Ligandability at the membrane interface of GPx4 revealed through a reverse micelle fragment screening platform

Abstract

AbstractWhile they account for a large portion of drug targets, membrane proteins (MPs) present a unique challenge for drug discovery. Peripheral membrane proteins (PMPs), a class of proteins that bind reversibly to membranes, are also difficult targets, particularly those that function only while bound to membranes. The protein-membrane interface in PMPs is often where functional interactions and catalysis occur, making it a logical target for inhibition. However, interfaces are underexplored spaces in inhibitor design and there is a need for enhanced methods for small-molecule ligand discovery. In an effort to better initiate drug discovery efforts for PMPs, this study presents a screening methodology using membrane-mimicking reverse micelles (mmRM) and NMR-based fragment screening to assess ligandability in the protein-membrane interface. The proof-of-principle target, glutathione peroxidase 4 (GPx4), is a lipid hydroperoxidase which is essential for the oxidative protection of membranes and thereby the prevention of ferroptosis. GPx4 inhibition is promising for therapy-resistant cancer therapy, but current inhibitors are generally covalent ligands with limited clinical utility. Presented here is the discovery of non-covalent small-molecule ligands for membrane-bound GPx4 revealed through the mmRM fragment screening methodology. The fragments were tested against GPx4 in bulk aqueous conditions and displayed little to no binding to the protein without embedment into the membrane. The 9 hits had varying affinities and partitioning coefficients and revealed properties of fragments that bind within the protein-membrane interface. Additionally, a secondary screen confirmed the potential to progress the fragments by enhancing the affinity from > 200 µM to ∼15 µM with the addition of certain hydrophobic groups. This study presents an advancement of screening capabilities for membrane associated proteins, reveals ligandability within the GPx4 protein-membrane interface, and may serve as a starting point for developing non-covalent inhibitors of GPx4.