Tankyrase Research Articles

Exploring the Effects of Chirality of 5-methyl-5-[4-(4-oxo-3H-quinazolin-2-yl) phenyl]imidazolidine-2,4-dione and its Derivatives on the Oncological Target Tankyrase 2. Atomistic Insights.

Tankyrases (TNKS) are homomultimers existing in two forms, viz. TNKS1 and TNKS2. TNKS2 plays a pivotal role in carcinogenesis by activating the Wnt//β-catenin pathway. TNKS2 has been identified as a suitable target in oncology due to its crucial role in mediating tumour progression. The discovery of 5-methyl-5-[4-(4-oxo-3H-quinazolin-2-yl) phenyl] imidazolidine-2,4-dione, a hydantoin phenylquinazolinone derivative which exists as a racemic mixture and in its pure enantiomer forms, has reportedly exhibited inhibitory potency towards TNKS2. However, the molecular events surrounding its chirality towards TNKS2 remain unresolved. Herein, we employed in silico methods such as molecular dynamics simulation coupled with binding free energy estimations to explore the mechanistic activity of the racemic inhibitor and its enantiomer forms on TNK2 at a molecular level Results: Favourable binding free energies were noted for all three ligands propelled by electrostatic and van der Waals forces. The positive enantiomer demonstrated the highest total binding free energy (-38.15 kcal/mol), exhibiting a more potent binding affinity to TNKS2. Amino acids PHE1035, ALA1038, and HIS1048; PHE1035, HIS1048 and ILE1039; and TYR1060, SER1033 and ILE1059 were identified as key drivers of TNKS2 inhibition for all three inhibitors, characterized by the contribution of highest residual energies and the formation of crucial high-affinity interactions with the bound inhibitors. Further assessment of chirality by the inhibitors revealed a stabilizing effect of the complex systems of all three inhibitors on the TNKS2 structure. Concerning flexibility and mobility, the racemic inhibitor and negative enantiomer revealed a more rigid structure when bound to TNKS2, which could potentiate biological activity interference. The positive enantiomer, however, displayed much more elasticity and flexibility when bound to TNKS2. Overall, 5-methyl-5-[4-(4-oxo-3H-quinazolin-2-yl) phenyl] imidazolidine-2,4-dione and its derivatives showed their inhibitory prowess when bound to the TNKS2 target via in silico assessment. Thus, results from this study offer insight into chirality and the possibility of adjustments of the enantiomer ratio to promote greater inhibitory results. These results could also offer insight into lead optimization to enhance inhibitory effects.

Therapeutic Path to Triple Knockout: Investigating the Pan-inhibitory Mechanisms of AKT, CDK9, and TNKS2 by a Novel 2-phenylquinazolinone Derivative in Cancer Therapy- An In-silico Investigation Therapy.

Blocking the oncogenic Wnt//β-catenin pathway has of late been investigated as a viable therapeutic approach in the treatment of cancer. This involves the multi-targeting of certain members of the tankyrase-kinase family; Tankyrase 2 (TNKS2), Protein Kinase B (AKT), and Cyclin- Dependent Kinase 9 (CDK9), which propagate the oncogenic Wnt/β-catenin signalling pathway. During a recent investigation, the pharmacological activity of 2-(4-aminophenyl)-7-chloro- 3H-quinazolin-4-one was repurposed to serve as a 'triple-target' inhibitor of TNKS2, AKT and CDK9. Yet, the molecular mechanism that surrounds its multi-targeting activity remains unanswered. As such, this study aims to explore the pan-inhibitory mechanism of 2-(4-aminophenyl)-7-chloro-3H-quinazolin- 4-one towards AKT, CDK9, and TNKS2, using in silico techniques. Results revealed favourable binding affinities of -34.17 kcal/mol, -28.74 kcal/mol, and -27.30 kcal/mol for 2-(4-aminophenyl)-7-chloro-3H-quinazolin-4-one towards TNKS2, CDK9, and AKT, respectively. Pan-inhibitory binding of 2-(4-aminophenyl)-7-chloro-3H-quinazolin-4-one is illustrated by close interaction with specific residues on tankyrase-kinase. Structurally, 2-(4-aminophenyl)-7-chloro- 3H-quinazolin-4-one had an impact on the flexibility, solvent-accessible surface area, and stability of all three proteins, which was illustrated by numerous modifications observed in the unbound as well as the bound states of the structures, which evidenced the disruption of their biological function. Prediction of the pharmacokinetics and physicochemical properties of 2-(4-aminophenyl)-7-chloro-3H-quinazolin-4- one further established its inhibitory potential, evidenced by the favourable absorption, metabolism, excretion, and minimal toxicity properties. The following structural insights provide a starting point for understanding the paninhibitory activity of 2-(4-aminophenyl)-7-chloro-3H-quinazolin-4-one. Determining the criticality of the interactions that exist between the pyrimidine ring and catalytic residues could offer insight into the structure-based design of innovative tankyrase-kinase inhibitors with enhanced therapeutic effects.

