discovery of tankyrase inhibiting flavones with increased potency and isoenzyme selectivity

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Article

Discovery of Tankyrase Inhibiting Flavones withIncreased Potency and Isoenzyme Selectivity

Mohit Narwal, Jarkko Koivunen, Teemu Haikarainen, Ezeogo Obaji, Ongey ElvisLegala, Harikanth Venkannagari, Päivi Joensuu, Taina Pihlajaniemi , and Lari Lehtiö

J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Sep 2013

Downloaded from http://pubs.acs.org on September 26, 2013

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1

Discovery of Tankyrase Inhibiting Flavones with Increased Potency

and Isoenzyme Selectivity

Mohit Narwal1,2

, Jarkko Koivunen3, Teemu Haikarainen

1, Ezeogo Obaji

1, Ongey E. Legala

1,

Harikanth Venkannagari1, Päivi Joensuu

4, Taina Pihlajaniemi

3 & Lari Lehtiö

1,*

1Department of Biochemistry and Biocenter Oulu, University of Oulu, Oulu, Finland

2Pharmaceutical Sciences, Department of Biosciences, Abo Akademi University, Turku, Finland 3Center for Cell-Matrix Research and Biocenter Oulu, Department of Medical Biochemistry and Molecular

Biology, University of Oulu 4Department of Chemistry, University of Oulu, Oulu, Finland

*Corresponding author: Lari Lehtiö ([email protected])

Running title: Inhibition of tankyrase by flavone derivatives

ABSTRACT

Tankyrases are ADP-ribosyltransferases that play key roles in various cellular pathways, including

the regulation of cell proliferation, and thus they are promising drug targets for the treatment of

cancer. Flavones have been shown to inhibit tankyrases and we report here the discovery of more

potent and selective flavone derivatives. Commercially available flavones with single substitutions

were used for structure activity relationship studies, and co-crystal structures of the 18 hit

compounds were analyzed to explain their potency and selectivity. The most potent inhibitors were

also tested in a cell-based assay, which demonstrated that they effectively antagonize Wnt signaling.

To assess selectivity, they were further tested against a panel of homologous human ADP-

ribosyltransferases. The most effective compound, 22 (MN-64), showed 6 nM potency against

tankyrase 1, isoenzyme selectivity, and Wnt signaling inhibition. This work forms a basis for

rational development of flavones as tankyrase inhibitors and guides the development of other

structurally related inhibitors.

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INTRODUCTION

Tankyrases belong to the diphtheria toxin-like ADP-ribosyltransferase (ARTD) enzyme family also

known as poly (ADP-ribose) polymerases (PARPs) (EC 2.4.2.30). These proteins share a common

catalytic ART domain, which in the case of tankyrases post-translationally poly ADP-ribosylates

itself or acceptor proteins. Tankyrases (ARTD5/PARP-5a/TNKS1 and ARTD6/PARP-5b/TNKS2)

are highly homologous and have redundant functions in cells.1 TNKS1 and TNKS2 are

multidomain proteins containing a sterile alpha motif (SAM), an ankyrin repeat region, and a C-

terminal ART domain. The SAM domain is required for multimerization of tankyrases, and ankyrin

repeats are required for interactions with the target proteins. A HPS region, which contains

histidine, proline, and serine repeats, is present only in the N-terminus of TNKS1 and its function is

unknown. Tankyrases are mainly found in nuclear pore complexes, the cytoplasm, mitotic

centrosomes and the Golgi apparatus and are involved in many critical cellular functions.2–8 Many

of these functions make tankyrases potential drug targets.9,10 One promising approach in this aspect

is inhibition of the Wnt signaling pathway through tankyrase inhibition.11

Wnt signaling is essential and controls many biological processes. Notably, it has been found to be

overactivated in many cancers.12,13 In Wnt signaling, β–catenin proteolysis is controlled by the β–

catenin destruction complex. This destruction complex is comprised of multiple proteins:

adenomatous polyposis coli (APC), glycogen synthase kinase 3 α/β (GSK3α/β), casein kinase Iα

and Axin. The β–catenin destruction complex phosphorylates β–catenin and regulates its

proteolysis. Tankyrases destabilize the destruction complex by ADP-ribosylating Axin, which is a

concentration limiting factor of the complex.14,15 ADP-ribosylation targets Axin for degradation and

subsequently leads to accumulation of β–catenin. This makes it possible to prevent β–catenin

signaling through tankyrase inhibition.

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Recently we optimized an assay for screening tankyrase inhibitors16 and identified 1 (flavone) as a

potent hit against TNKS1 in accordance with an earlier report.16,17 Flavones, which belong to the

large group of flavonoids, have antioxidant properties and are present in wide variety of food items.

