Department of Pharmaceutical Sciences and Center for Molecular Drug Targeting, College of Pharmacy, The University of Michigan, 428 Church St., Ann Arbor, MI 48109-1065, United States.
Antiviral Res. 2010 Mar;85(3):482-9. doi: 10.1016/j.antiviral.2009.12.003. Epub 2009 Dec 5.
Cidofovir (HPMPC) is a broad-spectrum antiviral agent, currently used to treat AIDS-related human cytomegalovirus retinitis. Cidofovir has recognized therapeutic potential for orthopox virus infections, although its use is hampered by its inherent low oral bioavailability. Val-Ser-cyclic HPMPC (Val-Ser-cHPMPC) is a promising peptide prodrug which has previously been shown by us to improve the permeability and bioavailability of the parent compound in rodent models (Eriksson et al., 2008. Molecular Pharmaceutics 5, 598-609). Puromycin-sensitive aminopeptidase was partially purified from Caco-2 cell homogenates and identified as a prodrug activating enzyme for Val-Ser-cHPMPC. The prodrug activation process initially involves an enzymatic step where the l-Valine residue is removed by puromycin-sensitive aminopeptidase, a step that is bestatin-sensitive. Subsequent chemical hydrolysis results in the generation of cHPMPC. A recombinant puromycin-sensitive aminopeptidase was generated and its substrate specificity investigated. The k(cat) for Val-pNA was significantly lower than that for Ala-pNA, suggesting that some amino acids are preferred over others. Furthermore, the three-fold higher k(cat) for Val-Ser-cHPMPC as compared to Val-pNA suggests that the leaving group may play an important role in determining hydrolytic activity. In addition to its ability to hydrolyze a variety of substrates, these observations strongly suggest that puromycin-sensitive aminopeptidase is an important enzyme for activating Val-Ser-cHPMPC in vivo. Taken together, our data suggest that puromycin-sensitive aminopeptidase makes an attractive target for future prodrug design.
西多福韦（HPMPC）是一种广谱抗病毒药物，目前用于治疗与艾滋病相关的人巨细胞病毒视网膜炎。西多福韦对正痘病毒感染具有公认的治疗潜力，但其应用受到固有低口服生物利用度的限制。缬氨酸-丝氨酸-环 HPMPC（Val-Ser-cHPMPC）是一种有前途的肽前药，我们之前已经证明它可以提高亲化合物在啮齿动物模型中的通透性和生物利用度（Eriksson 等人，2008. 分子药剂学 5,598-609）。Puromycin 敏感氨肽酶从 Caco-2 细胞匀浆中部分纯化，并被鉴定为 Val-Ser-cHPMPC 的前药激活酶。前药激活过程最初涉及一个酶促步骤，其中 puromycin 敏感氨肽酶去除 L-缬氨酸残基，这一步是 bestatin 敏感的。随后的化学水解导致 cHPMPC 的生成。生成了重组 puromycin 敏感氨肽酶并研究了其底物特异性。Val-pNA 的 k(cat)明显低于 Ala-pNA，表明某些氨基酸比其他氨基酸更受青睐。此外，与 Val-pNA 相比，Val-Ser-cHPMPC 的 k(cat)高三倍，这表明离去基团在确定水解活性方面可能起重要作用。除了能够水解各种底物外，这些观察结果强烈表明 puromycin 敏感氨肽酶是体内激活 Val-Ser-cHPMPC 的重要酶。总之，我们的数据表明 puromycin 敏感氨肽酶是未来前药设计的有吸引力的靶标。
