Cathepsin A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide phosphonoamidate prodrugs GS-7340 and GS-9131
Cathepsin A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide phosphonoamidate prodrugs GS-7340 and GS-9131
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DOI：
10.1128/aac.00968-06
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发表时间：
2007-02-01
影响因子：
4.9
通讯作者：
McDermott, Martin
中科院分区：
文献类型：
--
作者：
Birkus, Gabriel;Wang, Ruth;McDermott, Martin
GS-7340和GS-9131 {9-[（R）-2-[[（S）-[[（S）-1-[（S）-1-[[（S）-1-[（S）-1-[[（S）-1-[（S）-1-[（S）-1（异丙氧基羰基）乙基]氨基]苯氧基氧膦基]甲氧基]丙基]腺嘌呤和9-（R）-4 ′-（R）-[[[（S）-1-[（乙氧羰基）乙基]氨基]苯氧基氧膦基]甲氧基]-2 '-氟-1'-呋喃基腺嘌呤，分别是替诺福韦{9-R-[（2-膦酰甲氧基）丙基]腺嘌呤}（TFV）和环核苷酸类似物GS-9148（膦酰甲氧基-2 '-氟-2'，3 '-二脱氧二脱氢腺苷）。这两种前药都对野生型和耐药人类免疫缺陷病毒I型毒株表现出强效的抗逆转录病毒活性，并且具有出色的体内药代动力学特性。在本研究中，从人外周血单核细胞中分离了负责GS-7340和GS-9131细胞内活化初始步骤的主要酶活性，并鉴定为溶酶体羧肽酶A（组织蛋白酶A [CatA]; EC 3.4.16.5）。纯化的水解酶（天然复合物和催化亚基分子量分别为100和29 kDa，等电点[pI]为5.5）的生化特性相匹配的CatA。重组CatA和分离的前药水解酶显示出相同的亲和性抑制剂和相同的底物偏好对一组替诺福韦膦酰胺酯前药。这两种酶与C-14标记的GS-7340或[H-3]二氟膦酸酯的孵育导致相同的29-kDa催化亚基的共价标记。最后，与GS-7340和GS-9131孵育4小时后，在CatA阴性成纤维细胞中检测到的前药代谢物的细胞内浓度分别比正常对照成纤维细胞中检测到的浓度低约7.5倍和3倍。总的来说，这些数据证明了CatA在核苷酸膦酰胺酯前药的细胞内活化中的关键作用，并为进一步改进这类重要的抗病毒前药开辟了新的可能性。
GS-7340 and GS-9131 {9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy] propyl]adenine and 9-(R)-4'-(R)-[[[(S)-1-[(ethoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-2'-fluoro-1'-furanyladenine, respectively} are novel alkylalaninyl phenyl ester prodrugs of tenofovir {9-R-[(2-phosphonomethoxy)propyl]adenine} (TFV) and a cyclic nucleotide analog, GS-9148 (phosphonomethoxy-2'-fluoro-2', 3'-dideoxydidehydroadenosine), respectively. Both prodrugs exhibit potent antiretroviral activity against both wild-type and drug-resistant human immunodeficiency virus type I strains and excellent in vivo pharmacokinetic properties. In this study, the main enzymatic activity responsible for the initial step in the intracellular activation of GS-7340 and GS-9131 was isolated from human peripheral blood mononuclear cells and identified as lysosomal carboxypeptidase A (cathepsin A [CatA]; EC 3.4.16.5). Biochemical properties of the purified hydrolase (native complex and catalytic subunit molecular masses of 100 and 29 kDa, respectively; isoelectric point [pI] of 5.5) matched those of CatA. Recombinant CatA and the isolated prodrug hydrolase displayed identical susceptibilities to inhibitors and identical substrate preferences towards a panel of tenofovir phosphonoamidate prodrugs. Incubation of both enzymes with C-14-labelled GS-7340 or [H-3] difluorophosphonate resulted in the covalent labeling of identical 29-kDa catalytic subunits. Finally, following a 4-h incubation with GS-7340 and GS-9131, the intracellular concentrations of prodrug metabolites detected in CatA-negative fibroblasts were approximately 7.5- and 3-fold lower, respectively, than those detected in normal control fibroblasts. Collectively, these data demonstrate the key role of CatA in the intracellular activation of nucleotide phosphonoamidate prodrugs and open new possibilities for further improvement of this important class of antiviral prodrugs.