靶向1型神经降压素受体的细胞穿透肽蛋白减轻疼痛
Cell-penetrating pepducins targeting the neurotensin receptor type 1 relieve pain
摘要：
肽蛋白是细胞穿透性的、膜束缚的脂肽，其被设计为靶向G蛋白偶联受体（GPCR）的细胞内区域，以便变构地调节受体的信号输出。在这项概念验证研究中，我们探讨了来自1型神经降压素受体（NTS 1）的第一个细胞内环的pepducin系列的镇痛潜力，NTS 1是一种A类GPCR，介导了神经降压素（NT）十三肽的许多作用，包括低温、低血压和镇痛。我们使用基于BRET的生物传感器来确定pepducins参与与NTS 1激活相关的G蛋白信号通路的能力。我们在10 μM浓度下观察到部分Gq和G13活化，表明这些肽蛋白可能作为NTS 1的变构激动剂。此外，我们使用表面等离子体共振（SPR）作为一种无标记的检测方法，以监测稳定表达hNTS 1的CHO-K1细胞中的pepducin诱导的反应。这种全细胞整合测定使我们能够将我们的pepducin系列细分为三个特征响应组。为了确定pepducin的抗伤害感受潜力，我们随后在鞘内给予pepducin（275 nmol/kg）后，通过测量对热伤害感受刺激的尾部回缩潜伏期，在急性疼痛模型（甩尾试验）中筛选该系列。我们进一步评估了在强直性疼痛模型（福尔马林试验）以及神经性（慢性缩窄性损伤）和炎性（完全弗氏佐剂）慢性疼痛模型中有前景的肽蛋白。我们报道了一种肽调节素PP-001，即使在慢性疼痛范例中，也能持续降低大鼠的伤害性行为。总之，这些结果表明，NTS 1衍生的pepducins可能是一个有前途的战略，在疼痛缓解。
英文摘要：
Pepducins are cell-penetrating, membrane-tethered lipopeptides designed to target the intracellular region of a G protein-coupled receptor (GPCR) in order to allosterically modulate the receptor's signaling output. In this proof-of-concept study, we explored the pain-relief potential of a pepducin series derived from the first intracellular loop of neurotensin receptor type 1 (NTS1), a class A GPCR that mediates many of the effects of the neurotensin (NT) tridecapeptide, including hypothermia, hypotension and analgesia. We used BRET-based biosensors to determine the pepducins' ability to engage G protein signaling pathways associated with NTS1 activation. We observed partial Gq and G13 activation at a 10 μM concentration, indicating that these pepducins may act as allosteric agonists of NTS1. Additionally, we used surface plasmon resonance (SPR) as a label-free assay to monitor pepducin-induced responses in CHO-K1 cells stably expressing hNTS1. This whole-cell integrated assay enabled us to subdivide our pepducin series into three profile response groups. In order to determine the pepducins' antinociceptive potential, we then screened the series in an acute pain model (tail-flick test) by measuring tail withdrawal latencies to a thermal nociceptive stimulus, following intrathecal pepducin administration (275 nmol/kg). We further evaluated promising pepducins in a tonic pain model (formalin test), as well as in neuropathic (Chronic Constriction Injury) and inflammatory (Complete Freund's Adjuvant) chronic pain models. We report one pepducin, PP-001, that consistently reduced rat nociceptive behaviors, even in chronic pain paradigm. Altogether, these results suggest that NTS1-derived pepducins may represent a promising strategy in pain-relief.
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