Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

我们的工作重点是阐明atp结合盒（ABC）药物转运体在癌症多药耐药（MDR）发展中的作用，并开发新的治疗策略以提高癌症患者的化疗效率。在这些研究中，我们采用了创新的方法，包括生物物理技术，如连续波和脉冲双电子-电子共振ESR光谱，定向诱变，分子模型来阐明ATP水解催化循环和药物运输的分子机制。利用Fab的单克隆抗体和在催化循环的不同步骤中捕获的各种突变蛋白，使我们能够将转运蛋白固定在特定的构象中，从而通过x射线晶体学解析Pgp的结构，并通过冷冻电子断层扫描对单分子进行三维图像分析。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。为了监测ATP水解和药物运输过程中发生的构象变化，我们使用了EPR光谱和自旋标记方法。基于同源性模型，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ndd的区域。到目前为止，我们已经产生了25个双细胞和25个单细胞突变体。这些突变体在High-Five昆虫细胞中表达后被纯化，并发现其功能与野生型蛋白保持相同的水平。我们优化了用自旋标记MTSL在洗涤剂溶液中标记这些突变蛋白的条件，用于EPR光谱分析。我们已经开始使用连续波和脉冲双电子-电子共振（DEER） ESR光谱，与NIH资助的机构（康奈尔大学化学和化学生物学）的Jack Freed博士合作，监测药物底物和ATP存在和不存在时的构象变化。双cys突变体的DEER ESR研究也将使我们能够验证人类Pgp的同源性模型。DEER和化学交联研究的初步结果表明，人类Pgp是一个非常灵活的分子，nbd之间的距离比在已发表的小鼠Pgp结构中发现的更接近。在相关研究中，我们描述了ATP类似物5-氟磺酰基苯甲酰5-腺苷（FSBA）与Pgp的相互作用。我们首次观察到FSBA不仅在核苷酸结合域（nbd， ATP位点）上相互作用，而且在Pgp的转运-底物结合位点上相互作用。通过对FSBA标记的Pgp进行HPLC/质谱分析，我们发现这种ATP类似物与nbd中的残基交联，而不是与该转运体跨膜结构域的药物结合位点交联。我们相信FSBA将对了解Pgp的多特异性非常有用。我们以qz59sss结合形式的小鼠Pgp结构为模板，将环孢素A、塔奎达、维拉帕米和FSBA停靠在人Pgp的药物结合域。与这些底物/调节剂相互作用的残基已被半胱氨酸取代，以绘制药物结合位点。目前，我们正在描述这些单和双cys突变体在bacman杆状病毒转导的HeLa细胞中的转运功能。初步结果表明，这些突变体在HeLa细胞的质膜上以与野生型相似的水平表达。2. 开发有效的天然产物和其他无毒的ABC转运蛋白调节剂/抑制剂：我们继续验证天然产物，无毒调节剂在癌症中逆转耐药的使用。在与博士合作。Pornngarm Limtrakul和Mei-Lin Go，我们发现Kugacin J， stemofoline以及功能化的aurones调节Pgp和ABCG2转运蛋白的活性。我们继续研究酪氨酸激酶抑制剂（TKIs）作为ABC药物转运体抑制剂的潜在用途。我们已经证明，第二代TKI尼罗替尼（Tasigna）可以通过Pgp和ABCG2转运。此外，我们首次合成并表征了Tasigna的荧光衍生物（bodipy-Tasigna），这可能是一个有用的探针，用于肿瘤细胞中这些转运蛋白的功能分析和临床前研究。在与博士合作。Susan Bates （CCR， NCI内科肿瘤学分支）和zhes - sheng Chen (St. Johns university)，我们继续表征TKIs和其他小分子与ABC药物转运体的相互作用。有趣的是，我们发现西地那非（伟哥）是Pgp和ABCG2的有效调节剂，这表明这种已知副作用和药物-药物相互作用的临床批准药物可能用于提高抗癌药物的疗效。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7.5-10.0 mg的99%均质纯度的Pgp。布法罗Hauptman Woodward研究所的高通量筛选实验室设计了一种专门用于膜蛋白的结晶筛选，这是基于这样一个事实，即观察到膜蛋白形成的晶体接近用于形成蛋白质-洗涤剂复合物的洗涤剂的相分离边界。我们目前正在测试这些条件，在初始筛选试验中获得的命中值将帮助我们确定获得高质量人类Pgp晶体的最佳结晶条件。我们继续使用UIC2络合物的Pgp-Fab进行结晶。为了确定UIC2在Pgp上的表位，我们对UIC2的Fab进行了测序。此外，我们用Alexa荧光染料对FAB进行了标记，并证明FAB在完整HeLa细胞的细胞表面识别人类Pgp，类似于UIC2抗体。该抗体表位的鉴定将有助于了解UIC2的构象敏感性。4. 癌细胞中抗癌药物单步和多步选择的耐药分子机制：我们最近对多步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-。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（CSCs）特征的细胞在长期药物治疗后富集。根据这些发现，我们推测乳腺癌患者的长期药物治疗也可能导致具有高度化疗抗性的癌症干细胞样表型的细胞数量增加，甚至可能是癌症干细胞。5. 在患者肿瘤样本中评估ABC转运蛋白以及其他与耐多药相关的基因的表达谱：这些研究是与LCB的Michael Gottesmans博士小组合作进行的。我们用最先进的微流控TLDA芯片为基础的qRT-PCR方法评估了32例未配对的卵巢浆液性癌患者的耐多药相关转录组。这些研究确定了三个与总生存期、治疗反应和无进展生存期显著相关的基因特征。这些基因特征可能有助于优化卵巢癌积液患者的治疗。

Our work is focused on the elucidation of the role of ATP-binding cassette (ABC) drug transporters in the development of multidrug resistance (MDR) in cancers and on the development of new therapeutic strategies to increase efficiency of chemotherapy for cancer patients. For these studies we have employed innovative approaches including biophysical techniques such as continuous wave and pulse double electron-electron resonance ESR spectroscopy, directed mutagenesis, molecular modeling to elucidate molecular mechanisms of the ATP hydrolysis catalytic cycle and drug transport, the use of Fab of monoclonal antibodies and various mutant proteins arrested at various steps in the catalytic cycle to enable us to fix the transporter in a particular conformation for resolution of the structure of Pgp by X-ray crystallography and for 3-D image analysis of single molecules by cryo-electron tomography. 