The atypical protein kinase C-iota (PKC-ι) enzyme is implicated in various cancers and has been put forward as an attractive target for developing anticancer therapy. A high concentration biochemical screen identified pyridine fragment weakly inhibiting PKC-ι with IC50 = 424 μM. Driven by structure-activity relationships and guided by docking hypothesis, the weakly bound fragment was eventually optimized into a potent inhibitor of PKC-ι (IC50= 270 nM). Through the course of the optimization, an intermediate compound was crystallized with the protein, and careful analysis of the X-ray crystal structure revealed a unique binding mode involving the post-kinase domain (C-terminal tail) of PKC-ι.