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小组之间的合作研究项目。目的是设计和合成新的含有b-内酰胺的化合物，作为化学探针，更好地了解b-内酰胺类抗生素头孢曲松（CTX）激活谷氨酸转运蛋白亚型1 （GLT-1）的生物学特性，并研究GLT-1转运蛋白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，包括癫痫、中风、肌萎缩侧索硬化症、多发性硬化症、抑郁和成瘾（Rothstein et al., 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有力的方法，但研究不足，可用于管理广泛的谷氨酸相关疾病。考虑到这些疾病对社会、医疗、经济和犯罪的综合影响，以及目前缺乏安全、有效和方便的治疗方法，开发针对GLT-1转运蛋白的新颖和有吸引力的治疗方法可能有助于管理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无可争议的，但我们对GLT-1转运体激活的生物学基础以及GLT-1激活所产生的药理作用的了解尚缺乏。主要原因是缺乏激活GLT-1的化合物。这种观点最近发生了变化，在对1040种fda批准的药物和营养品进行筛选后，发现b-内酰胺类抗生素是唯一能够激活GLT-1转运蛋白的实用药物。使用CTX的临床前研究已经证明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激活应该作为一种强大的治疗方法进行探索。然而，考虑到长期使用抗生素引起的耐药和腹泻，以及体内所需的大剂量CTX (200mg /kg)，在临床处理过度谷氨酸传递引起的病理时，不太可能直接使用抗生素治疗。该建议通过设计和合成非抗生素、含有b-内酰胺的化合物来直接解决这一治疗限制，最终实现开发一种新型药物的最终目标，这种药物将在广泛的谷氨酸相关神经和病理疾病的临床管理中有用。最终开发潜在治疗实体的第一步需要对b-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，能够有效地穿过血脑屏障（BBB），将为未来化合物的进一步体内研究提供一个结构模板。因此，本提案的重点将放在设计具有三种特性的b-内酰胺探针上：增强血脑屏障穿透性；GLT-1活动;降低了抗生素的功效。满足这些条件的化学探针的体内GLT-1活性将通过阿片耐受、身体依赖和成瘾等生物学终点进行研究，所有这些终点都部分由增强的谷氨酸能传递介导。我们将测试整体假设，即非抗生素，含有b-内酰胺的化合物显示增强的脑穿透性和GLT-1活性，将通过GLT-1转运体激活阻止大鼠吗啡耐受性和身体依赖性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研究与谷氨酸相关的广泛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。天普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8.5亿美元的年度运营支出，创造或支持6439个工作岗位，10.9亿美元的员工支出，维持额外的5299个工作岗位。天普目前每年在建设项目上的支出为9550万美元，创造了868个就业岗位。天普毕业生为经济做出了贡献，总体经济影响达87亿美元，提供了42386个工作岗位。据估计，目前的提案除了在两年内提供约18.8万美元的购买力外，还将创造或保留相当于6-8个工作岗位。

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.