Molecular Analyses of Folate and Antifolate Transport

描述(申请人提供)：叶酸类药物用于治疗癌症和炎症性疾病已有数十年的历史。这些药物，通常被称为抗叶酸，在临床治疗中取得了成功，但由于它们对正常细胞的细胞毒作用而受到严重限制。一个最好的例子是经典的抗叶酸甲氨蝶呤(MTX)，它仍然在低剂量下被广泛用于治疗癌症、牛皮癣和关节炎。较高剂量的MTX会导致明显的副作用，包括脱发，因为MTX优先杀死快速分裂的细胞，无论是正常的还是非正常的，没有区别。理想情况下，可以开发出针对患病细胞而不影响正常细胞的特定抗叶酸。在研究叶酸转运时，研究人员发现，人类叶酸受体蛋白(HFRS)在许多肿瘤类型中过度表达，并在炎症条件(如关节炎)部位激活巨噬细胞。此外，肾综合征出血热在正常组织上的表达是局部的，因此不会暴露于静脉注射的药物中。总而言之，专为肾综合征出血热患者设计的抗叶酸可以静脉注射，对正常组织的毒性最小。我们的建议旨在通过广泛的结构、热力学、动力学和基于细胞的表征HFRS与叶酸和抗叶酸配体的相互作用来推进新型HFR特异性抗叶酸药物的开发。具体地说，我们的目标是：1)确定人膜叶酸受体1型和2型的X射线晶体结构，这些受体是实验性抗叶酸和叶酸偶联药物的组织选择性给药靶点，以帮助设计治疗癌症和炎症性疾病的新药；2)阐明人叶酸受体与配体结合和释放的生物物理参数，以最终描绘叶酸类药物所需的结构特征；以及3)分析人叶酸受体突变体，以确定叶酸和抗叶酸在原位运输和释放所需的FRS的关键决定因素。鉴于叶酸类药物在疾病治疗中的悠久历史，对HFR-配体相互作用缺乏明显的分子洞察力，充其量也是令人失望的。这些研究的完成将产生一套分子参数，这些参数将指导下一代抗叶酸的合理设计，从而深刻改善与癌症和慢性炎症性疾病有关的公共健康。 公共卫生相关性：我们提议的研究计划有可能通过大幅改善许多流行的人类癌症的治疗来改善公共健康。在肿瘤的化疗治疗中，由于化疗药物对正常细胞的毒性，使患者产生了许多不良反应。我们的工作应该导致专门针对癌细胞的新型药物的开发，从而改善患者的治疗，并增加副作用较少的好处。

DESCRIPTION (provided by applicant): Folate-based drugs have been employed for decades in the treatment of cancer and inflammatory disease. These drugs, commonly termed antifolates, have met with success in clinical treatments but are severely limited due to their cytotoxic effects on normal cells. A prime example is the classic antifolate methotrexate (MTX), which is still commonly used at low doses in treatment of cancer, psoriasis and arthritic disease. Higher doses of MTX lead to obvious side effects including loss of hair as MTX preferentially kills rapidly dividing cells, normal or otherwise, without discrimination. Ideally, specific antifolates that target diseased cells without affecting normal cells could be developed. In studying folate transport, researchers have discovered that human folate receptor proteins (hFRs) are overexpressed in many tumor types and also activated macrophages at the site of inflammatory conditions (e.g. arthritis). Furthermore, the expression of hFRs on normal tissue is localized such that is not exposed to drugs administered intravenously. Taken together, antifolates that are designed to be taken up specifically by hFRs could be administered intravenously with minimal toxicity to normal tissue. Our proposal seeks to advance the development of novel hFR-specific antifolate drugs through extensive structural, thermodynamic, kinetic and cell-based characterizations of the interaction of hFRs with folate and antifolate ligands. Specifically our aims are to: 1) Determine the X-ray crystallographic structures of human membrane folate receptor types 1 and 2, targets for tissue selective delivery of experimental antifolate and folate-conjugate drugs, to aid in the design of new drugs to treat cancer and inflammatory diseases; 2) Elucidate the biophysical parameters of ligand binding and release by human folate receptors to ultimately delineate the desired structural features for folate-based drugs; and 3) Analyze human folate receptor mutants to identify key determinants of FRs required for trafficking and release of folates and antifolates in situ. Given the long history of folate-based drugs in disease treatment, the obvious lack of molecular insight into hFR-ligand interaction is disappointing at best. Completion of these studies will lead to a set of molecular parameters that will guide the rational design of the next generation of antifolates, leading to profound improvements in public health relating to cancer and chronic inflammatory disease. PUBLIC HEALTH RELEVANCE: Our proposed research program has the potential to improve public health by substantially improving the treatment of many prevalent human cancers. During chemotherapy treatment for cancer, many adverse side effects are realized by the patient due to the toxicity of the chemotherapeutic drugs in normal cells. Our work should lead to the development of novel drugs that specifically target cancer cells and therefore improve patient therapy with the added benefit of fewer side effects.