Potent Tankyrase Inhibitor Research Articles

β-catenin inhibition using XAV939 nanoparticle promotes immunogenic cell death and suppresses tumor progression

Abstract Although the incidence of conjunctival melanoma is rare, it is a highly metastatic tumor with high recurrence and mortality rates. The current conjunctival melanoma treatment strategies, including surgical resection combined with chemo- or cryotherapy, have limited usefulness because of systemic side effects, recurrence, and metastases. Tumor-intrinsic Wnt/β-catenin pathway mediates immune exclusion in the tumor microenvironment (TME) and promotes tumorigenesis. Previous studies have shown that XAV939, a potent tankyrase inhibitor, can be used for the pharmacological inhibition of β-catenin. In this study, using tocopheryl polyethylene glycol 1000 succinate (TPGS)-based nanoparticle formulation for XAV939, we show that β-catenin inhibition suppresses melanoma cell proliferation and migration. Additionally, XAV939 nanoparticles promoted immunogenic cell death (ICD) of tumor cells with a significant extracellular release of ICD molecules, including HMGB1, calreticulin, and ATP. Further, using a mouse model of conjunctival melanoma, we show that β-catenin inhibition significantly suppresses conjunctival melanoma progression. Collectively, the local delivery of β-catenin represents a novel and safe approach to promoting ICD and activating anti-tumor immunity for combinatorial immune checkpoint inhibition therapies against vision and life-threatening ocular melanomas. This study was supported by research funding from the Intramural Grants Program, Auburn University Research Initiative in Cancer (AURIC), and start-up funds from the Department of Pathobiology, College of Veterinary Medicine, Auburn University.

Structural insights on 2-phenylquinazolin-4-one derivatives as tankyrase inhibitors through CoMFA, CoMSIA, topomer CoMFA and HQSAR studies

Tankyrase is an emerging target of 17 membered poly(ADP-ribose)polymerase enzyme family for the treatment of Wnt driven cancers. TNKS are associated with many cellular functions; Wnt–β-catenin signaling, telomere homeostasis, mitotic spindle formation etc. The inhibition of cell growth could be achieved by the inhibition of TNKS through Wnt-signaling mechanism. To develop novel lead molecules as tankyrase inhibitors, we performed 3D-QSAR studies on 37 reported 2-phenylquinazolin-4-one derivatives. Training set contained 29 while test set contained 8 molecules. Four different techniques, CoMFA, CoMSIA, Topomer CoMFA and HQSAR were used to generate the QSAR models. Distill alignment based CoMFA analysis showed q2 value of 0.605, r2ncv value of 0.958 and r2pred value of 0.553. Optimized CoMSIA analysis (SEHD) showed q2 value of 0.580, r2ncv value of 0.953 and r2pred value of 0.723. Topomer CoMFA analysis showed q2 value of 0.684, r2 (conventional correlation coefficient) value of 0.955 and r2pred value of 0.721. HQSAR analysis showed q2, r2 and r2pred values of 0.833, 0.951 and 0.753, respectively. Combined results of all studies provided significant output in terms of substitutions required for activity and can be used for the design of novel quinazolinone derivatives as potent tankyrase inhibitors.

Design, synthesis, and biological evaluation of isoquinolin-1(2H)-one derivates as tankyrase-1/2 inhibitors.

To investigate structure-activity relationships of tankyrase (TNKS) inhibitors, twelve new derivatives of isoquinolin-1(2 H )-one were designed and synthesized, and biological assessments were conducted. Several potent TNKS inhibitors with single- or double-digit nanomolar IC50 values were identified using enzymatic assays. Compound 11c was the most potent compound of this series and inhibited TNKS1 and TNKS2 at an IC50 of 0.009 and 0.003 μM, respectively, and showed an IC50 of 0.029 μM in a DLD-1 SuperTopFlash assay. Molecular docking results showed that compound 11c occupied a unique subpocket and formed a hydrogen bond with Glu1138 of TNKS2, which was not consistent with the patterns of known TNKS inhibitors and thus warrants further research.

