentration-limiting component of the complex, leading to its degradation.14 This leads to the dissociation of β-catenin Received: December 20, 2012 Published: April 10, 2013 3507

dx.doi.org/10.1021/jm3018783 | J. Med. Chem. 2013, 56, 3507−3517

Journal of Medicinal Chemistry

Article

Figure 1. ADP-ribosylation reaction catalyzed by tankyrases. In the screening assay, the automodification reaction is used where the acceptor protein is also TNKS.

inhibitors. We also screened a large flavonoids library (containing 500 molecules) and characterized the potency and selectivity of the active ones. Additionally, we describe the inhibition mechanism of the top hit compounds at molecular level using protein X-ray crystallography, thus elucidating the binding of flavone derivatives to tankyrases.

from the destruction complex, nuclear translocation of βcatenin, and activation of Wnt signaling. The inhibition of tankyrases stabilizes Axin and stimulates the degradation of βcatenin deactivating the Wnt pathway.14 Compound 1 (flavone) has been identified as a tankyrase inhibitor with a yeast-based screening system.15 Recently, we optimized an activity-based biochemical screening assay for TNKS116 and performed a validatory screening for TNKS1 inhibition using a natural chemical collection. Compound 1 was also here identified as a hit (with a potency value of 325 nM) along with flavone derivatives 2 (apigenin) and 3 (luteolin), although of lower potency (3.1 and 2.4 μM, respectively).16 The lower potency compound 2 exhibited higher selectivity toward tankyrases in comparison to ARTD1,16 implying that some flavonoids could be very selective tankyrase inhibitors. Flavonoids contain a benzene ring typically condensed with pyran or pyrone ring that is attached to a phenyl group. Flavonoids are further divided into various classes, such as flavones, flavonols, flavonones, anthocyanidins, and flavanols.17 They are secondary metabolites that are widely present in plant sources18,19 and in our daily diet,20 and they are commonly associated to the health enhancing effects of fruits and vegetables.21 Among many beneficial activities, it has been claimed that flavonoids show antioxidant, antiviral, antiinflammatory, neuroprotective, and anticancer properties.22−25 Ultimately, their in vivo effects depend on their bioavailability. It is assumed that flavonoids are absorbed as aglycones after the hydrolysis of the glycosides along the digestive tract, but the absorption process can also be modulated upon the transformation by the colonic microbiota. This way, gut microbial metabolism seems to impact the systemic effects of flavonoids. Although the bioavailability of dietary flavonoids is highly variable between individuals, data suggests that in general, it is low.21 However, these concerns do not diminish the fact that flavonoids are excellent templates for chemical elaborations and are highly valuable as chemical tools to investigate the molecular mechanisms of biological processes. In particular, for anticancer effects, a wide array of beneficial effects has been described for flavonoids. For instance, they have been shown to be cytotoxic against a variety of tumor cell lines.26 Often, their specific molecular targets are not clearly defined but computational efforts have recently been made to identify them.27 Our previous results16 indicated that flavonoids could offer selective scaffolds for the inhibition of tankyrases. In this contribution, we describe an assay for screening TNKS2

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RESULTS Inhibitor Screening. 1 was first identified as TNKS1 inhibitor in a cell-based assay,15 and we also previously identified it as a potent inhibitor of TNKS1 from an in-house natural product library together with a lower potency flavones 2 and 3.16 Although 2 displayed lower potency than 1, it was found to be highly selective toward TNKS1 over ARTD1. This prompted us to screen a larger library of flavonoids (500 compounds) to evaluate the usefulness of the flavone scaffold for the selective inhibition of tankyrases, as well as to establish structure−activity relationships that could guide further drug discovery efforts. Recently, we showed that TNKS2 ART domain fragment is active and easier to produce in larger quantities than the recombinant TNKS1 used earlier in the screening assay.28 Therefore, we decided to establish an assay for inhibitor screening against TNKS2. TNKS1 assay was modified for TNKS2 ART domain, and the conditions were optimized in a similar fashion as in our previous contribution.16 The activity, as judged by the substrate consumption, was used to test the optimal pH for the reaction. Similarly to TNKS1, the optimum pH was found to be 5.5, although the activity was very similar across the whole pH range tested (pH 5−8). pH 7 was selected for further studies as it is close to the physiological pH. The best buffering agent based on substrate consumption was Bis−Tris propane, similar to our prior findings with TNKS1. The effects on TNKS2 activity of selected buffer reagents, such as various cations, reducing agents, and detergents, were also measured. On the basis of these results, 1 mM TCEP and 2 mM NiCl2 were added to the final assay buffer. The assay was found to tolerate DMSO up to 3% (v/v) without significant change in the enzymatic activity. The performance for screening was evaluated using several statistical parameters (Table 1). The assay has a high signal-to-noise ratio and the calculated Z′ factor was 0.72 indicating that the assay would be suitable for inhibitor screening campaigns.29 Compound concentration used in screening of the compound library was 1 μM. Hit limit was set to 60% activity to identify compounds exhibiting equal or higher potency when 3508



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in the micromolar range for tankyrases, but it showed a high selectivity (33-fold) toward tankyrases over ARTD1. Compound 5 showed even higher selectivity (over 200-fold) toward tankyrases than 2, while retaining similar potency toward tankyrases as 1. Compared to 1, no dramatic improvements in the potencies against tankyrases were detected, except for compound 10, which had IC50 of 0.052 μM for TNKS1 and 500-fold selectivity for TNKS1 over ARTD1 (Figure 3). Most of the compounds showed very similar inhibitory potencies against TNKS1 and TNKS2. Interestingly, despite that TNKS1 and TNKS2 have a highly conserved nicotinamide binding site,30,31 compounds 9 and 10 had higher potency for TNKS1 than for TNKS2 (Figure 3). Chemical Space Populated by the Flavones. We used a computational method (ChemGPS-NP) to visualize the chemical space occupied by the compounds in the used flavonoids library. This allows global investigation of chemical properties of the large set of compounds. On the basis of a principle component analysis with eight dimensions describing 2D physical−chemical parameters (e.g., size, shape, aromaticity, lipophilicity, and flexibility) for a reference set, ChemGPS-NP allows mapping new compounds into the chemical space.32,33 As presented in Figure 4a, flavonoids occupy positive and negative regions of the principal component describing the size (PC1), indicating the presence of both small and large molecules as the molecular weight varied from 221 to 1031 Da. The compounds in general are aromatic because all but two molecules are located in the positive region of PC2. The entire library spanned through polar and lipophilic regions, as indicated by the coverage of negative and positive regions of PC3, respectively. The calculated log P varied from −0.87 to 11.66. When the flexibility of the structures (PC4) was taken into account as the third dimension instead of lipophilicity (Figure 4b), it could be noticed that roughly an equal amount of flavonoids had rigid or flexible structures (negative and positive regions of PC4, respectively) and the number of rotatable bonds varied from 1 to 26. Thus, even though the flavonoid collection was inherently limited to a defined

