Rational surface engineering of an arginine deiminase (an anti‐tumor enzyme) for increased PEGylation efficiency
用于提高PEG化效率的精氨酸脫亞胺酶（抗腫瘤酶）有理曲面工程化
Abstract
摘要
Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine‐auxotrophic tumors. However, its application as anti‐cancer drug is hampered by its poor stability under physiological conditions in blood stream. Commonly, random PEGylation is being used for increasing the stability of ADI and in turn the improved half‐life. However, the traditional random PEGylation usually leads to poor PEGylation efficiency due to the limited number of Lys on the protein surface. In order to boost the PEGylation efficiency and enhance the stability of ADI further, surface engineering of PpADI (an ADI from Pseudomonas plecoglossicida) to increase the suitable PEGylation sites was carried out. A new in silico approach for increasing the PEGylation sites was developed. The validation of this approach was performed on previously identified PpADI variant M31 to increase potential PEGylation sites. Four Arg residues on the surface of PpADI M31 were selected through three criteria and subsequently substituted to Lys, aiming for providing primary amines for PEGylation. Two out of the four substitutions (R299K and R382K) enhanced the stability of PEGylated PpADI in human serum. The average numbers of PEGylation sites were increased from ~12 (tetrameric PpADI M31, starting point) to ~20 (tetrameric PpADI M36, final variant). Importantly, the PEGylated PpADI M36 after PEGylation exhibited significantly improved T m values (M31: 40 °C; M36: 40 °C; PEG‐M31: 54 °C; PEG‐M36: 64 °C) and half‐life in human serum (M31: 1.9 days; M36: 2.0 days; PEG‐M31: 3.2 days; PEG‐M36: 4.8 days). These proved that the surface engineering is an effective approach to increase the PEGylation efficiency which therefore enhances the stability of therapeutic enzymes. Furthermore, the PEGylated PpADI M36 represents a highly attractive candidate for the treatment of arginine‐auxotrophic tumors.
精氨酸脫亞胺酶（ADI）是用于精氨酸營養缺陷型腫瘤的癌癥治療的治療性蛋白質。然而，其作為抗癌藥物的應用受到其在生理條件下血流中穩定性差的阻礙。通常，隨機PEG化用于增加ADI的穩定性，進而改善半衰期。然而，由于蛋白質表面上Lys的數量有限，傳統的隨機PEG化通常導致差的PEG化效率。為了進一步提高PEG化效率和增強ADI的穩定性，進行PpADI（一種來自假單胞菌的ADI）的表面工程化以增加合適的PEG化位點。一種用于增加PEG化位點的新的計算機方法被開發了。對先前鑒定的PpADI變體M31進行該方法的驗證以增加潛在的PEG化位點。通過三個標準選擇PpADI M31表面上的4個Arg殘基，隨后用Lys替換，為聚乙二醇化提供伯胺。四種取代中的兩種（R299K和R382K）增強了PEG化PpADI在人血清中的穩定性。PEG化位點的平均數量從~12（四聚體PpADI M31，起始點）增加至~20（四聚體PpADI M36，最終變體）。重要的是，PEG化后聚乙二醇化的PpADI M36表現出顯著改善的T m值（M31：40℃; M36：40℃; PEG-M31：54℃; PEG-M36：64℃）和人血清中的半衰期（M31：1.9天; M36：2.0天; PEG-M31：3.2天; PEG-M36：4.8天）。 這些證明表面工程是提高PEG化效率的有效方法，因此增強了治療酶的穩定性。 此外，PEG化的PpADI M36代表了用于治療精氨酸營養缺陷型腫瘤的極具吸引力的候選物。
首次發表: 2019-5-7 https://doi.org/10.1002/bit.27011