New molecular insights into dual inhibitors of tankyrase as Wnt signaling antagonists: 3D-QSAR studies on 4H-1,2,4-triazole derivatives for the design of novel anticancer agents

Genetic mutations in APC or CNTBB1 gene with aberrant canonical Wnt/β-catenin pathway are responsible for more than 90% of colorectal carcinogenesis. Tankyrases (TNKS) are known to downregulate Wnt signaling by stabilizing AXIN protein through poly(ADP ribose)polymerization or PARSylation process and subsequently, promoting degradation of intracellular β-catenin. Tankyrase enzymes are modulated by a range of known inhibitors that bind individually to any of the nicotinamide or induced adenosine pockets or as dual binding antagonists. Hence, for designing dual tankyrase inhibitors as Wnt signaling antagonist; we carried out 3D-QSAR studies using a data set of 51 molecules of reported 3,4,5-trisubstituted 4H-1,2,4-triazole derivatives. These reported 51 molecules were divided into a training set (39 molecules) and test set (12 molecules), aligned and subjected to generate CoMFA, CoMSIA, and HQSAR models. CoMFA analysis showed q2 value of 0.694, r2ncv value of 0.991 and r2pred value of 0.641. Optimized CoMSIA analysis (SEHA) showed q2 value of 0.624, r2ncv value of 0.909 and r2pred value of 0.850. Both internal and external validations were performed for generated models of CoMFA and CoMSIA (SEHA) and satisfactory results were obtained. HQSAR analysis showed q2, r2, and r2pred values of 0.781, 0.901, and 0.811, respectively. Applicability domain was also found to be satisfactory with all compounds falling within the range and no outlier was observed. Contour maps from all studies provided significant results with identification of desired spatial arrangement of different atoms or functional groups in a molecule. Triazole ring system-based molecules were reported as potent tankyrase inhibitors. These noteworthy results were employed for the design of different triazole derivatives as potent tankyrase inhibitors, wherein a series of 20 different molecules were designed for evaluation of their potentials as novel tankyrase inhibitors.

Design and Discovery of an Orally Efficacious Spiroindolinone-Based Tankyrase Inhibitor for the Treatment of Colon Cancer.

Tankyrases (TNKS/TNKS2) belong to the poly(ADP-ribose) polymerase family. Inhibition of their enzymatic activities attenuates the Wnt/β-catenin signaling, which plays an important role in cancer pathogenesis. We previously reported the discovery of RK-287107, a spiroindoline-based, highly selective, potent tankyrase inhibitor. Herein we describe the optimization process of RK-287107 leading to RK-582, which exhibits a markedly improved robust tumor growth inhibition in a COLO-320DM mouse xenograft model when orally administered. In addition to the dose-dependent elevation and attenuation of the levels of biomarkers AXIN2 and β-catenin, respectively, results of the TCF reporter and cell proliferation studies on COLO-320DM are discussed.

Discovery and Optimization of 2-Arylquinazolin-4-ones into a Potent and Selective Tankyrase Inhibitor Modulating Wnt Pathway Activity.

Tankyrases 1 and 2 (TNKS1/2) are promising pharmacological targets that recently gained interest for anticancer therapy in Wnt pathway dependent tumors. 2-Aryl-quinazolinones were identified and optimized into potent tankyrase inhibitors through SAR exploration around the quinazolinone core and the 4'-position of the phenyl residue. These efforts were supported by analysis of TNKS X-ray and WaterMap structures and resulted in compound 5k, a potent, selective tankyrase inhibitor with favorable pharmacokinetic properties. The X-ray structure of 5k in complex with TNKS1 was solved and confirmed the design hypothesis. Modulation of Wnt pathway activity was demonstrated with this compound in a colorectal xenograft model in vivo.

Inhibition of WNT/β-catenin signaling under serum starvation and hypoxia induces adipocytic transdifferentiation in human leiomyoma cells

Fatty metamorphosis is an uncommon alteration in uterine leiomyoma (i.e., lipoleiomyoma), and the pathogenetic mechanisms underlying this phenomenon remain poorly understood. Because a conditional deletion of β-catenin, a major transducer of the canonical Wingless/integrated (WNT) pathway, in the developing mouse uterus can induce adipogenesis in the myometrium, it is hypothesized that inhibition of the WNT/β-catenin signaling may be also involved in the development of fat cells within uterine leiomyoma. In the current study, which was performed to address this point, intracytoplasmic lipid droplets were detectable in cultured human leiomyoma cells by treatment with a potent tankyrase inhibitor, XAV939, which antagonizes β-catenin, in a serum-starved culture medium without additional adipogenesis-inducing agents or supplements, and showed increasing accumulation in a time-dependent manner. In addition, the induction of fat cells was greatly enhanced under hypoxic conditions (i.e., 2.5% O2)—recapitulating the local in vivo situation of uterine leiomyoma—in comparison to that under normoxic conditions (i.e., 21% O2). The marker genes of differentiated fat cells such as ADIPOQ and PLIN were highly expressed in leiomyoma cells that were treated with XAV939 under hypoxia and serum starvation, whereas the immunohistochemical expression of desmin—a cytoskeletal protein representing smooth muscle differentiation—was downregulated, which appears in line with the switch in differentiation. The results of our study suggest that the inhibition of canonical WNT/β-catenin signaling under the stress due to hypoxia and serum starvation can initiate adipocytic transdifferentiation or metaplasia in human uterine leiomyoma cells, which is potentially related to the development of lipoleiomyoma.

