Beta-lactam chemical probes for GLT-1 transporter-related pathologies

描述(由申请人提供)：本申请涉及广泛的挑战领域(06)使能技术和特定挑战主题06-GM-102*：化学家/生物学家合作促进工具开发。这是天普大学药学院药物化学和生物学小组的合作研究项目。目的是设计和合成新型的含有β-内酰胺的化合物作为化学探针，以更好地了解谷氨酸转运体亚型1(GLT-1)在被β-内酰胺类抗生素头孢曲松(CTX)激活后的生物学特性，并研究GLT-1转运体激活在谷氨酸相关神经和病理疾病的临床前模型中的影响，这些疾病包括癫痫、中风、肌萎缩侧索硬化症、多发性硬化症、抑郁症和成瘾(Rothstein等，2005)。这项研究对人类很重要，因为神经科学家普遍认为，GLT-1转运体激活是一种强大的-但尚未被研究-的方法，可以管理一系列与谷氨酸相关的疾病。考虑到这些疾病对社会、医疗、经济和刑事的综合影响，以及目前缺乏安全、有效和方便的治疗方法，针对GLT-1转运体的新的和有吸引力的治疗方法的开发可能有助于控制这些影响很大一部分人口的疾病。尽管GLT-1在谷氨酸生理和病理生理中的关键作用是毋庸置疑的，但我们对GLT-1转运体激活的生物学基础和GLT-1激活所产生的药理作用缺乏了解。主要原因是缺乏激活GLT-1的化合物。最近，当1040种FDA批准的药物和营养素筛选发现β-内酰胺类抗生素是唯一能够激活GLT-1转运体的实际药物时，这种观点发生了变化。使用CTX的临床前研究现已证明，GLT-1激活在谷氨酸相关病理的动物模型中是有效的。这些研究提供了关于GLT-1生物学的宝贵知识，并证明了GLT-1的激活应该被作为一种有效的治疗方法来探索。然而，考虑到长期使用抗生素导致的耐药性和腹泻，以及体内必需的大剂量环磷酰胺(200 mg/kg)，直接抗生素疗法不太可能用于临床治疗由过度谷氨酸传递引起的病理。这项建议通过设计和合成非抗生素、含有β-内酰胺的化合物，直接解决了这一治疗局限性，最终实现了开发一种新药的最终目标，该新药将用于广泛的谷氨酸相关神经和病理疾病的临床治疗。最终开发潜在的治疗实体的第一步需要对β-内酰胺衍生物进行结构-活性分析，以分离GLT-1和抗生素活性。GLT-1特异性药效团的建立，具有有效穿越血脑屏障(BBB)的能力，将为进一步体内研究的未来化合物的开发提供结构模板。因此，这项提案的重点将是设计具有三种特性的β-内酰胺探针：增强的血脑屏障穿透性；GLT-1活性；以及降低抗生素效力。然后将使用阿片耐受性、身体依赖和成瘾的生物终点来研究满足这些条件的化学探针在体内的GLT-1活性，所有这些都在一定程度上通过增强谷氨酸能传递来调节。我们将测试这一总体假设，即非抗生素、β-内酰胺类化合物显示出增强的脑渗透性和GLT-1活性，将通过GLT-1转运体激活来阻止大鼠对吗啡的耐受和身体依赖以及海洛因和可卡因的自我给药。综上所述，本文提出的实验将首次全面调查与谷氨酸相关的一系列神经和病理疾病相关的b-内酰胺化合物的化学和生物学特性。坦普尔大学为当地经济做出了巨大贡献，每年的经济影响总额为27亿美元。这包括每年8.5亿美元的运营支出，用于创造或支持6439个就业机会，以及10.9亿美元的员工支出，以维持额外的5299个就业机会。天普目前每年在建筑项目上花费9550万美元，创造了868个就业机会。坦普尔大学的毕业生为经济做出了贡献，总体经济影响为87亿美元，支持了42,386个工作岗位。据估计，目前的提案除了在两年内提供约188,000美元的购买力外，还将创造或保留相当于6-8个工作岗位。 与公共卫生有关：相当大比例的美国人口受到与谷氨酸有关的各种神经和病理疾病的影响，这些疾病目前缺乏有效和安全的治疗。谷氨酸转运体亚型1(GLT-1)的激活是管理此类疾病最有希望、但研究最少的策略之一。最近发现的β-内酰胺类抗生素是唯一能够激活GLT-1的实际药物，这表明，针对分离GLT-1和抗生素活性的b-内酰胺衍生物的结构-活性分析将导致鉴定一个或多个新的、非抗生素的b-内酰胺类参考探针，这将为未来有吸引力的GLT-1导向疗法的开发提供结构模板。

DESCRIPTION (provided by applicant): This application addresses broad challenge Area (06) Enabling Technologies and Specific Challenge Topic 06-GM-102*: Chemist/Biologist Collaborations facilitating tool development. This is a collaborative research project between the medicinal chemistry and biology groups at Temple University School of Pharmacy. The purpose is to design and synthesize novel b-lactam containing compounds to use as chemical probes to better understand the biology of glutamate transporter subtype 1 (GLT-1) upon activation by the b-lactam antibiotic ceftriaxone (CTX) and to investigate the impact of GLT-1 transporter activation in preclinical models of glutamate-related neurological and pathological disorders, including such as epilepsy, stroke, amyotrophic lateral sclerosis, multiple sclerosis, depression and addiction (Rothstein et al., 2005). This research is important to mankind because of the widely held belief among neuroscientists that GLT-1 transporter activation is a powerful - and understudied - approach for managing a broad range of glutamate-related diseases. Considering the combined social, medical, economic and criminal impact of these diseases and the lack of safe, efficacious, and convenient treatments currently available to treat them, the development of novel and attractive therapies targeting GLT-1 transporters might be useful in managing these disorders that affect a significant proportion of the population. Although a critical role for GLT- 1 in glutamate physiology and pathophysiology is indisputable, our knowledge of the biology underlying GLT-1 transporter activation and the pharmacological effects resulting from GLT-1 activation is lacking. The main reason is a lack of compounds that activate GLT-1. This perspective changed recently when a screen of 1040 FDA-approved drugs and nutritionals identified b-lactam antibiotics as the only practical pharmaceuticals capable of activating GLT-1 transporters. Preclinical studies using CTX have now demonstrated