Fragments vs GPx4 – in reverse micelles

Membrane proteins account for more than half of drug targets, but the fraction is far smaller for fragment-derived drugs. In part this is because biophysical methods, the mainstay of FBLD, have been harder to apply to membrane proteins. A recent (open-access) paper in JACS Au by Courtney Labrecque and Brian Fuglestad at Virginia Commonwealth University tackles this challenge.

 

The researchers use an approach called membrane-mimicking reverse micelles, or mmRM: tiny water-filled bubbles surrounded by lipids and suspended in an organic solvent. We last wrote about reverse micelles back in 2019, where they were being used to study high local concentrations of water-soluble proteins and ligands. Here, the researchers turned to membrane proteins.

 

There are actually two types of membrane proteins: integral membrane proteins and peripheral membrane proteins. The former, as their name implies, have at least part of the protein anchored in the membrane at all times; GPCRs are a prominent class. Peripheral membrane proteins are water soluble but associate with the membrane, and this interaction is often required for folding or function. One example is glutathione peroxidase 4 (GPx4), which reduces oxidized lipids. It is an intriguing but  challenging cancer target, with the only ligands being fairly reactive covalent modifiers. Thus the researchers turned to mmRMs, hoping these could both stabilize the protein in a biologically relevant state and also present binding opportunities unavailable in standard screens.

 

A library of 1911 fragments from Life Chemicals was screened against mmRM-encapsulated GPx4 using ¹⁵N-¹H HSQC protein-detected NMR. Fragments were chosen to have high aqueous solubility (at least 1 mM in PBS) and were screened in mixtures of 10 at 400 µM per fragment. After deconvolution, 14 hits were identified, and dose-response titrations revealed that 9 had apparent dissociation constants < 1 mM, with the most potent having a K_d = 105 µM.

 

Three fragments were studied in greater detail, and these were chosen to have a range of hydrophobicities from clogD = -2.1 (most polar) to clogD = 2.1 (most hydrophobic). Chemical shift perturbation (CSP) analyses suggested that the two more lipophilic fragments bind to the membrane-interacting region of the protein, while the more polar fragment likely binds to a water-exposed site. SAR-by-catalog was applied to find analogs, some of which had increased affinity for the protein, with the best being around K_d = 15 µM.

 

Interestingly, the fragments showed minimal binding to GPx4 under normal aqueous conditions (ie, in the absence of the mmRMs), even at very high fragment concentrations. The researchers suggest this is because the fragments are binding to the membrane-bound state of the protein found in mmRMs, which may adopt a different conformation than that in the absence of membranes. Perhaps. But as prior work shows, it is possible to detect extraordinarily low affinity interactions inside reverse micelles, so maybe these are just very weak binders. Ultimately it remains to be seen whether these fragments will have practical applications. I hope so, and look forward to seeing how they progress.