Alpha Folate Receptor Mediated GARFTase Inhibitors as Selective Antitumor Agents

描述（由申请人提供）：癌症化疗的主要障碍之一是药物无法选择性靶向肿瘤细胞。fr - α在卵巢、子宫内膜、肾脏、肺、间皮瘤、乳腺和脑等多种肿瘤表面的过表达，以及fr - β在髓性白血病表面的过表达，促使叶酸和翼石基部分作为fr - α表达肿瘤的选择性靶向药物发展起来。因此，叶酸和翼酸盐缀合物已被用于选择性地将毒素、脂质体、成像和细胞毒性药物递送到表达fr - α的肿瘤中，并取得了很高的成功。利用具有叶酸或翼油基片段的细胞毒性偶联物作为抗肿瘤剂的过程需要对偶联物进行额外的切割。这种分裂需要在肿瘤细胞内选择性地发生，以释放细胞毒素。细胞毒性药物的过早释放取消了选择性并导致毒性。设计一种本身选择性靶向fr - α而不被RFC明显吸收的细胞毒性药物，将提供高度选择性的药物来对抗表达fr - α的肿瘤，而不会产生严重的毒性。我们最近发现了两种独特的化合物AAG366和AAG344，它们是甘氨酸酰胺核糖核苷酸甲酰转移酶（GARFTase）的抑制剂，RFC对它们的吸收较差，能有效抑制表达fr - α的KB肿瘤细胞的生长，IC50值分别为2.5和2.9 nM。与不表达fr - α的细胞相比，AAG366和AAG344对表达fr - α的肿瘤细胞的抑制作用显著提高100-345倍。本课题的具体目的是：1)合成AAG366和AAG344的类似物，进行构效关系（SAR）研究，优化其抗肿瘤活性和对GARFTase的抑制活性，以及fr - α的高亲和力结合和摄取；2)在等基因（CHO， KB, SKOV3, ov3, CCRF-CEM）细胞系模型中检测类似物的细胞毒性，这些模型具有已建立的FR- α， RFC和folylpolyglutamate syntase的差异，通过放射性标记（甘氨酸，甲酸酯）生物合成前体的原位代谢标记，通过核苷和氨基吡唑羧酰胺保护细胞毒性来确定分子靶标，以确定FR结合的亲和力。并通过分离酶研究确定对目标酶GARFTase和其他叶酸代谢酶的抑制作用；3)评价AAG366、AAG344及所选类似物对表达fr - α的肿瘤的体内抗肿瘤活性。该研究将提供一个全面的SAR，并提供优化的类似物，在体外和体内对表达fr - α的肿瘤具有更高的抗肿瘤活性。这项研究还将进一步确定这些新型类似物的作用机制，并可能提供用于单一治疗或联合临床使用的药物，具有不同的抗肿瘤活性谱，并且比目前使用的毒性更低。

DESCRIPTION (provided by applicant): One of the major hurdles in cancer chemotherapy is the inability of the agent to selectively target tumor cells. The over expression of the FR-alpha on the surface of a number of tumors including ovarian, endometrial, kidney, lung, mesothelioma, breast and brain and FR-beta on the surface of myeloid leukemia has prompted the development of folic acid and the pteroyl moiety as selective targeting agents to FR-alpha expressing tumors. Thus, conjugates of folic acid and pteroates have been used to selectively deliver toxins, liposomes, imaging and cytotoxic agents to FR-alpha expressing tumors with a high degree of success. The process of utilizing a cytotoxic conjugate with the folic acid or pteroyl moiety as an antitumor agent requires the additional step of cleavage of the conjugate. This cleavage needs to occur selectively inside the tumor cell to release the cytotoxic agent. Premature release of the cytotoxic agent abrogates selectivity and leads to toxicity. The design of a cytotoxic agent that itself selectively targets the FR-alpha and is not appreciably taken up by the RFC would afford highly selective agents against FR-alpha expressing tumors without serious toxicity. We have recently discovered two compounds, AAG366 and AAG344 that are unique and are inhibitors of glycinamide ribonucleotide formyltransferase (GARFTase), are poorly taken up by the RFC and potently inhibits the growth of FR-alpha expressing KB tumor cells with IC50 values of 2.5 and 2.9 nM respectively. AAG366 and AAG344 are remarkable 100-345-fold more inhibitory to tumor cells expressing the FR-a compared to cells that do not express the FR-alpha. The Specific Aims of this proposal are: 1) to synthesize analogs of AAG366 and AAG344 to provide a structure-activity relationship (SAR) study to optimize the antitumor activity and the inhibitory activity against GARFTase as well as the high affinity binding and uptake by FR-alpha; 2) to test the analogs for cytotoxicity in isogenic (CHO, KB, SKOV3, OVAR3, CCRF-CEM) cell line models with established differences in FR-alpha, RFC and folylpolyglutamate synthetase, to identify the molecular targets by nucleoside and aminoimidazole carboxamide protection from cytotoxicity by in situ metabolic labeling with radiolabeled (glycine, formate) biosynthetic precursors to determine affinities for FR binding, and to determine the inhibition of target enzyme GARFTase and other folate metabolizing enzymes by studies with isolated enzymes; 3) to evaluate the in vivo antitumor activity of AAG366, AAG344 and selected analogs against FR-alpha expressing tumors. This study will provide a comprehensive SAR and should afford optimized analogs with increased antitumor activity against FR-alpha expressing tumors in vitro and in vivo. This study will also further define the mechanism(s) of action of these novel analogs and could provide agents to be used as monotherapy or in combination for clinical use with a different spectrum of antitumor activity and reduced toxicity than those currently in use.