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。为了监测ATP水解和药物运输过程中发生的构象变化，我们已经开始使用EPR光谱和自旋标记方法。在与博士合作。Stuart Durrell和Di Xia以Sav1866和小鼠mdr1b结构为模板构建了人类Pgp的同源性模型。基于该模型，我们在无cys Pgp的不同位置引入了单个或两个cys残基，包括来自细胞外环、跨膜结构域、细胞内环和nbd的区域。到目前为止，我们已经产生了30个单细胞和25个双细胞突变体。这些突变体在High-Five昆虫细胞中表达后，大多数已被鉴定。这些突变体将被纯化，用自旋标记，MTSL标记，并进行EPR光谱分析。在与John Golin（美国天主教大学）的合作中，我们正在使用一种遗传学方法来确定抑制突变的第二位点突变，这种突变导致酵母菌Pdr5p转运体的药物转运活性恢复，该转运体外排多种外源化合物。我们观察到，TM2中的抑制突变S558Y导致药物转运活性完全丧失，而不影响Pdr5p结合药物底物或结合和水解ATP的能力，这表明该突变体的药物转运和ATP酶活性是解耦的。有趣的是，恢复功能的第二位点突变都定位在NBD区。这些研究表明，采用遗传和生化相结合的方法将使我们能够绘制Pdr5p转运体中药物底物结合位点与ATP位点之间的信号通路。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：我们继续验证使用天然产物无毒调节剂姜黄素来逆转癌症的耐药。我们与博士合作。Bjorn Bauer和Anika Hartz（明尼苏达大学）利用大鼠脑毛细血管作为血脑屏障的离体模型，证明了纳米摩尔浓度的姜黄素可以阻断Abcg2和Pgp的功能。此外，通过Abcg2基因敲除小鼠，我们进一步证实口服姜黄素通过选择性抑制Abcg2功能提高了C-max和硫柳氮的相对生物利用度。我们建议，当药物吸收的限速步骤和/或组织分布受到ABC药物转运体的影响时，无毒浓度的姜黄素可以用来增强药物暴露。在与Rajendra Prasad博士（印度贾瓦哈拉尔尼赫鲁大学）的合作中，我们还证明了姜黄素也能调节白色念珠菌（一种真菌病原体）Cdr1p的活性。因此，姜黄素可以与某些常规抗真菌药物联合使用，以逆转致病性酵母的耐药性。我们继续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，用于治疗肾细胞癌和耐伊马替尼胃肠道肿瘤的舒尼替尼（Sutent, SU11248）可阻断Pgp和ABCG2的功能，这可能会影响与舒尼替尼合用药物的生物利用度。在与博士合作。陈哲生（St. Johns university）和傅立伟（中国中山大学）观察到另一种小分子FG020326（三芳基取代咪唑衍生物）在小鼠细胞培养和异种移植模型中都是Pgp的有效调节剂。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。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一个botryllamides文库，这些文库具有抑制ABCG2功能的能力。此外，我们还筛选了国家癌症研究所药物数据筛选数据库（与博士合作）。约翰·迪肯和苏珊·贝茨，NCI)，并确定了几种新的ABCG2底物和其他化合物，它们可能作为调节剂与这种转运体相互作用。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的浓度获得了7.5-10.0 mg的99%均质纯度的Pgp。为了改善P-gp的结晶，我们启动了另一种方法，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7-10 mg/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。我们已经成功地从杂交瘤细胞系HB1287中获得900-1000 mg纯化的UIC2抗体。这将允许我们准备250- 300毫克Fab用于测试新的结晶条件。4. 癌细胞单步和多步选择抗癌药物耐药的分子机制：为了了解临床条件下多药耐药（MDR）的机制，我们建立了使用极低浓度（14或21 nM）的单步选择阿霉素的MCF-7亚群。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体，这一点通过使用ABCG2- sirna得到了证实。包括CD44和CD24在内的干细胞标记物在低浓度阿霉素选择的MCF-7单步克隆中不富集。我们最近对多步骤选择的乳腺癌细胞系MCF-7/ADR的研究表明，这些细胞中有30%至50%是CD44+/CD24-。MCF-7/ADR是通过持续暴露于浓度不断增加的阿霉素而产生的。此外，这些细胞在体外三维培养中具有增强的自我更新、迁移和增殖能力，并在体内形成肿瘤。这些结果表明具有乳腺癌干细胞特征的细胞可以通过化疗选择(Calcagno et al.，手稿提交；工作是与dr。柳芭·瓦尔蒂科夫斯基和迈克尔·m·戈特斯曼)。5. 评估患者肿瘤样本中ABC转运蛋白以及与多药耐药相关的其他基因的表达谱：这些研究是与Dr.合作进行的[摘要被剪切为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. To monitor the conformational changes occurring during ATP hydrolysis and drug transport, we have begun to use the EPR spectroscopy and spin labeling approach. In collaboration with Drs. Stuart Durrell and Di Xia we have constructed homology model of human Pgp using Sav1866 and mouse mdr1b structure as a template. Based on this model we have introduced either single cys residue or two cys residues at various locations in the cys-less Pgp including regions from extracellular loops, transmembrane domains, intracellular loops, and NBDs. We have generated thirty single and twenty five double cys mutants so far. Most of these mutants after their expression in High-Five insect cells have been characterized. These mutants will be purified, labeled with spin label, MTSL and subjected to EPR spectroscopy analysis. In collaboration with John Golin (The Catholic University of America), we are using a genetic approach to identify second site mutations in a suppressor mutant which results in recovery of the drug transport activity of the yeast Pdr5p transporter that effluxes a variety of xenobiotic compounds. We observed that suppressor mutation S558Y in TM2 resulted in complete loss of drug transport activity without affecting the ability of Pdr5p to either bind drug substrates or to bind and hydrolyze ATP indicating that the drug transport and ATPase activities in this mutant are uncoupled. Interestingly the second site mutations that recover function are all localized to NBD region. These studies demonstrate that the use of combination of genetic and biochemical approach will allow us to map the signaling pathway between drug substrate binding sites and the ATP sites in Pdr5p transporter. 