Novel analgesics based on antagonism of TRPV1-AKAP79 binding

慢性疼痛由疼痛通路的外周感觉神经元的损伤或炎症触发。热敏离子通道TRPV 1是慢性疼痛的主要贡献者，并已成为许多制药公司开发新型镇痛药的目标。然而，直接阻断该通道会导致体温过高和对热刺激不敏感，这两种主要的副作用在很大程度上阻止了TRPV 1作为镇痛靶点的工作。在选定的研究实验室中的工作发现，炎症激活使TRPV 1敏感的激酶，即降低其热阈值，并且这些激酶通过支架蛋白AKAP 79定位在TRPV 1附近，AKAP 79直接与TRPV 1结合。已经鉴定了两种蛋白质上的结合位点，并且具有基于结合位点的序列的短竞争肽阻断结合，防止磷酸化，并且以这种方式完全防止TRPV 1的致敏。当与赋予细胞渗透性的达特域肽偶联时，这些肽在体内炎性和神经性疼痛的动物疼痛模型中的镇痛功效与TRPV 1的直接阻断剂相当，但没有高热和热不敏感性的主要副作用。在炎性疼痛和难治性人类疼痛病症糖尿病神经病变的动物模型中观察到优异的镇痛功效。该项目的目的是更详细地研究TRPV 1和AKAP 79之间的相互作用，并阐明拮抗TRPV 1-AKAP 79结合是否可能被证明是抑制体内慢性疼痛的新策略。我们已经表达并纯化了AKAP 79和TRPV 1的C端片段，并将使用这些纯化的蛋白进行NMR结构分析，以获得相互作用位点的三维结构。生物物理和生物化学测定将使我们能够确定AKAP 79和TRPV 1之间相互作用的亲和力和动力学。通过我们的NMR工作获得的知识将用于优化和构建修饰的肽抑制剂，其在体外结合试验和体内炎症性和神经性疼痛的动物模型中有效。此外，我们将扩展动物疼痛模型，以扩大我们对AKAP 79-TRPV 1相互作用的肽阻断剂在体内的功效的了解。拟议的研究项目将扩展我们对一个重要的蛋白质-蛋白质相互作用位点的了解，我们预计它还将提供信息，导致开发一种新的镇痛肽，该肽将来可用于治疗难治性人类疼痛，如糖尿病神经病变。

Chronic pain is triggered by injury or inflammation of peripheral sensory neurons of the pain pathway. The heat-sensitive ion channel TRPV1 is a major contributor to chronic pain and has been a target for the development of novel analgesics in many pharmaceutical companies. However, direct block of the channel causes hyperthermia and insensitivity to heat stimuli, both major side effects which have largely halted work on TRPV1 as an analgesic target. Work in the chosen research lab has found that inflammation activates kinases which sensitize TRPV1, i.e. lower its heat threshold, and that these kinases are positioned adjacent to TRPV1 by a scaffolding protein, AKAP79, which binds directly to TRPV1. The binding sites on both proteins have been identified, and short competitor peptides with a sequence based on the binding sites block the binding, preventing phosphorylation and in this way completely prevent sensitization of TRPV1. When coupled to a TAT domain peptide, which confers cell permeability, these peptides are comparable in their analgesic efficacy to direct blockers of TRPV1 in animal pain models of inflammatory and neuropathic pain in vivo, but without the major side effects of hyperthermia and thermal insensitivity. Excellent analgesic efficacy is observed in animal models of both inflammatory pain and of an intractable human pain condition, diabetic neuropathy. The aim of this project is to investigate the interaction between TRPV1 and AKAP79 in more detail and to elucidate whether antagonising TRPV1-AKAP79 binding may prove to be a novel strategy for inhibiting chronic pain in vivo. We have expressed and purified AKAP79 and the C-terminal fragment of TRPV1 and will use these purified proteins for NMR structural analysis in order to gain a 3-D-structure of the interaction site. Biophysical and biochemical assays will enable us to determine the affinity and kinetics of the interaction between AKAP79 and TRPV1. The knowledge gained by our NMR work will be used to optimize and construct modified peptide inhibitors effective with in vitro binding assays and in vivo animal models of inflammatory and neuropathic pain. Furthermore, we will extend the animal pain models to widen our knowledge of the efficacy of peptide blockers of the AKAP79-TRPV1 interaction in vivo. The proposed research project will extend our knowledge of an important protein-protein interaction site, and we anticipate that it will also provide information leading to the development of a new analgesic peptide which could in future be used to treat intractable human pain conditions such as diabetic neuropathy.