that GLT-1 activation is effective in animal models of glutamate-related pathologies. These studies have provided invaluable knowledge about GLT-1 biology and a proof-of-principle that GLT-1 activation should be explored as a powerful therapeutic approach. However, considering the resistance and diarrhea resulting from long-term antibiotic use, as well as the large doses of CTX (200 mg/kg) which are necessary in vivo, it is unlikely that direct antibiotic therapy will be used in the clinical management of pathologies caused by excessive glutamatergic transmission. This proposal directly addresses this therapeutic limitation by designing and synthesizing non-antibiotic, b-lactam containing compounds to eventually achieve the ultimate goal of developing a novel drug that will be useful in the clinical management of a broad range of glutamate-related neurological and pathological diseases. A primary step in the eventual development of potential therapeutic entities requires a structure-activity analysis of b-lactam derivatives towards a separation of GLT-1 and antibiotic activity. The establishment of a GLT-1-specific pharmacophore, with the ability to effectively cross the blood-brain barrier (BBB), will provide a structural template for the development of future compounds for further in vivo study. Therefore, emphasis in this proposal will be on designing b-lactam probes displaying three properties: enhanced BBB penetrability; GLT-1 activity; and reduced antibiotic efficacy. The in vivo GLT-1 activity of chemical probes satisfying these conditions will then be investigated using the biological endpoints of opiate tolerance, physical dependence and addiction, all of which are mediated in part by enhanced glutamatergic transmission. We will test the overall hypothesis that non-antibiotic, b-lactam containing compounds displaying enhanced brain penetrability and GLT-1 activity will block morphine tolerance and physical dependence and heroin and cocaine self-administration in rats through GLT-1 transporter activation. Taken together, experiments proposed herein will provide the first comprehensive investigation of the chemical and biological properties of b-lactam compounds as related to a broad range of glutamate-related neurological and pathological diseases. Temple University contributes substantially to the local economy, with a total annual economic impact of $2.7 billion. This includes annual operating spending of $850 million which creates or supports 6,439 jobs, $1.09 billion in employee spending that maintains an additional 5,299 jobs. Temple is currently spending $95.5 million on construction projects annually, creating 868 jobs. Temple graduates contribute to the economy, with an overall economic impact of $8.7 billion supporting 42,386 jobs. It is estimated the current proposal will create or retain the equivalent of 6-8 jobs in addition to providing purchasing power of ~$188,000 over two years. PUBLIC HEALTH RELEVANCE: A significant percentage of the United States population is affected by a broad range of glutamate-related neurological and pathological disorders which currently lack efficacious and safe treatments. One of the most promising - and understudied - strategies for managing such conditions is glutamate transporter subtype 1 (GLT-1) activation. The recent identification of b-lactam antibiotics as the only practical pharmaceuticals capable of activating GLT-1 indicates that a structure-activity analysis of b-lactam derivatives, directed towards a separation of GLT-1 and antibiotic activity, will result in the identification of one or more novel, non-antibiotic, b-lactam containing reference probes which will provide a structural template for the future development of attractive GLT-1-directed therapies.