Targeting the Proton-Coupled Folate Transporter for Selective Delivery of 6-Substituted Pyrrolo[2,3-d]Pyrimidine Antifolate Inhibitors of De Novo Purine Biosynthesis in the Chemotherapy of Solid Tumors
Targeting the Proton-Coupled Folate Transporter for Selective Delivery of 6-Substituted Pyrrolo[2,3-d]Pyrimidine Antifolate Inhibitors of De Novo Purine Biosynthesis in the Chemotherapy of Solid Tumors
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DOI：
10.1124/mol.110.065896
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发表时间：
2010-10-01
影响因子：
3.6
通讯作者：
Matherly, Larry H.
中科院分区：
文献类型：
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作者：
Desmoulin, Sita Kugel;Wang, Yiqiang;Matherly, Larry H.
质子偶联叶酸转运蛋白（PCFT）是一种叶酸-质子同向转运蛋白，具有酸性pH最佳值，接近实体瘤的微环境。我们测试了在桥区具有1至6个碳的6-取代的吡咯并[2，3-d]嘧啶抗叶酸剂对表达PCFT或还原叶酸载体（RFC）的同基因中国仓鼠卵巢（CHO）和HeLa细胞增殖的抑制。仅具有三个和四个桥碳的类似物（分别为N-{4-[3-2-氨基-4-氧代-4，7-二氢-3H-吡咯并[2，3-d]-嘧啶-6-基）丙基]苯甲酰基}-L-谷氨酸（化合物2）和N-{4-[4-2-氨基-4-氧代-4，7-二氢-3H-吡咯并[2，3-d]-嘧啶-6-基）丁基]苯甲酰基}-L-谷氨酸（化合物3）是抑制性的，其中2 >> 3。对RFC表达细胞的活性可忽略不计。化合物2和培美曲塞（Pmx）与[H-3]甲氨蝶呤竞争PCFT表达CHO（R2/hPCFT 4）细胞从pH 5.5至7.2的PCFT运输;抑制随pH值降低而增加。在非洲爪蟾卵母细胞中，PCFT cRNA显微注射，2的摄取，如Pmx，是产电的。在腺苷或5-氨基-4-咪唑甲酰胺存在下，2对R2/hPCFT 4细胞的细胞毒性被消除，表明从头嘌呤生物合成中的甘氨酰胺核糖核苷酸甲酰转移酶（GARFT酶）是主要靶点。化合物2使GTP和ATP库分别降低了50%和75%。通过原位GARFT酶测定，2对细胞内GARFT酶的抑制作用是对细胞生长或集落形成的20倍。化合物2不可逆地抑制克隆形成，尽管这需要至少4小时的暴露。我们的结果证明了化合物2的有效抗增殖活性，这归因于其被PCFT有效的细胞摄取，导致GARFT酶和嘌呤从头生物合成的抑制。此外，他们建立了通过PCFT而不是RFC进行选择性化疗药物递送的可行性，这是一个利用实体瘤独特生物学特征的过程。
The proton-coupled folate transporter (PCFT) is a folate-proton symporter with an acidic pH optimum, approximating the microenvironments of solid tumors. We tested 6-substituted pyrrolo[ 2,3-d]pyrimidine antifolates with one to six carbons in the bridge region for inhibition of proliferation in isogenic Chinese hamster ovary (CHO) and HeLa cells expressing PCFT or reduced folate carrier (RFC). Only analogs with three and four bridge carbons (N-{4-[3-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)propyl]benzoyl}-L-glutamic acid (compound 2) and N-{4-[4-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)butyl]benzoyl}-L-glutamic acid (compound 3), respectively) were inhibitory, with 2 >> 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [H-3]methotrexate for PCFT transport in PCFT-expressing CHO (R2/hPCFT4) cells from pH 5.5 to 7.2; inhibition increased with decreasing pH. In Xenopus laevis oocytes microinjected with PCFT cRNA, uptake of 2, like that of Pmx, was electrogenic. Cytotoxicity of 2 toward R2/hPCFT4 cells was abolished in the presence of adenosine or 5-amino-4-imidazolecarboxamide, suggesting that glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis was the primary target. Compound 2 decreased GTP and ATP pools by similar to 50 and 75%, respectively. By an in situ GARFTase assay, 2 was similar to 20-fold more inhibitory toward intracellular GARFTase than toward cell growth or colony formation. Compound 2 irreversibly inhibited clonogenicity, although this required at least 4 h of exposure. Our results document the potent antiproliferative activity of compound 2, attributable to its efficient cellular uptake by PCFT, resulting in inhibition of GARFTase and de novo purine biosynthesis. Furthermore, they establish the feasibility of selective chemotherapy drug delivery via PCFT over RFC, a process that takes advantage of a unique biological feature of solid tumors.