Kinome chemoproteomics characterization of pyrrolo[3,4-c]pyrazoles as potent and selective inhibitors of glycogen synthase kinase 3

Glycogen synthase kinase 3 has evolutionarily conserved roles in cell signaling and metabolism and is a recognized drug target in neurological pathologies, most prominently bipolar disorder. More recently it has been suggested that GSK3 may be a target for the treatment of trypanosomatid parasite infections, e.g. with T. brucei, due to the lethal phenotype observed in parasite GSK3 short RNAi knockdown experiments. Here we investigated the kinome selectivity of a library of pyrrolo[3,4-c]pyrazol inhibitors that were developed against T. brucei GSK3 but that also interact with the human orthologue and other protein kinases. We applied label-free MS-based kinome chemoproteomics profiling with kinobeads to obtain the selectivity profiles of all 39 library members against 217 human protein and lipid kinases. This allowed us to study the structure-activity relationship of the library members as well as the chemical genetic relationships between kinase targets. As a result, we identified a novel and highly selective HsGSK3 inhibitor containing a 2-chloroaniline-substituted squaric acid amide pharmacophore that confers low nanomolar (IC50 = 2.8 nM) and sub-micromolar potency against purified and cellular HsGSK3. The inhibitor will be useful as a new lead for GSK3 inhibitor development and as a chemical genetic probe to study roles of GSK3 in cell signaling.