Investigation of the ABC Half-Transporter ABCG2

Q141K单核苷酸多态的特征我们和其他人已经报道了ABCG2中Q141K SNP的功能受损，大多数研究人员发现细胞表面水平较低是损害的机制，我们的数据表明在纠正表面表达后存在额外的功能损害。这种SNP的临床影响已经得到证实，Q141K SNP患者的血浆中口服药物水平更高，包括口服拓扑替康和地氟莫替康(93，94)。与这种SNP相关的尿酸排泄减少与痛风有令人信服的联系。另一种可能性是Q141K SNP导致蛋白质错误折叠。如上所述，通常没有正确折叠的ABCG2蛋白会通过ERAD途径降解。石川观察到，Q141K SNP导致ERAD对蛋白质的识别。基于对囊性纤维化转运蛋白的研究，我们推测某些底物可以使Q141K ABCG2免于降解。事实上，我们能够证明秋水仙素和罗米地平都增加了细胞表面的Q141K ABCG2。这一策略可能被用来通过增加Q141K ABCG2的表达来调节吸入或摄入致癌物的外排增加。关于Q141K的一个值得注意的观察是，它的药理和生理影响在临床上很容易检测到。这与P-糖蛋白的变种形成了鲜明的对比，P-糖蛋白的变种一直备受争议。我们的实验室研究表明，即使当蛋白质到达细胞表面时，运输效率也会降低。我们与Suresh Ambukar博士和Suneet Shukla博士合作研究了这种多态的生物化学。基于125I-碘芳基氮杂氮唑的光交联数据表明，Q141K SNP不影响药物结合。只观察到了ATP水解率的微小差异，而且这些差异在生理上并不显著。半衰期的测量正在进行中，但减少并不令人惊讶，我们的假设将被我们用来证明人口贩运改善的相同代理人正常化。既没有检测到功能异常，也没有半衰期减少的发现，将表明Q141K的缺陷完全存在于细胞表面的表达水平。开发新的、有效的ABC转运蛋白抑制剂，用于中枢神经系统的临床应用虽然ABCG2在肠道和脑内皮细胞中的表达对正常生理起到保护作用，但在癌症治疗过程中可能是有害的。ABCG2的单独表达或与PGP联合表达已被证明限制了拓扑替康的口服生物利用度和脑渗透，以及几种靶向治疗，包括伊马替尼、达沙替尼、拉帕替尼、索拉非尼和埃洛替尼。由于肺癌和乳腺癌经常转移到大脑，ABCG2有可能限制这些疗法的大脑渗透，使它们的有效性降低。因此，通过血脑屏障增加靶向治疗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PGP或ABCG2单独被删除时，对大脑渗透的影响通常不大-但当两者都被删除时，影响会相当大。Polli和他的同事发现，在缺乏ABCG2和Pgp的小鼠中，拉帕替尼的大脑渗透率增加最多，这增加了Pgp和ABCG2双重抑制剂可能是必要的可能性。将ABCG2/PGP双重抑制剂elacridar(GF120918)与拓扑替康联合使用，可获得完全的口服生物利用度，并降低患者间的可变性。在小鼠身上的研究表明，伊拉克替尼和伊马替尼联合给药可以增加伊马替尼及其活性代谢物CGP74588的脑渗透率。达沙替尼也有类似的发现报道。我们之前已经证明，我们在临床试验中研究的Pgp调节剂Tariquidar也可以抑制ABCG2。目前，大多数可用的抑制剂对ABCG2缺乏效力或有毒性作用。为了开发有效的ABC转运蛋白抑制剂用于临床应用，我们与分子靶标实验室的James McMahon和Curtis Henrich博士合作，开发了一种高通量筛选ABCG2的新型抑制剂。筛选是基于ABCG2过表达的NCI-H460 MX20细胞中ABCG2特异性底物脱镁叶绿酸a的积累。筛选了NCI-DTP天然和合成化合物文库(7,325个化合物)以及NCI天然产物提取物文库(91,000个化合物)。天然化合物和合成化合物文库共得到5个先导化合物，其中NSC11668被选作进一步研究。天然产物提取物文库产生了一类新的与ABCG2相互作用的化合物--Botryllamide，从中选择了2个进行进一步研究，其中一个是Pgp和ABCG2的抑制剂。在William Figg博士实验室的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，正在测试botryllamide改善拉帕替尼吸收的能力。我们的目标是跟踪这项研究，如果是阳性的，通过涉及脑转移模型的概念前临床研究来证明。

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.