Molecular Mechanism of Mutational Disruption of DCLK1 Autoinhibition Provides a Rationale for Inhibitor Screening.

Doublecortin-like kinase 1 (DCLK1) is a prominent kinase involved in carcinogenesis, serving as a diagnostic marker for early cancer detection and prevention, as well as a target for cancer therapy. Extensive research efforts have been dedicated to understanding its role in cancer development and designing selective inhibitors. In our previous work, we successfully determined the crystal structure of DCLK1 while it was bound to its autoinhibitory domain (AID) at the active site. By analyzing this structure, we were able to uncover the intricate molecular mechanisms behind specific cancer-causing mutations in DCLK1. Utilizing molecular dynamics simulations, we discovered that these mutations disrupt the smooth assembly of the AID, particularly affecting the R2 helix, into the kinase domain (KD). This disruption leads to the exposure of the D533 residue of the DFG (Asp-Phe-Gly) motif in the KD, either through steric hindrance, the rearrangement of electrostatic interactions, or the disruption of local structures in the AID. With these molecular insights, we conducted a screening process to identify potential small-molecule inhibitors that could bind to DCLK1 through an alternative binding mode. To assess the binding affinity of these inhibitors to the KD of DCLK1, we performed calculations on their binding energy and conducted SPR experiments. We anticipate that our study will contribute novel perspectives to the field of drug screening and optimization, particularly in targeting DCLK1.