Poudel Pitambar, Shapovalov Ivan, Panday Shailesh Kumar, Nouri Kazem, Davies Peter L, Greer Peter A, Alexov Emil
Department of Physics & Astronomy, College of Science, Clemson University, Clemson, South Carolina 29634, United States.
Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario K7L 3N6, Canada.
J Chem Inf Model. 2025 Jun 9;65(11):5528-5543. doi: 10.1021/acs.jcim.5c00258. Epub 2025 May 20.
Dysregulated calpain-1 and calpain-2 protease activity linked to several diseases has encouraged efforts to explore inhibiting calpain to provide therapeutic benefits. However, there are currently no clinically approved drugs that specifically target calpain functionality. To address this unmet need, we carried out drug discovery efforts to identify small molecules capable of modulating calpain activity. Our approach is based on the observation that heterodimer formation of the calpain-2 catalytic (CAPN2) and regulatory (CAPNS1) subunits is needed for both proteolytic activity and CAPN2 stability. In recognition of this obligate protein-protein interaction (PPI), the CAPN2-CAPNS1 interface was targeted with nearly 3.6 million small molecules to find candidates that bind at the interface with high affinity and introduce steric clashes capable of altering heterodimerization or the conformation of CAPN2, thereby modulating proteolytic activity. Twenty small molecules predicted to disrupt the most hydrogen bonds at the CAPN2-CAPNS1 interface were validated experimentally. Five small molecules inhibited calpain activity by 53.6 ± 4.1, 36.8 ± 38.3, 31.1 ± 17.5, 69.8 ± 27.3, and 47.1 ± 18.5%, while two enhanced protease activity by 163.0 ± 41.9 and 129.2 ± 11.9%. Unexpectedly, the effects of these seven molecules on the CAPN2-CAPNS1 PPI assay did not correlate with their effects on protease activity. Molecular simulation showed that small molecules that modulate calpain activity without abolishing heterodimerization do so by altering the conformational changes needed for the activity. This apparent allosteric mechanism paves the way for developing novel therapeutic solutions for modulating the calpain activity in various diseases associated with calpain dysregulation.
与多种疾病相关的钙蛋白酶-1和钙蛋白酶-2蛋白酶活性失调，促使人们努力探索抑制钙蛋白酶以获得治疗益处。然而，目前尚无临床批准的专门针对钙蛋白酶功能的药物。为满足这一未被满足的需求，我们开展了药物研发工作，以鉴定能够调节钙蛋白酶活性的小分子。我们的方法基于这样的观察结果：钙蛋白酶-2催化亚基（CAPN2）和调节亚基（CAPNS1）的异二聚体形成对于蛋白水解活性和CAPN2稳定性都是必需的。鉴于这种必需的蛋白质-蛋白质相互作用（PPI），我们用近360万个小分子靶向CAPN2-CAPNS1界面，以寻找能够以高亲和力结合该界面并引入空间冲突从而改变异二聚化或CAPN2构象的候选分子，进而调节蛋白水解活性。实验验证了20个预计会破坏CAPN2-CAPNS1界面最多氢键的小分子。5个小分子将钙蛋白酶活性分别抑制了53.6±4.1%、36.8±38.3%、31.1±17.5%、69.8±27.3%和47.1±18.5%，而2个小分子使蛋白酶活性分别增强了163.0±41.9%和129.2±11.9%。出乎意料的是，这7个分子对CAPN2-CAPNS1 PPI分析的影响与其对蛋白酶活性的影响不相关。分子模拟表明，在不消除异二聚化的情况下调节钙蛋白酶活性的小分子是通过改变活性所需的构象变化来实现的。这种明显的变构机制为开发针对与钙蛋白酶失调相关的各种疾病调节钙蛋白酶活性的新型治疗方案铺平了道路。
