Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

1. 阐明ATP水解的催化循环和Pgp转运途径以及保守基序在ATP结合盒中的作用：我们正在继续对Pgp的催化循环和转运途径进行研究。基于热力学和动力学性质，我们确定了pgp介导的atp酶反应的ES和EP稳定反应中间体。最近，我们观察到Pgp的ES构象（之前在E556Q/E1201Q突变体中证实）可以通过使用不可水解的ATP类似物ATP-g- s与野生型蛋白一起获得。ATP-g- s与E556Q/E1201Q突变体中的ATP相似，在34-37C时被封闭在野生型Pgp nbd中，而在4C时则不被封闭。当Pgp被ATP-g-S阻断时，它对转运底物的亲和力降低。总的来说，这些数据为ATP驱动的二聚化和adp驱动的nbd解离提供了证据，尽管两个ATP分子可以启动二聚化，但只有一个被驱动到封闭的预水解中间状态。在全长ABC转运体中，如Pgp，其中一个nbd上的封闭核苷酸构象提供了转运-底物位点的能量行程。与John Golin（美国天主教大学）合作，我们表征了酵母Pdr5p转运体的atp酶活性，该转运体外排多种外源化合物。氯霉唑是一种有效的转运底物，但pdr5p特异性atp酶活性显示出完全的、浓度依赖性的抑制作用，然而GTPase和UTPase活性对该药物均具有相对抗性。我们的研究结果表明，这种抑制是非竞争性的，是由克霉唑与转运体在不同于atp结合结构域的位点相互作用引起的。我们提出Pdr5p通过使用多个核苷酸作为能量源来增加其转运底物的特异性。2. 开发有效的天然产物和其他ABC转运蛋白的无毒调节剂/抑制剂：为了开发能够抑制多种转运蛋白的调节剂，我们筛选了合成化合物和天然产物。ABCG2转运体对多种化疗药物具有耐药性。对抗这种转运体介导的耐多药的一种方法是开发抑制剂/调节剂，在无毒浓度下阻断其功能。我们发现萘醌类、维生素K3及其结构类似物白铅素是ABCG2的底物。因此，ABCG2可能在调节体内维生素K3水平中起作用。此外，一种能特异性杀死Pgp表达细胞的硫代氨基脲NSC73306被发现是ABCG2的有效调节剂。因此，NSC73306表现出相应的作用模式，可以通过消除过表达pgp的细胞来克服耐药性，并作为一种有效的调节剂，使表达ABCG2的癌细胞对化疗药物重新敏感。在与博士合作。Susan Bates, Curtis Henrich， Michael Dean和James McMahon (CCR和分子靶标开发计划，NCI)，我们用高通量分析筛选了7400个天然产物和合成化合物，以确定有效的ABCG2新抑制剂。我们也在研究酪氨酸激酶抑制剂作为ABC药物转运体抑制剂的潜在用途。我们发现新开发的酪氨酸激酶抑制剂AMN107（尼罗替尼）及其母体药物伊马替尼（格列卫）通过在药物-底物结合位点而不是像酪氨酸激酶抑制剂那样在ATP位点相互作用来调节Pgp和ABCG2的活性。我们与陈哲生博士（St. Johns university）合作，证明了Erlotinib （Tarceva）是一种EGFR酪氨酸激酶抑制剂，通过直接抑制这些转运蛋白的外排功能来逆转Pgp和abcg2介导的耐药性。3. 人类Pgp三维结构的分辨率：Pgp三维结构的分辨率是一个正在进行的项目，为此我们开发了一种纯化方案，以10-12 mg/ml的浓度产生了7.5-10 mg的总蛋白，纯度超过99%。为了改善Pgp的结晶，我们已经启动了另一种方法，即在构象敏感单克隆抗体的Fab中，在结晶过程中将UIC2与纯化的Pgp一起孵育。我们优化了生成UIC2 Fab的条件，蛋白浓度在5- 7mg /ml范围内。另外的实验表明，在类似的条件下，UIC2的Fab在洗涤剂溶液中与Pgp结合，用于几代晶体，这表明生成Pgp和UIC2-Fab的共晶体是可行的。目前我们正在从杂交瘤细胞系HB1287中产生400-500 mg的UIC2。这将允许我们准备150-200毫克Fab用于测试新的结晶条件。4. 肿瘤细胞中抗癌药物单步和多步选择耐药的分子机制：为了了解临床条件下多药耐药（MDR）的机制，我们开始研究治疗方案如何影响ABC药物转运蛋白在抗癌药物如阿霉素的单步和多步选择中的表达。我们建立了单步多柔比星选择MCF-7亚线，使用非常低的浓度，14或21 nM。我们发现ABCC2， ABCC4和ABCG2在mRNA水平上在这些单步选择的亚系中过表达。然而，只有ABCC4和ABCG2在蛋白水平上过表达。与亲本MCF-7细胞相比，14和21 nM单步阿霉素选择亚群对阿霉素的耐药性均接近5倍。然而，由于ABCC4不会产生对阿霉素的耐药性，因此ABCG2很可能是导致耐药性产生的主要转运蛋白。我们还通过染色质免疫沉淀法观察到，组蛋白超乙酰化促进了ABCG2的上调。有趣的是，包括CD44和CD24在内的干细胞标记物在低浓度（21 nM）阿霉素选择的乳腺癌细胞系MCF-7的单步克隆中不富集。这些结果表明，多药耐药表型是在低剂量、单步暴露于阿霉素后出现的，并进一步表明，通过表观遗传改变的ABCG2过表达可能介导了多药耐药发展的早期阶段。在另一项与Lyuba Varticovski （CCR， NCI）合作的相关研究中，我们试图确定癌症干细胞是否发生在brca1相关的乳腺癌中，并有助于治疗反应。我们观察到brca -1缺陷小鼠乳腺肿瘤含有异质的癌症干细胞群，CD44+/CD24-细胞代表了与人类乳腺癌干细胞相关的群体。5. ABCB1中单核苷酸多态性和单倍型的表征：与Michael Gottesman博士合作，我们发现在MDR1中，单倍型背景下的同义SNP，两个同义SNP （3435C b> T和1236C>T）和一个非同义SNP （2677G>T）与底物和抑制剂特异性的改变有关。进一步的近期工作与博士合作。Gottesman和Nussinov （SAIC, Frederick Inc.和CCR， NCI）建议long-[摘要被截断为7800个字符]

1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of Pgp and role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of Pgp. Based on the thermodynamic and kinetic properties, we have identified the ES and EP stable reaction intermediates of the Pgp-mediated ATPase reaction. Recently, we observed that the ES conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analog ATP-g-S. ATP-g-S, similar to ATP in E556Q/E1201Q mutant, is occluded into the wild-type Pgp NBDs at 34-37C but not at 4C. When Pgp is occluded with ATP-g-S, it exhibits reduced affinity for transport substrates. Collectively, these data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs and although, two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. It appears that in a full-length ABC transporter like Pgp, the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site. In collaboration with John Golin (The Catholic Univ. of America), we characterized the ATPase activity of the yeast Pdr5p transporter that effluxes a variety of xenobiotic compounds. