The initial structure of PIKfyve (PDB code: 7K2V, chain P) was prepared using the Schrödinger Protein Preparation Wizard. We used the standard settings in this program: adding and optimizing the positions of hydrogen atoms and adjusting residue ionization and tautomer states using PROPKA at pH 7, removing water molecules located more than 3 Å away from heteroatoms, performing restrained structural minimization using the OPLS4 force field, and converging the heavy atoms to a root mean square deviation (RMSD) of 0.3 Å. The 3D ligand structures were generated using Schrödinger LigPrep tool to sample all possible conformational models during the docking protocol. The Induced Fit Docking protocol was used to produce the initial model for the binding pose of 1 in the PIKfyve active site. To further refine the structural model of the protein-ligand complex, we performed molecular dynamics (MD) simulations using pmemd.cuda in Amber 22. We used a similar MD simulation protocol to that reported in our previous work. Briefly, the initial simulation system was set up with AmberTools (tleap and antechamber) and Amber forcefields (ff14SB, gaff2 and tip3p). The protein-ligand complex structure was solvated in a rectangular box and the minimum distance between box edges and complex was set to 12 Å. The total system charge was neutralized by adding chlorine ions. After initial energy equilibration, the system temperature was increased to 298 K using NVT ensemble over 100 ps with 10.0 kcal/(mol•Å 2 ) harmonic restraints added to the heavy atoms of the protein-ligand complex. The restraints were then gradually released over 1 ns in NPT ensembles. An additional 100 ns simulation was performed under a constant temperature (298 K) and constant pressure (1 bar) regulated by Langevin thermostat and Monte Carlo barostat, respectively. The simulation timestep was 2.0 fs and the SHAKE algorithm 66 was applied to constrain bonds involving hydrogens. The cutoff of nonbonded interactions was set to 10 Å and long-range electrostatic interactions were calculated with Particle mesh Ewald (PME) methods. The average structure of the 100 ns simulation was computed with cpptraj package and the simulation frame with the lowest RMSD from the averaged structure was selected as the refined model for further docking study.