Discovery of a Novel Triazolopyridine Derivative as a Tankyrase Inhibitor

More than 80% of colorectal cancer patients have adenomatous polyposis coli (APC) mutations, which induce abnormal WNT/β-catenin activation. Tankyrase (TNKS) mediates the release of active β-catenin, which occurs regardless of the ligand that translocates into the nucleus by AXIN degradation via the ubiquitin-proteasome pathway. Therefore, TNKS inhibition has emerged as an attractive strategy for cancer therapy. In this study, we identified pyridine derivatives by evaluating in vitro TNKS enzyme activity and investigated N-([1,2,4]triazolo[4,3-a]pyridin-3-yl)-1-(2-cyanophenyl)piperidine-4-carboxamide (TI-12403) as a novel TNKS inhibitor. TI-12403 stabilized AXIN2, reduced active β-catenin, and downregulated β-catenin target genes in COLO320DM and DLD-1 cells. The antitumor activities of TI-12403 were confirmed by the viability of the colorectal cancer cells and its lack of visible toxicity in DLD-1 xenograft mouse model. In addition, combined 5-FU and TI-12403 treatment synergistically inhibited proliferation to a greater extent than that in a single drug treatment. Our observations suggest that TI-12403, a novel selective TNKS1 inhibitor, may be a suitable compound for anticancer drug development.


