Novel Azaspirooxindolinone-Based PROTACs for Selective BTK Degradation and Enhanced Anticancer Activity

Abstract

Proteolysis targeting chimera (PROTAC) facilitates the degradation of specific endogenous proteins via the E3 ubiquitin ligase pathway. This study evaluates nine PROTAC derivatives of azaspirooxindolinone targeting IL-2 inducible T cell kinase (ITK) and Bruton’s Tyrosine Kinase (BTK), which are implicated in hematological malignancies, autoimmune diseases, allergies, and neuroinflammation. Among all the tested compounds, three (PROTACs 7, 14, and 25) exhibited high cytotoxicity (IC50 < 10 µM) against BTK- and ITK-positive cancer cell lines, while showing no cytotoxicity against non-cancer fibroblast cells and normal T/B-cell lymphocytes. Despite having the highest docking score of -12.1 kcal/mol, PROTAC 7 did not reduce BTK or ITK protein levels in treated cells. Similarly, PROTAC 14, with a docking score of -11.2 kcal/mol, and a high cytotoxic against RAMOS cells did not reduce BTK levels. PROTAC 25, also with a docking score of -11.2 kcal/mol, was notably effective in inducing BTK degradation in a proteasome-dependent manner, which was inhibited in the presence of bortezomib. PROTAC 25 degradation of BTK led to the inhibition of BTK phosphorylation and downstream activation of p38 in lipopolysaccharide and IgM-stimulated RAMOS cells. In conclusion, we report a PROTAC derivative (25) of azaspirooxindolinone that shows significant activity against BTK-high cells.