Flavonoids have also been shown to have antiproliferative properties in lung, prostate, colorectal,

pancreas, and ovarian cancer cells.18–22 Inhibition of TNKS1 with flavone and its antiproliferative

properties motivated us to screen a library of 500 naturally occurring flavonoids. This resulted, in

combination with protein X-ray crystallography, in an initial structure activity relationship analysis

of tankyrase inhibiting flavones.23 In order to further develop the potency and selectivity of the

flavone scaffold as a tankyrase inhibitor, we evaluated a collection of commercially available

synthetic flavones with single substitutions against TNKS1. We report here the resulting structure-

activity relationship study of these modified flavones. The biochemical potencies were measured,

potent hits were tested in a cell-based assay, and compounds inhibiting Wnt signaling were profiled

against other ARTD family members to gain insight into their target selectivity. Crystal structures of

the catalytic domain of TNKS2 in complex with all 18 new inhibitors were also solved. The results

revealed the modifications of the basic flavone core that improve the selectivity and potency of

flavones. This knowledge can also be applied to other inhibitors that bind to the nicotinamide

subsite of the tankyrase catalytic domain.

RESULTS

Identification of inhibitors

Flavone (1) has earlier been reported as a tankyrase inhibitor,16,17 and we have reported the binding

of flavone derivatives to TNKS2 elucidating the molecular details of the inhibition.23 Based on the

early results we selected commercially available compounds with single substitutions to the basic

scaffold. This makes it possible to pinpoint features leading to improved potency and isoenzyme

selectivity.

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The base compound 1 (2-phenyl-1-benzopyran-4-one) consists of a benzopyran ring attached to a

benzene ring (IC50 330 nM) (Table 1). We generated a library of commercially available compounds

having single substituents on the basic scaffold. Biochemical assay revealed that the modifications

at sites other than position 4´ abolished the inhibitory effect of the compound (Table 1) with some

exceptions. The introduction of a hydrophobic group or a halogen atom at position 3 in the

benzopyran ring (2-5) drastically lowered the potency (IC50 > 10 µM). Substitution at position 7 (6-

8) is also not favorable for inhibition. The introduction of a halogen atom at position 6 is tolerated,

but this does not improve the potency (9: IC50 210 nM, 10: IC50 595 nM). Hydrophobic

substitutions (11,12) led to only weak inhibition of TNKS1 (IC50 > 10 µM). A methoxy group at

position 2´ (13) in the phenyl ring completely abolished the inhibition (IC50 > 10 µM), whereas a

dioxolane ring connected to positions 3´ and 4´ of the phenyl group (14) results in an IC50 value of

360 nM, which is comparable to the base compound 1 (Table 1).

Substitutions to position 4´ were found to be most effective and in some cases led to significant

increases in the potency (Table 2). The introduction of halogen atoms (15-17) gave variable potency

depending on the halogen atom. Fluoride substitution (15) to position 4´ led to lower potency (IC50

700 nM), whereas, chloride (16: IC50 233 nM) and bromide (17: IC50 313 nM) substitutions did not

significantly affect the IC50 value in comparison to 1. Hydroxyl group (18) substitution and

carboxylate (19) were tolerated, but lowered the potency (IC50 788 nM and 850 nM, respectively).

Nitro group (20) substitution gave an improved IC50 value of 66 nM. Small hydrophobic groups

(21-22) increased the inhibition with IC50 values of 47 nM and 6 nM, respectively. The introduction

of dimethyl amine (23) or a methoxy group (24) improved the potency and gave similar IC50 values

of 67 nM and 71 nM, respectively. Ethyl methanoate (25) substitution did not improve the potency

significantly (IC50 272 nM). In comparison, a methyl methanoate group substitution (26) at position

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4´ has an improved potency (IC50 162 nM). Compound 27 has a larger methylpiperazine-1-carbonyl

substituent and is a better inhibitor of tankyrase 1 than flavone (IC50 146 nM). A cyano group (28)

substituent has a potency (IC50 145 nM) similar to that of flavone, but the introduction of

cyanomethyl acetate (29) improved the potency by two orders of magnitude (IC50 7 nM). Again a

larger tetrazol substitution (30) showed an IC50 value of 114 nM. Based on this analysis it can be

concluded that larger as well as hydrophobic substituents in the 4´ position improve the potency

against TNKS1, and these compounds were therefore included in further studies.

Inhibition of Wnt signaling

The most potent tankyrase inhibitors were tested in a cell-based assay using SuperTopFlash (STF)

construct, a firefly lusiferase TCF-reporter plasmid that responds to the Wnt/β-catenin pathway

activity. The IC50 value cut-off of 200 nM was used as the selection criterion for inclusion of the

compounds in the cell-based assay. The STF-plasmid was transiently transfected into HEK293 cells

and the Wnt/β-catenin pathway was activated with conditioned media from cells expressing Wnt3a.

Importantly, control conditioned medium had no significant effect on luciferase activity and the

compounds did not change the luciferase activity when control medium was used. Flavone

derivatives do not have a significant effect on β-galactosidase activity and accordingly, the

compounds do not alter HEK293 cell viability at the concentrations used.

