Centre for Advanced Imaging, The University of Queensland, Australia.
Institute for Molecular Bioscience, The University of Queensland, Australia.
Biochem Pharmacol. 2020 Nov;181:114148. doi: 10.1016/j.bcp.2020.114148. Epub 2020 Jul 12.
Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (Nas) play a critical role in this process. In particular, the Na subtype 1.7, has emerged as a promising universal target for the treatment of pain. Recently, a spider venom peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of Na1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a via sortase A-mediated ligation, providing avenues for further pre-clinical development. We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.
慢性疼痛是一种常见且常使人虚弱的病症。现有的治疗方法要么无效，要么伴有广泛的副作用。开发更安全、更有效的镇痛药的进展一直很缓慢，这在很大程度上是因为我们对不同疾病中疼痛的生理机制的理解有限。动作电位的产生和传播是疼痛感觉的一个核心组成部分，而电压门控钠离子通道（Nas）在这个过程中起着关键作用。特别是 Na 亚型 1.7，已成为治疗疼痛的有前途的通用靶点。最近，一种蜘蛛毒液肽 μ-TRTX-Pn3a 被发现是 Na1.7 的高度选择性抑制剂。在这里，我们报告了 Pn3a 在细菌宿主中的首次重组表达方法，这为生产提供了一种廉价的途径。此外，我们还开发了一种通过 sortase A 介导的连接对我们重组产生的 Pn3a 进行生物缀合的方法，为进一步的临床前开发提供了途径。我们展示了细菌中的异源表达如何使 Pn3a 易于进行同位素标记，这使我们能够使用最近开发的脂质纳米盘系统通过高分辨率溶液态核磁共振（NMR）光谱研究肽的膜结合特性。异核 NMR 数据表明，肽的 C 末端区域在与脂质结合时会发生构象变化。使用等温滴定量热法（ITC）进一步验证了 Pn3a 的膜结合特性，结果表明 Pn3a 与两性离子平面脂质双层结合的热力学主要由焓贡献驱动。
