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

项目总结 阿尔茨海默病(AD)是一种严重的公共卫生危机，目前尚无有效的治疗方法。载脂蛋白E(ApoE) 有三个主要的多态等位基因，分别表示为APOE2、ApoE3和ApoE4。ApoE4的纯合性是 阿尔茨海默病的最强遗传风险因素与患阿尔茨海默病的风险惊人地增加了12倍 在遗传ApoE3.1，2的个体中，ApoE4与ApoE3只有一种氨基酸不同，精氨酸而不是 半胱氨酸在第112位。这个微小的变化可能改变了蛋白质的构象，改变了它的 在许多生物途径中的活动导致功能的获得和丧失。1，3考虑到 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 内容物)，已经尝试识别与载脂蛋白E4结合并使其具有功能的小分子 类似于ApoE3，也就是所谓的“校正器”。然而，这一直是具有挑战性的：生产纯化的ApoE4蛋白 事实证明这很困难，因为这种蛋白质是出了名的粘性和容易聚集。C-基因的多个突变 末端区域是实现载脂蛋白E纯化和结构测定所必需的。4生物物理分析 这种纯化但经过修饰的蛋白质可以洞察单一氨基酸变化的后果， 表明ApoE4的热稳定性明显低于ApoE3.5。我们现在应对这一挑战 利用一种新兴技术在组织环境中分析蛋白质的热稳定性， 完整的细胞和称为细胞热位移(CETSA)的裂解产物。6人或 CETSA人源化载脂蛋白E转基因动物显示，脑载脂蛋白E4的热稳定性较差 与ApoE3相比。我们已经在转基因的人HEK细胞中复制了同样的表型，并通过 在载脂蛋白E4的N端设计HiBiT标签，产生分裂的纳米荧光素酶细胞热位移 适合于通过高通量(HTP)筛查识别新的校正子的化验(BITSA)7，8， 并推动随后的Hit领导努力。令人兴奋的是，我们发现最近发布的ApoE4修正器 (化合物8)，由AbbVie的科学家通过核磁共振片段筛选和结构引导化学发现 努力，以及之前发表的基因“校正器”--Arg61T突变，完全恢复了温度 ApoE4的稳定性，使其在BITSA试验中表现出与ApoE3相同的性能。化合物8具有Kd&lt；5μM9。我们 我已经制作了一个小型类比库，用一些潜在的洞察力再现了这种效果 改进。本申请的目的是1)利用ApoE BITSA分析来识别新的 ApoE4校正子，通过HTP筛选，2)推动化合物8的药物化学优化和 屏幕中识别的点击数和3)测试这些校正器可以改善载脂蛋白E4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的相关阿尔茨海默病表型 已经发展起来了。这些努力是朝着我们的总体长期目标迈出的第一步，发现 作为预防AD的治疗药物，ApoE4校正剂在类中名列前茅。

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.