Overcoming Ibrutinib Resistance: Discovery and Characterization of CB988, a Novel Non-Covalent Btk Inhibitor Targeting the C481S Mutant

The research introduces a novel non-covalent Bruton's tyrosine kinase (Btk) inhibitor, CB988, developed to address the challenge of ibrutinib resistance in B-cell malignancies. Ibrutinib, the first FDA-approved Btk inhibitor, has been effective in treating B-cell cancers, but the emergence of the C481S mutation in Btk has led to resistance against this therapy. To counter this, a new approach was needed.

The study involved the production of two Btk protein forms: activated (Btk[A]) and unactivated (Btk[U]), achieved through ATP treatment for autophosphorylation and Lambda Protein Phosphatase treatment for dephosphorylation, respectively. Cellular potency was evaluated in Ramos cells, a human Burkitt’s lymphoma cell line, using Western blotting to analyze Btk and PLC-γ phosphorylations. The anti-tumor efficacy was assessed in the ABC-type DLBCL cell line, OCI-Ly10. Additionally, an ibrutinib-resistant Btk[C481S] mutant was created to test the efficacy of the new inhibitor.

CB988 was identified through structure-activity relationship studies as a highly selective Btk inhibitor. It showed sub-nanomolar inhibitory potency against both Btk conformations, suggesting a preference for binding to the inactive form of Btk. CB988 significantly reduced Btk Tyr223 phosphorylation and PLC-γ phosphorylation in cellular assays and decreased the proliferation of OCI-Ly10 cells. Notably, CB988 effectively inhibited the ibrutinib-resistant Btk[C481S] mutant, demonstrating its potential to treat patients with relapsed or refractory B-cell malignancies to ibrutinib.

The study concludes that CB988, a non-covalent Btk inhibitor, has shown promise in inhibiting the Btk[C481S] mutant enzyme, suggesting its potential as a treatment for patients resistant to ibrutinib.