Casein kinase 1 (CK1) is an evolutionarily conserved protein kinase family among eukaryotes. Studies in non-plants have shown CK1-dependent divergent biological processes, but the collective knowledge regarding the biological roles of plant CK1 lags far behind other members of the Eukarya. One reason for this is that plants have many more genes encoding CK1 than do animals. To accelerate our understanding of the plant CK1 family, a strong CK1 inhibitor that efficiently inhibits multiple members of the CK1 protein family in vivo (i.e., in planta) is required. Here, we report a novel, specific, and effective CK1 inhibitor in Arabidopsis. Using circadian period-lengthening activity as an estimation of the CK1 inhibitor effect in vivo, we performed a structure–activity relationship study of analogues of the CK1 inhibitor PHA767491 (1,5,6,7-tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-one hydrochloride). A propargyl group at the pyrrole nitrogen atom (AMI-212) or a bromine atom at the pyrrole C3 position (AMI-23) had stronger CK1 inhibitory activity than PHA767491. A hybrid molecule of AMI-212 and AMI-23 (AMI-331) was about 100-fold more inhibitory than the parent molecule PHA767491. Affinity proteomics using an AMI-331 probe showed that the targets of AMI-331 inhibition are mostly CK1 kinases. As such, AMI-331 is a potent and selective CK1 inhibitor that shows promise in the research of CK1 in plants.
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