Table 1. Assay Performance

a

parameters

values

S/B S/N Z′ well-to-well CV (max, %) well-to-well CV (min, %) plate-to-plate CV (%)a day-to-day CV (%)a

2.62 ± 0.30 15.18 ± 1.43 0.72 ± 0.03 2.77 ± 0.61 7.64 ± 1.01 5.28 4.39

calculated from Z′.

compared to 1 (Figure 2). Ten hits were identified, and these were tested again in triplicates at 1 μM, and from this

Figure 2. Screening of TimTec natural product library with TNKS2. Screening was performed at 1 μM compound concentration using a hit limit of 60% activity.

reconfirmation test, three compounds were identified as false hits. Thus, seven hits with a common flavone scaffold were confirmed as actives. Inhibitor Potencies and Selectivity. Inhibitory potencies against TNKS1, TNKS2, and ARTD1 were measured for the seven hit compounds along with the three original hits (1, 2, and 3), and results are presented in Figure 3. IC50 value of 2 is

Figure 3. Structures of flavone derivatives and their IC50 (μM) and pIC50 ± SE values against tankyrases and ARTD1. 3509



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Figure 4. Exploration of the chemical space occupied by the flavonoids library, using ChemGPS-NP. The results were obtained using the first four dimensions of ChemGPS-NP. The data can be interpreted as follows: size increases in the positive direction of PC1, aromaticity, and conjugationrelated properties increase in the positive direction of PC2, compounds are increasingly lipophilic in the positive direction of PC3, and they are more flexible in the positive direction of PC4. Blue dots correspond to the active compounds, while in red only the other flavones are shown. The rest of the library is indicated with gray dots.

asymmetric unit, the binding mode is the same in both protein monomers. There are differences between the monomers only in the organization of the ordered water molecules interacting with the hydroxyl groups at the surface of the protein. This is likely due to slightly different environment within the crystal. The interactions made by the lower potency flavones 2 and 3 with tankyrases differ clearly from the more potent compounds, even though the binding site of the compounds is the same. There are three additional hydroxyl groups in 2 (4′,5,7trihydroxyflavone) when compared to 1. All these three hydroxyls form hydrogen bonds with the protein atoms or with the ordered water molecules (Figure 5b). The hydroxyl group at position 7 makes hydrogen bonds with a water molecule and the carboxyl group of Glu1138 (distance 2.5 Å). 4′-Hydroxyl forms a hydrogen bond with an ordered water molecule, which is also interacting with Ala1049. The oxygen from the benzopyran-4-one at position 5 forms a nonoptimal hydrogen bond with Ser1068 hydroxyl. However, its 4-hydroxyl moves further away from the same serine when compared to 1 (from 2.83 to 3.41 Å), disrupting the hydrogen bond present in 1. Ser1068 exhibits two different conformations in the structure, implying structural flexibility induced by the binding of the inhibitor. 3 (3′,4′,5,7-tetrahydroxyflavone) has four additional hydroxyl groups attached to the flavone scaffold. 3′- and 4′-hydroxy groups both form hydrogen bonds with water molecules (Figure 5c). The hydroxyl groups at positions 5 and 7 make identical interactions as in 2. Also, Ser1068 assumes two conformations in the structure as in the case of 2. Compared to 1, TNKS2 complexes with 2 and 3 show the flexibility of the serine, and the compounds do not bind as deeply as 1 to the nicotinamide pocket but are translated 0.6 and 0.8 Å away from the Ser1068 in 2 and 3, respectively. All the new compounds identified from the screening (4− 10) show similar potency toward tankyrases as 1, and they also bind in a nearly identical manner to the pocket. Most of the hits (4−8) contain varying patterns of hydroxyl substitutions at positions 7, 3′, and 4′. Hydroxyl group at position 7 is found in 4 (3′,7-dihydroxyflavone), 5 (7-hydroxy, 4′-methoxyflavone),

chemical class, the screened collection was physico-chemically diverse. As a subset of the overall library, the flavones (red dots in Figure 4) behave in a similar way when compared to the rest of the library (gray dots). Active hits, on the other hand (blue dots in the figure), were confined to a small molecular weight region with high aromaticity, but the hits were both polar or apolar molecules (Figure 3). The two most potent and selective TNKS1 inhibitors (9 and 10) had the highest lipophilicity within the actives (Log P 3.11 and 3.48, respectively). Binding of the Flavones to TNKS2. We determined the crystal structures of TNKS2 in complex with the compounds 1−10. The structures were solved at 1.75−2.3 Å resolution (Table 2). It should be noted that the catalytic domains of TNKS1 and TNKS2 are highly homologous and all the residues around the substrate binding site are conserved.30 Flavones bind to the same cleft between two active site tyrosines (Tyr1060 and Tyr1071) as the nicotinamide part of the substrate NAD+ as well as most of the ARTD inhibitors developed to date. All the compounds were clearly defined in the crystal structures. There is a π−π stacking especially with the pyran ring of 1 and the aromatic side chain of Tyr1071 (Figure 5a). All the compounds studied here contain the flavone scaffold and the interactions made by 1, and TNKS2 are shared between all the hit compounds 4−10, while the lower potency flavones, 2 and 3, identified in the earlier study, differ slightly in their binding mode. For 1 and the hit compounds, the oxygen from the benzopyran-4-one at position 4 makes hydrogen bonds with Ser1068 hydroxyl and the main chain amide of Gly1032 (Figure 5a). Although flavones have no amide at the position 3 opposed to typical ARTD inhibitors,34 the carbon at position 3 in the benzopyran-4-one ring is located within hydrogen bonding distance from the main chain carbonyl of Gly1032. The other molecules studied here contain various substitutions in the flavone scaffold, and they make various additional interactions with the protein and with the water molecules. There are one or two protein molecules in the asymmetric unit of the crystal depending on the space group, and for clarity only the first monomer will be described here unless otherwise stated. When there are two molecules in the 3510


reflections Rwork/Rfree no. of atoms protein ligand Zn PEG GOL SO4 water B-factors protein ligand Zn PEG GOL SO4 water rmsd bond lengths (Å) bond angles (deg)

