Department of Chemistry, University of Toronto, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada.
ACS Chem Biol. 2021 Jan 15;16(1):35-44. doi: 10.1021/acschembio.0c00684. Epub 2020 Dec 28.
Protein-based therapeutics are part of the next-generation arsenal of drugs being developed against proto-oncoprotein Myc. We designed protein to mimic the basic region/helix-loop-helix/leucine zipper (bHLHZ) domain of Max and Myc, which bind to the E-box motif (enhancer box, CACGTG). To make , we started with our rationally designed ME47, a hybrid of the Max basic region and E47 HLH, that effectively inhibited tumor growth in a mouse model of breast cancer. We used phage-assisted continuous evolution (PACE), which uncovered mutations at Arg12 that contact the DNA phosphodiester backbone. The Arg12 mutations improved ME47's stability. We replaced Cys29 with Ala to eliminate potential undesired disulfide formation and fused the designed FosW leucine zipper to mutated ME47 to increase the dimerization interface and E-box targeting activity. This "franken-protein" comprises the Max basic region, E47 HLH, and FosW leucine zipper. Compared with ME47, gives 2-fold stronger binding to E-box and 4-fold increased specificity for E-box over nonspecific DNA. The synergistic combination of rational design and PACE allowed us to make and demonstrates the power and utility of our two-pronged approach toward development of promising protein drugs with robust structure and DNA-binding function.
蛋白质类药物是针对原癌蛋白 Myc 开发的新一代药物中的一部分。我们设计了一种蛋白质，以模拟 Max 和 Myc 的碱性区域/螺旋-环-螺旋/亮氨酸拉链（bHLHZ）结构域，该结构域与 E 盒基序（增强子盒，CACGTG）结合。为了生成 ，我们从我们合理设计的 ME47 开始，它是 Max 碱性区域和 E47 HLH 的杂交体，在乳腺癌小鼠模型中有效地抑制肿瘤生长。我们使用噬菌体辅助连续进化（PACE），发现了与 DNA 磷酸二酯骨架接触的 Arg12 突变。Arg12 突变提高了 ME47 的稳定性。我们用丙氨酸取代 Cys29，以消除潜在的不需要的二硫键形成，并将设计的 FosW 亮氨酸拉链融合到突变的 ME47 上，以增加二聚化界面和 E 盒靶向活性。这种“弗兰肯蛋白”由 Max 碱性区域、E47 HLH 和 FosW 亮氨酸拉链组成。与 ME47 相比， 对 E 盒的结合强度提高了 2 倍，对 E 盒的特异性提高了 4 倍，而非特异性 DNA 的特异性提高了 4 倍。理性设计和 PACE 的协同组合使我们能够生成 ，并证明了我们双管齐下的方法在开发具有稳健结构和 DNA 结合功能的有前途的蛋白质药物方面的强大力量和实用性。
