Kidney Disease Center, First Affiliated Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058, China.
Alibaba-Zhejiang University Joint Research Center of Future Digital Healthcare, Hangzhou, Zhejiang, 310058, China.
Adv Sci (Weinh). 2021 Sep;8(17):e2101716. doi: 10.1002/advs.202101716. Epub 2021 Jul 11.
Transient receptor potential vanilloid 1 (TRPV1) ion channel is a nociceptor critically involved in pain sensation. Direct blockade of TRPV1 exhibits significant analgesic effects but also incurs severe side effects such as hyperthermia, causing failures of TRPV1 inhibitors in clinical trials. In order to selectively target TRPV1 channels that are actively involved in pain-sensing, peptidic positive allosteric modulators (PAMs) based on the high-resolution structure of the TRPV1 intracellular ankyrin-repeat like domain are de novo designed. The hotspot centric approach is optimized for protein design; its usage in Rosetta increases the success rate in protein binder design. It is demonstrated experimentally, with a combination of fluorescence resonance energy transfer (FRET) imaging, surface plasmon resonance, and patch-clamp recording, that the designed PAMs bind to TRPV1 with nanomolar affinity and allosterically enhance its response to ligand activation as it is designed. It is further demonstrated that the designed PAM exhibits long-lasting in vivo analgesic effects in rats without changing their body temperature, suggesting that they have potentials for developing into novel analgesics.
瞬时受体电位香草酸 1 型（TRPV1）离子通道是一种伤害感受器，在疼痛感觉中起着关键作用。TRPV1 的直接阻断显示出显著的镇痛效果，但也会引起严重的副作用，如发热，导致 TRPV1 抑制剂在临床试验中失败。为了选择性地靶向主动参与痛觉感知的 TRPV1 通道，基于 TRPV1 细胞内锚重复样结构域的高分辨率结构，从头设计了肽类正变构调节剂（PAMs）。热点中心方法针对蛋白质设计进行了优化；它在 Rosetta 中的使用提高了蛋白质结合物设计的成功率。实验证明，通过荧光共振能量转移（FRET）成像、表面等离子体共振和膜片钳记录的组合，设计的 PAMs 以纳摩尔亲和力与 TRPV1 结合，并以设计的方式变构增强其对配体激活的反应。进一步证明，所设计的 PAM 在不改变体温的情况下，在大鼠体内表现出持久的镇痛作用，这表明它们有可能开发成新型的镇痛药。
