Crystal Structures and Inhibitor Interactions of Mouse and Dog MTH1 Reveal Species-Specific Differences in Affinity.

MTH1 hydrolyzes oxidized nucleoside triphosphates, thereby sanitizing the nucleotide pool from oxidative damage. This prevents incorporation of damaged nucleotides into DNA, which otherwise would lead to mutations and cell death. The high level of reactive oxygen species in cancer cells leads to a higher level of oxidized nucleotides in cancer cells compared to that in nonmalignant cells, making cancer cells more dependent on MTH1 for survival. The possibility of specifically targeting cancer cells by inhibiting MTH1 has highlighted MTH1 as a promising cancer target. The progression of MTH1 inhibitors into the clinic requires animal studies, and knowledge of species differences in the potency of inhibitors is vitally important. We here show that the human MTH1 inhibitor TH588 is approximately 20-fold less potent with respect to inhibition of mouse MTH1 than the human, rat, pig, and dog MTH1 proteins are. We present the crystal structures of mouse MTH1 in complex with TH588 and dog MTH1 and elucidate the structural and sequence basis for the observed difference in affinity for TH588. We identify amino acid residue 116 in MTH1 as an important determinant of TH588 affinity. Furthermore, we present the structure of mouse MTH1 in complex with the substrate 8-oxo-dGTP. The crystal structures provide insight into the high degree of structural conservation between MTH1 proteins from different organisms and provide a detailed view of interactions between MTH1 and the inhibitor, revealing that minute structural differences can have a large impact on affinity and specificity.

Exploration of the nicotinamide-binding site of the tankyrases, identifying 3-arylisoquinolin-1-ones as potent and selective inhibitors in vitro.

Tankyrases-1 and -2 (TNKS-1 and TNKS-2) have three cellular roles which make them important targets in cancer. Using NAD(+) as a substrate, they poly(ADP-ribosyl)ate TRF1 (regulating lengths of telomeres), NuMA (facilitating mitosis) and axin (in wnt/ß-catenin signalling). Using molecular modelling and the structure of the weak inhibitor 5-aminoiso quinolin-1-one, 3-aryl-5-substituted-isoquinolin-1-ones were designed as inhibitors to explore the structure-activity relationships (SARs) for binding and to define the shape of a hydrophobic cavity in the active site. 5-Amino-3-arylisoquinolinones were synthesised by Suzuki-Miyaura coupling of arylboronic acids to 3-bromo-1-methoxy-5-nitro-isoquinoline, reduction and O-demethylation. 3-Aryl-5-methylisoquinolin-1-ones, 3-aryl-5-fluoroisoquinolin-1-ones and 3-aryl-5-methoxyisoquinolin-1-ones were accessed by deprotonation of 3-substituted-N,N,2-trimethylbenzamides and quench with an appropriate benzonitrile. SAR around the isoquinolinone core showed that aryl was required at the 3-position, optimally with a para-substituent. Small meta-substituents were tolerated but groups in the ortho-positions reduced or abolished activity. This was not due to lack of coplanarity of the rings, as shown by the potency of 4,5-dimethyl-3-phenylisoquinolin-1-one. Methyl and methoxy were optimal at the 5-position. SAR was rationalised by modelling and by crystal structures of examples with TNKS-2. The 3-aryl unit was located in a large hydrophobic cavity and the para-substituents projected into a tunnel leading to the exterior. Potency against TNKS-1 paralleled potency against TNKS-2. Most inhibitors were highly selective for TNKSs over PARP-1 and PARP-2. A range of highly potent and selective inhibitors is now available for cellular studies.

Targeting virulence regulation to disarm Acinetobacter baumannii pathogenesis.

The development of anti-virulence drug therapy against Acinetobacter baumannii infections would provide an alternative to traditional antibacterial therapy that are increasingly failing. Here, we demonstrate that the OmpR transcriptional regulator plays a pivotal role in the pathogenesis of diverse A. baumannii clinical strains in multiple murine and G. mellonella invertebrate infection models. We identified OmpR-regulated genes using RNA sequencing and further validated two genes whose expression can be used as robust biomarker to quantify OmpR inhibition in A. baumannii. Moreover, the determination of the structure of the OmpR DNA binding domain of A. baumannii and the development of in vitro protein-DNA binding assays enabled the identification of an OmpR small molecule inhibitor. We conclude that OmpR is a valid and unexplored target to fight A. baumannii infections and we believe that the described platform combining in silico methods, in vitro OmpR inhibitory assays and in vivo G. mellonella surrogate infection model will facilitate future drug discovery programs.

