Investigation of the ABC Half-Transporter ABCG2

ABC 半转运蛋白 ABCG2 的研究

• 批准号: 8937784
• 负责人: susan bates
• 金额: $ 20.06万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937784
• 关键词: ABCG2 gene; ATP Hydrolysis; ATP-Binding Cassette Transporters; Affect; Amino Acids; Antineoplastic Agents; Asians; BAY 54-9085; Basic Science; Binding; Biochemistry; Biological Assay; Biological Availability; Biological Factors; Blood - brain barrier anatomy; Blood Group Antigens; Brain; Breathing; Camptothecin Analogue; Carcinogens; Cell Maintenance; Cell surface; Cells; Clinic; Clinical; Clinical Pharmacology; Clinical Trials; Colchicine; Cystic Fibrosis; Dasatinib; Data; Databases; Defect; Development; Drug resistance; Endoplasmic Reticulum Degradation Pathway; Endothelium; Erlotinib; Evaluation; Excretory function; Exposure to; Gastrointestinal tract structure; Gefitinib; Genetic Polymorphism; Goals; Gout; HPPH; Half-Life; Human; Imatinib; Impairment; Investigation; Kidney; Laboratories; Laboratory Study; Lead; Libraries; Link; Malignant Neoplasms; Malignant neoplasm of lung; Measurement; Mediating; Mediator of activation protein; Metastatic malignant neoplasm to brain; Methotrexate; Mitoxantrone; Modeling; Molecular Target; Multi-Drug Resistance; Mus; Names; Neoplasm Metastasis; Normal tissue morphology; Oral; P-Glycoprotein; Pathway interactions; Patients; Penetration; Pharmaceutical Preparations; Photosensitizing Agents; Physiological; Physiology; Placenta; Plasma; Play; Population; Prevention; Proteins; Pump; Regulation; Reporting; Research Personnel; Role; SN-38; Scheme; Single Nucleotide Polymorphism; Source; Stem cells; Structure; Surface; Tariquidar; Topotecan; Toxic effect; Translational Research; Tyrosine Kinase Inhibitor; Urate; Uric Acid; Variant; Work; absorption; base; cancer cell; cancer therapy; chemotherapy; clinical application; clinically relevant; crosslink; fetal; functional disability; gene cloning; high throughput screening; human ABCG2 protein; improved; in vivo Model; inhibitor/antagonist; interest; lapatinib; malignant breast neoplasm; mouse model; novel; overexpression; pheophorbide a; pre-clinical; preclinical study; prevent; protein expression; protein function; protein misfolding; pyropheophorbide a; targeted delivery; trafficking; transport inhibitor; uptake

Characterization of the Q141K Single Nucleotide Polymorphism We and others have reported on the impaired functionality of the Q141K SNP in ABCG2, with most investigators identifying lower cell surface levels as a mechanism of impairment and our data suggesting an additional functional impairment after correction for surface expression. The clinical impact of this SNP has been confirmed, with higher plasma levels of orally administered drugs, including oral topotecan and diflomotecan, demonstrated in patients with the Q141K SNP (93,94). Reduced uric acid excretion linked to this SNP has been convincingly linked to gout. Another possibility is that the Q141K SNP results in a misfolded protein. As noted above, often ABCG2 proteins that are not properly folded are degraded by the ERAD pathway. Ishikawa observed that the Q141K SNP leads to protein recognition by ERAD. Based on work with the cystic fibrosis transporter, we postulated that certain substrates could "rescue" Q141K ABCG2 from degradation. Indeed, we were able to show that both colchicine and romidepsin increased Q141K ABCG2 at the cell surface. This strategy could potentially be used to mediate increased efflux of inhaled or ingested carcinogens via increased expression of Q141K ABCG2 in a prevention scheme. One of the remarkable observations regarding Q141K is that its pharmacologic and physiologic impact has been so readily detected in the clinic. This contrasts with variants of P-glycoprotein around which there has been much