Comparative conformational variation and flexibility analysis of binding site D-loop and its importance in designing of potential tankyrases inhibitors

The principle point of this study is to know the dynamics of D-loop affecting the inhibitory action of tankyrase inhibitors (TNKSIs). The knowledge of D-loop conformational variations, flexibility analysis and its significance in the binding of TNKSIs has not been explored earlier. This study at first focused on observing the conformational changes of D-loop in tankyrases crystal structures by overlapping of D-loop residues. To achieve this, relative residue fluctuation profile and molecular mechanics with generalised born surface area solvation (MM/GBSA) free energy calculations were performed to expose the more prominent binding strength of strong inhibitors and selectivity compared with other respective site inhibitors. Molecular dynamics simulation search revealed that residue fluctuation profile of D-loop of TNKSs affects the binding potential of inhibitors. Thus, conformational changes in the D-loop affected by inhibitors can be used to screen the potent TNKSIs. The molecular docking, D-loop flexibility analysis, molecular dynamics and MM/GBSA binding energy calculations were carried out.

The Anti-Tumor Activity of Succinyl Macrolactin A Is Mediated through the β-Catenin Destruction Complex via the Suppression of Tankyrase and PI3K/Akt.

Accumulated gene mutations in cancer suggest that multi-targeted suppression of affected signaling networks is a promising strategy for cancer treatment. In the present study, we report that 7-O-succinyl macrolactin A (SMA) suppresses tumor growth by stabilizing the β-catenin destruction complex, which was achieved through inhibition of regulatory components associated with the complex. SMA significantly reduced the activities of PI3K/Akt, which corresponded with a decrease in GSK3β phosphorylation, an increase in β-catenin phosphorylation, and a reduction in nuclear β-catenin content in HT29 human colon cancer cells. At the same time, the activity of tankyrase, which inhibits the β-catenin destruction complex by destabilizing the axin level, was suppressed by SMA. Despite the low potency of SMA against tankyrase activity (IC50 of 50.1 μM and 15.5 μM for tankyrase 1 and 2, respectively) compared to XAV939 (IC50 of 11 nM for tankyrase 1), a selective and potent tankyrase inhibitor, SMA had strong inhibitory effects on β-catenin-dependent TCF/LEF1 transcriptional activity (IC50 of 39.8 nM), which were similar to that of XAV939 (IC50 of 28.1 nM). In addition to suppressing the colony forming ability of colon cancer cells in vitro, SMA significantly inhibited tumor growth in CT26 syngenic and HT29 xenograft mouse tumor models. Furthermore, treating mice with SMA in combination with 5-FU in a colon cancer xenograft model or with cisplatin in an A549 lung cancer xenograft model resulted in greater anti-tumor activity than did treatment with the drugs alone. In the xenograft tumor tissues, SMA dose-dependently inhibited nuclear β-catenin along with reductions in GSK3β phosphorylation and increases in axin levels. These results suggest that SMA is a possible candidate as an effective anti-cancer agent alone or in combination with cytotoxic chemotherapeutic drugs, such as 5-FU and cisplatin, and that the mode of action for SMA involves stabilization of the β-catenin destruction complex through inhibition of tankyrase and the PI3K/Akt signaling pathway.

Design and discovery of 3-aryl-5-substituted-isoquinolin-1-ones as potent tankyrase inhibitors

The tankyrase proteins (TNKS, TNKS2) are attractive anti-cancer drug targets, particularly as inhibition of their catalytic activity has been shown to antagonise oncogenic WNT signalling.