Seven out of 10 compounds inhibited Wnt/β-catenin signaling at a concentration of 5 µM (Figure

1). Compound 21 displayed only 30% inhibition and notably 28, 29 and 30, although effective in

the enzymatic assay, did not show any inhibition of Wnt-signaling (Supplementary Figure 1). We

also did not observe any inhibition of Wnt-signaling in the STF assay by 1. This inefficacy of 1 has

also been reported earlier in a similar assay system.24

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The most effective compounds were also tested at lower concentrations and 20, 22, 23 and 27 were

effective Wnt/β-catenin inhibitors at a concentration of 1 µM (Figure 1). However, only one

compound, 22, inhibited STF luciferase activity at a concentration of 200 nM. Compound 22 is as

efficient as the known potent tankyrase inhibitors IWR1 and XAV939, which were used as control

compounds (Figure 1). The potency values measured XAV939 (5 nM) and IWR1 (64 nM) for

TNKS1, were in accordance with previously reported value in the literature (11 nM and 131 nM,

respectively).11 The results of the enzymatic assay, a single digit nanomolar potency (IC50 6 nM),

and efficacy in the STF-assay make 22 (MN-64) the most promising compound in the series.

Profiling of the best inhibitors

In order to assess selectivity, the compounds that inhibited TNKS1 both in vitro and in the STF-

assay were also profiled against several other human ARTD enzymes by using an activity assay

(Figure 2). From the previous studies, it was clear that the compounds will bind to the nicotinamide

pocket as do other flavones.23 This binding site is well conserved in other ARTD enzymes and

therefore we anticipated that there would be some off target effects. Overall, most of the compounds

did not inhibit other ARTD family members effectively at 1 µM concentration, which makes this a

suitable scaffold to achieve isoenzyme selectivity. The most potent compound, 22, was also the

most selective, as it did not significantly inhibit any other ARTDs.

To confirm the improvement in the selectivity of the hit compounds, IC50 values of the best

compounds 20, 21, 22 and a known tankyrase inhibitor XAV939 were measured for TNKS1,

TNKS2, ARTD1/PARP-1 and ARTD2/PARP-2 with the assay used here (Table 3). Flavones and

XAV939 resemble each other in shape (Figure 3) and have been shown to bind similarly to the

active site of tankyrases. This analysis revealed that compound 22 is indeed significantly more

selective towards tankyrases than XAV939 (Figure 2) (Table 3).

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Binding of inhibitors to TNKS2

The structure of 1 in complex with the TNKS2 catalytic domain has been solved earlier (PDB code

4HKI).23 All the structural work presented here was carried out using a crystal form of TNKS2,

which allows soaking of the compounds. It is therefore important to mention that TNKS1 and

TNKS2 catalytic domains are 94% identical to each other in sequence and all the residues are

conserved in the donor NAD+ binding site.25 1 binds to the nicotinamide binding site of TNKS2

(Figure 4a) and makes the typical interactions of ARTD inhibitors: π-π stacking with the active site

tyrosine Tyr1071 and hydrogen bonds formed between the oxygen from the benzopyran ring and

the main chain amide of Gly1032 and the hydroxyl of Ser1068 (Figure 4a). The exception to the

typical ARTD inhibitors is that in flavones there is no amide at position 3 to make a hydrogen bond

with the main chain carbonyl of Gly1032. However, the carbon at position 3 (C3) in the benzopyran

ring is within a hydrogen bonding distance of the main chain carbonyl of Gly1032. In all the

structures reported here, there is also a water molecule making a π-interaction with the phenyl

substituent (Figure 4).

Structural basis for inhibition

To analyze the inhibitor binding modes, we solved co-crystal structures of all the new 18 potent

inhibitors. The structures were solved at 1.7 Å – 2.3 Å resolutions (Supplementary Table 2). All

the compounds presented here have the same basic flavone scaffold and bind in a similar orientation

to the nicotinamide site. The compounds were clearly visible in the electron density maps except

10, which was present only in one protein monomer of the two monomers found in the asymmetric

unit. The substitutions in the analogs form additional interactions with the protein and with water

molecules, which explain the differences in potencies observed in the biochemical assays. The

potent compounds typically have substitutions at position 4´. The only other tolerated site analyzed

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here for substitutions is position 6 (Table 1 & 2), e.g. 9 has fluorine substituted at position 6 (IC50 of

210 nM). This fluorine atom does not make any hydrogen bonds with the protein molecule,

although there is a water molecule present at a distance of 4.9 Å (Figure 4b). The fluorine is also

close to the hydrophobic parts of the side chains of Glu1138 and Lys1067, and Ala1064 as well as

to the carbonyl group of Phe1061 (distances < 3.3 Å). Otherwise the interactions of 9 with protein

residues are similar to those of 1. There is enough space to accommodate the fluorine substitution at

this region of the active site cleft, which explains why 9 is able to inhibit tankyrases (Figure 4b).