controversy. Our laboratory studies suggested that even when the protein reached the cell surface the transport efficiency is reduced. We have collaborated with Dr. Suresh Ambudkar and Dr. Suneet Shukla to study the biochemistry of this polymorphism. Data based on photo-crosslinking with 125I-iodoarylazidoprazosin suggest that the Q141K SNP does not affect drug binding. Only slight differences in ATP hydrolysis were observed, and these were not felt to be physiologically significant. Measurement of half-life is ongoing, but a reduction would not be surprising, and we hypothesize will be normalized by the same agents we used to show improved trafficking. A finding that neither functional abnormalities can be detected, nor a reduced half-life would indicate that the defect in Q141K lies entirely in level of expression at the cell surface. Development of Novel, Potent ABC Transporter Inhibitors for Clinical Use in the CNS While expression of ABCG2 in the gut and brain endothelium serves a protective role in normal physiology, it can be detrimental during cancer treatment. Expression of ABCG2 alone or in combination with Pgp has been shown to limit oral bioavailability and brain penetration of topotecan as well as several targeted therapies including imatinib, dasatinib, lapatinib, sorafenib and erlotinib in mice. Since lung and breast cancers often metastasize to the brain, there is the possibility ABCG2 limits brain penetration of these therapies, rendering them less effective. Thus, a transport inhibitor that increased delivery of targeted therapies across the blood-brain barrier could be used to prevent or treat brain metastases. Interestingly, in mouse models, when either Pgp or ABCG2 alone is deleted, there is often a modest impact on brain penetration - but there is a considerable impact when both are deleted. Polli and colleagues found that the greatest increase in brain penetration of lapatinib occurred in mice lacking both Abcg2 and Pgp, raising the possibility that a dual inhibitor of Pgp and ABCG2 may be necessary. Administering the dual ABCG2/ Pgp inhibitor, elacridar (GF120918), with topotecan resulted in complete oral bioavailability and decreased inter-patient variability. Studies in mice have demonstrated that increased brain penetration of imatinib and its active metabolite, CGP74588, can be achieved by co-administration of elacridar with imatinib. Similar findings have been reported for dasatinib. We have previously shown that tariquidar, the Pgp modulator we have studied in clinical trials, also inhibits ABCG2. Currently, the majority of available inhibitors lack potency with respect to ABCG2 or have toxic effects. To develop potent ABC transporter inhibitors for clinical applications, we collaborated with Drs. James McMahon and Curtis Henrich of the Molecular Targets Laboratory to develop a high-throughput screen for novel inhibitors of ABCG2. The screen was based on accumulation of the ABCG2 specific substrate pheophorbide a in ABCG2-overexpressing NCI-H460 MX20 cells. The NCI-DTP natural and synthetic compound library (7,325 compounds) as well as the NCI natural products extracts library (91,000 compounds) were screened. The natural and synthetic compound library yielded 5 lead compounds, of which NSC11668 was selected for further study. The natural product extracts library yielded a new class of ABCG2 interacting compounds, the botryllamides, from which 2 were selected for further study, one of which is an inhibitor of both Pgp and ABCG2. Assay of the ability of the botryllamides to improve lapatinib uptake is ongoing in preclinical in vivo models of CNS uptake and oral drug bioavailability in the laboratory of Dr. William Figg. Our goal is to follow this study, if positive, with proof of concept pre-clinical studies involving brain metastasis models.