Abstract 2715: Discovery and characterization of a highly potent Wnt/ β -catenin pathway inhibitor targeting tankyrase

Abstract Background: Wnt/β-catenin pathway controls many biological processes including cell proliferation and tissue development. In Wnt pathway, β-catenin is a key downstream factor which interacts with transcription factor T-cell factor (TCF) in the nucleus and induces expression of TCF responsive genes. β-catenin degradation is promoted by a cytoplasmic complex of APC, Axin and GSK3β. In many cancers, constitutive activation of Wnt/β-catenin pathway is observed due to mutations of the related genes and overexpression of Wnt ligands and receptors. For example, truncating mutation of the tumor suppressor APC are the most prevalent in colon cancer. APC deficiency leads to accumulation of nuclear β-catenin and promotes the transcription of downstream target genes. Hence, Wnt/β-catenin pathway inhibitor can be an attractive therapeutic agent for cancer patients. Results: To screen for Wnt/β-catenin pathway inhibitor, a reporter-based screen using a colon cancer cell line harboring APC mutation was performed. We identified a Wnt/β-catenin pathway inhibitor K-756 with an IC50 of 110 nmol/L, which was a distinct chemical structure from previously reported Wnt/β-catenin pathway inhibitors. From target identification study, it became clear that K-756 was a selective tankyrase inhibitor (AACR-NCI-EORTC 2013). Further development of the derivatives of K-756 using a structure-based drug design approach led to synthesis of K-476. K-476 inhibited Wnt/β-catenin pathway in APC mutant DLD-1 cell line with an IC50 of 0.3 nmol/L. K-476 stabilized Axin proteins and decreased active β-catenin. Wnt downstream target gene inhibition was also observed by K-476. PARP family profile assay was performed to evaluate the selectivity of K-476. K-476 inhibited tankyrase 1 and 2 and did not inhibit other PARP family isoforms even at 1000 nmol/L. Kinase profile assay was also performed towards 448 kinase but K-476 did not inhibit any of the kinases at 1000 nmol/L. Therefore K-476 is a highly selective tankyrase inhibitor. K-476 inhibited Wnt pathway signal and cell growth in colon cancer cell lines with an GI50 of less than 5 nmol/L. Oral dosing of K-476 resulted in inhibition of the reporter activity and Wnt downstream genes in a colon cancer xenograft mouse model. Taken together, K-476 represents a highly potent tankyrase inhibitor and is anticipated to be an important compound for further development of antitumor agent targeting the Wnt/β-catenin pathway regulation. Citation Format: Ryoko Okada, Yuichi Takahashi, Keiichi Motosawa, Yasuo Watanabe, Asae Igarashi, Ran Okada, Masahiro Ikkaku, Hikaru Miyagi, Yusuke Miura, Kazuki Asanome, Noriaki Uesaka, Jun-ichi Saito, Hiroshi Ishida, Ryuichiro Nakai. Discovery and characterization of a highly potent Wnt/ β -catenin pathway inhibitor targeting tankyrase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2715. doi:10.1158/1538-7445.AM2014-2715

Evaluation and Structural Basis for the Inhibition of Tankyrases by PARP Inhibitors.

Tankyrases, an enzyme subfamily of human poly(ADP-ribosyl)polymerases, are potential drug targets especially against cancer. We have evaluated inhibition of tankyrases by known PARP inhibitors and report five cocrystal structures of the most potent compounds in complex with human tankyrase 2. The inhibitors include the small general PARP inhibitors Phenanthridinone, PJ-34, and TIQ-A as well as the more advanced inhibitors EB-47 and rucaparib. The compounds anchor to the nicotinamide subsite of tankyrase 2. Crystal structures reveal flexibility of the ligand binding site with implications for drug development against tankyrases and other ADP-ribosyltransferases. EB-47 mimics the substrate NAD(+) and extends from the nicotinamide to the adenosine subsite. The clinical ARTD1 inhibitor candidate rucaparib was the most potent tankyrase inhibitor identified (24 and 14 nM for tankyrases), which indicates that inhibition of tankyrases would affect the cellular responses of this compound.

Abstract B30: KRAS wild type tumors exhibit increased efficacy to the combination of AZ1 (a tankyrase inhibitor) + irinotecan in a patient-derived CRC explant model.

