Decoding the off-target potency of DB18: A comparative structural study of CLK1 and TRK kinases

The Tropomyosin Receptor Kinase (TRK) family comprises highly homologous proteins, but achieving homolog selectivity remains a significant challenge among the developed inhibitors. DB18, a quinazoline-based scaffold specifically optimized as a CLK inhibitor, exhibited noticeable TRKA specificity in the kinome scan. This work elucidates functional aspects of DB18 responsible for its off-target specificity towards TRKA through rigorous structure-based investigations. An initial sequence and structural comparison of the CLK1, TRKA, TRKB, and TRKC revealed high residue conservation resulting in similar pocket occupancy including the persistent hydrogen bonding of quinazolin-2-amine core of DB18 with their respective hinge residues (CLK1: L244, TRKA: M592, TRKB: M636, and TRKC: M620). The residue variation of R599 of TRKA to lysines in others, affected the binding mode of tail region due to altered charge densities. Beyond hinge anchorage, auxiliary interactions, especially hydrophobic and vdW determined the occupancy of the triazole tail. In TRKA complex, auxiliary interactions were abundant while lacking in TRKB resulting in diminished activity. In contrast, in TRKC complex, despite maintaining abundant auxiliary interactions, the rigid conformation caused by a distinct helix at the activation loop altered the orientation of the tail region, which is detrimental to the activity. This study thus identified a template compound that can be used to design homolog-specific inhibitors to resolve pan-TRK toxicity issues.