Introduction
WNT/β-catenin signaling plays crucial roles in embryo development and tissue homeostasis. A recent analysis by The Cancer Genome Atlas (TCGA) revealed that 93% of colorectal cancers (CRC) have genetic alterations of the WNT signaling pathway, which have been identified as biallelic inactivation mutations of APC regulator of WNT signaling pathway (APC), a negative regulator of β-catenin/CTNNB1, or activating mutations of CTNNB1 in approximately 80% of the cases [1]. Canonical WNT signaling is activated when Wnt ligands bind to the Frizzled (Fzd) receptor. In the absence of Wnt ligands, β-catenin is scaffolded by the 'destruction complex' consisting of AXIN, APC, casein kinase 1 (CK1), and glycogen synthase kinase 3β (GSK3β). β-catenin, which is sequentially phosphorylated by CK1 and GSK3β, is ubiquitinated by E3 ubiquitin ligase (β-transducin repeat-containing protein; β-TrCP) and degraded by the 26S proteasome. In the presence of Wnt ligands, Fzd and LRP5/6 receptors are activated, and disheveled (DVL) polymers are formed. The complex binds to AXIN, GSK3, and CK1 and inhibits GSK3, leading to β-catenin accumulation [2]. Accumulated β-catenin translocates to the nucleus and binds to the T-cell factor/lymphoid enhancement factor (TCF/LEF) transcription factor, triggering upregulation of target genes, such as MYC and AXIN2 [3]. However, loss-offunction of APC in the β-catenin destruction complex or gain-of function of CTNNB1 leads to aberrant accumulation of β-catenin and expression of its target genes. The inhibition of WNT/β-catenin signaling has known as an important therapeutic target over several decades. Despite of tremendous efforts in the development of inhibitors for WNT/βcatenin signaling, no drugs for clinical use have been promising yet.
Recently, TNKS inhibitors such as XAV939, IWR-1, G007-LK, and NVP-TNKS656 have been reported to show inhibition of cell proliferation in β-catenin-dependent CRC cells with APC mutations [7,[18][19][20][21]. Since E7449, a dual inhibitor of PARP 1/2 and TNKS, is the only drug currently under clinical trials, TNKS inhibitors need to be continuously developed and studied as anticancer drugs to elucidate the biological aspects of cancer cells [22]. Herein, we describe the identification of a novel small-molecule selective TNKS inhibitor, TI-12403, and suggest that TI-12403 is a potent TNKS candidate for the development of a novel TNKS inhibitor.
Several TNKS inhibitors have a triazolopyridazine functional group or a dihydrothiazolotriazole group with an X-ray crystal structure [23,24]. We superimposed the released TNKS1 X-ray crystal structure (PDB code: 4KRS) with the dihydrothiazolotriazole complex structure for TI-12403 (Figure 2A,B). The Ser1221 side chain of hydroxyl forms a hydrogen bond with triazololpyridine, and the Gly1185 backbone chain oxygen forms a hydrogen bond with the triazololpyridyl group. The amide linker of TI-12403 also forms a hydrogen bond with the carbonyl oxygen of Gly1185. Additional hydrophobic interactions exist between the cyanophenyl ring of TI-12403 and the binding pocket involving Pro1187, Phe1188, and Ile1204 of TNKS1. In case of the docking position between TI-12403 and TNKS2 (PDB code: 3P0Q), the amide linker of TI-12403 forms two hydrogen bonds with carbonyl oxygen and NH hydrogen of Gly1032 ( Figure 2C). However, the Ser1068 side chain of hydroxyl does not form a hydrogen bond with triazolopyridine at the intermolecular distance 3.14 Å. Our docking studies suggested that TI-12403 bound to both the nicotinamide pockets of TNKS1 and TNKS2. The IC 50 value of TNKS1 and TNKS2 is shown in Table 1.   , cMYC, and FGF20) were quantified using quantitative polymerase chain reaction (qPCR). XAV939 was used as a reference control. Data represent the mean ± standard deviation (SD) of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus respective DMSO-treated cells. (B) Whole cell lysates were subjected to immunoblotting for detection of active β-catenin (ABC), total β-catenin, TNKS1/2, and AXIN2. β-actin was used as a loading control. The density of each band was measured by Image J software and normalized to that of β-actin. Data represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 versus respective DMSO-treated cells. (C) COLO320DM and DLD-1 cells were treated with 10 μM TI-12403 and immunostained for AXIN2 (green) and β-catenin (red). Nuclear DNA was counterstained with DAPI (blue). Scale bar, 20 μm. (D) Quantification of the mean fluorescence intensity in Figure 1C. * p < 0.05, ** p < 0.01, *** p < 0.001 versus corresponding cells. (E) COLO320DM and DLD-1 cells were transfected with either TOP-(wild-type TCF binding sites) or FOP-(mutated TCF binding sites) flash reporter plasmid and then treated with 10 μM TI-12403 for 48 h. Transcriptional activity was measured with a luciferase reporter assay system and was calculated by dividing the TOP ratio by the FOP ratio , cMYC, and FGF20) were quantified using quantitative polymerase chain reaction (qPCR). XAV939 was used as a reference control. Data represent the mean ± standard deviation (SD) of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus respective DMSO-treated cells. (B) Whole cell lysates were subjected to immunoblotting for detection of active β-catenin (ABC), total β-catenin, TNKS1/2, and AXIN2. β-actin was used as a loading control. The density of each band was measured by Image J software and normalized to that of β-actin. Data represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 versus respective DMSO-treated cells. (C) COLO320DM and DLD-1 cells were treated with 10 µM TI-12403 and immunostained for AXIN2 (green) and β-catenin (red). Nuclear DNA was counterstained with DAPI (blue). Scale bar, 20 µm. (D) Quantification of the mean fluorescence intensity in Figure 1C. * p < 0.05, ** p < 0.01, *** p < 0.001 versus corresponding cells. (E) COLO320DM and DLD-1 cells were transfected with either TOP-(wild-type TCF binding sites) or FOP-(mutated TCF binding sites) flash reporter plasmid and then treated with 10 µM TI-12403 for 48 h. Transcriptional activity was measured with a luciferase reporter assay system and was calculated by dividing the TOP ratio by the FOP ratio (TOP/FOP ratio). Data represent the mean ± SD of three independent experiments. *** p < 0.001 versus respective DMSO-treated cells. (TOP/FOP ratio). Data represent the mean ± SD of three independent experiments. *** p < 0.001 versus respective DMSOtreated cells.

