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 Kd = 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 Kd
= 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.
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