Modulation of Tau Tubulin Kinases (TTBK1 and TTBK2) Impacts Ciliogenesis

Abstract

SUMMARYTau tubulin kinase 1 and 2 (TTBK1 and TTBK2) are highly homologous kinases that are expressed and mediate disease-relevant pathways predominantly in the brain. Distinct roles for TTBK1 and TTBK2 have been delineated. While efforts have been devoted to characterizing the impact to TTBK1 inhibition in diseases like Alzheimer’s disease and amyotrophic lateral sclerosis, TTBK2 inhibition has been less explored. TTBK2 serves a critical function during cilia assembly. Given the biological importance of these kinases, we designed a targeted library from which we identified several chemical tools that engage TTBK1 and TTBK2 in cells and inhibit their downstream signaling. Indolyl pyrimidinamine 10 significantly reduced the expression of primary cilia on the surface of human induced pluripotent stem cells (iPSCs). Furthermore, analog 10 phenocopies TTBK2 KO in iPSCs, confirming an essential role for TTBK2 in ciliogenesis.

Identification of a chemical probe for lipid kinase phosphatidylinositol-5-phosphate 4-kinase gamma (PI5P4Kγ)

“…Based on the assay formats used, which both rely on displacement of an ATP-competitive molecule, we hypothesize that our chemical probe binds to the ATP site of PI5P4Kγ. This is also supported by crystal structures of this scaffold in other kinases (PDB codes: 7JXX, 4IDV, 7ZHQ, and 7ZHP) (Potjewyd et al ., 2022). Comparison of the structures of compounds 2 and 4 confirms that they are closely structurally related, making them a suitable probe – negative control pair.…”

Identification and Utilization of a Chemical Probe to Interrogate the Roles of PIKfyve in the Lifecycle of β-Coronaviruses

“… 17 − 20 Although they involve an unrelated human protein kinase that shares only 21% sequence identity with PIKfyve, recently solved cocrystal structures of AMG28 bound to TTBK1 (PDB: 7JXX and 7ZHN ) demonstrate that the aminopyrimidine ring in AMG28 makes essential hydrogen bonds with the hinge of TTBK1. 21 , 22 These studies suggest that aminopyrimidine could also bind the hinge of PIKfyve. It was suggested by the authors of the Biogen TTBK1 paper that the three methylene groups of the seven-membered ring help maintain the planarity of the molecule rather than making key contacts with the protein.…”

“…In comparison, analogue 25 (methyl, ethyl derivative) and AMG28 demonstrated comparable S 10 (1 μM) scores. A thorough analysis of analogue 26 was driven by interest in its inhibition of off-target kinases, revealing that analogue 26 lacks the requisite selectivity to be considered as a probe candidate . Finally, analogues 20 , 23 , and 24 exhibited intermediate selectivity (11–14 of 403 WT human kinases with PoC < 10 when profiled at 1 μM).…”

“…Compounds were designed to cap the propargylic alcohol of AMG28 with a methyl group (20) and reduce the bulk on the carbon to which the alcohol is attached (21). Next, three analogues that project the alcohol further into the PIKfyve binding pocket via insertion of a methylene group were selected without bulk (22), with maintained substitution of AMG28 (23), and with a sterically hindered alcohol (24). Finally, two derivatives with increased bulk around the carbon bearing the propargylic alcohol were prepared, one with methyl, ethyl substitution (25) and another with diethyl substitution (26).…”

“…A thorough analysis of analogue 26 was driven by interest in its inhibition of off-target kinases, revealing that analogue 26 lacks the requisite selectivity to be considered as a probe candidate. 22 PIKfyve NanoBRET assay. It is worth noting that for analogue 20 there was a larger drop-off in potency when moving from the PIKfyve enzymatic assay to the PIKfyve NanoBRET assay when compared to structurally similar analogues.…”