Compound 10 has a chlorine atom in this position instead of a fluorine atom and the larger size of

the chorine atom is likely the reason for the lower potency of 9 (IC50 595 nM). In the crystal

structure the compound was found only in one monomer (Figure 4c). This result, however, does not

readily explain why a methyl derivative, 12, would be very inactive (IC50 > 10 µM) (Table 1).

Despite this, a comparison of the flavone binding mode and the chemical structures of the

compounds may explain why some of the compounds are inactive. Compounds 2-5 are not potent

inhibitors because there is no space to accommodate substitution at position 3 as this leads to a

clash with Gly1032 (Figure 4). This would also disrupt the hydrogen bonding between the oxygen

of the benzopyran ring and the main chain amide of Gly1032 and the hydroxyl group of Ser1068.

The introduction of functional groups like methoxy, oxyethanenitrile and oxypentanenitrile at

position 7 interfere with Glu1138, which explains the inactivity of compounds 6-8. Small

substituents may be tolerated here as we have shown previously.23

The introduction of a methoxy (11) or a methyl group (12) at position 6 is not favorable because

these substitutions will again interfere with Glu1138 and with the main chain of Phe1061. The

introduction of a methoxy group at position 6´ is not favorable as it would impinge on the side

chain of His1031 and on Gly1032 or Tyr1050. The conformation observed in the crystal structure

will also not be energetically favorable for the compounds having ortho-substitutions on the phenyl

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ring. Substitution of cyclic dioxolane to positions 4´ and 5´ is tolerated (14) as there is space in the

binding groove to accommodate this modification (Figure 4d).

The substitutions at position 4´ point away from the binding site towards the solvent, which

explains why substitutions here are generally well tolerated. The benefit of this point is also that the

substitutions extend towards a less conserved region of the active site potentially leading to better

selectivity.

The introduction of halogens at position 4´ gave variable potencies depending on the halogen atom.

A chlorine (16) or a bromine (17) atom increased the potency more than did a fluorine atom (15)

(Table 2) (Figure 5a,b,c). Chlorine and bromine atoms are larger than fluorine atoms and are

involved in nonelectrostatic interactions with the hydrophobic residues Pro1034 and Phe1035. This

is the reason for the better potency of 16 (IC50 233 nM) and 17 (IC50 313 nM) over 15 (IC50 700

nM). The hydroxyl (18) and carboxyl groups (19) are surrounded by the hydrophobic residues

Pro1034 and Phe1035, that contribute to their lower potency (18: IC50 788 nM and 19: IC50 850

nM) (Figure 5d,e). Compound 19 also forms hydrogen bonds to two water molecules. The water

molecule bound to Ala1049 and Ile1051 is also present in 20. This compound contains a nitro

group, which, despite a similar shape, is in a different orientation than the carboxylate in 19 (Figure

5e,f). The rotation brings the oxygen atoms from the nitro group close to the main chain carbonyl of

Ala1049 (Figure 5f).

Compound 21 (PDB code 4HLK)23 has a methyl and 22 has an isopropyl group at position 4´.

These compounds have IC50 values 1 or 2 orders of magnitude better than 1 (21: IC50 47 nM and 22:

IC50 6 nM). As the 4’ position faces the solvent, the increased potency of 21 and 22 results from the

efficient hydrophobic interactions with Pro1034 and Phe1035 (Figure 5g,h).

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The dimethyl amine substitution of 23 (IC50 47 nM) has the same shape as the carboxyl group in 19

and the nitro group in 20, but the dimethyl amine group is found in yet another orientation in the

crystal, likely driven by the more hydrophobic nature of the substituent (Figure 5i). Dimethyl

amine forms an additional hydrogen bond with a nearby water molecule further connected to two

other water molecules. One of the water molecules is hydrogen bonded to the main chain carbonyl

of Tyr1071, and the second water molecule forms a hydrogen bond with the main chain amide of

Ile1075 (Figure 4i). The binding of 23 also induces a change in the side chain of Phe1035, which is

rotated away from the compound (movement of 1.1 Å).

Compound 24 has a methoxy group at position 4´. The methoxy group is located in the vicinity of a

hydrophobic Phe1035 and close (3.2 Å) to the main chain carbonyl of Ala1049 (Figure 5j).

Compound 25 has an ethyl methanoate group substituted at position 4´. The ethyl group is located

between the hydrophobic residues, Pro1034 and Phe1035, and forms hydrophobic interactions

(Figure 5k). The carbonyl from the methanoate moiety is within the hydrogen bond distance (2.8

Å) from the main chain carbonyl of Ala1049. This unfavorable interaction appears to counteract the

hydrophobic interactions of the substituent (IC50 272 nM).

Compound 26 (IC50 162 nM) is also very similar to 25 as it contains a methyl methanoate group

substituted at position 4´. However, the orientation of the group is different in 26 (Figure 5l). The

carbonyl of the methyl methanoate group forms a hydrogen bond with a water molecule similar to

that observed for 19 and 20 (Figure 5e,f). The compound also forms a hydrophobic interaction with

Ile1075, which is, however, in a double conformation in this crystal structure (Figure 5l).