TI-12403 Inhibits Human CRC Cell Growth In Vitro and In Vivo
We assessed whether TI-12403-mediated inhibition of Wnt/β-catenin signaling affects CRC cell proliferation. COLO320DM and DLD-1 cells were treated with TI-12403, and growth inhibition was determined using a colony formation assay. TI-12403 inhibited the viability of COLO320DM and DLD-1 cells ( Figure 3A,B). Consistent with these findings, TI-12403 treatment significantly inhibited the viability of COLO320DM and DLD-1 cells, as determined by MTT assay ( Figure 3C).
We next determined whether the in vitro antitumor effect of TI-12403 could be translated to an in vivo xenograft mouse model. BALB/c-nu/nu mice were subcutaneously implanted with DLD-1 cells in the right hind leg. When the tumors were palpable (average diameter approximately 100 mm 3 ; 7 days post-implantation), mice were intraperitoneally administered with 20 mg/kg TI-12403 or DMSO (control vehicle) once per day for 14 days. Compared to the vehicle, TI-12403 inhibited DLD-1 cell-derived tumor growth by 53% ( Figure 4A). We did not observe any differences in body weights between control mice and TI-12403-treated mice ( Figure 4B). Therefore, these results suggested that TI-12403 exhibited antitumor activity in APC-mutated CRCs both in vitro and in vivo. Hydrogen bonds with relevant active site residues are shown as red dashes.

TI-12403 Inhibits Human CRC Cell Growth In Vitro and In Vivo
We assessed whether TI-12403-mediated inhibition of Wnt/β-catenin signaling affects CRC cell proliferation. COLO320DM and DLD-1 cells were treated with TI-12403, and growth inhibition was determined using a colony formation assay. TI-12403 inhibited the viability of COLO320DM and DLD-1 cells ( Figure 3A,B). Consistent with these findings, TI-12403 treatment significantly inhibited the viability of COLO320DM and DLD-1 cells, as determined by MTT assay ( Figure 3C).
We next determined whether the in vitro antitumor effect of TI-12403 could be translated to an in vivo xenograft mouse model. BALB/c-nu/nu mice were subcutaneously implanted with DLD-1 cells in the right hind leg. When the tumors were palpable (average diameter approximately 100 mm 3 ; 7 days post-implantation), mice were intraperitoneally administered with 20 mg/kg TI-12403 or DMSO (control vehicle) once per day for 14 days. Compared to the vehicle, TI-12403 inhibited DLD-1 cell-derived tumor growth by 53% ( Figure 4A). We did not observe any differences in body weights between control mice and TI-12403-treated mice ( Figure 4B). Therefore, these results suggested that TI-12403 exhibited antitumor activity in APC-mutated CRCs both in vitro and in vivo.

TI-12403 Decreases β-Catenin Levels and Increases AXIN2 Levels in DLD-1 Xenograft Mouse Tumors without Inducing Intestinal Toxicity
Wnt/β-catenin signaling plays an important role in maintaining normal tissue homeostasis in the intestine [25,26]. Therefore, high doses of TNKS inhibitors induce intestinal toxicity [19,20]. To determine TI-12403 toxicity, we examined the small intestine of mice treated with TI-12403 for 21 days in DLD-1 xenograft tumor efficacy study. The small intestine tissue was embedded in formalin-fixed paraffin (FFPE) and stained with the proliferation marker Ki67. Ki67 expression in the small intestine of TI-12403-treated mice was similar to that observed in the control mice ( Figure 4C). Additionally, the TI-12403-treated small intestine did not appear to have damaged crypts or villi, and the small intestine was similar to that observed in the control group. Twenty-one days after TI-12403 treatment, we analyzed β-catenin and AXIN2 levels in tumor tissues of DLD-1 xenograft mice by immunohistochemistry assay. TI-12403-treated tumors had significantly increased AXIN2 protein expression levels and inhibited β-catenin levels compared to that in the controls ( Figure 4D), suggesting that TI-12403 exhibited antitumor activity and did not have intestinal toxicity in vivo.

