Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel.
The National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel.
Bioconjug Chem. 2022 May 18;33(5):795-806. doi: 10.1021/acs.bioconjchem.2c00059. Epub 2022 Apr 21.
Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of the matrix metalloproteinase (MMP) family of proteins, whose members are key regulators of the proteolysis of extracellular matrix components and hence of multiple biological processes. In particular, imbalanced activity of matrix metalloproteinase-14 (MMP-14) may lead to the development of cancer and cardiovascular and other diseases. This study aimed to engineer TIMP2, one of the four homologous TIMPs, as a potential therapeutic by virtue of its ability to bind to the active-site Zn of MMP-14. However, the susceptibility to degradation of TIMP2 and its small size, which results in a short circulation half-life, limit its use as a therapeutic. PEGylation was thus used to improve the pharmacokinetic profile of TIMP2. PEGylation of the MMP-targeting N-terminal domain of TIMP2 (N-TIMP2), via either cysteine or lysine residues, resulted in a significant decrease in N-TIMP2 affinity toward MMP-14 or multisite conjugation and conjugate heterogeneity, respectively. Our strategy designed to address this problem was based on incorporating a noncanonical amino acid (NCAA) into N-TIMP2 to enable site-specific mono-PEGylation. The first step was to incorporate the NCAA propargyl lysine (PrK) at position S31 in N-TIMP2, which does not interfere with the N-TIMP2-MMP-14 binding interface. Thereafter, site-specific PEGylation was achieved via a click chemistry reaction between N-TIMP2-S31PrK and PEG-azide-20K. Inhibition studies showed that PEGylated N-TIMP2-S31PrK did indeed retain its inhibitory activity toward MMP-14. The modified protein also showed improved serum stability vs non-PEGylated N-TIMP2. In vivo pharmacokinetic studies in mice revealed a significant 8-fold increase in the elimination half-life of PEGylated N-TIMP2 vs the non-PEGylated protein. This study shows that site-specific bioorthogonal mono-PEGylation extends the half-life of N-TIMP2 without impairing its biological activity, thereby highlighting the advantage of this strategy for generating potent PEGylated proteins.
组织金属蛋白酶抑制剂（TIMP）是基质金属蛋白酶（MMP）家族蛋白的天然抑制剂，其成员是细胞外基质成分蛋白水解的关键调节剂，因此也是多种生物学过程的关键调节剂。特别是，基质金属蛋白酶 14（MMP-14）活性的失衡可能导致癌症和心血管等疾病的发生。本研究旨在通过 TIMP2 与 MMP-14 活性位点 Zn 的结合能力，将其作为一种有潜力的治疗方法进行工程改造。然而，TIMP2 易降解及其较小的尺寸导致其循环半衰期较短，限制了其作为治疗药物的应用。因此，使用聚乙二醇（PEG）化来改善 TIMP2 的药代动力学特性。通过半胱氨酸或赖氨酸残基对 TIMP2 的 MMP 靶向 N 端结构域（N-TIMP2）进行 PEG 化，分别导致 N-TIMP2 对 MMP-14 的亲和力显著降低或多部位缀合和缀合物异质性。我们设计的解决此问题的策略是基于将非天然氨基酸（NCAA）掺入 N-TIMP2 中，以实现定点单 PEG 化。第一步是将 NCAA 炔丙基赖氨酸（PrK）掺入 N-TIMP2 的 S31 位，这不会干扰 N-TIMP2-MMP-14 结合界面。此后，通过 N-TIMP2-S31PrK 与 PEG-叠氮化物-20K 之间的点击化学反应实现定点 PEG 化。抑制研究表明，PEGylated N-TIMP2-S31PrK 确实保留了其对 MMP-14 的抑制活性。与非 PEGylated N-TIMP2 相比，修饰后的蛋白也显示出改善的血清稳定性。在小鼠体内药代动力学研究中，PEGylated N-TIMP2 的消除半衰期与非 PEGylated 蛋白相比显著增加了 8 倍。本研究表明，定点生物正交单 PEG 化延长了 N-TIMP2 的半衰期而不损害其生物学活性，从而突出了该策略在生成有效 PEGylated 蛋白方面的优势。
Zotero 插件