Small-Molecule Chemical Probe Rescues Cells from Mono-ADP-Ribosyltransferase ARTD10/PARP10-Induced Apoptosis and Sensitizes Cancer Cells to DNA Damage.

Members of the human diphtheria toxin-like ADP-ribosyltransferase (ARTD or PARP) family play important roles in regulating biological activities by mediating either a mono-ADP-ribosylation (MARylation) of a substrate or a poly-ADP-ribosylation (PARylation). ARTD10/PARP10 belongs to the MARylating ARTDs (mARTDs) subfamily, and plays important roles in biological processes that range from cellular signaling, DNA repair, and cell proliferation to immune response. Despite their biological and disease relevance, no selective inhibitors for mARTDs are available. Here we describe a small-molecule ARTD10 inhibitor, OUL35, a selective and potent inhibitor for this enzyme. We characterize its selectivity profile, model its binding, and demonstrate activity in HeLa cells where OUL35 rescued cells from ARTD10 induced cell death. Using OUL35 as a cell biology tool we show that ARTD10 inhibition sensitizes the cells to the hydroxyurea-induced genotoxic stress. Our study supports the proposed role of ARTD10 in DNA-damage repair and provides a tool compound for selective inhibition of ARTD10-mediated MARylation.

Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei.

BACKGROUND: Poly(ADP-ribose) (PAR) metabolism participates in several biological processes such as DNA damage signaling and repair, which is a thoroughly studied function. PAR is synthesized by Poly(ADP-ribose) polymerase (PARP) and hydrolyzed by Poly(ADP-ribose) glycohydrolase (PARG). In contrast to human and other higher eukaryotes, Trypanosoma brucei contains only one PARP and PARG. Up to date, the function of these enzymes has remained elusive in this parasite. The aim of this work is to unravel the role that PAR plays in genotoxic stress response. METHODS: The optimal conditions for the activity of purified recombinant TbPARP were determined by using a fluorometric activity assay followed by screening of PARP inhibitors. Sensitivity to a genotoxic agent, H2O2, was assessed by counting motile parasites over the total number in a Neubauer chamber, in presence of a potent PARP inhibitor as well as in procyclic transgenic lines which either down-regulate PARP or PARG, or over-express PARP. Triplicates were carried out for each condition tested and data significance was assessed with two-way Anova followed by Bonferroni test. Finally, PAR influence was studied in cell death pathways by flow cytometry. RESULTS: Abolition of a functional PARP either by using potent inhibitors present or in PARP-silenced parasites had no effect on parasite growth in culture; however, PARP-inhibited and PARP down-regulated parasites presented an increased resistance against H2O2 treatment when compared to their wild type counterparts. PARP over-expressing and PARG-silenced parasites displayed polymer accumulation in the nucleus and, as expected, showed diminished resistance when exposed to the same genotoxic stimulus. Indeed, they suffered a necrotic death pathway, while an apoptosis-like mechanism was observed in control cultures. Surprisingly, PARP migrated to the nucleus and synthesized PAR only after a genomic stress in wild type parasites while PARG occurred always in this organelle. CONCLUSIONS: PARP over-expressing and PARG-silenced cells presented PAR accumulation in the nucleus, even in absence of oxidative stress. Procyclic death pathway after genotoxic damage depends on basal nuclear PAR. This evidence demonstrates that the polymer may have a toxic action by itself since the consequences of an exacerbated PARP activity cannot fully explain the increment in sensitivity observed here. Moreover, the unusual localization of PARP and PARG would reveal a novel regulatory mechanism, making them invaluable model systems.

Hypoxic Signaling and the Cellular Redox Tumor Environment Determine Sensitivity to MTH1 Inhibition.

Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific nononcogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in nonmalignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathologic upregulation of the HIF1α response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance. Cancer Res; 76(8); 2366-75. ©2016 AACR.

