Beauveriolide-Derived Cyclodepsipeptides as a New Class of Anti-Alzheimer's Drugs

Beauveriolide 衍生的环缩酚肽作为一类新型抗阿尔茨海默病药物

• 批准号: 7758245
• 负责人: PAUL WENTWORTH
• 金额: $ 19.98万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7758245
• 关键词: Acyl Coenzyme A; Affect; Alzheimer' s Disease; Americas; Amino Acids; Amyloid; Antibodies; Atherosclerosis; Biological; Biological Assay; Biological Factors; Brain; Brain region; C-terminal; Cell Culture Techniques; Cell Line; Cells; Chinese Hamster Ovary Cell; Cholesterol; Cholesterol Homeostasis; Complex; Computer Simulation; Cost of Illness; Cyclodepsipeptides; Dementia; Development; Disease; Emotional; Enzyme-Linked Immunosorbent Assay; Event; Exhibits; Generations; Genetic; Gifts; Grant; Human; Impaired cognition; In Vitro; Investigation; Libraries; Life Expectancy; Lipids; Liposomes; Measurement; Measures; Memory impairment; Methods; Microscopy; Modification; Molecular Probes; Mus; Neuroblastoma; Neurons; Oral; Peptides; Personality; Pharmaceutical Preparations; Phase; Population; Production; Property; Proteins; Public Health; Quality of life; Randomized; Reporting; Scanning; Side; Solid; Solutions; Specific qualifier value; Staging; Staining method; Stains; Sterol O-Acyltransferase; Structure; Switzerland; Testing; Therapeutic; Virtual Library; Western Blotting; Work; base; cost; design; drug discovery; in vivo; inhibitor/antagonist; macrophage; member; oil red O; prevent; psychologic; public health relevance; response; secretase; small molecule libraries; social