Abstract Background: Dysregulation of the canonical Wnt signaling pathway has been implicated in colorectal cancer (CRC) development as well as incipient stages of malignant transformation. An important regulator of the Wnt pathway are tankyrases that function in reducing Axin2 levels which is an essential protein in the β-catenin destruction complex. Targeting this pathway with a potent tankyrase inhibitor presents a plausible approach in drug development. In this study, we evaluated the antitumor effects AZ1 (a novel tankyrase 1/2 inhibitor which also carries PARP1 activity) as a single agent and in combination with irinotecan in our patient derived CRC explant xenograft models. Methods: Twenty patient derived CRC explant xenografts were treated with vehicle, AZ1 (50mg/kg/day), irinotecan (30mg/kg/week) or AZ1 (50mg/kg/day) + irinotecan (30mg/kg/week) for 28 days. Pharmacokinetic effect of AZ1 was determined in plasma and tumor over 24h. Both pharmacodynamic effects (Axin2 levels) and changes in biomarkers relevant to the Wnt pathway were determined by Western blotting. Intra-nuclear β-catenin levels were evaluated by immunohistochemistry. A tumor growth index (TGI) was calculated as the ratio in tumour volumes in the treated animals and the control animals (T/C) on the last day of drug treatment to determine efficacy. TGI≤ 50% was considered sensitive and TGI > 50% was resistant. Results: Three out of 20 CRC explants showed some tumour growth inhibition with AZ1. In animals bearing CRC40 xenografts, AZ1 showed a Tmax of 1 hour in plasma and 30 minutes in tumor. In addition, Axin2 stabilization post AZ1 started 15 minutes after drug administration, with the maximum stabilization observed at 8 hours. Treatment with AZ1 + irinotecan resulted in a greater anti-tumor effect (achieving stasis or better) than either agent alone in 4 out of 20 CRC xenografts. A significant association (fisher exact test: p= 0.007) was identified between combinational sensitivity and KRAS wild type. Evaluation of tumors after treatment showed marked stabilization of Tankyrase 1/2 and Axin2; however, active β-catenin levels were not changed. Also, there were no changes to intra-nuclear β-catenin (IHC) levels after treatment. Interestingly, NuMa, a protein involved in maintenance of the mitotic spindle and a target of Tankyrase 1/2 increased following irinotecan in 3 models, 2 showing a combination effect and 1 which did not. Numa levels decreased with the combination of AZ1 and irinotecan compared to irinotecan alone in 2 models showing a significant combination effect. Conclusion: Combination AZ1 and irinotecan achieved greater anti-tumor effect compared to monotherapy in 4 out of 20 CRC xenografts. Activity was limited to CRC xenografts with KRAS WT status. Further studies are warranted to validate these findings in KRAS WT patients and to understanding the specific mechanisms in tumors that responded to combinational therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B30. Citation Format: WM Tai, Kevin Quackenbush, Alicia Purkey, Stacey Bagby, Wells Messersmith, Eun Kee Song, Todd Pitts, John J. Arcaroli. KRAS wild type tumors exhibit increased efficacy to the combination of AZ1 (a tankyrase inhibitor) + irinotecan in a patient-derived CRC explant model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B30.

Para‐Substituted 2‐Phenyl‐3,4‐dihydroquinazolin‐4‐ones As Potent and Selective Tankyrase Inhibitors

Human tankyrases are attractive drug targets, especially for the treatment of cancer. We identified a set of highly potent tankyrase inhibitors based on a 2-phenyl-3,4-dihydroquinazolin-4-one scaffold. Substitutions at the para position of the scaffold's phenyl group were evaluated as a strategy to increase potency and improve selectivity. The best compounds displayed single-digit nanomolar potencies, and profiling against several human diphtheria-toxin-like ADP-ribosyltransferases revealed that a subset of these compounds are highly selective tankyrase inhibitors. The compounds also effectively inhibit Wnt signaling in HEK293 cells. The binding mode of all inhibitors was studied by protein X-ray crystallography. This allowed us to establish a structural basis for the development of highly potent and selective tankyrase inhibitors based on the 2-phenyl-3,4-dihydroquinazolin-4-one scaffold and outline a rational approach to the modification of other inhibitor scaffolds that bind to the nicotinamide site of the catalytic domain.

Discovery of Novel, Induced-Pocket Binding Oxazolidinones as Potent, Selective, and Orally Bioavailable Tankyrase Inhibitors

Tankyrase (TNKS) is a poly-ADP-ribosylating protein (PARP) whose activity suppresses cellular axin protein levels and elevates β-catenin concentrations, resulting in increased oncogene expression. The inhibition of tankyrase (TNKS1 and 2) may reduce the levels of β-catenin-mediated transcription and inhibit tumorigenesis. Compound 1 is a previously described moderately potent tankyrase inhibitor that suffers from poor pharmacokinetic properties. Herein, we describe the utilization of structure-based design and molecular modeling toward novel, potent, and selective tankyrase inhibitors with improved pharmacokinetic properties (39, 40).