Tankyrase 2 promotes lung cancer cell malignancy.

Tankyrase 2 (TNKS2) is a potential candidate molecular target for the prognosis and treatment of non-small cell lung cancer (NSCLC), but its biological functions are unclear. To investigate the biological functions of TNKS2 in NSCLC. Using a lentiviral vector, we generated H647 model cells with TNKS2 knockdown by RNA interference and A549 model cells with TNKS2 overexpression by transfection with a TNKS2 overexpressing plasmid. Increased and decreased expression levels of TNKS2 in the two cell lines were verified using real-time reverse transcriptase-polymerase chain reaction and Western blot analyses. Cell apoptosis, proliferation, and migration were determined using flow cytometry, carboxyfluorescein succinimidyl ester staining, and scratch assay, respectively. Immunofluorescence staining was conducted to examine TNKS2 and β-catenin expression levels in the two transfected cell lines and the non-transfected cells. TNKS2 mRNA and protein expression was significantly higher in the highly malignant NCI-H647 cells, while it remained at a low level in the less malignant A549 cells. Lentivirus-mediated overexpression of TNKS2 in A549 cells resulted in a 3-fold increase in gene expression and a 1.7-fold increase in protein expression (P < 0.01). Conversely, shRNA interference targeting TNKS2 Led to an 8-fold decrease in gene expression and a 3-fold decrease in protein expression (P < 0.01) in NCI-H647 cells. Furthermore, the cell apoptosis rate was significantly reduced (50%) and cell migration rate was increased (35%) in the TNKS2 overexpression group than in the control group (P < 0.05). In contrast, shTNKS2 promoted apoptosis by more than one fold and reduced migration by 60% (P < 0.05). Immunofluorescence analysis revealed enhanced nuclear localization of β-catenin fluorescence signal associated with high TNKS2 expression levels. Western blot analysis investigating TNKS2/β-catenin-related proteins indicated consistent changes between TNKS2 and β-catenin expression in lung cancer cells, whereas Axin displayed an opposite trend (P < 0.05). The obtained results revealed that TNKS2 may serve as an adverse prognostic factor and a potential therapeutic target in NSCLC.

Design, synthesis and biological activity evaluation of novel inhibitors targeting TNKS2, based on docking, virtual screening and molecular dynamics simulation studies

In recent years, tankyrases (TNKS) have garnered substantial interest as a promising new drug target. An escalating amount of research has been dedicated to the development of TNKS-targeting drugs, highlighting the substantial potential in anti-cancer therapeutics. In this study, a potential TNKS2 lead compound, ZINC11726230, was identified through docking-based virtual screening, molecular dynamics simulation and bioassay. Structural modification of the lead compound, ZINC11726230, yielded two new TNKS2 inhibitors (compound 1-2), which exhibited remarkable inhibitory activity, with an IC50 value of 0.013 μM, outperforming the positive control compound G007-LK (IC50 = 0.035 μM). This study offers unique insights into the discovery of effective TNKS2 inhibitors.

Tankyrase-1 regulates RBP-mediated mRNA turnover to promote muscle fiber formation.

Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.

