Tau Tubulin Kinase 1 (TTBK1) is an emerging therapeutic target for neurodegenerative diseases because it promotes the pathological phosphorylation of tau and TDP-43, key drivers of Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). However, the development of selective TTBK1 inhibitors is hindered by the high structural similarity between TTBK1 and the essential paralog TTBK2, making ATP-competitive strategies challenging. Here, we report the discovery of computationally supported type II TTBK1 inhibitors by targeting the inactive DFG-out kinase conformation. A druggable DFG-out model was generated using metadynamics and molecular dynamic simulations and employed for virtual screening of the MBC chemical library. This approach identified novel chemotypes that were validated as selective TTBK1 inhibitors with improved discrimination over TTBK2. The most promising compounds also displayed neuroprotective activity in SH-SY5Y neuroblastoma cells. These findings demonstrate that targeting the inactive TTBK1 conformation is a viable strategy to achieve isoform selectivity and establish a new framework for developing disease-modifying therapies for ALS, AD, and other TTBK1-driven neurodegenerative disorders.
