In vivo structure-activity relationship studies support allosteric targeting of a dual specificity phosphatase.

Dual specificity phosphatase 6 (DUSP6) functions as a feedback attenuator of fibroblast growth factor signaling during development. In vitro high throughput chemical screening attempts to discover DUSP6 inhibitors have yielded limited success. However, in vivo whole-organism screens of zebrafish identified compound 1 (BCI) as an allosteric inhibitor of DUSP6. Here we designed and synthesized a panel of analogues to define the structure-activity relationship (SAR) of DUSP6 inhibition. In vivo high-content analysis in transgenic zebrafish, coupled with cell-based chemical complementation assays, identified structural features of the pharmacophore of 1 that were essential for biological activity. In vitro assays of DUSP hyperactivation corroborated the results from in vivo and cellular SAR. The results reinforce the notion that DUSPs are druggable through allosteric mechanisms and illustrate the utility of zebrafish as a model organism for in vivo SAR analyses.