A novel TNKS/USP25 inhibitor blocks the Wnt pathway to overcome multi-drug resistance in TNKS-overexpressing colorectal cancer

Modulating Tankyrases (TNKS), interactions with USP25 to promote TNKS degradation, rather than inhibiting their enzymatic activities, is emerging as an alternative/specific approach to inhibit the Wnt/β-catenin pathway. Here, we identified UAT-B, a novel neoantimycin analog isolated from Streptomyces conglobatus, as a small-molecule inhibitor of TNKS–USP25 protein–protein interaction (PPI) to overcome multi-drug resistance in colorectal cancer (CRC). The disruption of TNKS–USP25 complex formation by UAT-B led to a significant decrease in TNKS levels, triggering cell apoptosis through modulation of the Wnt/β-catenin pathway. Importantly, UAT-B successfully inhibited the CRC cells growth that harbored high TNKS levels, as demonstrated in various in vitro and in vivo studies utilizing cell line-based and patient-derived xenografts, as well as APCmin/+ spontaneous CRC models. Collectively, these findings suggest that targeting the TNKS–USP25 PPI using a small-molecule inhibitor represents a compelling therapeutic strategy for CRC treatment, and UAT-B emerges as a promising candidate for further preclinical and clinical investigations.

Tankyrase: a promising therapeutic target with pleiotropic action.

Tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) enzymes belong to the poly (ADP-ribose) polymerase (PARP) family participates in process of poly-ADP-ribosylation of different target proteins which leads to ubiquitin-mediated proteasomal degradation. Tankyrases are also involved in the pathophysiology of many diseases, especially cancer. Their functions include cell cycle homeostasis (primarily in mitosis), telomere maintenance, Wnt signaling pathway regulation, and insulin signaling (particularly GLUT4 translocation). Studies have implicated that genetic changes, mutations in the tankyrase coding sequence, or up regulation and down regulation of tankyrase are reflected in the numerous disease conditions. Investigations are pursued to develop putative molecules that target tankyrase in various diseases such as cancer, obesity, osteoarthritis, fibrosis, cherubism, and diabetes, thereby providing a new therapeutic treatment option. In the present review, we described the structure and function of tankyrase along with its role in different disease conditions. Furthermore, we also presented cumulative experimental evidences of different drugs acting on tankyrase.

Identification and evaluation of a novel PARP1 inhibitor for the treatment of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a particularly invasive subtype of breast cancer and usually has a poor prognosis due to the lack of effective therapeutic targets. Approximately 25% of TNBC patients carry a breast cancer susceptibility gene1/2 (BRCA1/2) mutation. Clinically, PARP1 inhibitors have been approved for the treatment of patients with BRCA1/2-mutated breast cancer through the mechanism of synthetic lethality. In this study, we identified compound 6 {systematic name: 2-[2-(4-Hydroxy-phenyl)-vinyl]-3H-quinazolin-4-one} as a novel PARP1 inhibitor from established virtual screening methods. Compound 6 exerted stronger PARP1 inhibitory activity and anti-cancer activity as compared to olaparib in BRCA1-mutated TNBC cells and TNBC patient-derived organoids. Unexpectedly, we found that compound 6 also significantly inhibited cell viability, proliferation, and induced cell apoptosis in BRCA wild-type TNBC cells. To further elucidate the underlying molecular mechanism, we found that tankyrase (TNKS), a vital promoter of homologous-recombination repair, was a potential target of compound 6 by cheminformatics analysis. Compound 6 not only decreased the expression of PAR, but also down-regulated the expression of TNKS, thus resulting in significant DNA single-strand and double-strand breaks in BRCA wild-type TNBC cells. In addition, we demonstrated that compound 6 enhanced the sensitivity of BRCA1-mutated and wild-type TNBC cells to chemotherapy including paclitaxel and cisplatin. Collectively, our study identified a novel PARP1 inhibitor, providing a therapeutic candidate for the treatment of TNBC.

Abstract 5811: Epigenetic regulation of chromosomal instability in triple-negative breast cancer