2. Development of potent natural product and other non-toxic modulators/inhibitors of ABC transporters: We continue to validate the use of natural product non-toxic modulator curcumin to reverse the drug resistance in cancer. We in collaboration with Drs. Bjorn Bauer and Anika Hartz (University of Minnesota, MN) use rat brain capillaries as an ex vivo model of blood-brain barrier to demonstrate that curcumin at nanomolar concentration blocks the function of both Abcg2 and Pgp. In addition, by using Abcg2 knockout mice we further confirmed that oral curcumin increased C-max and relative bioavailability of sulfasalazine by selectively inhibiting ABCG2 function. We propose that non-toxic concentrations of curcumin may be used to enhance drug exposure when the rate-limiting step of drug absorption and/or tissue distributions impacted by ABC drug transporter. In collaboration with Dr. Rajendra Prasad (Jawaharlal Nehru University, India), we also demonstrated that curcumin also modulates the activity of Cdr1p of Candida albicans, a fungal pathogen. Thus, curcumin may be used in combination with certain conventional antifungal drugs to reverse drug resistance in pathogenic yeast. We continue to study the tyrosine kinase inhibitors for their potential use as inhibitors of ABC drug transporters. We found that Sunitinib (Sutent, SU11248), which is used in the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal tumors blocks the function of Pgp and ABCG2 and this may affect the bioavailability of drugs co-administered with sunitinib. In collaboration with Drs. Zhe-Sheng Chen (St. Johns Univ.), and L-W Fu (Sun Yat-Sen Univ., China) we observed that another small molecule, FG020326 (triaryl-substituted imidazole derivative) is a potent modulator of Pgp in both cell culture and xenograft model in mice. 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. 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 botryllamides, which are for their ability to inhibit ABCG2 function. In addition, we also screened the National Cancer Institute drug data screen data base (in collaboration with Drs. John Deeken and Susan Bates, NCI) and identified several novel ABCG2 substrates and other compounds, which interact possibly as modulators with this transporter. 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 7.5-10.0 mg of > 99% homogeneously pure Pgp at 10-12 mg/ml concentration. For improving the crystallization of P-gp, 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 7-10 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. We have been successful in obtaining 900-1000 mg of purified UIC2 antibody from the hybridoma cell line HB1287. This will allow us to prepare 250--300 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 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, which is confirmed by using ABCG2-SiRNA. The stem cell markers including CD44 and CD24 are not enriched in single-step clones of MCF-7 selected with low concentrations of doxorubicin. Our recent work with the multi-step selected breast cancer cell line MCF-7/ADR, which was generated by continuous exposure to increasing concentrations of doxorubicin demonstrates that 30 to 50% of these cells are CD44+/CD24-. In addition, these cells have enhanced capacity to self-renew, migrate and proliferate in three-D cultures in vitro and form tumors in vivo. These results suggest that cells with characteristics of breast cancer stem cells can be selected by chemotherapy (Calcagno et al., manuscript submitted; work was done in collaboration with Drs. Lyuba Varticovski and Michael M. Gottesman). 5. Evaluation of expression profiles of ABC transporters as well as other genes linked with multidrug resistance in patient tumor samples: These studies are carried out in collaboration with Dr. [summary truncated at 7800 characters]