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.

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.

Structural basis and selectivity of tankyrase inhibition by a Wnt signaling inhibitor WIKI4.

Recently a novel inhibitor of Wnt signaling was discovered. The compound, WIKI4, was found to act through tankyrase inhibition and regulate ß-catenin levels in many cancer cell lines and human embryonic stem cells. Here we confirm that WIKI4 is a high potency tankyrase inhibitor and that it selectively inhibits tankyrases over other ARTD enzymes tested. The binding mode of the compound to tankyrase 2 was determined by protein X-ray crystallography to 2.4 Å resolution. The structure revealed a novel binding mode to the adenosine subsite of the donor NAD(+) binding groove of the catalytic domain. Our results form a structural basis for further development of potent and selective tankyrase inhibitors based on the WIKI4 scaffold.

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 tankyrase inhibiting flavones with increased potency and isoenzyme selectivity.

Tankyrases are ADP-ribosyltransferases that play key roles in various cellular pathways, including the regulation of cell proliferation, and thus, they are promising drug targets for the treatment of cancer. Flavones have been shown to inhibit tankyrases and we report here the discovery of more potent and selective flavone derivatives. Commercially available flavones with single substitutions were used for structure-activity relationship studies, and cocrystal structures of the 18 hit compounds were analyzed to explain their potency and selectivity. The most potent inhibitors were also tested in a cell-based assay, which demonstrated that they effectively antagonize Wnt signaling. To assess selectivity, they were further tested against a panel of homologous human ADP-ribosyltransferases. The most effective compound, 22 (MN-64), showed 6 nM potency against tankyrase 1, isoenzyme selectivity, and Wnt signaling inhibition. This work forms a basis for rational development of flavones as tankyrase inhibitors and guides the development of other structurally related inhibitors.

Structural basis of selective inhibition of human tankyrases.

Tankyrases are poly(ADP-ribose) polymerases that have many cellular functions. They play pharmaceutically important roles, at least in telomere homeostasis and Wnt signaling, by covalently ADP-ribosylating target proteins and consequently regulating their functions. These features make tankyrases potential targets for treatment of cancer. We report here crystal structures of human tankyrase 2 catalytic fragment in complex with a byproduct, nicotinamide, and with selective inhibitors of tankyrases (IWR-1) and PARPs 1 and 2 (olaparib). Binding of these inhibitors to tankyrase 2 induces specific conformational changes. The crystal structures explain the selectivity of the inhibitors, reveal the flexibility of a substrate binding loop, and explain existing structure-activity relationship data. The first crystal structure of a PARP enzyme in complex with a potent inhibitor, IWR-1, that does not bind to the widely utilized nicotinamide-binding site makes the structure valuable for development of PARP inhibitors in general.

Homogeneous screening assay for human tankyrase.

Tankyrase, a member of human PARP protein superfamily, catalyzes a covalent post-translational modification of substrate proteins. This modification, poly(ADP-ribos)ylation, leads to changes in protein interactions and modifies downstream signaling events. Tankyrase 1 is a potential drug target due to its functions in telomere homeostasis and in Wnt signaling. We describe here optimization and application of an activity-based homogenous assay for tankyrase inhibitors in a high-throughput screening format. The method measures the consumption of substrate by the chemical conversion of the remaining NAD(+) into a stable fluorescent condensation product. Conditions were optimized to measure the enzymatic auto-modification of a recombinant catalytic fragment of tankyrase 1. The fluorescence assay is inexpensive, operationally easy and performs well according to the statistical analysis (Z'= 0.7). A validatory screen with a natural product library confirmed suitability of the assay for finding new tankyrase inhibitors. Flavone was the most potent (IC(50)=325 nM) hit from the natural compounds. A flavone derivative, apigenin, and isopropyl gallate showed potency on the micromolar range, but displayed over 30-fold selectivity for tankyrase over the studied isoenzymes PARP1 and PARP2. The assay is robust and will be useful for screening new tankyrase inhibitors.

Inhibition of poly(ADP-ribose) polymerase interferes with Trypanosoma cruzi infection and proliferation of the parasite.

Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.