Drugs to Elevate Enkephalin as Novel Opiate Analgesics

提高脑啡肽水平的药物作为新型阿片类镇痛药

• 批准号: 6786438
• 负责人: TERRY REISINE
• 金额: $ 15.76万
• 依托单位: AMERICAN LIFE SCIENCE PHARMACEUTICALS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-15 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6786438
• 关键词: analgesia; analgesics; biotechnology; chemical registry /resource; chromaffin cells; cysteine endopeptidases; drug discovery /isolation; drug screening /evaluation; enkephalins; enzyme activity; high throughput technology; inhibitor /antagonist; protease inhibitor; protein biosynthesis; serine proteinases; small molecule; stimulant /agonist; technology /technique development; tissue /cell culture

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.

描述（由申请人提供）：疼痛是一种严重的、使人衰弱的病症，由于目前使用的阿片类药物（如吗啡）的剂量限制性副作用，治疗不足。脑啡肽是一种内源性阿片肽，可引起镇痛，但与吗啡不同，它几乎没有副作用（如果有的话）。重要的是，它不具有阿片生物碱的滥用潜力。由于脑啡肽不能穿过血脑屏障，因此脑啡肽本身不能用于治疗。然而，能够增加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。 脑啡肽是通过一系列蛋白水解事件产生的，这些蛋白水解事件将大的蛋白质前体脑啡肽原裂解为较小的具有生物活性的脑啡肽五肽。我们已经确定了分泌囊泡中用于生成脑啡肽的主要脑啡肽原裂解活性，并且它已被证明是半胱氨酸蛋白酶组织蛋白酶 L。组织蛋白酶 L 在适当的脑啡肽原加工位点进行裂解，与分泌囊泡中的脑啡肽共定位，并且组织蛋白酶 L 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 对于产生活性脑啡肽的重要性。值得注意的是，组织蛋白酶 L 的活性在体内受到内源性丝氨酸蛋白酶抑制剂内皮素 2 的强烈抑制。我们建议可以开发药物来阻止内皮素 2 抑制组织蛋白酶 L，从而增加组织蛋白酶 L 处理脑啡肽原的活性，从而提高中枢神经系统 (CNS) 中的脑啡肽并增强镇痛作用以缓解疼痛。 在这个为期 6 个月的第一阶段项目中，第一个具体目标将开发和优化基于荧光的高通量筛选 (HTS) 测定，以测量内皮素 2 存在下的组织蛋白酶 L 活性，作为具体目标 1。这种高通量测定将允许筛选化学库，以识别增加组织蛋白酶 L 活性的化合物，这将通过中断组织蛋白酶 L 的内皮素 2 调节来实现。第二个具体目标将利用这些 HTS 在内皮素 2 存在的情况下，筛选小分子库中激活组织蛋白酶 L 活性的化合物，以增加脑啡肽的产生。在未来的研究（II 期 SBIR）中，可以将先导化合物开发成新药，以增加脑啡肽的产生，从而缓解疼痛，而不会产生目前使用的阿片类麻醉药的副作用。