3511

47.7 32.3

44.5 33.8 0.012 1.49

0.012

1.56

1.60

0.013

30.7 29.9 28.9

28.2 22.6 25.8 49.8

15 83

20 246

21.4 14.4 21.7 50.5 29.0 38.1 22.9

1692 21 1

3372 40 2 14

3368 34 2 14 12 20 211

16753 0.19/0.23

36975 0.18/0.22

27679 0.19/0.23

1.53

0.012

17.1 13.9 19.1 30.9 35.7 31.5 13.8

1669 19 1 7 6 10 39

12517 0.21/0.26

67.3, 67.3, 117.01 58−2.30 2.36−2.30 0.15 (0.35) 8.63 (2.64) 99.7 (99.6) 3.5 (2.7)

93.8, 95.2, 118.0 20−1.95 2.00−1.95 0.062 (0.61) 14.35 (2.15) 99.5 (99.9) 3.7 (3.7)

93.2, 98.9, 120.0 44−2.15 2.21−2.15 0.11 (0.56) 12.11 (2.19) 99.2 (99.7) 3.7 (3.7)

68.1, 68.7, 120.2 44−2.05 2.10−2.05 0.082 (0.74) 16.04 (2.70) 99.7 (99.9) 7.1 (3.7)

Bruker Microstar 1.54058 P41212

ESRF ID23−2 0.87260 P41212

ESRF ID23−2 0.87260 C2221

ESRF ID14−1 0.93340 C2221

beamline wavelength (Å) space group cell dimensions a, b, c (Å) resolution (Å) (resolution) (Å) Rmerge I/σI completeness (%) redundancy

4 4HMH

3 4HKN

2 4HKK

4HKI

PDB code

1

Table 2. Data Collection and Refinement Statistics for the Crystal Structures

1.41

0.011

41.8 26.9

26.0 25.9 23.2 39.1

10 78

1669 20 1 7

67.1, 67.1, 117.2 47−2.20 2.25−2.20 0.16 (0.82) 9.19 (2.06) 99.6 (94.9) 7.6 (7.5) Refinement 13548 0.21/0.24

4HL5 Data ESRF ID23−2 0.87260 P41212

5

1.52

0.012

20.8 18.6 18.7 46.0 15.6 34.6 26.6

3361 40 2 14 12 20 300

27821 0.17/0.21

1.50

1.43

0.011

26.7 36.2 30.4

20.1 38.2 29.3 0.012

25.0 23.7 22.4

6 20 255

6 20 232 26.1 21.2 25.6

3358 38 2

37069 0.18/0.21

3380 36 2

33741 0.19/0.23

1.50

0.009

20.1 15.9 19.1 45.2 18.7 29.7 27.9

3372 36 2 7 6 25 338

50782 0.18/0.21

1.51

0.012

22.1 16.2 21.3 52.1 17.4 34.4 25.8

3363 36 2 7 6 20 239

33863 0.18/0.21

92.4, 98.4, 119.8 44−2.00 2.05−2.00 0.13 (0.84) 10.47 (1.88) 98.2 (78.1) 4.2 (4.2)

90.8, 98.1, 117.6 20−1.95 2.00−1.95 0.077 (0.70) 13.96 (2.31) 99.5 (97.9) 4.8 (4.8)

90.8, 98.1, 118.5 49−1.75 1.80−1.75 0.081 (0.79) 14.15 (2.20) 99.8 (98.4) 5.5 (4.8)

91.7, 97.6, 117.4 20−2.00 2.12−2.00 0.096 (0.69) 14.68 (2.48) 99.4 (98.1) 4.8 (4.8)

ESRF ID23−2 0.87260 C2221

ESRF ID14−4 0.93927 C2221

ESRF ID14−4 0.93927 C2221

ESRF ID14−4 0.93927 C2221

ESRF ID23−2 0.87260 C2221 91.2, 97.8, 118.9 49−2.15 2.21−2.15 0.11 (0.51) 11.05 (2.64) 99.7 (99.5) 4.2 (4.2)

4HLK

10

4HLH

9

4HLM

8

4HLG

7

4HLF

6

Journal of Medicinal Chemistry Article



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Interestingly, these compounds are the ones showing the best potency or the best selectivity toward tankyrases (Figure 3).

