Drugs to Elevate Enkephalin as Novel Opiate Analgesics

描述(申请人提供)：疼痛是一种严重的、虚弱的情况，由于目前使用的阿片类药物如吗啡的剂量限制副作用而得不到治疗。脑啡肽是一种内源性阿片肽，具有镇痛作用，但与吗啡不同，它几乎没有副作用。重要的是，它没有阿片类生物碱的滥用潜力。因为脑啡肽不能穿过血脑屏障，所以它本身不能用于治疗。然而，可以提高神经系统中脑啡肽水平的药物可能是一种新的疼痛治疗方法，与目前使用的麻醉剂相比具有重大优势。 脑啡肽是由一系列蛋白降解事件产生的，这些事件将大的蛋白前体前脑啡肽裂解成具有生物学活性的较小的脑啡肽五肽。我们已经确定了分泌囊泡中产生脑啡肽的主要前脑啡肽裂解活性，并已证明它是半胱氨酸蛋白酶组织蛋白L，组织蛋白酶L在适当的脑啡肽加工位点裂解，与脑啡肽共存于分泌囊泡中，组织蛋白酶L基因敲除小鼠的脑内脑啡肽水平降低。这些结果证明了组织蛋白酶L在活性脑啡肽产生中的重要作用。值得注意的是，组织蛋白酶L的活性在体内被内源性丝氨酸蛋白酶抑制剂内毒素2抑制。我们建议可以开发出阻止内毒素2抑制组织蛋白酶L的药物，从而增加组织蛋白酶L的活性，以处理前脑啡肽来升高中枢神经系统中的脑啡肽，并增强镇痛作用。 在这个为期6个月的第一阶段项目中，第一个特定目标将开发和优化一种基于荧光的高通量筛选方法，以在内毒素2存在的情况下测量组织蛋白酶L的活性，作为特定目标1。作为特定目标1，该高通量筛选将允许筛选一个化学文库以识别增加组织蛋白酶L活性的化合物，这将通过阻断内毒素2对组织蛋白酶L的调节而发生。第二个特定目标将利用这些高通量筛选方法来筛选在内毒素2存在的情况下激活组织蛋白酶L活性的化合物的小分子文库，以增加脑啡肽的产生。铅HITS可以在未来的研究中(第二阶段SBIR)发展成新的药物，以增加脑啡肽的产生，从而提供缓解疼痛的效果，而不会出现目前使用的阿片类麻醉剂的副作用。

DESCRIPTION (provided by applicant): Pain is a serious, debilitating condition that is under-treated because of the dose-limiting side effects of currently used opiate drugs such as morphine. Enkephalin is an endogenous opiate peptide, which induces analgesia, but unlike morphine it has few, if any side effects. Importantly, it does not have the abuse potential of opiate alkaloids. Because it does not cross the blood brain barrier, enkephalin itself cannot be employed therapeutically. However, drugs that could increase the levels of enkephalin in the nervous system could be a new treatment for pain with major advantages over presently used narcotics. Enkephalins are produced by a series of proteolytic events that cleave the large protein precursor, proenkephalin, to the smaller biologically active enkephalin pentapeptide. We have identified the major proenkephalin cleaving activity in secretory vesicles for the generation of enkephalin, and it has been demonstrated as the cysteine protease cathepsin L. Cathepsin L cleaves at appropriate proenkephalin processing sites, is colocalized with enkephalin in secretory vesicles, and brain levels of enkephalin are reduced in cathepsin L knockout mice. These results demonstrate the importance of cathepsin L for production of active enkephalin. Notably, cathepsin L activity is tonically inhibited in vivo by the endogenous serpin protease inhibitor endopin 2. We propose that drugs can be developed to prevent endopin 2 from inhibiting cathepsin L, thereby increasing cathepsin L activity for processing proenkephalin to elevate enkephalin in the central nervous system (CNS) and enhance analgesia for pain relief. In this 6-month Phase I project, the first specific aim will develop and optimize a fluorescent-based high throughput screening (HTS) assay to measure cathepsin L activity in the presence of endopin 2, as specific aim 1. This high throughput assay will allow screening of a chemical library to identify compounds that increase cathepsin L activity, which would occur by interrupting endopin 2 modulation of cathepsin L. The second specific aim will utilize these HTS assays to screen a small molecule library for compounds that activate cathepsin L activity, in the presence of endopin 2, to increase enkephalin production. Lead hits can be developed in future studies (phase II SBIR) into novel drugs to increase enkephalin production that would provide pain relief without the side effects of presently used opiate narcotics.