In the TNKS2 - 27 complex crystal structure, a nitrogen from the methylpiperazine-1-carbonyl

substitution forms a hydrogen bond with a water molecule connected to another water molecule and

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nitrogen of the main chain carbonyl of Ile1051 (Figure 5m). The core of the compound is in a

slightly different orientation than in the other derivatives, and is rotated approximately 5 ° towards

Tyr1071, which brings both C3 and C6´ to a hydrogen bond distance from the main chain carbonyl

of Gly1032. Binding of 27 also causes a shift in the D-loop lining the binding site. Residues 1046-

1051 of the D-loop move 0.9 Å in order to accommodate the bulky substituent (Figure 5m). This

loosens the packing of the D-loop with the active site helix 1059-1062, causes a rotation of

His1048, and disrupts the hydrogen bond between His1048 and Asp1045 (not shown).

Compound 28 has a cyano group attached at position 4´. Although the potency of 28 is better (IC50

145 nM) than that of 1, the substituent does not form any significant interactions with the protein

(Figure 5n). The only compound in this series that forms an additional direct hydrogen bond with

the protein is 29. The nitrogen of the cyanomethyl acetate substituent forms hydrogen bonds with

the main chain amide of Ile1051 and a water molecule, which is further hydrogen bonded with the

main chain amide of Gly1052 (Figure 5o). The additional hydrogen bond is the reason for the good

in vitro potency of the compound (IC50 7 nM).

A nitrogen from a tetrazol substitution (30) forms a hydrogen bond with a water molecule in a

fashion similar to that of 19, 20, and 26 (Figure 5e,f,l,p) and the potency of the compound is also at

a similar level (IC50 114 nM).

Although most of the interactions of the substituents with the protein are mediated by water

molecules, the compounds are complementary to the binding site of the TNKS2, where the D-loop

closes the donor site. Some inhibitors have been shown to open the cavity upon binding to the

preformed crystals.26,27, but the protein conformations observed in all the complexes described here

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are nearly identical. A notable exception is the TNKS2-27 complex where the D-loop has moved to

accommodate the inhibitor (Figure 5m).

DISCUSSION

In order to improve the flavone scaffold as a tankyrase inhibitor, we tested inhibition of TNKS1 by

commercially available flavone derivatives using a homogenous activity assay. Nineteen

compounds were selected for potency measurements on the basis of their inhibition at 1 µM. IC50

values were measured and co-crystal structures with TNKS2 were solved for all the hit compounds.

TNKS1 and 2 have catalytic domains that are nearly identical in sequence and completely identical

around the binding site of the compounds. Despite this the potency of these compounds in general is

lower for TNKS2 and for TNKS1 (Table 3). Soaking of the inhibitors is possible for the TNKS2

crystals as the crystal packing allows large rearrangement of the D-loop lining the binding site, and

it was therefore used for structural analysis.26,28

Position 4´ was the best position for substitutions as it extends from the binding site towards the

solvent. An exception to this was position 6, where small substituents were tolerated, but these did

not significantly improve the potency of the compound (Table 1). Modifications to position 4´ led

to more potent and selective inhibitors. Hydrophobic substitutions as in 21 and 22 were potent

together with the cyanomethyl acetate derivative, 29, which is the only compound that formed an

additional direct hydrogen bond with the protein. This compound, however, was not effective in the

β–catenin reporter assay. There could be multiple potential reasons behind the inactivity of

compounds in the cell-based assay such as cell permeability, non-specific binding, compound

solubility and stability and therefore it was not surprising that the reporter assay results did not

correlate directly with the potency in the enzymatic assay.

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As the compounds bind to the nicotinamide subsite, which is conserved in the ARTD enzyme

family, we wanted to establish a selectivity profile for the compounds that were effective in the

STF-reporter assay. Interestingly the small hydrophobic substituents in 21 and 22, with only 1 or 3

additional heavy atoms, respectively, were found to be the most selective. This small modification

in 22 improves the potency of the compound 55-fold yielding a potency similar to that of XAV939,

the model compound for a tankyrase inhibitor. In contrast to XAV939, 22 has also improved the

selectivity towards tankyrases over other ARTD family members. Most of the compounds studied

here show some selectivity (Figure 2), but 22 stands out as the most potent and most selective

inhibitor among them. The isopropyl substitution makes 22 the most hydrophobic in the series, but

the ligand efficiency of the compound is still the best and the lipophilic efficiency is at the same

level with the other potent compounds that antagonized Wnt signaling (Table 2).

We anticipated that larger substituents at the 4´ position would display higher selectivity as it has

been often mentioned that the compounds extending to this direction would interfere with the α-

helical regulatory domain present in ARTD1-4, but not in ARTD5-ARTD17.23,28 It was therefore

interesting to note that this was not the case. The largest compound, 27, also significantly inhibited

ARTD1 at 1 µM concentration (Figure 2). It is possible that some of these compounds could form

interactions with the more polar binding cleft of ARTD1-429 and perhaps therefore also interact with

the regulatory domain present in these enzymes.