Abstract Metastasis is the leading cause of cancer-related death among women with breast cancer. Chromosomal instability (CIN) has emerged as a hallmark of triple-negative breast cancer (TNBC) as it has recently been shown to promote metastasis. However, the underlying molecular mechanisms by which CIN drives metastasis are not completely understood. We have identified a discrete population of highly metastatic SOX2/OCT4+ cells expressing elevated levels of epigenetic regulator EZH2 associated with increased CIN in both human and mouse TNBC. EZH2 histone methyl transferase (HMT) is the catalytic subunit of the Polycomb repressive complex 2 (PRC2), represses target genes through trimethylation of Histone 3 at lysine 27 (H3K27me3). Importantly, genetic and pharmacologic inhibition of EZH2 lead to reduction of CIN and impaired metastasis. These findings led to the hypothesis that EZH2-mediated CIN constitutes a novel mechanism of metastasis regulation. To directly demonstrate epigenetic regulation of CIN, we used genome wide-Cleavage Under Targets and Release Using Nuclease (CUT&amp;RUN) in parallel with RNA-seq. Gene set enrichment of EZH2-repressed target genes directly implicated the spindle formation pathway network. The central core of this network comprised of tankyrase (TNKS), a multifunctional poly (ADP-ribose) polymerase (PARP) previously implicated in DNA repair, telomere function and centrosome maturation. ChIP-PCR confirmed that EZH2 directly binds to the TNKS promoter, and CRISPR knockout of TNKS abrogated the ability of EZH2 inhibition in suppressing CIN, consistent with pharmacological inhibition of TNKS. More specifically, dysregulation of TNKS by EZH2 in OCT4/SOX2+ cells lead to increased numbers of centrosomes and multipolar mitosis. To directly demonstrate the role of aberrant H3K27me3 in regulating chromosomal segregation during mitosis, we used dCas9-EZH2 or dCas9-EZH2 catalytically dead mutant together with chromosome-specific CRISPR guides to ectopically enhance H3K27 trimethylation at the pericentromeric region of specific chromosomes. Ectopic deposition of EZH2 at pericentromeric regions lead to increased CIN. Conceptually, our work provides an unappreciated link between epigenetic regulation and CIN which have been hitherto studied in isolation. From a clinical perspective, demonstrating epigenetic regulation of CIN has opened the possibility for the development of first CIN suppressive therapeutic strategies targeting TNBC metastasis. Citation Format: Yang Bai, Albert Agustinus, Cem Meydan, Dylan R. McNally, Shira Yomtoubian, Liron Yoffe, Ari M. Melnick, Samuel Bakhoum, Vivek Mittal. Epigenetic regulation of chromosomal instability in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5811.

New molecular insights for 4H-1,2,4-triazole derivatives as inhibitors of tankyrase and Wnt-signaling antagonist: a molecular dynamics simulation study

Tankyrase (TNKS) enzymes remained central biotargets to treat Wnt-driven colorectal cancers. The success of Olaparib posited the druggability of PARP family enzymes depending on their role in tumor proliferation. In this work, an MD-simulation-based comparative assessment of the protein–ligand interactions using the best-docked poses of three selected compounds (two of the designed and previously synthesized molecules obtained through molecular docking and one reported TNKS inhibitor) was performed for a 500 ns period. The PDB:ID-7KKP and 3U9H were selected for TNKS1 and TNKS2, respectively. The Molecular Mechanics Generalized Born Surface Area (MM-GBSA) based binding energy data exhibited stronger binding of compound-15 (average values of −102.92 and −104.32 kcal/mol for TNKS1 and TNKS2, respectively) as compared to compound-22 (average values of −82.99 and −85.68 kcal/mol for TNKS1 and TNKS2, respectively) and the reported compound-32 (average values of −81.89 and −74.43 kcal/mol for TNKS1 and TNKS2, respectively). Compound-15 and compound-22 exhibited comparable or superior binding to both receptors forming stable complexes when compared to that of compound-32 upon examining their MD trajectories. The key contributors were hydrophobic stacking and optimum hydrogen bonding allowing these molecules to occupy the adenosine pocket by interfacing D-loop residues. The results of bond distance analysis, radius of gyration, root mean square deviation, root mean square fluctuation, snapshots at different time intervals, LUMO–HUMO energy differences, electrostatic potential calculations, and binding free energy suggested better binding efficiency for compound-15 to TNKS enzymes. The computed physicochemical and ADMET properties of compound-15 were encouraging and could be explored further for drug development. Communicated by Ramaswamy H. Sarma