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DISCUSSION TNKS1 inhibition by 1 has been earlier described with a yeastbased screening method,15 and we also identified 1 as a TNKS1 inhibitor from a small natural compound library along with two other natural flavones, 2 and 3.16 This raised the questions whether flavonoids (and more specifically which ones) would inhibit tankyrases and what would be their inhibition mechanism. Here we have attempted to shed light into these questions by demonstrating that flavones are indeed the most potent tankyrase inhibitors from a nature-inspired, comprehensive, and chemically diverse flavonoid library. Derivatives of chalcones, flavanones, dihydroflavonols, flavonols, flavans, anthocyanins, isoflavonoids, and neoflavonoids did not significantly inhibit tankyrase (over 40%) when tested at 1 μM concentration. Screening was conducted with a recombinantly expressed TNKS2 fragment as we identified during our earlier structural work that the ART domain of TNKS2 alone is active.28 The main benefit of using TNKS2 ART domain is that the protein is very easy to express in Escherichia coli and purification can be carried out by two simple steps. This makes it feasible to screen large compound libraries despite the requirement of relatively high protein concentration used in the assay. The performance of the assay is good according to statistical analysis (Table 1). It should be noted that the catalytic domains of TNKS1 and TNKS2 are very similar and the sequence of the region where the compounds interact is identical in the ART domains used for structural studies.30 We were interested in identifying the best inhibitors from the library, and therefore the screening was done at low concentration (1 μM). Flavones do not contain a characteristic nicotinamide motif found in most ARTD inhibitors as the basic scaffold lacks an amide, a hydrogen bond donor typically thought to be required for binding to the nicotinamide binding site of the protein.34 However, the crystal structure of TNKS2 in complex with 1 (TNKS1/TNKS2 IC50: 0.32 μM/0.14 μM) revealed that the inhibitor binds to the same place as canonical ARTD inhibitors. Compound 1 stacks between two tyrosines and forms hydrogen bonds with the glycine and serine residues at the bottom of the cavity in a similar fashion as nicotinamide (Figure 6a). The amide of the nicotinamide also forms a hydrogen bond with the carbonyl of Gly1032. In the case of flavones, there is no traditional hydrogen bond in the corresponding position. Despite this, flavone and XAV939, a potent tankyrase inhibitor with a similar overall shape (TNKS1/TNKS2 IC50: 0.011 μM/ 0.004 μM), superpose very well when bound to TNKS2 (Figure 6b).31 The interaction between the carbon at position 3 and the glycine carbonyl is based on the partial charge distribution character of the hydrogen and the C3 carbon. The distance between C3 and the carbonyl oxygen is between 2.96 and 3.15 Å in the crystal structures, indicating a clear and defined interaction and explaining the good potency of flavones toward tankyrases. The potencies of 2 (TNKS1/TNKS2 IC50: 3.1 μM/2.9 μM) and 3 (TNKS1/TNKS2 IC50: 2.4 μM/1.1 μM) toward tankyrases are significantly lower when compared to the rest of the compounds. The additional hydroxyl at the benzopyran4-one at position 5 leads to the disruption of the hydrogen bond between 4-hydroxyl and Ser1068 hydroxyl and to the conformational flexibility of the residue. This explains the lower

Figure 5. Structures of TNKS2−inhibitor complexes. The view is identical in all panels. Tyr1060 was removed for clarity. Hydrogen bonds between the protein and the compounds and ordered water molecules are shown.

and 6 (3′,4′,7-trihydroxyflavone), and in all cases it forms a hydrogen bond with the catalytic Glu1138 (Figure 5d−f). Additional ordered water molecules were observed in complex crystal structures with compounds containing hydroxyl groups at positions 3′ and 4′. The additional hydroxyl group at position 3′ present in compounds 4, 6, 7 (3′-hydroxyflavone), and 8 (3′,4′-dihydroxyflavone) forms a hydrogen bond with a water molecule bridged between the compound, His1031 and Ser1033 (Figure 5d,f−h). The hydroxyl group at position 4′ causes a variable pattern in the surrounding water molecules, which is also not always identical in both protein monomers in the crystal structures (Figure 5c,f,h). The density for the water molecule bound to 3′- and 4′-hydroxyls of 8 in monomer B in the asymmetric unit is not as clear and well-defined as in the monomer A (Figure 5h). Three of the hit compounds also contained other type of substitutions at 4′ position. In 5, there is a methoxy group, in 9 a fluorine, and in 10 a methyl substituent. These substituents mainly appear to form hydrophobic interactions with Pro1034 and Phe1035 in the vicinity of the substituent (Figure 5e,i,j). 3512



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(Figure 3). Unexpected exceptions to this are 9 (TNKS1/ TNKS2 IC50: 0.27 μM/1.1 μM) and 10 (TNKS1/TNKS2 IC50: 0.052 μM/1.1 μM), which are more potent TNKS1 inhibitors. As the catalytic domains are nearly identical, current structural data does not provide an explanation for this discrepancy. Our current view is that the SAM domain, responsible for the multimerization of tankyrases,35 may interact with the binding site in some fashion and enhance the hydrophobic interactions of the methyl and fluoryl substitutions at the 4′-position, potentially through Phe1035 and Pro1034. The additional domains may also affect protein flexibility affecting the binding of the inhibitor. It is also possible that parts of the adjacent catalytic domains in a multimeric tankyrase would affect this binding site, but further structural studies are required to elucidate this. It is, however, an observation that needs to be taken into account when analyzing and comparing compound interactions with the catalytic fragments of ARTDs. Compounds 2 and 5 show the highest selectivity (33-fold and 200-fold, respectively) for tankyrases over ARTD1. Compound 5 contains a methoxy substituent, which would extend toward the regulatory domain in ARTD1. Glu763 and Asp766, especially, would come close to the methoxy group and interfere with the binding of the compound (Figure 6b). In 2, the 4′ substituent is a hydroxyl group. As compounds 3, 6, and 8 also have 4′ hydroxyl-substituents (Figure 3), we cannot explain the selectivity of 2 from current structural data. Potentially, the distortion caused by hydroxyl at position 5 in 2 is tolerated better in tankyrases than ARTD1. This could then be compensated in 3 (ARTD1 IC50: 4.2 μM) by additional interactions of 3′-hydroxyl in ARTD1. Comparison of the crystal structures would not directly support this hypothesis as the 3′-hydroxyl is interacting with His1031 and Ser1033 via a water molecule in TNKS2 (Figure 5c,d,f,g,h), and these residues are conserved in ARTD1 (Figure 6c). Flavonoids have been found to have many effects at the cellular level, and importantly they have also been described as antiproliferative agents.26,36 However, it is not always clear what is the cellular target of the compounds. Tankyrases are potential drug targets, especially toward cancer,37 suggesting that some of the reported effects of flavones might be due to inhibition of tankyrases. Although flavones in general show selectivity to tankyrases over ARTD1, it cannot be ruled out that the effects could also come from the inhibition of other ARTDs, or other intracellular targets. Experimental evidence suggests that flavonoids have pleiotropic effects and modulate signal transduction pathways at various stage of carcinogenesis.38 Distribution of the hit compounds in the chemical space makes it possible to better understand the factors influencing the inhibition of tankyrases by flavones. On the basis of our analysis, the flavones inhibiting tankyrases would be small and aromatic (Figure 7). Small substitutions, such as hydroxylation, observed in many natural compounds are well tolerated (Figure 7). Analysis of the compounds not identified as hits, but clustering together with the hit compounds revealed that they are typically flavone derivatives. The compounds show some inhibition and are close to the hit limit in the screening (Supporting Information Table 1) or they contain substitutions that make them less potent tankyrase inhibitors. The crystal structures reported here provide molecular details of the interactions of TNKS2 with flavones, and although the compounds clustering close to the hits contain multiple substitutions, the unfavorable effect of these can be explained by the crystal structures, such as the substitutions at positions 5,