TI-12403 Decreases β-Catenin Levels and Increases AXIN2 Levels in DLD-1 Xenograft Mouse Tumors without Inducing Intestinal Toxicity
Wnt/β-catenin signaling plays an important role in maintaining normal tissue homeostasis in the intestine [25,26]. Therefore, high doses of TNKS inhibitors induce intestinal toxicity [19,20]. To determine TI-12403 toxicity, we examined the small intestine of mice treated with TI-12403 for 21 days in DLD-1 xenograft tumor efficacy study. The small intestine tissue was embedded in formalin-fixed paraffin (FFPE) and stained with the proliferation marker Ki67. Ki67 expression in the small intestine of TI-12403-treated mice was similar to that observed in the control mice ( Figure 4C). Additionally, the TI-12403-treated small intestine did not appear to have damaged crypts or villi, and the small intestine was similar to that observed in the control group. Twenty-one days after TI-12403 treatment, we analyzed β-catenin and AXIN2 levels in tumor tissues of DLD-1 xenograft mice by immunohistochemistry assay. TI-12403-treated tumors had significantly increased AXIN2 protein expression levels and inhibited β-catenin levels compared to that in the controls ( Figure 4D), suggesting that TI-12403 exhibited antitumor activity and did not have intestinal toxicity in vivo.   [26,27]. We evaluated whether treatment with a combination of 5-FU and TI-12403 produced synergistic effects. COLO320DM and DLD-1 cells were treated with the indicated concentrations of TI-12403 and 5-FU, and cell viability was assessed using a colony formation assay. Compared to TI-12403 or 5-FU treatment alone, combination treatment showed a stronger synergistic effect than XAV939 in COLO320DM ( Figure 5A,B) and DLD-1 cells (Figure 5C,D). These results indicated that TI-12403 and 5-FU combination treatment synergistically inhibited COLO320DM and DLD-1 cell growth.  [26,27]. We evaluated whether treatment with a combination of 5-FU and TI-12403 produced synergistic effects. COLO320DM and DLD-1 cells were treated with the indicated concentrations of TI-12403 and 5-FU, and cell viability was assessed using a colony formation assay. Compared to TI-12403 or 5-FU treatment alone, combination treatment showed a stronger synergistic effect than XAV939 in COLO320DM ( Figure 5A,B) and DLD-1 cells (Figure 5C,D). These results indicated that TI-12403 and 5-FU combination treatment synergistically inhibited COLO320DM and DLD-1 cell growth.

Discussion
Although dysregulated Wnt/β-catenin signaling is one of the distinguishing features of CRC, efforts continue to be carried out to identify targets that inhibit this pathway for clinical use. TNKS, which degrades AXIN in the β-catenin destruction complex, has been suggested as an attractive target for cancer therapy [5]. Here, we identified two commercial compounds through virtual screening, based on which we designed and synthesized 17 compounds. We discovered that TI-12403, a small-molecule compound, was the most potent among the compounds identified by in vitro screening and strongly inhibited TNKS1/2 but not PARP-1 activity. TI-12403 stabilized AXIN2, reduced active β-catenin, and showed anticancer effects in human APC-mutant CRC cells, thus making it a potential TNKS1 inhibitor.
Tanaka et al. reported that the APC mutation length predicts the sensitivity of CRC cells to TNKS inhibitors [21]. COLO320DM cells have an APC mutation that lacks all 20-