MicroRNA-9-1 Attenuates Influenza A Virus Replication via Targeting Tankyrase 1

An unstable influenza genome leads to the virus resistance to antiviral drugs that target viral proteins. Thus, identification of host factors essential for virus replication may pave the way to develop novel antiviral therapies. In this study, we investigated the roles of the poly(ADP-ribose) polymerase enzyme, tankyrase 1 (TNKS1), and the endogenous small noncoding RNA, miR-9-1, in influenza A virus (IAV) infection. Increased expression of TNKS1 was observed in IAV-infected human lung epithelial cells and mouse lungs. TNKS1 knockdown by RNA interference repressed influenza viral replication. A screen using TNKS1 3′-untranslation region (3′-UTR) reporter assays and predicted microRNAs identified that miR-9-1 targeted TNKS1. Overexpression of miR-9-1 reduced influenza viral replication in lung epithelial cells as measured by viral mRNA and protein levels as well as virus production. miR-9-1 induced type I interferon production and enhanced the phosphorylation of STAT1 in cell culture. The ectopic expression of miR-9-1 in the lungs of mice by using an adenoviral viral vector enhanced type I interferon response, inhibited viral replication, and reduced susceptibility to IAV infection. Our results indicate that miR-9-1 is an anti-influenza microRNA that targets TNKS1 and enhances cellular antiviral state.

Inhibition of GSK3β Promotes Proliferation and Suppresses Apoptosis of Porcine Muscle Satellite Cells

As the global population increases, interest in cultured meat (a new research field) is gradually increasing. The main raw material for the production of cultured meat is muscle stem cells called satellite cells isolated from livestock. However, how to mass proliferate and maintain satellite cells in vitro without genetic manipulation remains unclear. In the present study, we isolated and purified porcine muscle satellite cells (PMSCs) from the femur of a 1-day-old piglet and cultured PMSCs by treating them with an inhibitor (XAV939, Tankyrase (TNKS) inhibitor) or an activator (CHIR99021, glycogen synthase kinase 3 beta (GSK3β) inhibitor) of Wnt signaling. The CHIR group treated with 3 μM CHIR99021 showed a significantly increased proliferation rate of PMSCs compared to the SC group (control), whereas the XAV group treated with 1 μM XAV939 showed a significantly decreased proliferation rate of PMSCs. CHIR99021 also inhibited the differentiation of PMSCs by reducing the expression of MyoD while maintaining the expression of Pax7 and suppressed apoptosis by regulating the expression of apoptosis-related proteins and genes. RNA sequencing was performed to obtain gene expression profiles following inhibition or activation of the Wnt signaling pathway and various signaling mechanisms related to the maintenance of satellite cells were identified. Our results suggest that inhibition of GSK3β could dramatically improve the maintenance and mass proliferation ability of PMSCs in vitro by regulating the expression of myogenic markers and the cell cycle.

Tankyrase Inhibition Attenuates Cardiac Dilatation and Dysfunction in Ischemic Heart Failure.

Hyperactive poly(ADP-ribose) polymerases (PARP) promote ischemic heart failure (IHF) after myocardial infarction (MI). However, the role of tankyrases (TNKSs), members of the PARP family, in pathogenesis of IHF remains unknown. We investigated the expression and activation of TNKSs in myocardium of IHF patients and MI rats. We explored the cardioprotective effect of TNKS inhibition in an isoproterenol-induced zebrafish HF model. In IHF patients, we observed elevated TNKS2 and DICER and concomitant upregulation of miR-34a-5p and miR-21-5p in non-infarcted myocardium. In a rat MI model, we found augmented TNKS2 and DICER in the border and infarct areas at the early stage of post-MI. We also observed consistently increased TNKS1 in the border and infarct areas and destabilized AXIN in the infarct area from 4 weeks onward, which in turn triggered Wnt/β-catenin signaling. In an isoproterenol-induced HF zebrafish model, inhibition of TNKS activity with XAV939, a TNKSs-specific inhibitor, protected against ventricular dilatation and cardiac dysfunction and abrogated overactivation of Wnt/β-catenin signaling and dysregulation of miR-34a-5p induced by isoproterenol. Our study unravels a potential role of TNKSs in the pathogenesis of IHF by regulating Wnt/β-catenin signaling and possibly modulating miRNAs and highlights the pharmacotherapeutic potential of TNKS inhibition for prevention of IHF.

Tankyrase inhibition attenuates cardiac dilatation and dysfunction in ischemic heart failure

Background and Aims : Hyperactive poly(ADP-ribose) polymerases (PARP) promote ischemic heart failure (IHF) after myocardial infarction (MI). However, the role of Tankyrases (TNKSs), members of PARP family, in the progression of IHF remains unknown. We aimed to explore the role of TNKSs in pathophysiological process of IHF and the therapeutic potential of TNKS inhibition for IHF.