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is known to be a potent transport substrate, however both GTPase and UTPase activities are relatively resistant to this drug. Our results indicate that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. We propose that Pdr5p increases its transport substrate specificity by using more than one nucleotide as an energy source. 2. Development of potent natural product and other non-toxic modulators/inhibitors of ABC transporters: To develop modulator(s) that will inhibit multiple transporters we screened synthetic compounds as well as natural products. The ABCG2 transporter confers resistance to multiple chemotherapeutic agents. One approach to combat MDR mediated by this transporter is the development of inhibitors/modulators that block its function at non-toxic concentrations. We found that napthoquinones, vitamin K3 and its structural analogue plumbagin are substrates of ABCG2. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, a thiosemicarbazone, NSC73306, which kills specifically Pgp expressing cells, was found to be a potent modulator of ABCG2. Thus, NSC73306 exhibits due mode of action that can be exploited to overcome drug resistance by eliminating Pgp-overexpressing cells and by acting as a potent modulator to resensitize ABCG2 expressing cancer cells to chemotherapeutics. In collaboration with Drs. Susan Bates, Curtis Henrich, Michael Dean and James McMahon (CCR and Molecular Targets Development Program, NCI) we have screened with a high-throughput assay a library of 7400 natural products and synthetic compounds to identify potent new inhibitors of ABCG2. We are also studying tyrosine kinase inhibitors for their potential use as inhibitors of ABC drug transporters. We found that the newly developed tyrosine kinase inhibitor AMN107 (nilotinib), and its parent drug imatinib (Gleevec) modulates activity of Pgp and ABCG2 by interacting at the drug-substrate binding sites instead of ATP sites as is the case with tyrosine kinase inhibitors. In collaboration with Dr. Zhe-Sheng Chen (St. Johns Univ.), we have demonstrated that Erlotinib (Tarceva), which is an EGFR tyrosine kinase inhibitor, reverses Pgp and ABCG2-mediated resistance by directly inhibiting the efflux function of these transporters. 