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. To monitor the conformational changes occurring during ATP hydrolysis and drug transport, we are using an EPR spectroscopy and spin labeling approach. Based on a homology model, we have introduced either a single cys residue or two cys residues at various locations in cys-less Pgp, including regions from extracellular loops, transmembrane domains, intracellular loops, and NBDs. We have generated 25 double- and 25 single-cys mutants so far. These mutants, after their expression in High-Five insect cells, were purified and found to retain function to the same level as wild-type protein. We have optimized the conditions for labeling of these mutant proteins in detergent solution with the spin label MTSL for EPR spectroscopy analysis. We have begun to use continuous wave and pulse double electron-electron resonance (DEER) ESR spectroscopy in collaboration with Dr. Jack Freed at an NIH funded facility (Chemistry and Chemical Biology, Cornell University) to monitor conformational changes in the presence and absence of drug-substrate and ATP. The DEER ESR spectroscopy studies with the double cys mutants will also allow us to validate the homology model of human Pgp. Preliminary results of DEER and chemical crosslinking studies suggest that human Pgp is a very flexible molecule and the NBDs are much closer to each other than found in the published mouse Pgp structure. In related studies, we have characterized the interaction of an ATP analog, 5-fluorosulfonylbenzoyl 5-adenosine (FSBA) with Pgp. For the first time, we observed that FSBA interacts not only at the nucleotide-binding domains (NBDs, ATP sites) but also at the transport-substrate binding sites of Pgp. By HPLC/Mass spec analysis of FSBA labeled Pgp, we found that this ATP analog crosslinks to residues in NBDs, but not in the drug-binding sites in the transmembrane domains of this transporter. We believe that FSBA will be very useful for understanding the polyspecificity of Pgp. We have docked cyclosporine A, tariquidar, verapamil and FSBA in the drug-binding domain of human Pgp using the structure of mouse Pgp in QZ59SSS-bound form as a template. The residues interacting with these substrates/modulators have been substituted with cysteine to map the drug-binding sites. Currently, we are characterizing the transport function of these single and double cys mutants in bac-mam baculovirus-transduced HeLa cells. The preliminary results suggest that these mutants are expressed, at a similar level as in the wild-type, at the plasma membrane of HeLa cells. 