Labrecque Courtney L, Fuglestad Brian
Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284, United States.
Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, Virginia 23219, United States.
JACS Au. 2024 Jun 26;4(7):2676-2686. doi: 10.1021/jacsau.4c00427. eCollection 2024 Jul 22.
While they account for a large portion of drug targets, membrane proteins present a unique challenge for drug discovery. Peripheral membrane proteins (PMPs), a class of water-soluble proteins that bind to membranes, are also difficult targets, particularly those that function only when 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, protein-membrane 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 at the protein-membrane interface. The proof-of-principle target, glutathione peroxidase 4 (GPx4), is a lipid hydroperoxidase that 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 noncovalent small-molecule ligands for membrane-bound GPx4 revealed through the mmRM fragment screening methodology. The fragments were tested against GPx4 under 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 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 noncovalent inhibitors of GPx4.
虽然膜蛋白占药物靶点的很大一部分，但它们给药物研发带来了独特的挑战。外周膜蛋白（PMPs）是一类与膜结合的水溶性蛋白，也是难以攻克的靶点，尤其是那些仅在与膜结合时才发挥功能的蛋白。PMPs中的蛋白质-膜界面通常是功能相互作用和催化发生的地方，使其成为抑制的合理靶点。然而，在抑制剂设计中，蛋白质-膜界面是未被充分探索的领域，需要改进小分子配体发现方法。为了更好地启动针对PMPs的药物研发工作，本研究提出了一种筛选方法，使用膜模拟反胶束（mmRM）和基于核磁共振的片段筛选来评估蛋白质-膜界面的配体结合能力。原理验证靶点谷胱甘肽过氧化物酶4（GPx4）是一种脂质氢过氧化物酶，对膜的氧化保护以及预防铁死亡至关重要。抑制GPx4有望用于治疗耐药性癌症，但目前的抑制剂通常是共价配体，临床应用有限。本文展示了通过mmRM片段筛选方法发现的膜结合型GPx4的非共价小分子配体。这些片段在大量水相条件下针对GPx4进行了测试，在没有嵌入膜的情况下，它们与该蛋白几乎没有或没有结合。这9个命中片段具有不同的亲和力和分配系数，并揭示了在蛋白质-膜界面内结合的片段的特性。此外，二次筛选证实了通过添加某些疏水基团将片段的亲和力从>200 μM提高到~15 μM从而推进这些片段研究的潜力。本研究提高了膜相关蛋白的筛选能力，揭示了GPx4蛋白质-膜界面内的配体结合能力，并可能作为开发GPx4非共价抑制剂的起点。