Figure 6. Superposition of the binding sites of nicotinamide and XAV939 with flavones and potential interactions with ARTD1. (a) Comparison of nicotinamide (orange, PDB 3U9H) and 1 (blue) binding to TNKS2 (gray and magenta, respectively). Hydrogen bonds are shown in black and gray dotted lines for 1 and nicotinamide, respectively. (b) Comparison of 1 with XAV939 (orange, PDB 3KR8) bound to TNKS2 (gray and magenta, respectively).31 Hydrogen bonds are shown in black and gray dotted lines for 1 and XAV939, respectively. (c) Superposition of TNKS2 1 complex and ARTD1 (pink). Conserved residues between TNKS2 and ARTD1 as well as ARTD regulatory domain (ARD) are shown. Labeling is according to ARTD1 sequence numbers.

potency of these two compounds. Apart from the hydroxyl residues in the positions 4 and 5 in the benzopyran-4-one, which lower the potency toward tankyrases, other hydroxyl substituents interacting with the catalytic glutamate and water molecules do not appear to have a high impact on the potency of the compounds. As expected on the basis of the sequence and structural similarities of TNKS1 and TNKS2, potencies of the hit compounds are comparable between TNKS1 and TNKS2 3513



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Figure 7. Structure−activity relationship of the identified inhibitors. Analysis is based on the chemical space analysis and the cocrystal structures with TNKS2. All compounds inhibiting tankyrases are shown as observed in the superposed crystal structures. Chemical Space Analysis. The chemical space occupied by the screened library was studied using the principal component analysisbased chemical space navigation tool ChemGPS-NP,32,33 which is freely available online (http://www.chemgps.bmc.uu.se/) and particularly suited for the exploration of the space occupied by compounds of natural origin.32 This analysis is made from 2D descriptors (total of 35) that describe physical−chemical properties of the compounds and are calculated from SMILES. SMILES notations were obtained from the commercial supplier (TimTec). If errors were encountered by ChemGPS-NP, the skeletal 2D structures of the compounds were manually drawn using ChemSketch v.12.01 (Advanced Chemistry Development Inc., ACD Laboratories, 2010) and converted to canonical smiles. Analysis was focused on the first four dimensions of ChemGPS-NP (PC1 to PC4). Visualization of the chemical space was done in a two-dimensional or a tridimensional space, and graphs were constructed using Grapher for OSX (version 10.4). To identify the nonactive compounds populating similar chemical space as the hits, euclidean distances (ED) were computed between 1−10 and the entire flavonoid collection. EDs were calculated between points P = (p1, p2,..., p8) and Q = (q1, q2,..., q8) in Euclidean eight-dimensional space provided by the ChemGPS-NP coordinates, as follows: ED = √((p1−q1)2 + (p2−q2)2 +...+ (p8−q8)2). Averages of the EDs toward compounds 1−10 were computed, and close compounds were defined as those having an average of EDs lower than 1.1. Protein Production. Recombinant human ARTD1 was purchased from Trevigen. Expression constructs of the catalytic fragment of human TNKS1 consisting of SAM and ART domain (1030−1317) and TNKS2 ART domain (946−1161) were a generous gift from Structural Genomics Consortium (Stockholm, Sweden). The proteins were cloned in pNIC28-Bsa4 plasmid, which contains an N-terminal 6xHis-tag followed by a TEV protease cleavage site (MHHHHHHSSGVDLGTENLYFQ*SM) before the tankyrase sequence. TNKS2 protein was expressed in E. coli Rosetta2 (DE3) cells in Terrific Broth autoinduction media containing trace elements (ForMedium) supplemented with 8 g/L glycerol and antibiotics (34 μg/mL chloramphenicol and 50 μg/mL kanamycin). Cultures were kept in an incubator shaker at 37 °C/200 rpm until culture turbidity, i.e., OD600 reached 1. Then, the cultures were moved to an incubator set at 18 °C/180 rpm and incubated overnight. The cells were collected by centrifugation (4 °C, 5020g, 40 min), resuspended in lysis buffer (100 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 10 mM imidazole and 0.5 mM TCEP), and stored at −20 °C. Expression and purification of TNKS1 was done as previously reported.16

6, or 2′. A hydroxyl group at position 5 leads to lower potency as observed for 2 and 3, substitutions at position 6 may not fit to the defined nicotinamide binding pocket, and substitutions at 2′ would not allow the planar conformation of the compounds observed in the crystal structures. Our results, therefore, provide molecular details of tankyrase inhibitors and can be used to predict inhibition and identify potential routes to enhance the activity of flavones and related compounds toward tankyrases.

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CONCLUSIONS We described here an activity-based assay for screening of inhibitors of TNKS2. The assay was used to screen a commercial flavonoid library, and we identified seven potent tankyrase inhibitors. The inhibitory potencies were compared between TNKS1, TNKS2, and ARTD1, which allowed us to rationalize which modifications would make inhibitors more selective toward tankyrases. The study was complemented by 10 cocrystal structures of the compounds with the catalytic domain of human TNKS2, clearly defining the molecular mechanism of tankyrase inhibition by flavones. Modifications in the 4′ position could provide additional interactions and increase the potency as seen for 10. The chemical space analysis and structural studies made it possible to assess which kind of flavones would inhibit tankyrases and offer potential explanations for some of the cellular effects of the compounds.