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 modulators to reverse drug resistance in cancer. In collaboration with Drs. Pornngarm Limtrakul and Mei-Lin Go, we found that Kugacin J, stemofoline as well as functionalized aurones modulate activities of Pgp and ABCG2 transporters. We continue to study tyrosine kinase inhibitors (TKIs) for their potential use as inhibitors of ABC drug transporters. We have demonstrated that the second generation TKI nilotinib (Tasigna) is transported by both Pgp and ABCG2. In addition, for the first time we have synthesized and characterized a fluorescent derivative of Tasigna (bodipy-Tasigna), which may be a useful probe for functional analysis of these transporters in cancer cells and also in preclinical studies. In collaboration with Drs. Susan Bates (Medical Oncology Branch, CCR, NCI) and Zhe-Sheng Chen (St. Johns Univ.), we continue to characterize interaction of TKIs and other small molecules with ABC drug transporters. Interestingly we found that sildenafil (Viagra) is a potent modulator of Pgp and ABCG2, suggesting that this clinically approved drug with known side effects and drug-drug-interactions may be used to increase the efficacy of anticancer drugs. 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 of 7.5-10.0 mg of > 99% homogeneously pure Pgp at 10-12 mg/ml concentration. The high-throughput screening laboratory at Hauptman Woodward Institute, Buffalo has designed a crystallization screen specifically for membrane proteins based on the fact that membrane proteins have been observed to form crystals close to the phase separation boundaries of the detergent used to form the protein-detergent complex. We are currently testing these conditions and the hits obtained in the initial screening assays will assist us in identifying crystallization conditions optimal for obtaining good quality crystals of human Pgp. We continue to use Pgp-Fab of UIC2 complex for crystallization. To identify the epitope of UIC2 on Pgp, we have sequenced the Fab of UIC2. In addition, we have labeled the FAB with Alexa fluorescent dye and demonstrated that the Fab recognizes human Pgp, similar to UIC2 antibody, at the cell surface in intact HeLa cells. The identification of an epitope of this antibody will be of use to understand the conformation-sensitivity of UIC2. 4. Molecular mechanism of drug resistance in single- and multi-step selection with anticancer agents in cancer cells: 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 (CSCs) are enriched following prolonged drug treatment. From these findings, we speculate that prolonged drug treatment of patients with breast cancer may also result in increased numbers of cells with a highly chemotherapy-resistant cancer stem cell-like phenotype that may even be cancer stem cells. 5. Evaluation of expression profiles of ABC transporters as well as other genes linked with MDR in patient tumor samples: These studies are carried out in collaboration with Dr. Michael Gottesmans group in LCB. We have assessed the MDR-linked transcriptome in 32 unpaired ovarian serous carcinoma patients with the state-of-the art microfluidic TLDA chip-based qRT-PCR assay. These studies resulted in identification of three gene signatures with a significant correlation with overall survival, response to treatment and progression free survival. These gene signatures may be useful for optimizing the treatment for ovarian cancer patients with effusions.