Investigation of the ABC Half-Transporter ABCG2

我们的实验室对非pgp介导的耐药机制有着长期的兴趣，已经建立了几种以ABC半转运体ABCG2为重点的耐药细胞系模型。我们成功地从耐米托蒽醌的结肠癌细胞系中克隆了ABCG2，该细胞系表现出atp依赖性的药物积累减少。该基因包括6个跨膜结构域和一个ATP结合结构域，编码一个半转运蛋白分子，其活性需要二聚化。ABCG2的过表达使细胞在体外对米托醌、喜树碱、拓扑替康和SN-38（伊立替康的活性代谢物）产生耐药性。已经发现了大量的ABCG2底物和抑制剂，并且各种底物和抑制剂与p -糖蛋白相媲美。由于药物转运体在介导癌症耐药中的作用从未被定义，我们的小组已经研究了靶向这种蛋白质的其他方面。我们研究了一种变异蛋白，它可能与ABCG2底物暴露增加有关，特别是与选定的致癌物有关。ABCG2在限制药物的口服吸收和通过血脑屏障的表达限制脑吸收方面也有作用（参见项目1）。总的来说，这项工作的最好特点是努力理解和利用ABCG2作为治疗靶点来改善抗癌治疗。我们研究了蛋白质的结构和功能关系。我们和其他人报道了在氨基酸141上携带单核苷酸多态性的细胞中运输受损，该多态性将谷氨酰胺转变为赖氨酸。一些证据表明，这种多态变异可能与ABCG2底物药物暴露增加有关，或者与已知的ABCG2底物致癌物亚群暴露增加有关，包括烟草烟雾中的致癌物。这种变异首先与痛风的易感性增加有关，因为尿酸排泄减少，血液中尿酸水平相应增加。同样，胃肠道吸收也与肠上皮中ABCG2的表达有关，这项工作的一个含义是Q141K SNP可能与底物药物如拓扑替康、伊马替尼或伊立替康的吸收增加有关。我们重点了解了这种由错误折叠和靶向聚合体导致的变异的贩运和功能。我们还发现，靶向细胞表面的蛋白质破坏了运输活动，我们正在与Suresh Ambudkar博士合作进行研究，以了解这种异常功能。基于我们早期对ABCG2二聚化基序的研究，我们通过添加底物研究了ABCG2的药理学拯救作用。事实上，最好的修复介质是罗米地辛，显然是由于至少三个机制：增加RNA表达，减少向进攻性转移，以及由于改善折叠而改善表面定位。ABCG2在脑内皮细胞中表达，该蛋白的另一个重要作用是作为血脑屏障的组成部分保护中枢神经系统。这一发现的一个含义是，绕开ABCG2和血脑屏障的化合物可能在治疗或预防中枢神经系统转移方面具有更高的功效。随着我们在新药物如小分子酪氨酸激酶抑制剂中识别ABCG2底物，ABCG2在血脑屏障中定位的重要性将会增加。在Michael Dean博士和James McMahon博士领导的分子靶标开发项目的合作下，ABCG2过表达细胞被用于筛选ABCG2抑制剂。这是一项重要的工作，因为无论ABCG2在肿瘤耐药中是否重要，其调节口服药物吸收和中枢神经系统摄取的潜在能力都将是重要的。分子靶标开发项目的柯蒂斯·亨利希博士和柯克·古斯塔夫森博士，已经确定了一些我们现在确认为ABCG2抑制剂的靶点，其中最有趣的是一个家族的三烯酰胺化合物。这些化合物在我们的实验室和Suresh Ambudkar博士的实验室中进行了二次筛选评估，以优先进行进一步的临床前开发。我们的计划是与威廉·费格博士的团队合作，将这些化合物用于动物研究。特别是，我们希望评估这些抑制剂是否会增加CNS中ABCG2和Pgp药物底物的积累。

PROJECT SUMMARY Our laboratory has a long-standing interest in non-Pgp mediated mechanisms of drug resistance, having established several cell line models of resistance focusing on the ABC half-transporter ABCG2. We successfully cloned ABCG2 from a mitoxantrone-resistant colon cancer cell line, that exhibited an ATP-dependent reduction in drug accumulation. Comprising 6 transmembrane domains and a single ATP binding domain, the gene encodes a half-transporter molecule and dimerization is required for activity. Overexpression of ABCG2 renders cells resistant to mitoxantrone and to the camptothecins, topotecan and SN-38 (the active metabolite of irinotecan) in vitro. A large number of both substrates and inhibitors of ABCG2 have been discovered, and the variety of both substrates and inhibitors rivals that described for P-glycoprotein. Since a role for drug transporters in mediating cancer drug resistance has never been defined, our group has studied other aspects of targeting this protein. We have studied a variant protein that may be associated with increased exposure to ABCG2 substrates, and particularly selected carcinogens. There is also a role for ABCG2 in limiting the oral absorption of pharmacologic agents and in limiting the brain uptake through expression in the blood brain barrier (See also project #1). Collectively, this work is best characterized as an effort to both understand and exploit ABCG2 as a therapeutic target to improve anticancer therapy. We have worked on structure and function relationships in the protein. We and others reported impaired transport in cells bearing a single nucleotide polymorphism at amino acid 141 that changes glutamine to lysine. Several lines of evidence suggest that this polymorphic variant could be associated with increased exposure to drugs that are substrates of ABCG2, or to a subset of carcinogens that are also known to be substrates of ABCG2, including those in tobacco smoke. This variant was first linked with an increased susceptibility to gout due to reduced excretion of urate and a corresponding increase in blood levels of urate. Similarly, gastrointestinal absorption has also been related to ABCG2 expression in the intestinal epithelium, and one implication of this work is that the Q141K SNP could be associated with increased absorption of substrate drugs such as topotecan, imatinib, or irinotecan. We have focused on understanding the trafficking and function of this variant that results from misfolding and targeting to the aggresome. We have also found that the protein that is targeted to the cell surface has impaired transport activity, and studies are underway in collaboration with Dr. Suresh Ambudkar to understand this abnormal function. Based on our earlier work with the dimerization motif in ABCG2, we have studied the pharmacologic rescue of ABCG2 through the addition of substrates. In fact, the best mediator of rescue was romidepsin, apparently due to at least three mechanisms: increased RNA expression, reduced transfer to the aggressome, and improved surface localization due to improved folding. ABCG2 is expressed in the endothelial cells in the brain and another important role for the protein is in protection of the CNS as a component of the blood-brain barrier. An implication of this finding is that compounds that circumvent ABCG2 and thus, the blood-brain barrier, could have increased efficacy in treating or preventing CNS metastases. The importance of localization of ABCG2 in the blood brain barrier will increase as we recognize ABCG2 substrates in new agents such as small molecule tyrosine kinase inhibitors. In collaboration with Dr. Michael Dean and the Molecular Targets Development Program, led by Dr. James McMahon, ABCG2-overexpressing cells have been used to screen for inhibitors of ABCG2. This is an important undertaking since the potential ability to modulate oral drug absorption and CNS uptake will be important whether or not ABCG2 proves important in oncologic drug resistance. Dr. Curtis Henrich and Kirk Gustafson, of the Molecular Targets Development Program, has identified a number of hits that we have now confirmed as ABCG2 inhibitors, most interesting of which is a family of botryllamide compounds. These compounds were evaluated in secondary screens in our laboratory and in that of Dr. Suresh Ambudkar, to prioritize for further preclinical development. Our plan is to take these compounds into animal studies in collaboration with Dr. William Figg's group. In particular, we hope to assess whether any of these inhibitors will increase CNS accumulation of ABCG2 and Pgp drug substrates.