Discussion
Although dysregulated Wnt/β-catenin signaling is one of the distinguishing features of CRC, efforts continue to be carried out to identify targets that inhibit this pathway for clinical use. TNKS, which degrades AXIN in the β-catenin destruction complex, has been suggested as an attractive target for cancer therapy [5]. Here, we identified two commercial compounds through virtual screening, based on which we designed and synthesized 17 compounds. We discovered that TI-12403, a small-molecule compound, was the most potent among the compounds identified by in vitro screening and strongly inhibited TNKS1/2 but not PARP-1 activity. TI-12403 stabilized AXIN2, reduced active β-catenin, and showed anticancer effects in human APC-mutant CRC cells, thus making it a potential TNKS1 inhibitor.
Tanaka et al. reported that the APC mutation length predicts the sensitivity of CRC cells to TNKS inhibitors [21]. COLO320DM cells have an APC mutation that lacks all 20amino acid repeats (20-AAR), which are highly dependent on β-catenin signaling and sensitive to TNKS inhibitors. DLD-1 cells are relatively less sensitive compared to COLO320DM cells to TNKS inhibitors, despite the cells having mutant APCs with two 20-AARs. Consistent with this report, we observed that TI-14203 reduced COLO320DM cell viability more effectively compared to DLD-1 cells (Figure 3). Although TNKS inhibitors reduce APC mutation-dependent cell viability, it is worthy to mention that TI-12403 inhibit proliferation of DLD-1 cells. TNKS is involved in other oncoproteins networks, including Hippo-Yes associated protein (YAP)/TAZ (PDZ-binding motif) signaling pathway. Inhibition of TNKS stabilizes the AMOT family of proteins by inhibiting RNF146 axis-mediated degradation, thereby inhibiting YAP oncogenic function [25]. Notably, TI-12403 inhibited YAP target gene expression in DLD-1 cells. However, we did not observe the expression of YAP target genes in COLO320DM cells with either TI-12403 or DMSO treatment (Supplementary Figure S3). Since E-cadherin can positively regulate YAP signaling, E-cadherin-deficient COLO320DM cells did not appear to express the YAP target genes [28]. We speculated that the anticancer effects of TI-12403 on DLD-1 cells might be due to TI-12403-mediated inhibition of YAP signaling. Therefore, TI-12403 is expected to have a therapeutic effect in a wider variety of cancers where YAP signaling is upregulated.
A few selective TNKS inhibitors are being developed; however, most of their evaluation remains preclinical. Since β-catenin is a key in maintaining intestinal homeostasis, TNKS inhibitors such as G007-LK, XAV939, and G-631 have side effects leading to intestinal toxicity or severe weight loss in mice [19,20,29]. TI-12403 showed reduced intestinal toxicity or body weight change ( Figure 4). Additionally, TI-12403 had excellent metabolic stability in human liver microsomal and plasma and did not show cytochrome P450 inhibitory activity (Supplementary Table S3). Altogether, TI-12403 exerted no significant toxicity due to its high metabolic stability in mice. We postulate that TI-12403 requires further evaluation for effective drug development but has potential as a therapeutic agent against cancer.
The current first-line therapeutic agent used clinically in CRC is 5-FU and it has improved the overall survival rate of patients with CRC; however, its clinical use is limited due to its toxicity and chemoresistance. Combination treatment is an effective clinical strategy for anticancer therapy in CRC [26,30]. Previous studies have reported that APC mutations contribute to 5-FU resistance in CRC cells [29,31]. Recently, it has been suggested that TNKS inhibitors reduced 5-FU resistance in APC mutant cells [29]. Consistent with the aforementioned report, we found that combination treatment with TI-12403 and 5-FU significantly inhibited COLO32DM and DLD-1 cell viability ( Figure 5). Thus, TNKS inhibitors can be considered as therapeutic agents for combination treatment in CRC.
In summary, TI-12403 exhibited potent TNKS inhibitor activity and cytotoxicity toward CRC cells. TI-12403 induced AXIN2 expression and downregulated β-catenin, increasing the sensitivity of cancer cells. Moreover, TI-12403 and 5-FU combination treatment considerably inhibited cell proliferation. Therefore, the novel TNKS inhibitor TI-12403 may be effective in the treatment of APC-mutant CRC and could have further potential as an adjuvant when used in combination with 5-FU.

In Vitro Enzyme Assay
TNKS1 and TNKS2 activities of the compounds were measured using colorimetric activity assays (BPS Bioscience, San Diego, CA, USA) according to the manufacturer's protocol, and their IC 50 values were determined based on the TNKS1 and TNKS2 activities.

