Figure 1

LRRK2-IN-1 inhibits DCLK1 kinase activity. An in vitro kinase assay was performed using Purified active DCLK1 kinase (0.25 μg) with 2.5 μg of autocamtide II substrate, 1 μM ATP, and either DMSO, 0.6, 2.5, 5, 10, or 50 nM LRRK2-IN-1 (A). Using relative luminescent units (RLU) data, a sigmoidal-dose response curve was plotted in GraphPad Prism 6.0 (adj. R² = 0.952) revealing an IC₅₀ value of 2.61 nM (B). AsPC-1 cells were treated with LRRK2-IN-1 at varying concentrations for 48 h. Following treatment cells were lysed, protein was isolated and quantified by BCA assay, and immunoblotting was performed with α-phospho-DCLK1. The ratio of phospho-DCLK1 to total DCLK1 (Figure 4B; 48 h) was determined and demonstrated decreased phosphorylation of DCLK1 (p < 0.05) following treatment (C). Schematic demonstrating the shared protein kinase domain between DCLK1 isoforms referenced in Uniprot [Swiss-Prot: O15075] (D). Three dimensional view of LRRK2-IN-1 binding site in DCLK-long-β revealing predicted interactions with residues of the hinge region, catalytic loop (C-loop), activation loop (A-loop), αC-helix (αC), and the highly conserved lysine (Lys112) of the kinase catalytic domain suggesting that LRRK2-IN-1 competes with ATP for the DCLK1 kinase binding pocket. Dashed lines mark the hydrogen bond formed with the conserved aspartate (“D” of the “DFG” motif) of the activation loop (E).