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EXPERIMENTAL SECTION

Chemical Collection. A commercial flavonoids compound library with 500 compounds (www.timtec.net) was used. The collection consists of both naturally occurring and synthetic flavonoid derivatives with nine different basic cores (flavanones, flavones, chalcones, flavonols, dihydroflavonols, flavans, anthocyanins, isoflavonoids, and neoflavonoids). The list of tested compounds is presented in Supporting Information Table 1. The average molecular weight of the compounds in the collection was 342.8 g/mol, and the average of predicted log P and log S were 3.49 and −4.46, respectively, as provided by the supplier. Identity of the compounds was confirmed by NMR (300 MHz or higher) and LC/MS, while purity of all the tested compounds was measured by the suppliers using HPLC and ensured to be over 95%. Compounds 1, 2, and 3 used in this study were purchased from Sigma-Aldrich. 3514



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Purification of TNKS2. The cell suspension was supplemented with 2 mM TCEP, approximately 0.2 mg lysozyme, 250 U benzonase (Sigma-Aldrich), and an EDTA-free protease inhibitor tablet (Roche). Cell suspension was sonicated 6 × 20 s using MSE Sonirep 150 (Sanyo). Solution was cleared by centrifugation (31000g, 40 min, 4 °C), and supernatant was filtered through 0.45 μm filters. Cleared lysate was loaded on a HisTrap HP column (GE Healthcare), which was pre-equilibrated with binding buffer (30 mM HEPES pH 7.5, 500 mM NaCl, 10% (w/v) glycerol, 10 mM imidazole, 0.5 mM TCEP) at 4 °C. After loading the lysate, the column was washed with wash buffer (30 mM HEPES pH 7.5, 500 mM NaCl, 10% (w/v) glycerol, 25 mM imidazole, 0.5 mM TCEP) and protein was eluted with the same buffer containing 250 mM imidazole. Protein was further purified with gel filtration. The column (HiLoad 16/600 Superdex 75 prep grade) was pre-equilibrated with gel filtration buffer (30 mM HEPES pH 7.5, 500 mM NaCl, 10% (w/v) glycerol, 0.5 mM TCEP). Fractions containing TNKS2 were analyzed with SDS-PAGE and tested for activity. The protein was frozen in liquid nitrogen immediately after purification and stored in aliquots in −70 °C. Homogenous Activity Assay. TNKS2 assay was modified from our previously reported TNKS1 assay.16 The enzyme modifies, ADPribosylates, itself through automodification and in the process consumes NAD+. The amount of the leftover substrate was measured by converting the NAD+ to a fluorophore. Heating of the plate during the chemical reaction was found not be necessary, making the assay simpler. Briefly, the enzymatic reaction was carried out at room temperature in 96-well plates (Greiner bio-one U-shaped black plates). Reaction was stopped by adding 20 μL of 20% acetophenone in ethanol and 2 M KOH. Plate was incubated for 10 min at room temperature, and 90 μL of 100% formic acid was added. Plates were further incubated for 20 min before reading with Fluoroskan Ascent FL (Labsystems) using an excitation wavelength of 355 nm and emission wavelength of 460 nm. Assay conditions were optimized by testing the effect of pH and different reagents to the protein activity. Final optimal buffer used in the screening contained 50 mM BisTris propane pH 7, 1 mM TCEP, and 2 mM NiCl2. Substrate (NAD+) was included at 500 nM concentration. Performance of the assay was tested by setting the plates with maximal and minimal signal wells. Maximal signal was NAD+ and buffer solution, and minimal signal was NAD+ and protein solution. Assay performance was evaluated following the signal-to-noise (S/N) and Z′ factor. S/N and Z′ values are calculated using the following equations:

(Sartorius Stedim Biotech). Protein concentration was measured using a NanoDrop (Thermo Fisher Scientific Inc.) using the extinction coefficient 0.972 calculated from the amino acid sequence (http:// web.expasy.org/protparam/). Protein preparations were divided to small aliquots, flash frozen in liquid nitrogen, and stored at −70 °C. TNKS2 was crystallized using sitting drop vapor diffusion method. Equal amounts of protein solution (5.8 mg/mL) and well solution (0.2 M Li2SO4, 0.1 M Tris-HCl pH 8.5, 24−26% PEG 3350) were mixed, and the plate was incubated at 4 °C. Crystals appeared within a week and grew to a size of 50 × 50 × 50 μm3 in one week. The inhibitor complexes were prepared by soaking the crystals in well solution supplemented with 100 μM of the inhibitor and 250 mM NaCl for 24 h. Crystals were cryoprotected by soaking them quickly in well solution supplemented with 100 μM of the inhibitor, 250 mM NaCl, and 22% glycerol and flash frozen in liquid nitrogen for data collection. Data Collection, Processing, and Refinement. Diffraction data were collected at ESRF Grenoble on beamlines ID23−2, ID14−1, and ID14−4 and at the home source Bruker Microstar X-ray generator with PLATINUM135 CCD detector. XDS40 and Proteum2 suite (Bruker) were used for data processing. Space group of the crystals was either C2221 or P41212. Molecular replacement was done using Molrep.41 Nicotinamide bound and apo structures of TNKS2 (PDB: 3U9H and 3KR7) were used as templates. Refmac542 was used from CCP443 program suite for refinement and Coot44 was used for manual building of the model. Data and refinement statistics are shown in Table 2. CCP4mg45 was used for making structural figures, and Marvin was used for drawing chemical structures (Marvin 5.7.0, 2011, ChemAxon, http://www.chemaxon.com).

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* Supporting Information

Details of the compound library and the normalized enzymatic activity observed in the screening. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

Coordinates and structure factors are deposited to the Protein Data Bank with codes 4HMH, 4HLM, 4HLK, 4HLH, 4HLG, 4HLF, 4HL5, 4HKN, 4HKK, and 4HKI.

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AUTHOR INFORMATION

Corresponding Author

Z′ = 1 − (3SDcontrol + 3SDreaction )/(Xcontrol − X reaction) 2

ASSOCIATED CONTENT

S

*Phone: +358 2 9448 1169. E-mail: lari.lehtio@oulu.fi. Notes

2

S/N = (Xcontrol − X reaction)/√((SDcontrol ) + (SDreaction ) )

The authors declare no competing financial interest.

