Pirtobrutinib preclinical characterization: a highly selective, non-covalent (reversible) BTK inhibitor

• Published in: Blood 2023-02
• Main Authors: Gomez, Eliana B, Ebata, Kevin, Randeria, Hetal S, Rosendahl, Mary S, Cedervall, Ernst Peder, Morales, Tony H, Hanson, Lauren M, Brown, Nicholas E, Gong, Xueqian, Stephens, Jennifer Rachelle, Wu, Wenjuan, Lippincott, Isabel, Ku, Karin S., Walgren, Richard A, Abada, Paolo B, Ballard, Joshua A, Allerston, Charles K, Brandhuber, Barbara J
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.2022018674

Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a major therapeutic target for B-cell driven malignancies. However, approved covalent BTK inhibitors (cBTKi) are associated with treatment limitations due to off-target side effects, suboptimal oral pharmacology, and development of resistance mutations (eg, C481) that prevent inhibitor binding. Here we describe the preclinical profile of pirtobrutinib, a potent, highly selective, non-covalent (reversible) BTK inhibitor. Pirtobrutinib binds BTK with an extensive network of interactions to BTK and water molecules in the adenosine triphosphate (ATP)-binding region and shows no direct interaction with C481. As a result, pirtobrutinib inhibits both BTK and BTK C481 substitution mutants in enzymatic and cell-based assays with similar potencies. In differential scanning fluorimetry studies, BTK bound to pirtobrutinib exhibited a higher melting temperature than cBTKi-bound BTK. Pirtobrutinib, but not cBTKi, prevented Y551 phosphorylation in the activation loop. These data suggest pirtobrutinib uniquely stabilizes BTK in a closed, inactive conformation. Pirtobrutinib inhibits BTK signaling and cell proliferation in multiple B-cell lymphoma cell lines and significantly inhibits tumor growth in human lymphoma xenografts in vivo. Enzymatic profiling showed pirtobrutinib was highly selective for BTK in >98% of the human kinome, and in follow-up cellular studies pirtobrutinib retained >100-fold selectivity over other tested kinases. Collectively, these findings suggest pirtobrutinib represents a novel BTK inhibitor with improved selectivity and unique pharmacologic, biophysical and structural attributes with the potential to treat B-cell driven cancers with improved precision and tolerability. Pirtobrutinib is being tested in phase 3 clinical studies for a variety of B-cell malignancies.