Tankyrases inhibit innate antiviral response by PARylating VISA/MAVS and priming it for RNF146-mediated ubiquitination and degradation

During viral infection, sensing of viral RNA by retinoic acid-inducible gene-I-like receptors (RLRs) initiates an antiviral innate immune response, which is mediated by the mitochondrial adaptor protein VISA (virus-induced signal adaptor; also known as mitochondrial antiviral signaling protein [MAVS]). VISA is regulated by various posttranslational modifications (PTMs), such as polyubiquitination, phosphorylation, O-linked β-d-N-acetylglucosaminylation (O-GlcNAcylation), and monomethylation. However, whether other forms of PTMs regulate VISA-mediated innate immune signaling remains elusive. Here, we report that Poly(ADP-ribosyl)ation(PARylation) is a PTM of VISA, which attenuates innate immune response to RNA viruses. Using a biochemical purification approach, we identified tankyrase 1 (TNKS1) as a VISA-associated protein. Viral infection led to the induction of TNKS1 and its homolog TNKS2, which translocated from cytosol to mitochondria and interacted with VISA. TNKS1 and TNKS2 catalyze the PARylation of VISA at Glu137 residue, thereby priming it for K48-linked polyubiquitination by the E3 ligase Ring figure protein 146 (RNF146) and subsequent degradation. Consistently, TNKS1, TNKS2, or RNF146 deficiency increased the RNA virus-triggered induction of downstream effector genes and impaired the replication of the virus. Moreover, TNKS1- or TNKS2-deficient mice produced higher levels of type I interferons (IFNs) and proinflammatory cytokines after virus infection and markedly reduced virus loads in the brains and lungs. Together, our findings uncover an essential role of PARylation of VISA in virus-triggered innate immune signaling, which represents a mechanism to avoid excessive harmful immune response.

Unravelling the Structural Mechanism of Action of 5-methyl-5-[4-(4-oxo-3H-quinazolin-2-yl)phenyl]imidazolidine-2,4-dione in Dual-Targeting Tankyrase 1 and 2: A Novel Avenue in Cancer Therapy.

Tankyrase (TNKS) belonging to the poly(ADPribose) polymerase family, are known for their multi-functioning capabilities, and play an essential role in the Wnt β-catenin pathway and various other cellular processes. Although showing inhibitory potential at a nanomolar level, the structural dual-inhibitory mechanism of the novel TNKS inhibitor, 5-methyl-5-[4-(4-oxo-3H-quinazolin-2-yl)phenyl]imidazolidine-2,4-dione, remains unexplored. By employing advanced molecular modeling, this study provides these insights. Results of sequence alignments of binding site residues identified conserved residues; GLY1185 and ILE1224 in TNKS-1 and PHE1035 and PRO1034 in TNKS-2 as crucial mediators of the dual binding mechanism of 5-methyl-5-[4-(4-oxo-3H-quinazolin-2-yl)phenyl]imidazolidine-2,4-dione, corroborated by high per-residue energy contributions and consistent high-affinity interactions of these residues. Estimation of the binding free energy of 5-methyl-5-[4-(4-oxo-3H-quinazolin-2-yl)phenyl]imidazolidine-2,4-dione showed estimated total energy of -43.88 kcal/mol and -30.79 kcal/mol towards TNKS-1 and 2, respectively, indicating favorable analogous dual binding as previously reported. Assessment of the conformational dynamics of TNKS-1 and 2 upon the binding of 5-methyl-5-[4-(4-oxo-3H-quinazolin-2-yl)phenyl]imidazolidine-2,4-dione revealed similar structural changes characterized by increased flexibility and solvent assessible surface area of the residues inferring an analogous structural binding mechanism. Insights from this study show that peculiar, conserved residues are the driving force behind the dual inhibitory mechanism of 5-methyl-5-[4-(4-oxo-3H-quinazolin-2-yl)phenyl]imidazolidine-2,4-dione and could aid in the design of novel dual inhibitors of TNKS-1 and 2 with improved therapeutic properties.

Knockdown of tankyrase 1 inhibits the progression of gastric adenocarcinoma via regulating human telomerase reverse transcriptase and telomeric repeat binding factor 1.

