Development and Characterization of Type I, Type II, and Type III LIM-Kinase Chemical Probes

LIMKs are important regulators of actin and microtubule
dynamics,
and they play essential roles in many cellular processes. Deregulation
of LIMKs has been linked to the development of diverse diseases, including
cancers and cognitive disabilities, but well-characterized inhibitors
known as chemical probes are still lacking. Here, we report the characterization
of three highly selective LIMK1/2 inhibitors covering all canonical
binding modes (type I/II/III) and the structure-based design of the
type II/III inhibitors. Characterization of these chemical probes
revealed a low nanomolar affinity for LIMK1/2, and all inhibitors 1 (LIMKi3; type I), 48 (TH470; type II), and 15 (TH257; type III) showed
excellent selectivity in a comprehensive scanMAX kinase selectivity
panel. Phosphoproteomics revealed remarkable differences between type
I and type II inhibitors compared with the allosteric inhibitor 15. In phenotypic assays such as neurite outgrowth models
of fragile X-chromosome, 15 showed promising activity,
suggesting the potential application of allosteric LIMK inhibitors
treating this orphan disease.