Stabilizing apolipoprotein E4 with fragments

Among fragment-derived drugs that have entered the clinic, BACE1 inhibitors are well-represented. Sadly, multiple drugs targeting this protein have failed to show efficacy against Alzheimer’s disease. That said, every drug that has been thrown at Alzheimer’s has failed to slow the disease, so perhaps we need to think more boldly. An example was published recently in J. Med. Chem. by Andrew Petros, Eric Mohler, and colleagues at AbbVie.

The researchers were interested in apolipoprotein E4 (apoE4), one of three isoforms found in humans. Folks who have two apoE4 alleles are at increased risk for Alzheimer’s, suggesting that the protein might make a good drug target. Unfortunately, although it is known to be a lipid carrier, its precise function is unclear. What is known is that apoE4 is less stable to thermal denaturation than apoE2 or apoE3, so the team set out to find molecules that would stabilize the protein. This being AbbVie, they used two-dimensional NMR to find fragments.

The methyl groups of all the isoleucine, leucine, methionine, and valine residues in apoE4 were ¹³C labeled, and the researchers looked for changes in the ¹³C-HSQC spectra upon addition of fragments; just over 4000 were screened in pools of 12, each at 1 mM. Of the dozen or so hits, compound 1 was among the best.

NMR titration studies revealed an affinity just under 1 mM, while SPR suggested slightly stronger binding. As hoped, compound 1 raised the melting temperature of apoE4. Adding the fragment also altered the kinetics of liposome breakdown, causing the protein to behave more like apoE2 and apoE3. Although this assay isn’t necessarily physiologically relevant, the reasoning is that causing apoE4 to behave more like the other isoforms may be useful.

A crystal structure of compound 1 bound to apoE4 revealed the fragment to be binding in a small pocket, and growing led to compound 2, with a slightly improved affinity. Introduction of polar substituents to interact with a nearby aspartic acid side chain led to compound 8, with low micromolar affinity (assessed by NMR). This molecule also stabilized apoE4 with respect to thermal denaturation.

As noted above, it is not entirely clear why apoE4 is associated with Alzheimer’s, but researchers had previously found that overexpression in a neuronal cell line caused release of the inflammatory cytokines IL-6 and IL-8. When human induced pluripotent stem cell (iPSC)-derived astrocytes carrying two copies of apoE4 were treated with compound 8, release of IL-6 and IL-8 cytokines was reduced to levels similar to those from iPSC-derived astrocytes carrying two copies of apoE3. The compound also showed no toxicity, even at relatively high concentrations (100 µM).

There is still a tremendous amount to do: affinity needs to be improved considerably, and permeability is also mentioned as an issue. Moreover, the highly polar nature of compound 8 will likely make transport across the blood-brain barrier challenging. Optimizing activity against a target whose function is poorly understood will present a host of problems. But if it were easy, Alzheimer’s disease would not be the scourge that it is. Practical Fragments salutes thinking outside the box, and wishes those involved the best of luck.