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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.

项目摘要阿尔茨海默病（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）有三个主要的多态性等位基因，表示为ApoE 2，ApoE 3和ApoE 4。ApoE 4的纯合性是最强的遗传风险因素，发展为AD的风险增加了12倍， ApoE 4与ApoE 3只有一个氨基酸不同，即一个精氨酸，而不是一个精氨酸。 112位的半胱氨酸。这种微小的变化可能改变了蛋白质的构象，改变了其在许多生物途径中的活性导致功能的获得和丧失。 ApoE 4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，突触内容），已经尝试鉴定与ApoE 4结合并使其在功能上类似于ApoE 3，一种所谓的“校正剂”。然而，这一直是具有挑战性的：产生纯化的ApoE 4蛋白，这被证明是困难的，因为蛋白质是众所周知的粘性和容易聚集。C-中的多个突变需要末端区域来进行ApoE纯化和结构测定。这种纯化但修饰的蛋白质使人了解单个氨基酸变化的结果，这表明ApoE 4的热稳定性明显低于ApoE 3.5。利用新兴技术来分析组织中蛋白质的热稳定性，完整细胞和裂解物称为细胞热位移（CETSA）。通过CETSA的人源化ApoE转基因动物显示脑ApoE 4热稳定性较低与ApoE 3相比我们在转染的人HEK细胞中复制了相同的表型，将HiBiT标签工程化到ApoE 4的N-末端上，产生分裂的纳米荧光素酶细胞热位移，测定（BiTSA）7，8，其适合于通过高通量（HTP）筛选鉴定新的校正剂，并推动后续的打击，以领导努力。令人兴奋的是，我们发现，最近发表的ApoE 4校正（化合物8），由AbbVie的科学家通过NMR片段筛选和结构指导化学发现努力，和先前发表的遗传“校正”-Arg 61 T突变，完全恢复温度 ApoE 4的稳定性使得其在BiTSA测定中表现得像ApoE 3。化合物8具有<5 μ M 9的KD。我们我已经制作了一个小型的类似物库，再现了这种效应，并对改进.本申请的目的是1）利用ApoE BiTSA测定来鉴定新的ApoE BiTSA。 ApoE 4校正剂通过HTP筛选，2）驱动化合物8的药物化学优化， 3）测试这些校正剂可以改善ApoE 4的假设我们和其他人使用细胞和小鼠模型在体外和体内研究了相关的阿尔茨海默氏症表型，已经发展。这些努力代表了我们实现总体长期目标的初步步骤，作为预防AD的治疗剂的第一类ApoE 4校正剂。