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功能受损的表征，大多数研究人员将较低的细胞表面水平识别为损伤机制，我们的数据表明表面表达后校正后有其他功能障碍。该SNP的临床影响已得到证实，在Q141K SNP的患者中，血浆口服药物的水平较高，包括口服拓扑替克和Diflomotecan（93,94）。与此SNP相关的尿酸排泄减少与痛风有关。另一种可能性是Q141K SNP导致错误折叠的蛋白质。如上所述，通常无法正确折叠的ABCG2蛋白会被ERAD途径降解。 Ishikawa观察到Q141K SNP导致Erad识别蛋白质。根据与囊性纤维化转运蛋白的工作，我们假设某些底物可以从降解中“拯救” Q141K ABCG2。确实，我们能够证明秋水仙碱和romidepsin都增加了细胞表面的Q141K ABCG2。该策略可能可用于通过在预防方案中增加Q141K ABCG2的表达来介导增加吸入或摄入的致癌物的外排。关于Q141K的显着观察之一是，在诊所中很容易检测到其药理和生理影响。这与P-糖蛋白的变体形成鲜明对比，围绕该变异引起了很多争议。我们的实验室研究表明，即使蛋白质到达细胞表面时，运输效率也会降低。我们已经与Suresh Ambudkar博士和Suneet Shukla博士合作研究了这种多态性的生物化学。基于与125-iodoylazidoprazosin的光链接的数据表明，Q141K SNP不会影响药物结合。仅观察到ATP水解的略有差异，并且这些感觉在生理上没有意义。正在进行半衰期的测量，但是减少并不奇怪，我们假设我们用来表现出改善贩运的代理人将标准化。这一发现表明，无法检测到功能异常，也无法降低半衰期，这表明Q141K中的缺陷完全在于细胞表面的表达水平。开发新型的，有效的ABC转运蛋白抑制剂用于CNS临床使用，而肠道和脑内皮中ABCG2的表达在正常生理学中起保护作用，在癌症治疗过程中可能是有害的。单独或与PGP结合使用ABCG2的表达已被证明可以限制拓扑替康的口服生物利用度和脑部渗透，以及几种针对性的疗法，包括伊马替尼，达西替尼，拉帕替尼，索拉非尼和erlotinib在小鼠中。由于肺部和乳腺癌经常转移到大脑，因此ABCG2可能会限制这些疗法的脑部渗透，从而使它们的效果降低。因此，可以使用靶向疗法在血脑屏障上增加的转运抑制剂可用于预防或治疗脑转移。有趣的是，在鼠标模型中，仅删除了PGP或ABCG2时，通常会对脑穿透性产生适度的影响 - 但是当两者都被删除时会产生相当大的影响。 Polli及其同事发现，在缺乏ABCG2和PGP的小鼠中，Lapatinib脑穿透的最大增加，这增加了可能需要PGP和ABCG2双重抑制剂的可能性。给予双ABCG2/ PGP抑制剂ELACRIDAR（GF120918），带有拓扑替康，导致了完全的口服生物利用度和患者间的变异性。在小鼠中的研究表明，可以通过与伊替尼共同给药来实现伊马替尼及其活性代谢产物CGP74588的大脑渗透。据报道达沙替尼的类似发现。我们先前已经表明，在临床试验中研究的PGP调节剂Tariquidar也抑制ABCG2。目前，大多数可用抑制剂在ABCG2方面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。该分子目标实验室的詹姆斯·麦克马洪（James McMahon）和柯蒂斯·亨里希（Curtis Henrich）为ABCG2的新型抑制剂开发了高通量屏幕。该屏幕基于ABCG2特异性底物质体A中ABCG2过表达的NCI-H460 MX20细胞的积累。筛选了NCI-DTP天然和合成化合物库（7,325种化合物）以及NCI天然产品提取物库（91,000种化合物）。天然和合成化合物库产生5种铅化合物，其中选择了NSC11668进行进一步研究。天然产物提取物库产生了一类新的ABCG2相互作用化合物，即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。在威廉·菲格（William Figg）博士的实验室中，在CNS摄取和口服药物生物利用度的临床前体内模型中，botryllamides改善拉帕替尼摄取的能力的测定正在进行中。我们的目标是通过涉及脑转移模型的概念前研究的概念证明，遵循这项研究。