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，我们正在研究人类p -糖蛋白（Pgp, ABCB1）和ABCG2，并采用了创新的方法，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。最近，为了三维结构的分辨率，我们开始了小鼠Pgp （mdr1a）的研究。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、细胞内环和核苷酸结合结构域（nbd）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是一种非常灵活的分子，其nbd之间的距离比已发表的小鼠Pgp结构更接近。为了提高DEER数据的质量，我们已经开始使用重组成纳米圆盘的Pgp，这使得无需进一步操作即可访问细胞外和细胞内区域。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。在Y307C/Q725C和V982C三突变体中，我们发现环孢素A、tariquar和valinomycin都不能抑制IAAP的标记，这表明这些药物已经失去了与主要药物结合位点的结合能力。然而，这些药物仍然通过在另一个位点结合来调节突变Pgp的atp酶活性和转运功能。其他研究表明，Pgp在与底物和调节剂相互作用时表现出优异的化学柔韧性。2.开发有效的ABC转运蛋白的无毒小分子调节剂/抑制剂：我们继续研究酪氨酸激酶抑制剂（TKIs）作为ABC药物转运蛋白抑制剂的潜在用途。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。陈哲生和Tanaji Talele （St. Johns University）以及傅立武（Li-wu Fu，中国广州中山大学）继续表征TKIs和其他小分子与ABC药物转运体的相互作用。几种用于临床或处于II/III期临床试验的tki包括Ponatinib， saracatinib和neratinib与Pgp相互作用并有效调节其转运功能。这些研究强烈提示，在常规化疗药物中联合使用一种或两种tki可能有助于提高癌症患者的化疗效率。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。位于纽约布法罗的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，该筛选基于这样一个事实，即已经观察到膜蛋白在形成蛋白质-洗涤剂复合物的洗涤剂的相分离边界附近形成晶体。最近，我们从昆虫细胞中大量纯化了小鼠Pgp (mdr1a) (10-12 mg蛋白/ml)，使用了纯化人类Pgp的条件并启动了结晶研究。我们加入了NIH-FEI活体实验室项目，利用单粒子冷冻电子显微镜研究获得了人类Pgp的高分辨率结构。4. 细胞表面Pgp内化和降解途径的阐明：我们发现Pgp在结肠癌细胞系HCT-15细胞表面的半衰期为24-26小时，用溶酶体/吞噬体抑制剂巴菲霉素治疗后，Pgp在细胞表面的滞留时间延长（半衰期为32-36小时），表明细胞表面Pgp在溶酶体/吞噬体中被降解。当用蛋白酶体抑制剂MG132处理细胞时，蛋白质的半衰期没有改变，这表明蛋白酶体途径在细胞表面Pgp的降解中没有显著作用。这些研究可能通过加速细胞表面转运蛋白的降解，为逆转耐药性提供一个或多个治疗靶点。5. 在患者肿瘤样本中评估ABC转运蛋白以及与耐多药相关的其他基因的表达谱：这些研究是与Michael Gottesman？LCB的s组。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。当将基因表达添加到四个协变量（年龄、分期、CA125水平和手术减体积）时，我们发现了一个11个基因的特征，它为总体生存预测提供了重大改进。这种11个基因的特征使得卡铂和紫杉醇治疗的卵巢浆液性癌患者的预后更加精确。在另一项与同一组的合作研究中，我们比较了耐多药相关的380个基因在组织培养癌细胞系和从患者肿瘤样本中分离的细胞中的表达谱。大多数基因的表达谱在肿瘤样本中与在组织培养中生长的细胞系有很大的不同，这表明在培养中长时间生长的细胞系可能无法模仿体内的癌症微环境。

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 are working with human P-glycoprotein (Pgp, ABCB1) and ABCG2 and 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. Recently, for resolution of the 3-D structure, we have initiated studies with mouse Pgp (mdr1a). 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 nucleotide-binding domains (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 that its NBDs are much closer to each other than those in the published mouse Pgp structure. To improve the quality of DEER data, we have begun to use Pgp reconstituted into nanodiscs, which allows access to both extra- and intracellular regions without further manipulations. 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. We found that neither cyclosporine A, tariquidar nor valinomycin were able to inhibit labeling with IAAP in the Y307C/Q725C and V982C triple mutant, indicating that the drugs had lost the ability to bind to the primary drug-binding site. However, these drugs still modulate the ATPase activity and transport function of mutant Pgp by binding at an alternate site. Additional studies suggest that Pgp exhibits exceptional chemical flexibility for interaction with substrates and modulators. 2.Development of potent non-toxic small molecule modulators/inhibitors of ABC 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. Zhe-Sheng Chen and Tanaji Talele (St. Johns University), and Li-wu Fu (Sun Yet Sen University, Guangzhou, China) we continue to characterize the interaction of TKIs and other small molecules with ABC drug transporters. Several TKIs that are used in the clinic or are in phase II/III clinical trials including Ponatinib, saracatinib and neratinib interact with Pgp and potently modulate its transport function. These studies strongly suggest that combining one or two of these TKIs conventional chemotherapeutic drugs may help to increase the efficiency of chemotherapy in cancer patients.3. Resolution of the 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, New York, 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. Recently, we have purified mouse Pgp (mdr1a) in large amounts (10-12 mg protein/ml) from insect cells using conditions developed for purification of human Pgp and initiated crystallization studies. We have joined the NIH-FEI living lab program to obtain the high-resolution structure of human Pgp by using single particle cryo-electron microscopy studies. 4. Elucidation of pathways involved in internalization and degradation of cell surface Pgp: We found that the half-life of Pgp at the cell surface in the colon cancer cell line HCT-15 is in the range of 24-26 hrs and treatment with the lysosomal/phagosomal inhibitor bafilomycin results in prolonged retention at the cell surface (half-life 32-36 hrs), indicating that the cell suface Pgp is degraded in lysosomes/phagosomes. When cells were treated with the proteasomal inhibitor MG132, the half-life of the protein was not altered, suggesting that the proteasomal pathway does not play a significant role in the degradation of cell surface Pgp. These studies may provide one or more therapeutic targets for the reversal of drug resistance by accelerating the degradation of cell surface transporters. 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 Gottesman?s 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. When gene expression was added to four covariates (age, stage, CA125 level and surgical debulking, we found an 11-gene signature that provides a major improvement in overall survival prediction. This 11-gene signature allows a more precise prognosis for patients with sereous cancer of the ovary treated with carboplatin- and paclitaxel-based therapy. In another collaborative study with same group we compared the expression profile of MDR-linked 380 genes in tissue culture cancer cell lines and cells isolated from patient tumor samples. The expression profile of a majority of genes was quite different in tumor samples compared to cell lines grown in tissue culture, suggesting that cell lines grown for extended periods of time in culture may not mimic the in vivo cancer microenvironment.