Investigation of the ABC Half-Transporter ABCG2

Q141K单核苷酸多态性的表征我们和其他人已经报道了ABCG 2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损伤机制，我们的数据表明在校正表面表达后还有额外的功能损伤。该SNP的临床影响已得到证实，在Q141 K SNP患者中，口服药物（包括口服拓扑替康和二氟替康）的血浆水平较高（93，94）。与此SNP相关的尿酸排泄减少已令人信服地与痛风有关。另一种可能性是Q141K SNP导致错误折叠的蛋白质。如上所述，通常未正确折叠的ABCG 2蛋白被ERAD途径降解。石川观察到Q141 K 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 ABCG 2免于降解。事实上，我们能够证明秋水仙碱和罗米地辛都增加了细胞表面的Q141K ABCG 2。这种策略可能被用来介导通过增加Q141K ABCG2的表达，在预防方案中增加吸入或摄入的致癌物的外排。关于Q141K的一个显著观察结果是，它的药理学和生理学影响在临床上很容易被检测到。这与P-糖蛋白的变体形成对比，围绕P-糖蛋白的变体存在很多争议。我们的实验室研究表明，即使蛋白质到达细胞表面，运输效率也会降低。我们与Suresh Ambudkar博士和Suneet Shukla博士合作研究这种多态性的生物化学。基于与125 I-碘芳基叠氮吡唑嗪的光交联的数据表明，Q141 K SNP不影响药物结合。仅观察到ATP水解的轻微差异，并且认为这些差异不具有生理学意义。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改善贩运的相同药剂来正常化。既不能检测到功能异常，也不能检测到半衰期缩短的发现表明Q141K的缺陷完全在于细胞表面的表达水平。开发新型、有效的ABC转运蛋白抑制剂用于CNS的临床应用虽然ABCG 2在肠和脑内皮中的表达在正常生理学中起保护作用，但在癌症治疗期间可能是有害的。ABCG 2单独或与Pgp组合的表达已显示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，ABCG 2可能会限制这些疗法的大脑渗透，使其效果降低。因此，增加靶向治疗穿过血脑屏障的递送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ABCG 2单独缺失时，通常对脑渗透有适度的影响-但当两者都缺失时，影响相当大。Polli及其同事发现，拉帕替尼脑渗透的最大增加发生在缺乏Abcg2和Pgp的小鼠中，这增加了Pgp和ABCG2双重抑制剂可能是必要的。给予ABCG2/Pgp双重抑制剂依克立达（GF120918）和托泊替康导致完全的口服生物利用度和患者间变异性降低。小鼠研究表明，依克立达与伊马替尼联合给药可增加伊马替尼及其活性代谢产物CGP 74588的脑渗透。达沙替尼也报告了类似的结果。我们之前已经表明，我们在临床试验中研究的Pgp调节剂tariquidar也抑制ABCG 2。目前，大多数可用的抑制剂对ABCG 2缺乏效力或具有毒性作用。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ABCG 2新型抑制剂的方法。该筛选基于ABCG 2特异性底物脱镁叶绿酸a在ABCG 2过表达NCI-H460 MX20细胞中的积累。筛选了NCI-DTP天然和合成化合物文库（7，325种化合物）以及NCI天然产物提取物文库（91，000种化合物）。天然和合成化合物库产生5个先导化合物，其中NSC11668被选择用于进一步研究。天然产物提取物库产生了一类新的ABCG 2相互作用化合物，botryllamides，从中选择2个用于进一步研究，其中一个是Pgp和ABCG 2的抑制剂。在William Figg博士的实验室中，正在CNS摄取和口服药物生物利用度的临床前体内模型中测定葡萄糖酰胺改善拉帕替尼摄取的能力。我们的目标是遵循这项研究，如果是积极的，与涉及脑转移模型的临床前研究的概念证明。

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.