WIKI4 is a potent inhibitor of Wnt/β-catenin signaling and tankyrase 2 (TNKS2).

WIKI4 inhibitsWnt/?-catenin signaling in all tested cell lines, including A375, DLD1 colorectal cancer, NALM6 B, U2OS osteosarcoma and hESCs. WIKI4 increases the steady-state abundance of theWnt/?-catenin inhibitory protein, AXIN1 by inhibiting tankyrase activity, and thus preventing the ubiquitylation and degradation of AXIN proteins.[1]

Recombinant human wild-type and mutant ALK kinase domain proteins (amino acids 1093–1411) are produced in-house using baculoviral expression, preactivated via autophosphorylation with MgATP, and assayed for kinase activity using a microfluidic mobility shift assay. The reactions contained 1.3 nM wild-type ALK or 0.5 nM mutant ALK (appropriate to produce 15-20% phosphorylation of peptide substrate after 1 h of reaction), 3 μM 5-FAM-KKSRGDYMTMQIG-CONH2), 5 mM MgCl2, and the Km level of ATP in 25 mM Hepes, pH 7.1. The inhibitors are shown to be ATP-competitive from kinetic and crystallographic studies. The Ki values are calculated by fitting the conversion (%) to a competitive inhibition equation. ROS1 enzyme is assayed as described above for ALK, except using 0.25 nM recombinant human ROS1 catalytic domain (amino acids 1883-2347). Kinase inhibitor selectivity is evaluated using a 206-kinase panel.