The results presented here highlight that it is possible to create selective tankyrase inhibitors based

on the flavone scaffold. The compounds bind to the the conserved nicotinamide binding site and the

achieved selectivity is an improvement over the previously characterized compounds binding to this

subsite. This guides the future compound development of flavones as well as structurally related

scaffolds.

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EXPERIMENTAL SECTION

The compounds tested were purchased from Maybridge, Mir Biotech, SigmaAldrich and

Szintekon, Ltd. The purity of all the compounds was measured with RP HPLC using an X-Bridge

C18 2.1 x 50 mm column, eluted with a 15 min linear gradient of 10% methanol/90% water

containing 0.1% formic acid to 100% methanol. Detectors were a PDA detector scanning from 210

to 400 nm and a LCT time of flight mass spectrometer measuring m/z 100-1000. The purity of all

the compounds was ≥ 95% except for 5, 18, and 25, which were 91 %, 92%, and 84% pure,

respectively.

Cloning of TNKS2

ARTD6/TNKS2 cDNA was obtained from DNASU (Clone ID: HsCD00302726) and the catalytic

fragment (residues 873-1161) was cloned into a pNIC-ZB vector using overlap extension PCR

cloning. The construct was verified by sequencing, and transformed into Rosetta2(DE3) competent

cells for expression

Protein expression and Protein purification

ARTD6/TNKS2 was purified using Ni-affinity as described earlier for ARTD5/TNKS116. Cation

exchange choromatography was used as an additional purification step (HiLoad SP HP, GE

healthcare; Elution buffer: 20 mM Hepes pH 7.5, 1 M NaCl, 10% glycerol, 0.5 mM TCEP, 0.01%

triton-X100). Other human ARTD enzymes were expressed and purified as described earlier. The

constructs used encoded full-length human ARTD1 and ARTD227, ARTD4 (residues 250-565)27,

ARTD5/TNKS1 (residues 1030-1317)16, ARTD7 (residues 460-656)30, ARTD10 (residues 809-

1017)30 and ARTD12 (residues 489-684)27.

Homogeneous activity assay

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Assays were conducted as reported earlier.16,27,31 Reactions were carried out in 96-well plates

(Greiner bio-one U-shaped) at room temperature. At the completion of the incubation 20 µL of 20%

acetophenone in ethanol and 20 µL of 2 M KOH were added. The plate was incubated for another

10 minutes and then 90 µL of formic acid was added. The fluorescence was read after 20 minutes of

incubation using a Fluoroskan (excitation/emission, 355/460) or Tecan Infinity M1000

(excitation/emission, 372 nm/444 nm). The buffer used for TNKS1 contained 50 mM BisTris

propane pH 7, 0.5 mM TCEP and 0.01% Triton-X-100.

Screening of inhibitors and measurement of inhibitor potencies

Flavone derivatives with only one substitution were identified by searching commercially available

compound libraries and purchased from different vendors through Molport (Tables 1 and 2). The

compounds were stored at -20 ºC in DMSO and were diluted in the TNKS1 assay buffer

(Supplementary Table 2) before adding them into the reaction mixtures. The compounds were tested

at 10 µM and 1 µM concentrations in duplicate. Compound controls were used in this screening to

exclude the effect of compound fluorescence and quenching. Inhibition potencies were measured

for the inhibitors that had IC50 values below 10 µM based on the two point initial screening. IC50

values were measured using half log dilutions and reactions were carried out three individual times

in quadruplicate for TNKS1. The incubation time was adjusted so that substrate conversion was

more than 50% in the case of screening and less than 30% in the case of IC50 measurement. Dose

response curves were fitted using 4-parameters with Graphpad Prism (version 5.0 for windows).

Cell culture and reporter assays

HEK293, L Wnt-3a and L cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium

supplemented with 10% fetal bovine serum, penicillin and streptomycin at 37°C in a 5% CO2

atmosphere. Wnt3a and control conditioned medium were prepared as described previously.32

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HEK293 cells (1×105 cells/well) were plated on 24-well plates 16 hours before transfection. The

plated cells were transfected with SuperTopFlash reporter plasmids together with pCMV-β-

galactosidase plasmids (Clontech) using the FuGENETM 6 Transfection Reagent (Roche) according

to the manufacturer’s instructions. After transfection, the cells were treated with 30% Wnt3a-CM or

control-CM in serum free DMEM containing compounds or DMSO vehicle at the indicated

concentrations. The DMSO concentration was below 0.05% in all experiments. After 24 h, the cells

were lysed and luciferase activity was measured using the Luciferase assay system (Promega)

according to manufacturer’s protocol. In order to normalize the transfection efficiency, β-

galactosidase activity was measured using the β-galactosidase enzyme assay system (Promega). The

results are the mean of at least three independent experiments ± SEM. The SuperTopFlash

construct, originally designed in R. Moon’s laboratory33, was a gift from Professor S.Vainio.