Q141K 单核苷酸多态性的表征 我们和其他人报告了 ABCG2 中 Q141K SNP 的功能受损，大多数研究人员将较低的细胞表面水平确定为受损机制，并且我们的数据表明在校正表面表达后存在额外的功能受损。该 SNP 的临床影响已得到证实，在具有 Q141K SNP 的患者中，口服药物（包括口服拓扑替康和双氟莫替康）的血浆水平较高 (93,94)。与该 SNP 相关的尿酸排泄减少已令人信服地与痛风相关。另一种可能性是 Q141K SNP 导致蛋白质错误折叠。如上所述，未正确折叠的 ABCG2 蛋白通常会被 ERAD 途径降解。 Ishikawa 观察到 Q141K SNP 导致 ERAD 识别蛋白质。基于对囊性纤维化转运蛋白的研究，我们假设某些底物可以“拯救”Q141K ABCG2 免于降解。事实上，我们能够证明秋水仙碱和罗米地辛都能增加细胞表面的 Q141K ABCG2。在预防方案中，该策略可通过增加 Q141K ABCG2 的表达来介导增加吸入或摄入的致癌物的流出。关于 Q141K 的显着观察之一是它的药理和生理影响很容易在临床中检测到。这与存在很多争议的 P-糖蛋白变体形成鲜明对比。我们的实验室研究表明，即使蛋白质到达细胞表面，运输效率也会降低。我们与 Suresh Ambudkar 博士和 Suneet Shukla 博士合作研究了这种多态性的生物化学。基于 125I-碘芳基叠氮哌唑嗪光交联的数据表明，Q141K SNP 不会影响药物结合。仅观察到 ATP 水解的轻微差异，并且这些差异不具有生理意义。半衰期的测量正在进行中，但减少并不令人惊讶，我们假设将通过我们用来显示运输改善的相同药物来正常化。如果既没有检测到功能异常，也没有半衰期缩短，则表明 Q141K 的缺陷完全在于细胞表面的表达水平。开发用于中枢神经系统临床应用的新型强效 ABC 转运蛋白抑制剂 虽然肠道和脑内皮中 ABCG2 的表达在正常生理学中发挥保护作用，但在癌症治疗过程中可能有害。 ABCG2 的单独表达或与 Pgp 联合表达已被证明会限制托泊替康以及几种靶向疗法（包括伊马替尼、达沙替尼、拉帕替尼、索拉非尼和厄洛替尼）在小鼠中的口服生物利用度和脑渗透。由于肺癌和乳腺癌经常转移到大脑，因此 ABCG2 有可能限制这些疗法的大脑渗透性，从而降低它们的效果。因此，增加靶向治疗穿过血脑屏障的转运抑制剂可用于预防或治疗脑转移。有趣的是，在小鼠模型中，当单独删除 Pgp 或 ABCG2 时，通常会对大脑渗透产生适度的影响，但当两者都删除时，则会产生相当大的影响。 Polli 及其同事发现，拉帕替尼脑渗透率的最大增加发生在同时缺乏 Abcg2 和 Pgp 的小鼠中，这提高了可能需要 Pgp 和 ABCG2 双重抑制剂的可能性。 ABCG2/Pgp 双重抑制剂 elacridar (GF120918) 与拓扑替康联合使用可实现完全口服生物利用度并降低患者间变异性。小鼠研究表明，将 elacridar 与伊马替尼联合给药可增加伊马替尼及其活性代谢物 CGP74588 的脑渗透性。达沙替尼也有类似的发现。我们之前已经证明，我们在临床试验中研究的 Pgp 调节剂 tariquidar 也能抑制 ABCG2。目前，大多数可用的抑制剂对 ABCG2 缺乏效力或具有毒性作用。为了开发用于临床应用的有效 ABC 转运蛋白抑制剂，我们与 Drs. 合作。分子靶标实验室的 James McMahon 和 Curtis Henrich 开发了针对 ABCG2 新型抑制剂的高通量筛选。该筛选基于 ABCG2 特异性底物脱镁叶绿酸 a 在 ABCG2 过表达的 NCI-H460 MX20 细胞中的积累。筛选了NCI-DTP天然和合成化合物库（7,325种化合物）以及NCI天然产物提取物库（91,000种化合物）。天然和合成化合物库产生了 5 个先导化合物，其中 NSC11668 被选择进行进一步研究。天然产物提取物文库产生了一类新的 ABCG2 相互作用化合物——Botryllamides，从中选择了 2 种进行进一步研究，其中一种是 Pgp 和 ABCG2 的抑制剂。 William Figg 博士的实验室正在对中枢神经系统摄取和口服药物生物利用度的临床前体内模型中进行Botryllamides 改善拉帕替尼摄取的能力进行测定。我们的目标是通过涉及脑转移模型的临床前研究概念验证来跟踪这项研究（如果结果积极）。