Wednesday, January 15, 2025
1/15/2025
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 We and others have shown that analysis of such purified protein in a Protein Thermal Shift assay (PTS) shows that ApoE4 is >4oC less thermally stable than ApoE3.5 We have extended this finding by now demonstrating that lysates from the brains of humans or humanized ApoE transgenic animals when analyzed by an analogous technique, a CEllular Thermal Shift Assay (CETSA)6 also show that brain ApoE4 is less thermally stable compared to ApoE3. As CETSA is a low throughput assay, we engineered a HiBiT tag on to the N-terminus of ApoE to derive a split Nano-luciferase HiBiT cellular thermal Shift Assay (BiTSA)7,8 in HEK 293 cells, and miniaturized this assay to a 384 well format. Excitingly, we show that the thermal stability of ApoE4 measured by BiTSA can be “corrected” to that of ApoE3 by a recently published ApoE4 corrector (compound 8, EC50 <3uM) 9), discovered by scientists at AbbVie and a previously published genetic “corrector” – the Arg61T mutation. We have synthesized 30 analogs of compound 8 in a Hit to Lead effort and demonstrate nascent SAR and potential paths forward to optimize this series for in vivo use. Our research plan has three aims: 1) identify new ApoE4 corrector by screening 250,000 compounds deploying the ApoE4 PTS assay as a primary assay and the ApoE4 BiTSA as a secondary assay 2) drive medicinal chemistry optimization of compound 8 and hits identified in the HTP 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 for AD.
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