DESCRIPTION (provided by applicant): The pathogenic event common to all forms of Alzheimer's disease (AD) is the abnormal accumulation of the amyloid 2-peptide (A2) in specific regions of the brain. This accumulation of A2 is considered a key pathological event in AD and is the basis of the so-called amyloid hypothesis of the disease. Therefore, therapeutic approaches that reduce the accumulation of A2 are currently being sought. It has been shown definitively that the generation and clearance of A2 in the CNS is regulated by cholesterol homeostasis. Compounds that perturb cellular free cholesterol homeostasis such as acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors have been shown both in vitro and in vivo to reduce A2 production and secretion. However, it is generally the case that ACAY inhibitors exhibit poor oral activity. The beauveriolides are a new class of fungal metabolites that have been shown to be orally active ACAT inhibitors that are currently being investigated as potential therapeutics for atherosclerosis. NB No studies have been reported to date on the investigation of the A2-inhibitory effect of the natural product beauveriolides or beauveriolide-inspired compounds. The hypothesis being tested in this R21 is that the natural product beauveriolides and libraries of beauveriolide-inspired compounds should reduce A2- production and secretion in vitro via inhibition of ACAT and perturbation of cholesterol homeostasis. They would in such a case be a NEW CLASS of potential AD therapeutics working via an established mechanism of action and hence this proposal is entirely within the remit of the PA-06-261 titled Grants for Alzheimer's Disease Drug Discovery (R21). We will investigate this hypothesis under the remit of the following two specific aims: Specific aim #1. Synthesis of beauveriolide-derived cyclodepsipeptide and cyclopeptide libraries In this specific aim libraries of beauveriolide-inspired structures are proposed (L1/L1', L2, L3 and L4). The key structural aspects being optimized are the macrolactone ring, the lipid side chain and the three hydrophobic amino acid components of the ring. Prior to selection for synthesis, all virtual library members will first be interrogated using in silico methods (the QikProp v3.0 suite of Maestro v 8.0) for favorable properties including Lipinkski's rule of 5, log BB and oral absorbtion. Only those members that fulfill the criterion will be synthesized. The synthesis is proposed to be a mixed solid- phase/solution phase approach. Library synthesis and optimization is proposed to occur in two stages, the first being randomization of the macrolactone and lipid side chain (libraries L1/L1') which will then be screened for biological activity as outlined in specific aim #2. The active compounds from L1/L1' will then be further refined through structural modifications of the hydrophobic amino-acids (libraries L2, L3 and L4) using positional scanning. Specific aim #2: To determine whether synthetic beauveriolide-inspired libraries reduce the levels of 2-secretase cleavage products of APP, including A2 in vitro In this specific aim there are three sub-aims each being a cell-culture assay designed to assess the ability of the library members synthesized in specific aim #1 to either 1. reduce lipid loading or 2. reduce A2 production and secretion. All analyses will be correlated with measurements of total protein production (BCA analysis) and free and esterified cholesterol (Amplex Red assay, Molecular Probes). Sub-aim 2.1 Measuring lipid loading in culture macrophages. Lipid droplet formation will be measured in a cultured murine macrophage cell line (J774.1) that has been treated with cholesterol-loaded liposomes. Quantification of lipid loading will be performed by microscopy of fixed and stained (H&E and oil-red O) cells. Sub-aim 2.2 ELISA assay to measure A2 secretion in cell-culture For this specific aim the CHO cell-lines stably transfected with human APP751 (7WD10 cell line and AC29, gifts from Prof. Ta-Yuan Chang) will be treated with library members from specific aim #1. The levels of A240 and A242 -secreted will be determined using a commercially-available ELISA kit (The Genetics Company, Switzerland). Sub-aim 2.3 Measurement of APP 2-cleavage product (2-APP-CTF) in cell culture In order to test the response of human neuronal cells to the chemical libraries synthesized in specific aim # 1 we will analyze the levels of 2-APP-CTF generated by the SH-SY5Y neuroblastoma cell line (ATCC, VA, US) using Western blot analysis with commercially available antibodies. These cells do not generate a detectable level of A2, but do generate detectable levels of 2-C-terminal fragments (2-APP-CTF), which have been analyzed in previous studies of ACAT inhibition and are indicative of 2-cleavage products in general, one of which is A2. NB CP-113,818 will be studied as a control ACAT inhibitor in this R21 proposal studies (gift from Pfizer Inc., c/o Mr.D. W. Owens, Pfizer Compound transfer manager). PUBLIC HEALTH RELEVANCE: Alzheimer's disease is now the most common form of dementia, affecting up to 15 million people worldwide. As a result of the increase in life expectancy, it is anticipated that by 2050 approximately 25 % of the world population will be over 65, of which one third are likely to develop AD. AD is a complex and genetically heterogeneous disease, characterized by progressive memory deficit, cognitive impairment and personality changes accompanied by specific structural abnormalities in the brain. In monetary terms, AD is already America's third most expensive disease - costing over $100 billion per year. Add to this the social, psychological and emotional costs of sufferers' and carers' diminishing quality of life and it becomes clear that inhibiting the development of Alzheimer's would have a hugely beneficial impact on public health and wealth. This project describes the development of a new class of compounds that may prevent and/or AD.

