Discovery and development of apoE4 correctors for the treatment of Alzheimer's disease

发现和开发用于治疗阿尔茨海默病的 apoE4 校正剂

基本信息

批准号：
10901029
负责人：
BRADLEY T. HYMAN
金额：
$ 91.91万
依托单位：
SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-15 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10901029
关键词：
Alleles Alzheimer&apos s Disease Alzheimer&apos s disease risk Alzheimer&apos s disease therapeutic Amino Acids Amyloid deposition Apolipoprotein E Arginine Astrocytes Automobile Driving Binding Binding Sites Biological Biological Assay Biological Markers Biology Blood - brain barrier anatomy Brain C-terminal Cell model Cells Chemistry Clinical Trials Complex Cysteine DNA Sequence Alteration Data Defect Development Disease Docking Dose Drug Kinetics Emerging Technologies Engineering Functional disorder Genetic Genetic Risk Genotype Human In Vitro Individual Inherited Laboratories Lead Length Libraries Lipid Binding Lipids Luciferases Mediating Medical Membrane Modeling Molecular Conformation Mus Mutate Mutation N-terminal Neurofibrillary Tangles Pathway interactions Patients Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacodynamics Phenotype Positioning Attribute Property Protein Conformation Protein Isoforms Proteins Public Health Publishing Recombinants Relative Risks Risk Route Scientist Structure Synapses Temperature Testing Therapeutic Tissues Toxic effect Transfection Transgenic Animals Transgenic Mice Transgenic Model Transgenic Organisms Variant analog apolipoprotein E-3 apolipoprotein E-4 biophysical analysis blood-brain barrier crossing blood-brain barrier function blood-brain barrier penetration design effective therapy experimental study gain of function genetic risk factor high throughput screening improved in silico in vivo induced pluripotent stem cell innovation insight loss of function melting mouse model nanoluciferase novel prevent response scaffold small molecule

项目摘要

PROJECT SUMMARY Alzheimer's disease (AD) is a major public health crisis with no effective treatments. Apolipoprotein E (ApoE) has three major polymorphic alleles, denoted ApoE2, ApoE3, and ApoE4. Homozygosity for ApoE4 is the strongest genetic risk factor for AD with an astonishing 12-fold increased risk of developing AD compared with individuals who inherit ApoE3.1,2 ApoE4 differs from ApoE3 by a single amino acid, an arginine instead of cysteine at position 112. This small change presumably alters the conformation of the protein, altering its activity in many biological pathways resulting in both gain and loss of function.1,3 Given the dramatic impact of ApoE4 on AD biology (including increased amyloid deposition, faster rate of progression, decreased synaptic content), there have been attempts to identify a small molecule that binds to ApoE4 and makes it functionally similar to ApoE3, a so called “corrector”. Yet this has been challenging: Generating purified ApoE4 protein has proven to be difficult, as the protein is notoriously sticky and readily aggregates. Multiple mutations in the C- terminal region are required to enable ApoE purification and structural determination.4 Biophysical analysis of this purified but modified protein gives insight into the consequences of the single amino acid change, suggesting that the thermal stability of ApoE4 is notably less than ApoE3.5 We now approach this challenge to take advantage of an emerging technology to analyze thermal stability of proteins in the context of tissues, intact cells and lysates called cellular thermal shift (CETSA).6 Analysis of lysates from the brains of humans or humanized ApoE transgenic animals by CETSA showed that brain ApoE4 is less thermally stable compared to ApoE3. We have reproduced this same phenotype in transfected human HEK cells and by engineering a HiBiT tag on to the N-terminus of ApoE4, creating a split Nano-luciferase cellular thermal shift assay (BiTSA)7,8 that is suitable for both identifying novel correctors, through a high throughput (HTP) screen, and driving subsequent hit to lead efforts. Excitingly, we show that both a recently published ApoE4 corrector (compound 8), discovered by scientists at AbbVie by an NMR fragment screen and structure guided chemistry effort, and a previously published genetic “corrector” – the Arg61T mutation, fully restore the temperature stability of ApoE4 such that it performed like ApoE3 in the BiTSA assay. Compound 8 has a KD <5 μM9. We have already made a small library of analogues, reproducing the effect with some potential insight into improvements. The aims of this application are to 1) leverage the ApoE BiTSA assay to identify novel ApoE4 correctors, through an HTP screen, 2) drive medicinal chemistry optimization of compound 8 and hits identified in the screen and 3) test the hypothesis that these correctors can ameliorate ApoE4 related Alzheimer phenotypes both in vitro and in vivo using cell and murine models that we and others have developed. These efforts represent initial steps towards our overall long-term objective, discovering first in class ApoE4 correctors as therapeutics to prevent AD.

项目概要阿尔茨海默病 (AD) 是一种重大的公共卫生危机，目前尚无有效的治疗方法。具有三个主要的多态性等位基因，表示为 ApoE2、ApoE3 和 ApoE4，ApoE4 的纯合性是。 AD 最强的遗传风险因素，与相比，患 AD 的风险增加了 12 倍与继承 ApoE3.1,2 的个体相比，ApoE4 与 ApoE3 的区别在于一个氨基酸，即精氨酸而不是 112位上的半胱氨酸。这个微小的变化可能会改变蛋白质的构象，从而改变其许多生物途径中的活性导致功能的获得和丧失。1,3 鉴于 ApoE4 对 AD 生物学的影响（包括淀粉样蛋白沉积增加、进展速度加快、突触减少内容），已经尝试鉴定一种与 ApoE4 结合并使其发挥功能的小分子与 ApoE3 类似，一种所谓的“校正器”，但这一直具有挑战性：生成纯化的 ApoE4 蛋白已成为现实。事实证明这很困难，因为该蛋白质非常粘并且很容易在 C- 中聚集多个突变。需要末端区域才能实现 ApoE 纯化和结构测定。4 这种纯化但经过修饰的蛋白质可以深入了解单个氨基酸变化的后果，表明 ApoE4 的热稳定性明显低于 ApoE3.5 我们现在应对这一挑战利用新兴技术来分析组织中蛋白质的热稳定性，完整的细胞和裂解物称为细胞热位移 (CETSA)。6 对人类大脑裂解物的分析 CETSA 人源化 ApoE 转基因动物表明大脑 ApoE4 热稳定性较差与 ApoE3 相比，我们在转染的人 HEK 细胞中复制了相同的表型。将 HiBiT 标签设计到 ApoE4 的 N 末端，产生分裂的纳米荧光素酶细胞热位移测定 (BiTSA)7,8 适合通过高通量 (HTP) 筛选鉴定新型校正剂，令人兴奋的是，我们展示了最近发布的 ApoE4 校正器。（化合物 8），由艾伯维科学家通过 NMR 片段筛选和结构引导化学发现努力，加上之前发表的基因“校正器”——Arg61T突变，完全恢复温度 ApoE4 的稳定性，使其在 BiTSA 测定中的表现类似于 ApoE3，其 KD <5 μM9。已经制作了一个小型类似物库，通过一些潜在的洞察力再现了效果该应用的目的是 1) 利用 ApoE BiTSA 检测进行新的鉴定。 ApoE4 校正器，通过 HTP 筛选，2) 驱动化合物 8 和的药物化学优化屏幕中识别出的命中结果以及 3) 检验这些校正器可以改善 ApoE4 的假设使用我们和其他人的细胞和小鼠模型在体外和体内研究相关的阿尔茨海默病表型这些努力代表了我们实现总体长期目标的初步步骤，即发现。首个 ApoE4 校正剂作为预防 AD 的治疗药物。