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.

中文翻译：

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具有镇痛作用的靶向 TRPV1 的肽正变构调节剂的从头设计

瞬时受体电位香草素 1 (TRPV1) 离子通道是一种与痛觉密切相关的伤害感受器。直接阻断 TRPV1 具有显着的镇痛作用，但也会引起严重的副作用，例如高热，导致 TRPV1 抑制剂在临床试验中失败。为了选择性地靶向积极参与疼痛感知的 TRPV1 通道，从头设计了基于 TRPV1 细胞内锚蛋白重复样结构域的高分辨率结构的肽正变构调节剂 (PAM)。以热点为中心的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粘合剂设计的成功率。结合荧光共振能量转移 (FRET) 成像、表面等离子共振和膜片钳记录，通过实验证明了这一点，设计的 PAM 以纳摩尔亲和力与 TRPV1 结合，并在设计时变构增强其对配体激活的反应。进一步证明，所设计的 PAM 在大鼠体内表现出持久的镇痛作用，而不会改变其体温，表明它们具有发展成为新型镇痛剂的潜力。