Figure 4.

ITCH inhibitions by structural analogs of Clomipramine. (a–e) GST-ITCH was incubated with the indicated concentrations of the indicated structural analogs (A1 to A17) of clomipramine (CL) in ubiquitylation assay buffer. The reactions were resolved by SDS-PAGE and stained with Coomassie. As a control, E2 was excluded from the reaction. The intensity of nonubiquitylated GST-ITCH band was quantified. (f) Clomipramine molecular moieties important for ITCH inhibition. The presence of a Chloride (circle in red) on the Benzene ring increases the inhibitory activity of the compounds tested by roughly fivefold or more. The length of the side chain is also critical. Shorter or longer chains dramatically decrease the inhibitor effect. The Amine number 1 can be substituted by a C=C double bond, without affecting the inhibition capacity of the compound. The extent of methylation of amine number 2 affects the inhibitor effect of the compounds tested. A dimethylamine group in this position decreases the inhibitory potency by a factor of two compared with a monomethylamine