Selective inhibition of human tankyrases

Tankyrases belong to the Diphtheria toxin-like ADP-ribosyltransferase (ARTD) enzyme superfamily, also known as poly(ADP-ribose) polymerases (PARPs). They catalyze a covalent post-translational modification reaction where they transfer ADP-ribose units from NAD+ to target proteins. Tankyrases are involved in many cellular processes and their roles in telomere homeostasis, Wnt signaling and in several diseases including cancers have made them interesting drug targets. In this thesis project, selective inhibition of human tankyrases was studied. A homogeneous fluorescence-based assay was developed to screen the compound libraries. The assay is inexpensive, operationally easy, and performs well according to the statistical analysis. Assay suitability was confirmed by screening a natural product library. Flavone was identified as the most potent inhibitor in the library and this motivated us to screen a larger flavonoid library. Results showed that flavones were indeed the best inhibitor of tankyrases among flavonoids. To further study the structure-activity relationship, a small library of flavones containing single substitution was screened and potency measurements allowed us to generate structure-activity relationship. Compounds containing substitutions at 4´-position were more potent in comparison to other substitutions, and importantly, hydrophobic groups improved isoenzyme selectivity as well as the potency. A flavone derivative containing a hydrophobic isopropyl group (compound 22), displayed 6 nM potency against TNKS1, excellent isoenzyme selectivity and Wnt signaling inhibition. Protein interactions with compounds were studied by solving complex crystal structures of the compounds with TNKS2 catalytic domain. A novel tankyrase inhibitor (IWR-1) was also crystallized in complex with TNKS2 catalytic domain. The crystal structure of TNKS2 in complex with IWR-1 showed that the compound binds to adenosine site and it was the first known ARTD inhibitor of this kind. To date, there is no structural information available about the substrate binding with any of the ARTD family members; therefore NAD+ was soaked with TNKS2 catalytic domain crystals. However, analysis of crystal structure showed that NAD+ was hydrolyzed to nicotinamide. Also, a co-crystal structure of NAD+ mimic compound, EB-47, was solved which was used to deduce some insights about the substrate interactions with the enzyme. Like EB-47, other ARTD1 inhibitors were also shown to inhibit tankyrases. It indicated that selectivity of the ARTD1 inhibitors should be considered as some of the effects in cells could come from tankyrase inhibition. In conclusion, the study provides novel information on tankyrase inhibition and presents new insight into the selectivity and potency of compounds

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

TbPARG in Trypanosoma brucei. A) TbPARG localization in untreated (control) and in procyclic cultures exposed to 500 μM H2O2 for 10 min. IFI was carried out as reported in our previous work [33]. TbPARG was identified with our home-made antibody against TcPARG [33]; and PAR was identified with a commercial antibody against PAR (BD). White bar represents 50 μm. B) Western blot analysis of 40 μg protein per lane revealed with a commercial anti-PARG antibody (Antibody Verify) in T. brucei procyclic (PC) and bloodstream (BST) forms. The arrow indicates the band with the expected molecular weight (approximately 60 kDa). The membrane stained with Red Ponceau was used as a loading control. (TIF 4272 kb

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

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/β-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.</p

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

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

Digitaliseringens inverkan på redovisningsarbetet : En kvalitativ studie om redovisningsproduktens kvalité

Digitaliseringen är ett aktuellt ämne i dagens samhälle, och den snabba utvecklingen gör att redovisningsbranschen utmanas i allt större utsträckning än tidigare. Till följd av detta har det uppstått omfattande förändringar i redovisningskonsulternas dagliga arbete vilket påverkar redovisningsbranschen i stort. Syftet med studien var därmed att undersöka och analysera om en ökad digitalisering av redovisningsarbetet försämrar kvalitén av den produkt som redovisningskonsulter skapar. En empirisk undersökning har genomförts med hjälp av kvalitativ metod, där intervjuer med erfarna redovisningskonsulter skapat grunden för arbetet. För att ytterligare stärka det empiriska underlaget samlades sekundärdata in från de aktuella redovisningsbyråernas hemsidor. En jämförelse med den tidigare forskningen inom området gjordes sedan, där studiens resultat analyserades och tolkningar gavs. Resultaten ansågs inte ge tillräckligt starka indikationer för att digitaliseringen påverkar kvalitén av den produkt som redovisningskonsulter skapar. Därav är slutsatsen för denna studie att redovisningsproduktens kvalité inte tenderar att försämras av de digitaliserade processerna inom redovisningsyrket.The digitization is a current topic in today's society, and the rapid progress creates greater challenges than before for the accounting industry. Due to this, significant changes have occurred in the accountants daily tasks which affects the industry in general. Thus, the purpose of the study was to investigate and analyze whether an increased digitization of the accounting profession deteriorates the quality of the product that accountants create. An empirical research has also been conducted using qualitative methods, where interviews with senior accountants create the basis for the study. To further strengthen the empirical data, secondary data was collected from the accounting bureaus websites. A comparison was then made with the previous research on the subject, whereupon the results of the study were analyzed and interpreted. The results of the study were not considered strong enough to provide indications that digitization affects the quality of the products that accountants create. Therefore, the conclusion of the study is that the product of the accounting does not seem to be impaired by the digitization within the accounting profession

Potency of WIKI4 against TNKS1.

<p>The <i>in vitro</i> dose response curves were measured three times with a fluorescence-based homogenous activity assay.</p

Structure of TNKS2 ARTD domain.

<p>Acceptor and donor NAD⁺ binding sites, including nicotinamide subsite (NI) and adenosine subsite (ADE) are labelled.</p

Profiling of inhibitor selectivity.

<p>The selectivity of WIKI4 against 8 ARTDs polymerases was measured at 10 µM concentration. XAV939 and IWR-1 were used as controls.</p