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

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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.