3. Resolution of three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of Pgp is an ongoing project and for this we have developed a purification scheme that has yielded total protein 7.5-10 mg of more than 99% homogeneously pure Pgp at 10-12 mg/ml concentration. For improving the crystallization of Pgp, we have initiated another approach where in the Fab of the conformation-sensitive monoclonal antibody, UIC2 is incubated along with the purified Pgp during crystallization. We have optimized the conditions to generate Fab of UIC2 with protein concentration in the range of 5-7 mg/ml. Additional experiments demonstrate that Fab of UIC2 binds to Pgp in detergent solution under similar conditions that are used for generations of crystals indicating that it is feasible to generate co-crystals of Pgp and UIC2-Fab. At present we are in the process of generating 400-500 mg of UIC2 from the hybridoma cell line HB1287. This will allow us to prepare 150-200 mg of Fab for testing new crystallization conditions. 4. Molecular mechanism of drug resistance in single- and multi-step selection with anticancer agents in cancer cells: To understand the mechanism of multidrug resistance (MDR) under clinical conditions, we have begun to examine how treatment regimens affect the expression of ABC drug transporters in single- and multi-step selection with anticancer drugs such as doxorubicin. We established single-step doxorubicin-selected MCF-7 sublines using very low concentrations, 14 or 21 nM. We have found that ABCC2, ABCC4 and ABCG2 were overexpressed at the mRNA level in these single-step selected sublines. Yet, only ABCC4 and ABCG2 were overexpressed at the protein level. Both 14 and 21 nM single-step doxorubicin-selected sublines exhibit nearly 5-fold resistance to doxorubicin compared to parental MCF-7 cells. However, as ABCC4 does not confer resistance to doxorubicin it is most likely that ABCG2 is the major transporter responsible for the development of resistance. We also observed by using chromatin immunoprecipitation assay that the upregulation of ABCG2 is facilitated by histone hyperacetylation. Interestingly, stem cell markers including CD44 and CD24 are not enriched in single-step clones of breast cancer cell line MCF-7 selected with low concentration (21 nM) of doxorubicin. These results indicate that the MDR phenotype arises following lose-dose, single-step exposure to doxorubicin, and further suggest that overexpression of ABCG2 by epigenetic changes may mediate early stages of MDR development. In another related study in collaboration with Lyuba Varticovski (CCR, NCI), we sought to determine whether cancer stem cells occur in BRCA1-associated breast cancer and contribute to therapeutic response. We observed that Brca-1-deficient mouse mammary tumors harbor heterogeneous cancer stem cell populations, and CD44+/CD24- cells represent a population that correlates with human breast cancer stem cells. 5. Characterization of Single nucleotide polymorphisms and haplotypes in ABCB1: In collaboration with Dr. Michael Gottesman we showed that in MDR1, synonymous SNPs in the context of a haplotype, with two synonymous (3435C>T & 1236C>T) and one non-synonymous (2677G>T) SNP, were associated with altered substrate and inhibitor specificity. Further recent work in collaboration with Drs. Gottesman and Nussinov (SAIC, Frederick Inc., and CCR, NCI) suggests that long- [summary truncated at 7800 characters]