布鲁顿酪氨酸激酶（BTK）是一种非受体酪氨酸激酶，是治疗B细胞驱动的恶性肿瘤的主要治疗靶点。然而，已批准的共价BTK抑制剂（cBTKi）由于非特异性的副作用、口服药理学的次优性以及耐药突变的发展（如C481），导致治疗限制。在这里，我们描述了拮抗剂皮托布鲁替尼的临床前资料，该药具有高选择性及强效的非共价（可逆）BTK抑制作用。皮托布鲁替尼在BTK和ATP结合区域与BTK和水分子形成广泛的相互作用，并且没有直接与C481发生相互作用。因此，皮托布鲁替尼可以在酶促和细胞基础试验中同时抑制BTK和BTK C481置换突变，并具有相似的效力。在差示扫描荧光法研究中，与cBTKi结合的BTK相比，与皮托布鲁替尼结合的BTK表现出更高的熔点。皮托布鲁替尼可以阻止Y551的磷酸化，而cBTKi无法起到此作用。这些数据表明，皮托布鲁替尼可以独特地稳定BTK在关闭的、非活性构形中。皮托布鲁替尼可以抑制多种B细胞淋巴瘤细胞系的信号：BTK信号和细胞增殖，并显著抑制体内人源性淋巴瘤移植瘤的生长。酶促调查显示，在人kinome中，皮托布鲁替尼对BTK具有高度选择性，在后续的细胞研究中，皮托布鲁替尼与其他测试激酶相比保留了100倍以上的选择性。总之，这些发现表明，皮托布鲁替尼是一种具有改进选择性、独特药理学、生物物理学和结构属性的新型BTK抑制剂，它具有提高治疗B细胞驱动癌症的精确性和耐受性的潜力。皮托布鲁替尼正在进行第3期临床研究，用于治疗多种B细胞恶性肿瘤。Copyright © 2023 American Society of Hematology。

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.Copyright © 2023 American Society of Hematology.