A virtual screening procedure was applied to identify new tankyrase inhibitors. Through pharmacophore screening of a compounds collection from the SPECS database, the methoxy[l]benzothieno[2,3-c]qui-nolin-6(5H)-one scaffold was identified as nicotinamide mimetic able to inhibit tankyrase activity at low micromolar concentration. In order to improve potency and selectivity, tandem structure-based and scaffold hopping approaches were carried out over the new scaffold leading to the discovery of the 2-(phenyl)-3H-benzo[4,5]thieno[3,2-d]pyrimidin-4-one as powerful chemotype suitable for tankyrase in-hibition. The best compound 2-(4-tert-butyl-phenyl)-3H-benzo[4,5]thieno[3,2-d]pyrimidin-4-one (23) displayed nanomolar potencies (IC 50s TNKS-1 ¼ 21 nM and TNKS-2 ¼ 29 nM) and high selectivity when profiled against several other PARPs. Furthermore, a striking Wnt signaling, as well as cell growth in-hibition, was observed assaying 23 in DLD-1 cancer cells.