Role of Cathepsin A and Lysosomes in the Intracellular Activation of Novel Antipapillomavirus Agent GS-9191
Role of Cathepsin A and Lysosomes in the Intracellular Activation of Novel Antipapillomavirus Agent GS-9191
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DOI：
10.1128/aac.01603-10
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发表时间：
2011-05-01
影响因子：
4.9
通讯作者：
Cihlar, Tomas
中科院分区：
文献类型：
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作者：
Birkus, Gabriel;Kutty, Nilima;Cihlar, Tomas
GS-9191是一种核苷酸类似物9-(2-phosphonylmethoxyethyl)-N(6)-cyclopropyl-2，6-diaminopurine(CPrPMEDAP)的双酰胺类前药，设计用于治疗乳头瘤病毒相关的增殖性疾病，如生殖器疣。在本研究中，我们研究了GS-9191转化为cPrPMEDAP的机制。我们观察到GS-9191在溶酶体羧肽酶组织蛋白A(CATA)的存在下在体外被水解，并且在CATA缺陷的成纤维细胞中的代谢效率低于对照细胞。此外，用小干扰RNA(SiRNA)敲除CATA可减少GS-9191代谢物在细胞内的积累。然而，细胞内CATA水平与测试细胞系对GS-9191的敏感性并不相关，这表明CATA步骤不太可能是GS-9191激活的速率限制。进一步的分析表明，在GS-9191的一个酰胺基段上的羧酸酯键被水解后，未被掩蔽的羧基取代了另一个酰胺基上的L-苯丙氨酸2-甲基丙酯。形成的cPrPMEDAP-L-苯丙氨酸偶合物(cPrPMEDAP-Phe)不被组氨酸三核苷酸结合蛋白1(组氨酸三核苷酸结合蛋白1)磷酰胺酶代谢，但在酸性pH下自发降解为cPrPMEDAP，加入SiHA细胞提取物可显着增强cPrPMEDAP-Phe的活性。用巴菲霉素A或氯喹处理SIHA细胞后，细胞内cPrPMEDAP-Phe代谢物浓度增加8倍，GS-9191代谢物在溶酶体/内涵体部分积聚。综上所述，这些观察表明，在溶酶体中，GS-9191通过CATA介导的酯裂解转化为cPrPMEDAP，随后cPrPMEDAP被释放，最有可能的是通过酶驱动和自发pH驱动的cPrPMEDAP-Phe中间体的水解。
GS-9191, a bis-amidate prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl)-N(6)-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), was designed as a topical agent for the treatment of papillomavirus-associated proliferative disorders, such as genital warts. In this study, we investigated the mechanism of conversion of GS-9191 to cPrPMEDAP. We observed that GS-9191 is hydrolyzed in the presence of the lysosomal carboxypeptidase cathepsin A (CatA) in vitro and is less efficiently metabolized in CatA-deficient fibroblasts than in control cells. In addition, knockdown of CatA by small interfering RNA (siRNA) reduced the intracellular accumulation of GS-9191 metabolites. However, intracellular CatA levels did not correlate with the susceptibility of tested cell lines to GS-9191, indicating that the CatA step is unlikely to be rate limiting for the activation of GS-9191. Further analysis showed that upon the hydrolysis of the carboxylester bond in one of the GS-9191 amidate moieties, the unmasked carboxyl group displaces L-phenylalanine 2-methylpropyl ester from the other amidate moiety. The cPrPMEDAP-L-phenylalanine conjugate (cPrPMEDAP-Phe) formed is not metabolized by Hint1 (histidine triad nucleotide binding protein 1) phosphoramidase but undergoes spontaneous degradation to cPrPMEDAP in acidic pH that can be significantly enhanced by the addition of SiHa cell extract. Pretreatment of SiHa cells with bafilomycin A or chloroquine resulted in an 8-fold increase in the intracellular concentration of cPrPMEDAP-Phe metabolite and the accumulation of GS-9191 metabolites in the lysosomal/endosomal fraction. Together, these observations indicate that the conversion of GS-9191 to cPrPMEDAP occurs in lysosomes via CatA-mediated ester cleavage, followed by the release of cPrPMEDAP, most likely through the combination of enzyme-driven and spontaneous pH-driven hydrolysis of a cPrPMEDAP-Phe intermediate.