Optimization of a Screening Hit toward M2912, an Oral Tankyrase Inhibitor with Antitumor Activity in Colorectal Cancer Models.

Constitutive activation of the canonical Wnt signaling pathway, in most cases driven by inactivation of the tumor suppressor APC, is a hallmark of colorectal cancer. Tankyrases are druggable key regulators in these malignancies and are considered as attractive targets for therapeutic interventions, although no inhibitor has been progressed to clinical development yet. We continued our efforts to develop tankyrase inhibitors targeting the nicotinamide pocket with suitable drug-like properties for investigating effects of Wnt pathway inhibition on tumor growth. Herein, the identification of a screening hit series and its optimization through scaffold hopping and SAR exploration is described. The systematic assessment delivered M2912, a compound with an optimal balance between excellent TNKS potency, exquisite PARP selectivity, and a predicted human PK compatible with once daily oral dosing. Modulation of cellular Wnt pathway activity and significant tumor growth inhibition was demonstrated with this compound in colorectal xenograft models in vivo.

In silico insights on tankyrase protein: A potential target for colorectal cancer.

The Wnt/ß-catenin pathway plays an important regulatory role in cancer signaling and cell regenerative mechanisms. Its suppression has long been considered as an important challenge of anticancer treatment and management. The poly(ADP-ribose) polymerase (PARP) family represented as a new class of therapeutic targets with diverse potential disease indications. Tankyrase (TNKS) is considered to be a potential target for the intervention of various cancers. The main objective of the work is to explore the molecular and quantum mechanics of the drug-like compounds and to identify the potential inhibitors for TNKS protein using the structure and ligand-based virtual screening from several databases and to explore the binding pocket and interactions of active residues. The screened compounds were further filtered using binding-free energy calculation and molecular dynamics simulation studies. The results have provided a strong molecular knowledge of TNKS and offered top hit potent inhibitors. The identified lead compounds LC_40781, LC_40777, LC_39767, LC_8346, NCI_682438, and NCI_721141 were observed to have potent activity against TNKS protein. The hydrogen bonding of compounds with Asp1198, His1201, Tyr1203 in TNKS1 and Gly1032, Ser1068 in TNKS2 are the key interactions plays a major role in binding energy. Therefore, the outcome of the study would help for further validation and provides valuable information to guide the future TNKS-specific inhibitor designing. Communicated by Ramaswamy H. Sarma.

In silico studies on potential TNKS inhibitors: a combination of pharmacophore and 3D-QSAR modelling, virtual screening, molecular docking and molecular dynamics.

Maladjustment of the Wnt-signalling pathway can lead to a variety of cancers. Axin can be expressed stably through the Tankyrases (TNKS) inhibitor, thus inducing the degradation of ß-catenin, antagonizing the Wnt-signalling pathway and consequently inhibiting tumour growth. Thus, TNKS has become a hot research target for anticancer drugs, and a systematic study of tetrazoloquinoxaline analogues as TNKS inhibitors is of considerable value. In this paper, three-dimensional (3D)-quantitative structure-activity relationship (QSAR), molecular docking and DISCOtech were applied to study a series of tetrazoloquinoxaline and establish a good comparative molecular field analysis (CoMFA) (q2 = 0.701, r2 = 0.968 and rpred2 = 0.754), comparative molecular similarity index analysis (CoMSIA) (q2 = 0.572, r2 = 0.991 and rpred2 = 0.721) and Topomer CoMFA (q2 = 0.692, r2 = 0.979 and rpred2 = 0.532) models, which offer high predictability. The effect of steric hindrance, electrostatic interaction, hydrophobic interaction and hydrogen-bonding acceptor of the molecular group on molecular activity was revealed through contour map. Molecular docking revealed the binding pattern between acceptor and ligand and determined that the effect of hydrogen-bond interaction between the inhibitor and residue GLY1032 was critical to the molecular activity. The pharmacophore features were consistent with the contour map and the molecular-docking result. Through filtering the ZINC database (including 8777 micro-molecule structures), we obtained candidate compounds TS1 and TS2, which exhibit a novel scaffold structure and potential inhibitory activities against TNKS. Molecular docking and molecular dynamics studies of compounds TS1 and TS2 were used to confirm that the binding interaction between ligand and receptor is mainly hydrogen bonding between the compound and residue GLY1032, as well as the hydrophobic interaction with TYR1071. Molecular dynamics studies showed that compounds TS1 and TS2 can stably bind with receptors. These results provide favourable theoretical guidance for the development of novel TNKS inhibitors.

Identification by Inverse Virtual Screening of magnolol-based scaffold as new tankyrase-2 inhibitors.

The natural product magnolol (1) and a selection of its bioinspired derivatives 2-5, were investigated by Inverse Virtual Screening in order to identify putative biological targets from a panel of 308 proteins involved in cancer processes. By this in silico analysis we selected tankyrase-2 (TNKS2), casein kinase 2 (CK2) and bromodomain 9 (Brd9) as potential targets for experimental evaluations. The Surface Plasmon Resonance assay revealed that 3-5 present a good affinity for tankyrase-2, and, in particular, 3 showed an antiproliferative activity on A549 cells higher than the well-known tankyrase-2 inhibitor XAV939 used as reference compound.

Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach.

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC50 values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.

Discovery of AZ0108, an orally bioavailable phthalazinone PARP inhibitor that blocks centrosome clustering.

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca's collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

Discovery of potent and selective nonplanar tankyrase inhibiting nicotinamide mimics.

Diphtheria toxin-like ADP-ribosyltransferases catalyse a posttranslational modification, ADP-ribosylation and form a protein family of 17 members in humans. Two of the family members, tankyrases 1 and 2, are involved in several cellular processes including mitosis and Wnt/ß-catenin signalling pathway. They are often over-expressed in cancer cells and have been linked with the survival of cancer cells making them potential therapeutic targets. In this study, we identified nine tankyrase inhibitors through virtual and in vitro screening. Crystal structures of tankyrase 2 with the compounds showed that they bind to the nicotinamide binding site of the catalytic domain. Based on the co-crystal structures we designed and synthesized a series of tetrahydroquinazolin-4-one and pyridopyrimidin-4-one analogs and were subsequently able to improve the potency of a hit compound almost 100-fold (from 11 µM to 150 nM). The most potent compounds were selective towards tankyrases over a panel of other human ARTD enzymes. They also inhibited Wnt/ß-catenin pathway in a cell-based reporter assay demonstrating the potential usefulness of the identified new scaffolds for further development.

The antimitotic potential of PARP inhibitors, an unexplored therapeutic alternative.

ADP-ribosylation or PARsylation is one of the most abundant modifications of proteins and DNA. Although the usual context for PARsylation involves the detection and repair of DNA damage in the cell, poly(ADP-ribose) polymerases are known to regulate a number of biological processes besides maintaining genome integrity. One of these processes is the assembly and maintenance of the mitotic spindle where the presence of PARP-1 and tankyrase 1 (TNKS1), two of the best-characterized members of the PARP superfamily, is of critical importance. Here, we recapitulate the biological implications of the absence of poly(ADP-ribose) polymerases and depletion of PARsylation occurrence in mitosis in order to better understand the antimitotic effects of PARP inhibitors. In this regard, we also present an overview of the existing and more relevant molecules, with a special attention to the historical development of their pharmacological properties and structures, as well as a brief summary of clinical trials involving PARP inhibitors.

Screening and structural analysis of flavones inhibiting tankyrases.

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

A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor.

The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.