Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.
【저자키워드】 Drug development, molecular dynamics, VEGF, Vascular endothelial growth factor, designed ankyrin repeat protein, DARPin, ensovibep, abicipar pegol, thermostability, N-terminal capping repeat, drug engineering, DARPins, designed ankyrin repeat proteins, HER2, human epidermal growth factor receptor 2, hGABP, human guanine-adenine-binding protein, HSA, human serum albumin, IR, internal repeat, 【초록키워드】 Mutation, antibody, drug, Molecular dynamics simulation, improvement, Protein, temperature, in silico analysis, inhibitor, endothelial, Vascular, domains, growth, SARS-CoV-2 entry, domain, clinical development, N-terminal, electrostatic repulsion, FIVE, flexible, IMPROVE, responsible, clinically, less, replaced, reduce, Ala, Ile, Thr, transferred, Val, was increased, 【제목키워드】 drug, Protein, thermostable, block,