Department of Bioorganic Chemistry, Wrocław University of Science and Technology, Wrocław, Poland.
Faculty of Chemistry, Jagiellonian University, Kraków, Poland.
Protein Sci. 2024 Aug;33(8):e5106. doi: 10.1002/pro.5106.
Miniproteins constitute an excellent basis for the development of structurally demanding functional molecules. The engrailed homeodomain, a three-helix-containing miniprotein, was applied as a scaffold for constructing programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) interaction inhibitors. PD-L1 binders were initially designed using the computer-aided approach and subsequently optimized iteratively. The conformational stability was assessed for each obtained miniprotein using circular dichroism spectroscopy, indicating that numerous mutations could be introduced. The formation of a sizable hydrophobic surface at the inhibitor that fits the molecular target imposed the necessity for the incorporation of additional charged amino acid residues to retain its appropriate solubility. Finally, the miniprotein effectively binding to PD-L1 (K = 51.4 nM) that inhibits PD-1/PD-L1 interaction in cell-based studies with EC = 3.9 μM, was discovered.
小分子蛋白是构建结构要求高的功能分子的理想基础。含有三个螺旋的 engrailed 同源结构域被用作构建程序性细胞死亡蛋白 1/程序性死亡配体 1（PD-1/PD-L1）相互作用抑制剂的支架。最初使用计算机辅助方法设计 PD-L1 结合物，然后进行反复迭代优化。使用圆二色性光谱法评估每个获得的小分子蛋白的构象稳定性，表明可以引入许多突变。抑制剂上形成一个适合分子靶标的大的疏水面，这就需要引入额外的带电荷的氨基酸残基来保持其适当的溶解度。最后，发现了一种能够有效结合 PD-L1（Kd=51.4 nM）的小分子蛋白，它能够在细胞水平上抑制 PD-1/PD-L1 相互作用，EC50 为 3.9 μM。