描述（由申请人提供）：所有形式的阿尔茨海默氏病（AD）常见的致病事件是淀粉样蛋白2肽（A2）在大脑的特定区域中的异常积累。 A2的积累被认为是AD中的关键病理事件，是该疾病所谓的淀粉样假说的基础。因此，目前正在寻求减少A2积累的治疗方法。已经明确地表明，CNS中A2的产生和清除受胆固醇稳态调节。已经在体外和体内显示了促尿液胆固醇稳态的化合物，例如酰基辅酶A：胆固醇酰基转移酶（ACAT）抑制剂，以减少A2的产生和分泌。但是，通常情况下，Acay抑制剂表现出较差的口服活性。 Beauveriolides是一种新的真菌代谢产物，已被证明是口服活性的ACAT抑制剂，目前正在研究为动脉粥样硬化的潜在治疗剂。 NB迄今尚无研究对天然产物Beauveriolides或Beauveriolide启发化合物的A2抑制作用的研究。在此R21中检验的假设是，天然产物Beauveriolides和Beauveriolide启发化合物的库应通过抑制ACAT和胆固醇稳态扰动而在体外减少A2-产生和分泌。在这种情况下，它们将是通过既定的作用机理起作用的新型潜在AD治疗剂，因此该提案完全在PA-06-261的派遣之内，名为“阿尔茨海默氏病药物发现的赠款”（R21）。我们将根据以下两个特定目的的列表进行调查：特定目标＃1。提出了Beauveriolide衍生的环肽二肽和环肽库的合成（在Beauveriolide启发的结构的特定目的库中）（L1/L1'，L1，L2，L3和L4）。要优化的关键结构方面是大分子环，脂质侧链和该环的三个疏水氨基酸成分。在选择合成之前，所有虚拟库成员将首先使用Silico方法（Maestro V 8.0的Qikprop v3.0套件）进行询问，以包括包括Lipinkski 5的5，log bb和口服吸收的规则，包括Lipinkski规则。只有那些符合标准的成员才能合成。该合成被认为是混合的固相/溶液相方法。提出了库的合成和优化分为两个阶段，第一个是大分子和脂质侧链（库L1/L1'）的随机化，然后将筛选出生物学活性，如特定AIM＃2中所述。然后，通过使用位置扫描的疏水性氨基酸（库L2，L3和L4）的结构修饰，将进一步改进来自L1/L1'的活性化合物。具体目的2：确定合成的BeauVeriolide启发的库是否会降低APP的2-分泌酶裂解产品的水平，包括在该特定目标中的A2体外，每个子艾滋病中的三个是一个细胞培养分析，旨在评估在特定目标中评估特定目标＃1的能力至1。减少Lipid Positing of Docaling of。降低了Lipid Poading ofering和2。分泌和2。所有分析都将与总蛋白质产生（BCA分析）和游离和酯化的胆固醇（Amplex Red Assay，Molecular Probes）相关。 Sub-aim 2.1测量培养巨噬细胞中的脂质负荷。脂质液滴的形成将在已用胆固醇载荷的脂质体处理的培养的鼠巨噬细胞系（J774.1）中测量。脂质负荷的定量将通过固定和染色（H＆E和油红色O）细胞的显微镜进行。 Sub-aim 2.2 ELISA测定法测量该特定目的的细胞培养中A2分泌的CHO细胞线（7WD10细胞系和AC29）稳定转染的CHO细胞线（来自Ta-Yuan Chang教授的礼物）将与图书馆成员一起从特定目标＃1中与图书馆成员进行治疗。 A240和A242分泌的水平将使用市售的ELISA KIT（瑞士遗传学公司）确定。 Sub-aim 2.3在细胞培养中测量APP 2裂解产品（2-APP-CTF），以测试人类神经元细胞对特定目的＃1合成的化学文库的响应，我们将分析由SH-SY5Y NeuroBlastoma Cell Line（ATCC，VA，VA，VA，US）的2-APP-CTF的水平与SH-SY5Y NeuroCT-CTF产生的水平。这些细胞不会产生可检测到的A2水平，但确实会产生可检测到的2-C末端片段（2-APP-CTF）的水平，这些水平已在先前的ACAT抑制研究中进行了分析，并且总体上指示了2裂产物，其中之一是A2。 NB CP-113,818将在此R21提案研究中作为对照ACAT抑制剂进行研究（辉瑞公司（Pfizer Inc.公共卫生相关性：阿尔茨海默氏病现在是最常见的痴呆症形式，影响了全球多达1500万人。由于预期寿命的增加，预计到2050年，大约25％的世界人口将超过65，其中三分之一可能会发展AD。 AD是一种复杂且遗传上异质性的疾病，其特征是进行性记忆缺陷，认知障碍和人格变化，伴随着大脑中特定的结构异常。就货币而言，广告已经是美国第三昂贵的疾病 - 每年耗资超过1000亿美元。除此之外，患者和照顾者的社会，心理和情感成本降低了生活质量，很明显，抑制阿尔茨海默氏症的发展将对公共卫生和财富产生巨大的有益影响。该项目描述了可能预防和/或AD的新化合物的开发。

项目成果

The natural products beauveriolide I and III: a new class of beta-amyloid-lowering compounds.

• DOI: 10.1002/cbic.200900139
• 发表时间: 2009-05-25
• 期刊: CHEMBIOCHEM
• 影响因子: 3.2
• 作者: Witter, Daniel P.;Chen, Yanping;Rogel, Joseph K.;Boldt, Grant E.;Wentworth, Paul, Jr.
• 通讯作者: Wentworth, Paul, Jr.