Potent and Cell Active 5-Azaindole-based Tau Tubulin Kinase Inhibitors Disrupt Ciliogenesis

We have characterized a potent and cell active tau tubulin kinase 1 and 2 (TTBK1 and TTBK2) inhibitor, 13. Analysis of the in-cell, kinome-wide selectivity of compound 13 revealed it to only bind five of 300 kinases with >50% occupancy at 1 μM: CDK11B, CDK11A, CK1δ, CK1ε, and EPHA2. Extensive dose−response follow-up studies in cells refined the off-target kinases of compound 13 to include CDK11B, CDK11A, CK1δ, and PIKfyve based on their NanoBRET IC50 values being <1 μM. As part of our medicinal chemistry campaign, we also identified a structurally similar negative control, compound 5, which lacks in-cell affinity for TTBK1 and TTBK2. TTBK2 is an effector of ciliogenesis, acting in concert with CEP164, CP110, and CEP83 to initiate the biogenesis of primary cilia. We demonstrate that compound 13 ablates TTBK2 autophosphorylation and significantly disrupts ciliogenesis in hTERT-RPE-1 cells, making it a powerful tool compound for ciliary studies.