Profiling of the inhibitors

The best tankyrase inhibitors identified were also profiled against six other human ARTD family

members using the homogenous activity assay described above. Incubation times and conditions

varied for each enzyme based on optimization carried out previously (Supplementary Table 1).27

The substrate NAD+ concentration was 250 nM or 500 nM in the profiling assays. In order to obtain

a robust signal the incubation time was adjusted so that substrate conversion was more than 50% in

each case. In order to efficiently evaluate the selectivity of the compounds, they were profiled at 1

µM concentration. DMSO, compound and protein controls were used with all the enzymes to

exclude or correct for the effects of DMSO, autofluorescence and quenching of the fluorescence.

Crystallization, data collection and refinement

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The TNKS2 catalytic domain was crystallized as previously reported.26 For crystallization,

chymotrypsin was added to the protein solution. Protein was concentrated to 5.8 mg/mL, aliquoted

in small volumes, and flash frozen. Protein crystallization was done using the sitting drop vapor

diffusion method in TTP labtech iQ plates. The well solution contained 0.2 M Li2SO4, 0.1 M Tris-

HCl pH 8.5, and 24-26% PEG 3350, and the well solution and protein solution were pipetted into

the crystallization wells in different ratios to 300 nL drops. Crystals appeared within a week.

Inhibitors were soaked for 24 h into the crystals in the well solution supplemented with the

compound (100 µM) and 250 mM NaCl. The crystals were dipped into cryo solution supplemented

with 20% glycerol and flash cooled in liquid nitrogen.

Data collection was done at ESRF, Grenoble, on beam lines ID14-1, ID14-4, ID23-2 and in a

Diamond Light source beamline I04-1. XDS34 was used for data processing. The structures were

solved using the molecular replacement method. Programs from CCP4 suite35 were used for

molecular replacement (MOLREP)36 and refinement (REFMAC5)37. TNKS2 structure with

nicotinamide (3U9H) was used as a template for molecular replacement. Coot was used for

visualization and manual building of the model.38 Data collection and refinement statistics are

reported in Supplementary Table 1. Marvin was used for drawing chemical structures and

InstantJChem for logD calculations (Marvin 5.7.0, 2011, ChemAxon, http://www.chemaxon.com).

Supporting information available: Supplementary Figure 1 contains the results of the cell-based

assay of all the compounds tested at 5 µM concentration. Supplementary Figure 2 contains the

experimental curves of the key compounds. Supplementary Table 1 contains the data collection

and refinement statistics for the crystal structures. Supplementary Table 2 contains the conditions

used for profiling the ARTD isoenzymes. Supplementary Table 3 contains supplier information of

the tested compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding author

*Phone: +358 2 9448 1169; E-mail: [email protected]

ACKNOWLEDGEMENTS

The work was funded by Biocenter Oulu, Sigrid Jusélius Foundation and Centre of Excellence

Grant of the Academy of Finland (251314). M.N. is supported by the National Doctoral Programme

of Informational and Structural Biology. This work was carried out with the support of the Diamond

Light Source (Didcot, UK) and the European Synchrotron Radiation Facility (ESRF, Grenoble,

France). We are grateful to Local Contacts at ESRF and at Diamond for providing assistance in

using beamlines ID14-1, ID14-4, ID23-2 and I04-1. The research leading to these results has

received funding from the European Community's Seventh Framework Programme (FP7/2007-

2013) under BioStruct-X (grant agreement N°283570). We also thank Erik Rannaste and Sari Ek for

the analysis of the compounds.

ABBREVIATIONS

APC, adenomatous polyposis coli; ART, ADP-ribosyltransferase; axin, axis inhibition protein;

GSK3β, glycogen synthase kinase 3 β; HPS, Histidine Proline Serine; NAD+, nicotinamide adenine

dinucleotide; PAR, poly(ADP-ribose); PARP, poly- (ADP-ribose) polymerase; PC, principal

component; SAM, sterile α motif; TCF, Transfection grade T-cell factor; TNKS, tankyrase; STF,

SuperTopFlash

PDB CODES

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Coordinates and structure factors are deposited to the protein data bank with codes 4L2G, 4L2F,

4L2K, 4KZL, 4L0V, 4KZU, 4KZQ, 4L09, 4L0T, 4BS4, 4L0B, 4L10, 4L0I, 4L31, 4L32, 4L0S,

4L33 and 4L34.

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FIGURE LEGENDS

Figure 1. Inhibition of Wnt signaling as measured by the SuperTopFlash assay. Compounds along

with a control inhibitors, XAV939 and IWR1, were tested at 5 µM, 1 µM and 0.2 µM. Inhibition %

± SEM is shown.

Figure 2. Profiling of the hit compounds with other members of the ARTD family. The profiling

was done using 1 µM concentration to highlight the differences between enzymes. Inhibition % ±

SD are presented.