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where SD is standard deviation and X is average signal. Screening of Inhibitors. The flavonoids library was screened with TNKS2 catalytic domain using the optimized assay. Compounds were stored at 10 mM concentration in dry (100%) DMSO at −20 °C and were diluted in the assay buffer before using them in the reaction. Compounds were tested at final concentration of 1 μM in duplicates with the same batch of protein. Hits were retested in triplicates to confirm the screening results. Controls with inhibitors and NAD+ were used in all the reactions to correct for possible fluorescence of the compounds. Measurement of Inhibitor Potencies. Inhibitor potencies were measured for the hit compounds identified from the screening. Half log dilutions of inhibitors were used, and the reaction time was set to achieve less than 30% and 20% conversion with tankyrases and ARTD1, respectively. Controls with protein and inhibitor (tankyrases) and inhibitor only (ARTD1) were made for the compounds showing autofluorescence. Reactions were done in quadruplicates. IC50 curves were fitted using sigmoidal dose response curve (four variables) in GraphPad Prism version 5.04 for Windows (GraphPad Software). Assay buffer used with ARTD1 was 50 mM Tris, pH 8, 4 mM Mg2+, and 20 μg/mL activated DNA.39 Crystallization. For crystallization, chymotrypsin and 2 mM TCEP was added to the TNKS2 protein solution and the protein was concentrated to 5.8 mg/mL using VIVASPIN 20 concentrators

ACKNOWLEDGMENTS The work was funded by Biocenter Oulu and the Academy of Finland (grant no. 128322 to L.L.). M.N. is a member of the National Doctoral Programme of Informational and Structural Biology. A.F. and P.V acknowledge the support received from the Drug Discovery and Chemical Biology network of Biocenter Finland. We are grateful to local contacts at ESRF for providing assistance in using beamlines ID23-2, ID14-1, and ID14-4 and to University of Oulu and Biocenter Finland for the local data collection facility. We also thank Janne Isojärvi for technical assistance.

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ABBREVIATIONS USED APC, adenomatous polyposis coli; ART, ADP-ribosyltransferase; axin, axis inhibition protein; ED, Euclidean distance; GSK3β, glycogen synthase kinase 3 β; NAD+, nicotinamide adenine dinucleotide; PAR, poly(ADP-ribose); PARP, poly(ADP-ribose) polymerase; PC, principal component; SAM, sterile α motif; TNKS, tankyrase; TRF1, telomer repeat binding factor 1 3515



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(20) Benavente-García, O.; Castillo, J.; Marin, F. R.; Ortuño, A.; Del Río, J. A. Uses and Properties of Citrus Flavonoids. J. Agric. Food Chem. 1997, 45, 4505−4515. (21) Van Duynhoven, J.; Vaughan, E. E.; Jacobs, D. M.; Kemperman, R. A.; Van Velzen, E. J. J.; Gross, G.; Roger, L. C.; Possemiers, S.; Smilde, A. K.; Doré, J.; Westerhuis, J. A.; Van de Wiele, T. Metabolic fate of polyphenols in the human superorganism. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (Suppl 1), 4531−4538. (22) Middleton, E., Jr.; Kandaswami, C.; Theoharides, T. C. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 2000, 52, 673−751. (23) Galati, G.; Teng, S.; Moridani, M. Y.; Chan, T. S.; O’Brien, P. J. Cancer chemoprevention and apoptosis mechanisms induced by dietary polyphenolics. Drug Metabol. Drug Interact. 2000, 17, 311−349. (24) Yang, C. S.; Landau, J. M.; Huang, M. T.; Newmark, H. L. Inhibition of carcinogenesis by dietary polyphenolic compounds. Annu. Rev. Nutr. 2001, 21, 381−406. (25) Birt, D. F.; Hendrich, S.; Wang, W. Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacol. Ther. 2001, 90, 157−177. (26) Kandaswami, C.; Lee, L.-T.; Lee, P.-P. H.; Hwang, J.-J.; Ke, F.C.; Huang, Y.-T.; Lee, M.-T. The antitumor activities of flavonoids. In Vivo 2005, 19, 895−909. (27) Chen, H.; Yao, K.; Nadas, J.; Bode, A. M.; Malakhova, M.; Oi, N.; Li, H.; Lubet, R. A.; Dong, Z. Prediction of molecular targets of cancer preventing flavonoid compounds using computational methods. PLoS One 2012, 7, e38261. (28) Narwal, M.; Venkannagari, H.; Lehtiö, L. Structural basis of selective inhibition of human tankyrases. J. Med. Chem. 2012, 55, 1360−1367. (29) Zhang, J. H.; Chung, T. D.; Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J. Biomol. Screening 1999, 4, 67−73. (30) Lehtiö, L.; Collins, R.; Van den Berg, S.; Johansson, A.; Dahlgren, L.-G.; Hammarströ m, M.; Helleday, T.; HolmbergSchiavone, L.; Karlberg, T.; Weigelt, J. Zinc binding catalytic domain of human tankyrase 1. J. Mol. Biol. 2008, 379, 136−145. (31) Karlberg, T.; Markova, N.; Johansson, I.; Hammarström, M.; Schütz, P.; Weigelt, J.; Schüler, H. Structural basis for the interaction between tankyrase-2 and a potent Wnt-signaling inhibitor. J. Med. Chem. 2010, 53, 5352−5355. (32) Larsson, J.; Gottfries, J.; Muresan, S.; Backlund, A. ChemGPSNP: tuned for navigation in biologically relevant chemical space. J. Nat. Prod. 2007, 70, 789−794. (33) Rosén, J.; Lövgren, A.; Kogej, T.; Muresan, S.; Gottfries, J.; Backlund, A. ChemGPS-NP(Web): chemical space navigation online. J. Comput.-Aided Mol. Des. 2009, 23, 253−259. (34) Ferraris, D. V. Evolution of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors. From concept to clinic. J. Med. Chem. 2010, 53, 4561−4584. (35) De Rycker, M.; Price, C. M. Tankyrase polymerization is controlled by its sterile alpha motif and poly(ADP-ribose) polymerase domains. Mol. Cell. Ther. 2004, 24, 9802−9812. (36) Asensi, M.; Ortega, A.; Mena, S.; Feddi, F.; Estrela, J. M. Natural polyphenols in cancer therapy. Crit. Rev. Clin. Lab. Sci. 2011, 48, 197− 216. (37) Lau, T.; Chan, E.; Callow, M.; Waaler, J.; Boggs, J.; Blake, R. A.; Magnuson, S.; Sambrone, A.; Schutten, M.; Firestein, R.; Machon, O.; Korinek, V.; Choo, E.; Diaz, D.; Merchant, M.; Polakis, P.; Holsworth, D. D.; Krauss, S.; Costa, M. A Novel Tankyrase Small-molecule Inhibitor Suppresses APC Mutation-driven Colorectal Tumor Growth. Cancer Res. 2013, DOI: 10.1158/0008-5472.CAN-12-4562. (38) Nishiumi, S.; Miyamoto, S.; Kawabata, K.; Ohnishi, K.; Mukai, R.; Murakami, A.; Ashida, H.; Terao, J. Dietary flavonoids as cancerpreventive and therapeutic biofactors. Front. Biosci. (Schol. Ed.) 2011, 3, 1332−1362. (39) Putt, K. S.; Hergenrother, P. J. An enzymatic assay for poly(ADP-ribose) polymerase-1 (PARP-1) via the chemical quantita-

