title: Homogeneous screening assay for human tankyrase.
abstract: 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.
authors.affiliation: Abo Akademi University, FI, Turku, Finland.
authors.forename: Mohit
authors.initials: M
authors.lastname: Narwal
authors.forename: Adyary
authors.initials: A
authors.lastname: Fallarero
authors.forename: Pia
authors.initials: P
authors.lastname: Vuorela
authors.forename: Lari
authors.initials: L
authors.lastname: Lehtiö
mesh_terms: D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D054852:Small Molecule Libraries / Q000494:pharmacology; D038501:Tankyrases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D038501:Tankyrases