Figure 3. Comparison of (a) 22 and (b) XAV939 structures.

Figure 4. Inhibitor binding modes. Cocrystal structures of TNKS2 with (a) 1, (b) 9, (c) 10 and (d)

14. Hydrogen bonds between the compound and protein and solvent as well as the molecular

surface of the catalytic domain are shown.

Figure 5. Binding of 4´ substituted flavones to TNKS2. Binding mode observed in a cocrystal

structure of TNKS2 and (a) 15, (b) 16, (c) 17, (d) 18, (e) 19, (f) 20, (g) 21, (h) 22, (i) 23, (j) 24, (k)

25, (l) 26, (m) 27, (n) 28, (o) 29 and (p) 30 to the TNKS2 ART domain. Hydrogen bonds that the

compound makes are shown as dashed lines.

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Table 1. Evaluation of flavone derivatives as tankyrase inhibitors. Potency (pIC50 ± SEM, n = 3) was measured for 1, 9, 10 and 14 using the biochemical assay. For the less potent compounds the potency is based on the screening data of two concentrations.

Structure IC50 Structure IC50

1

330 nM (6.49 ± 0.19)

8 >10 µM

2

>10 µM

9

210 nM (6.68 ± 0.05)

3

>10 µM

10

595 nM (6.23 ± 0.09)

4

>10 µM

11

>10 µM

5

>10 µM

12

>10 µM

6

~5 µM

13

>10 µM

7

>10 µM

14

360 nM (6.44 ± 0.10)

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Table 2a. Potencies of 4´-substituted flavones as tankyrase inhibitors. IC50 and corresponding pIC50

± SEM (n=3) are reported. Ligand efficiency (LE = pIC50/HA) and Lipophilic efficiency (LiPE = pIC50-LogD) are also calculated for the compounds.

Structure IC50 LogD

pH 7.4

LE LiPE

15

700 nM (6.16 ± 0.06)

3.11 0.34 3.1

16

233 nM (6.63 ± 0.04)

3.57 0.37 3.1

17

313 nM (6.50 ± 0.11)

3.74 0.36 2.76

18

788 nM (6.10 ± 0.13)

2.64 0.33 3.5

19

850 nM (6.07 ± 0.11)

-0.58 0.30 6.7

20

66 nM (7.18 ± 0.08)

2.91 0.36 4.2

21

47 nM (7.33 ± 0.18)

3.48 0.41 3.9

22

6 nM (8.22 ± 0.07)

4.21 0.41 4.0

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Table 2b. Potencies of 4´-substituted flavones as tankyrase inhibitors. IC50 and corresponding pIC50±SEM (n=3) are reported. Ligand efficiency (LE = pIC50/HA) and Lipophilic efficiency (LiPE = pIC50-LogD) are also calculated for the compounds.

Structure IC50 LogD

pH 7.4

LE LiPE

23

67 nM (7.18 ± 0.06)

3.08 0.36 4.1

24

71 nM (7.15 ± 0.06)

2.81 0.38 4.3

25

272 nM (6.56 ± 0.02)

3.33 0.30 3.2

26

162 nM (6.79 ± 0.22)

2.97 0.32 3.8

27

146 nM (6.84 ± 0.08)

2.11 0.26 4.7

28

145 nM (6.84 ± 0.02)

2.82 0.36 4.0

29

7 nM (8.13 ± 0.07)

2.51 0.37 5.6

30

114 nM (6.94 ± 0.18)

0.65 0.32 6.3

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Table 3. Potency measurements of the selected hit compounds and XAV939 against TNKS1, TNKS2, ARTD1 and ARTD2. IC50 values and pIC50 ± SEM (n=3) are reported.

Compound TNKS1 TNKS2 ARTD1 ARTD2

20 66 nM (7.18 ± 0.08)

140 nM (6.85 ± 0.04)

2.6 µM (5.57 ± 0.22)

8.3 µM (5.08 ± 0.1)

21 47 nM (7.33 ± 0.18)

190 nM (6.72 ± 0.19)

7.1 µM (5.15 ± 0.09)

27.8 µM (4.56 ± 0.07)

22 6 nM (8.22 ± 0.07)

72 nM (7.14 ± 0.05)

19.1 µM (4.72 ± 0.10)

34.9 µM (4.46 ± 0.06)

XAV939 5 nM (8.31 ± 0.04)

2 nM (8.63 ± 0.15)

5.5 µM (5.25 ± 0.05)

479 nM (6.32 ± 0.06)

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Figure 1

213x186mm (300 x 300 DPI)

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Figure 2

138x120mm (300 x 300 DPI)

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Figure 3

114x163mm (600 x 600 DPI)

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Figure 4

115x80mm (600 x 600 DPI)

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Figure 5

158x150mm (300 x 300 DPI)

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discovery of tankyrase inhibiting flavones with increased potency and isoenzyme selectivity

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