To gain insight into the protein-ligand interaction patterns and their relationship to measured binding affinities, we performed a comparative analysis of the docking poses for the alkyne, indole, and aminopyrimidine analogues (Figure ). SGC-PIKFYVE-1 and 1 were found to adopt very similar conformations with coplanarity of the aminopyrimidine and indole rings, supporting their nearly identical binding affinities for PIKfyve. Additionally, the docking model for our inactive compound 39 supports our hypothesis that a bulky group on the alkyne terminus is not tolerated by the back, buried portion of the PIKfyve pocket. Interestingly, our modeling suggests that the flexibility of the amide on the indole analogue bearing a 2-position alkyl amide, 13, allows it to shift into the solvent while maintaining the coplanarity between the pyrimidine and indole rings. This finding explains its nearly equivalent PIKfyve binding affinity compared to that of compound 1. Finally, examination of the docking pose of aminopyrimidine analogue 26 revealed that the methoxy group on the aminopyrimidine ring creates steric clash with hinge residues that is proposed to weaken the key hinge binding interaction. To gain a better understanding of the docking models and their correlation with the measured binding data for the indolyl pyrimidinamine series and TTBK1, we conducted additional in silico studies. We performed a structural alignment of the kinase domains of PIKfyve (PDB code: 7K2V and AlphaFold DB: AF-Q9Y2I7-F1; residues 1822-2085) and the kinase domain of TTBK1 bound to 2 (PDB code: 7ZHO; residues 21-317) (Figure ). This structural alignment allowed us to identify co-located residues in the binding sites of PIKfyve and TTBK1, despite the low amino acid sequence conservation between PIKfyve and other proteins, including TTBK1. Examination of the crystal structure of TTBK1 bound to 2 shows that Gln108 and Gln110 make key backbone hinge hydrogen bonds with the aminopyrimidine core. Our analyses suggested that in PIKfyve, main chain atoms from residues Glu1938 and Leu1940 could support similar interactions to 2, indicating that ligands within this chemical series may have equivalent binding modes to these two non-homologous kinases. Furthermore, the deep, buried portion of the pocket in TTBK1, which accommodates binding of groups on the alkyne terminus, includes the residues Lys63, Glu77, Val105, Met107, and Phe177. Within this pocket, 2 engages TTBK1 via hydrogen bonds to the side chain of Glu77 and the main chain of Phe177. A structurally equivalent back pocket is found in PIKfyve, composed of Lys1877, Ile2038, Leu1935, Met1937, and Tyr2037. Our in silico analyses suggested that the main chain of Tyr 2037 in PIKfyve may engage 2 via hydrogen bonds, akin to the interaction facilitated by Phe177 in TTBK1. Nevertheless, the second hydrogen bond facilitated by the side chain of TTBK1 back pocket residue Glu77 would not be supported by the structurally equivalent residue in PIKfyve, Ile2038. The insertion of hydrophobic groups into the back pocket of TTBK1 43 resulted in enhanced binding, suggesting that the placement of the thioether in this pocket may help explain the high PIKfyve affinity of 40.