Cell Proliferation Assay
Cells were seed in 96-well plates at a density of 1 × 10 3 cells/well in triplicate, treated with 10 µM TI-12304 or XAV939 for 72 h. Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay (Sigma-Aldrich) according to the manufacturer's recommendations. Briefly, 10 µL of MTT (0.5 mg/mL) was added to the culture medium and incubated for 2 h, and the absorbance at 540 nm was determined by a Multiskan EX plate reader (Thermo LabSystems, Waltham, MA, USA). For colony formation assay, cells were seeded at 500 cells per 60 mm dish and incubated at 37 • C and 5% CO 2 . After 24 h, the cells were treated with 10 µM TI-12403 or XAV939. In the combination treatment experiment, cells were treated with the indicated dose of TI-12403 for 2 h before treatment with the indicated doses of 5-fluorouracil (5-FU). After 10 days, the colonies were fixed and stained with 1.5% methylene blue (Sigma Aldrich) in methanol solution for visualization. Colonies containing >50 cells were counted.

Immunoblot Analysis
Cell lysates were prepared by extracting proteins with TNN buffer (40 mM Tris-Cl pH 8.0, 0.2% NP-40, 120 mM NaCl) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). Western blot analysis was performed as previously described [32]. Details of the primary antibodies used in this study are provided in the Supplementary Materials and Methods section.

RNA Extraction and Quantitative Polymerase Chain Reaction (qPCR) Analysis
RNA was extracted using TRIzol ® RNA isolation reagent (Thermo Fisher Scientific). Reverse transcription of RNA to cDNA was performed using AccuPower ® CycleScript™ RT PreMix (Bioneer, Daejeon, Korea). The cDNA was quantified using real-time PCR with SYBR Green/fluorescein qPCR master mix (Thermo Scientific, Carlsbad, CA, USA) on a Lightcycler 96 system (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. The sequences of the primers used are provided in the Supplementary Materials and Methods section.

Immunofluorescence Staining
COLO320DM and DLD-1 cells were fixed in 4% paraformaldehyde for 15 min at 25 • C and permeabilized with 0.1% Triton X-100 in PBS for 20 min. The cells were then incubated with a 1:100 dilution of anti-anti-active β-catenin (ABC) and Axin2 antibody overnight at 4 • C. Next, the cells were incubated with Alexa Fluor 488-conjugated antimouse IgG antibody (Abcam, Cambridge, UK) and Alexa Fluor 594-conjugated anti-rabbit IgG antibody at a 1:400 dilution for 1 h at room temperature. The slides were mounted in mounting medium (DAKO, Santa Clara, CA, USA) with DAPI (Thermo Fisher Scientific) before imaging. Images were acquired using an LSM880 laser scanning microscope (ZEISS, Jena, Germany). Fluorescence images were captured using appropriate filters. Images were analyzed using ImageJ and ZEN software.

Tumor Xenograft Mouse Models
All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Korea Institute of Radiological and Medical Sciences (Kirams2018-0063). DLD-1 cells (2 × 10 6 ) were implanted subcutaneously into the thigh of the right hind leg of six-week-old mice. When tumor volumes reached approximately 100 mm 3 , TI-12403 (20 mg/kg) was administered intraperitoneally once per day for 14 days. The body weights of the mice were measured once a week.

Immunohistochemistry (IHC)
Mice treated with DMSO or TI-12403 were sacrificed, and the intestines and tumors were dissected. The tissues were fixed in 4% paraformaldehyde (in PBS solution) overnight and embedded in paraffin. After sectioning, the intestines were stained with Ki67. The tumor tissues were stained with an anti-β-catenin antibody and anti-Axin2 antibody.

Statistical Analysis
Results are shown as the mean ± standard deviation (SD). Data were analyzed using two-tailed Student's t-tests. Differences between groups with p-values < 0.05 were considered statistically significant.