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不能选择性靶向肿瘤细胞。FR-α在许多肿瘤（包括卵巢、子宫内膜、肾、肺、间皮瘤、乳腺和脑）表面上的过表达以及FR-β在髓性白血病表面上的过表达促使叶酸和蝶酰基部分作为FR-α表达肿瘤的选择性靶向剂的开发。因此，叶酸和蝶酸盐的缀合物已被用于选择性地递送毒素、脂质体、成像剂和细胞毒性剂至表达FR-α的肿瘤，具有高度的成功。利用具有叶酸或蝶酰基部分的细胞毒性缀合物作为抗肿瘤剂的方法需要缀合物的额外裂解步骤。这种裂解需要在肿瘤细胞内选择性地发生以释放细胞毒性剂。细胞毒性剂的过早释放消除了选择性并导致毒性。设计本身选择性靶向FR-α并且不明显被RFC吸收的细胞毒性剂将提供针对FR-α表达肿瘤的高度选择性试剂，而没有严重毒性。我们最近发现了两种独特的化合物AAG 366和AAG 344，它们是甘氨酰胺核糖核苷酸甲酰转移酶（GARFT酶）的抑制剂，RFC吸收较差，并有效抑制表达FR-α的KB肿瘤细胞的生长，IC 50值分别为2.5和2.9 nM。与不表达FR-α的细胞相比，AAG 366和AAG 344对表达FR-α的肿瘤细胞的抑制性显著高100-345倍。本发明的具体目的是：1）合成AAG 366和AAG 344的类似物，以提供结构-活性关系（SAR）研究，从而优化抗肿瘤活性和对GARFT酶的抑制活性以及FR-α的高亲和力结合和摄取; 2）测试类似物在同基因组中的细胞毒性。（CHO、KB、SKOV 3、OVAR 3、CCRF-CEM）细胞系模型，其具有FR-α、RFC和叶酰聚谷氨酸合成酶的确定差异，通过放射性标记的原位代谢标记，（甘氨酸、甲酸盐）生物合成前体，以确定对FR结合的亲和力，并通过用分离的酶的研究来确定对靶酶GARFT酶和其它叶酸代谢酶的抑制; 3）评估AAG 366、AAG 344和所选类似物针对表达FR-α的肿瘤的体内抗肿瘤活性。本研究将提供全面的SAR，并应提供优化的类似物，其在体外和体内对表达FR-α的肿瘤具有增加的抗肿瘤活性。本研究还将进一步定义这些新型类似物的作用机制，并可提供用作单一疗法或组合用于临床用途的药剂，其具有与目前使用的药剂不同的抗肿瘤活性谱和降低的毒性。

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.