Gastric cancer is one of the most lethal cancers. Aberrant expression levels of genes are frequently associated with cell immortalization and the occurrence of tumors. In this study, we aimed to investigate the role of tankyrase 1 (TANK1) in gastric adenocarcinoma and clarify the underlying mechanism. The messenger RNA (mRNA) levels of TANK1, human telomerase reverse transcriptase (h-TERT), and telomeric repeat binding factor 1 (TRF1) in clinical specimens and SGC-7901 cells were measured via real-time quantitative polymerase chain reaction (RT-qPCR). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immunohistochemistry (IHC) assays were utilized to observe the cell apoptosis as well as Ki67 and h-TERT expression in tumor-bearing models. The effects of TANK1 antisense oligonucleotides (TANK1 ASODN) on viability and apoptosis of SGC 7901 cells were evaluated by cell counting kit-8 and flow cytometry analysis. We found that TANK1 and h-TERT were both increased in gastric adenocarcinoma, while TRF1 was decreased. Tumor-bearing models demonstrated that TANK1 ASODN appeared to be effective in inhibiting tumor growth and decreasing the expression of h-TERT. Additionally, TANK1 ASODN inhibited the viability and promoted apoptosis of SGC-7901 cells. Moreover, the mRNA levels of h-TERT and TRF1 were modulated by TANK1 ASODN. This study revealed that TANK1 ASODN inhibits the proliferation and induced the apoptosis of gastric adenocarcinoma cells via manipulating the expression levels of h-TERT and TRF1.

The zinc-binding motif in tankyrases is required for the structural integrity of the catalytic ADP-ribosyltransferase domain.

Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on the activity, stability and structure of human tankyrases. We generated mutants of human tankyrase (TNKS) 1 and TNKS2, abolishing the zinc-binding capabilities, and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive, probably due to introduction of major structural defects, the TNKS2 mutant remained active and displayed an altered activity profile compared to the wild-type.

Combinatorial Virtual Screening Revealed a Novel Scaffold for TNKS Inhibition to Combat Colorectal Cancer.

The abnormal Wnt signaling pathway leads to a high expression of β-catenin, which causes several types of cancer, particularly colorectal cancer (CRC). The inhibition of tankyrase (TNKS) activity can reduce cancer cell growth, invasion, and resistance to treatment by blocking the Wnt signaling pathway. A pharmacophore search and pharmacophore docking were performed to identify potential TNKS inhibitors in the training databases. The weighted MM/PBSA binding free energy of the docking model was calculated to rank the databases. The reranked results indicated that 26.98% of TNKS inhibitors that were present in the top 5% of compounds in the database and near an ideal value ranked 28.57%. The National Cancer Institute database was selected for formal virtual screening, and 11 potential TNKS inhibitors were identified. An enzyme-based experiment was performed to demonstrate that of the 11 potential TNKS inhibitors, NSC295092 and NSC319963 had the most potential. Finally, Wnt pathway analysis was performed through a cell-based assay, which indicated that NSC319963 is the most likely TNKS inhibitor (pIC50 = 5.59). The antiproliferation assay demonstrated that NSC319963 can decrease colorectal cancer cell growth; therefore, the proposed method successfully identified a novel TNKS inhibitor that can alleviate CRC.

Tankyrase-1-mediated degradation of Golgin45 regulates glycosyltransferase trafficking and protein glycosylation in Rab2-GTP-dependent manner

Altered glycosylation plays an important role during development and is also a hallmark of increased tumorigenicity and metastatic potentials of several cancers. We report here that Tankyrase-1 (TNKS1) controls protein glycosylation by Poly-ADP-ribosylation (PARylation) of a Golgi structural protein, Golgin45, at the Golgi. TNKS1 is a Golgi-localized peripheral membrane protein that plays various roles throughout the cell, ranging from telomere maintenance to Glut4 trafficking. Our study indicates that TNKS1 localization to the Golgi apparatus is mediated by Golgin45. TNKS1-dependent control of Golgin45 protein stability influences protein glycosylation, as shown by Glycomic analysis. Further, FRAP experiments indicated that Golgin45 protein level modulates Golgi glycosyltransferease trafficking in Rab2-GTP-dependent manner. Taken together, these results suggest that TNKS1-dependent regulation of Golgin45 may provide a molecular underpinning for altered glycosylation at the Golgi during development or oncogenic transformation.