REFERENCES

(1) Hottiger, M. O.; Hassa, P. O.; Lüscher, B.; Schüler, H.; KochNolte, F. Toward a unified nomenclature for mammalian ADPribosyltransferases. Trends Biochem. Sci. 2010, 35, 208−219. (2) Smith, S.; De Lange, T. Tankyrase promotes telomere elongation in human cells. Curr. Biol. 2000, 10, 1299−1302. (3) Chi, N. W.; Lodish, H. F. Tankyrase is a golgi-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles. J. Biol. Chem. 2000, 275, 38437−38444. (4) Kaminker, P. G.; Kim, S. H.; Taylor, R. D.; Zebarjadian, Y.; Funk, W. D.; Morin, G. B.; Yaswen, P.; Campisi, J. TANK2, a new TRF1associated poly(ADP-ribose) polymerase, causes rapid induction of cell death upon overexpression. J. Biol. Chem. 2001, 276, 35891− 35899. (5) Lyons, R. J.; Deane, R.; Lynch, D. K.; Ye, Z. S.; Sanderson, G. M.; Eyre, H. J.; Sutherland, G. R.; Daly, R. J. Identification of a novel human tankyrase through its interaction with the adaptor protein Grb14. J. Biol. Chem. 2001, 276, 17172−17180. (6) Cook, B. D.; Dynek, J. N.; Chang, W.; Shostak, G.; Smith, S. Role for the related poly(ADP-Ribose) polymerases tankyrase 1 and 2 at human telomeres. Mol. Cell. Biol. 2002, 22, 332−342. (7) Leung, A. K. L.; Vyas, S.; Rood, J. E.; Bhutkar, A.; Sharp, P. A.; Chang, P. Poly(ADP-ribose) regulates stress responses and microRNA activity in the cytoplasm. Mol. Cell 2011, 42, 489−499. (8) Chiang, Y. J.; Hsiao, S. J.; Yver, D.; Cushman, S. W.; Tessarollo, L.; Smith, S.; Hodes, R. J. Tankyrase 1 and tankyrase 2 are essential but redundant for mouse embryonic development. PLoS One 2008, 3, e2639. (9) Riffell, J. L.; Lord, C. J.; Ashworth, A. Tankyrase-targeted therapeutics: expanding opportunities in the PARP family. Nature Rev. Drug Discovery 2012, 11, 923−936. (10) Donigian, J. R.; De Lange, T. The role of the poly(ADP-ribose) polymerase tankyrase1 in telomere length control by the TRF1 component of the shelterin complex. J. Biol. Chem. 2007, 282, 22662− 22667. (11) Seimiya, H.; Muramatsu, Y.; Ohishi, T.; Tsuruo, T. Tankyrase 1 as a target for telomere-directed molecular cancer therapeutics. Cancer Cell 2005, 7, 25−37. (12) Chang, W.; Dynek, J. N.; Smith, S. NuMA is a major acceptor of poly(ADP-ribosyl)ation by tankyrase 1 in mitosis. Biochem. J. 2005, 391, 177−184. (13) Chang, P.; Coughlin, M.; Mitchison, T. J. Interaction between Poly(ADP-ribose) and NuMA contributes to mitotic spindle pole assembly. Mol. Biol. Cell 2009, 20, 4575−4585. (14) Huang, S.-M. A.; 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, 614−620. (15) Yashiroda, Y.; Okamoto, R.; Hatsugai, K.; Takemoto, Y.; Goshima, N.; Saito, T.; Hamamoto, M.; Sugimoto, Y.; Osada, H.; Seimiya, H.; Yoshida, M. A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor. Biochem. Biophys. Res. Commun. 2010, 394, 569−573. (16) Narwal, M.; Fallarero, A.; Vuorela, P.; Lehtiö, L. Homogeneous screening assay for human tankyrase. J. Biomol. Screening 2012, 17, 593−604. (17) Harborne, J. B.; Williams, C. A. Advances in flavonoid research since 1992. Phytochemistry 2000, 55, 481−504. (18) Aron, P. M.; Kennedy, J. A. Flavan-3-ols: nature, occurrence and biological activity. Mol. Nutr. Food Res. 2008, 52, 79−104. (19) Landete, J. M. Updated knowledge about polyphenols: functions, bioavailability, metabolism, and health. Crit. Rev. Food Sci. Nutr. 2012, 52, 936−948. 3516



Article

tion of NAD(+): application to the high-throughput screening of small molecules as potential inhibitors. Anal. Biochem. 2004, 326, 78−86. (40) Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125−132. (41) Vagin, A.; Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 1997, 30, 1022−1025. (42) Murshudov, G. N.; Skubák, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 355−367. (43) Dodson, E. J.; Winn, M.; Ralph, A. Collaborative Computational Project, number 4: providing programs for protein crystallography. Methods Enzymol. 1997, 277, 620−633. (44) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−2132. (45) McNicholas, S.; Potterton, E.; Wilson, K. S.; Noble, M. E. M. Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 386−394.

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Screening and Structural Analysis of Flavones Inhibiting Tankyrases

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