Discovery and optimization of ApoE4 correctors for the treatment of Alzheimer's Disease

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