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，过渡金属离子福斯特共振能量转移（tmFRET），化学交联，定向诱变和分子建模，以阐明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，我们已经能够通过x射线晶体学获得从昆虫细胞中纯化的小鼠Pgp的7至8埃分辨率结构。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。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系）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。此外，过渡金属离子福斯特共振能量转移（tmFRET）是一种新的生物物理方法，用于在极低浓度下测定蛋白质不同位置的短距离（5 - 20埃）和小距离。使用这种基于荧光的灵敏方法，我们已经开始确定与载脂蛋白和P-gp的封闭（ATP/Vi捕获）构象相关的距离变化。利用tmFRET，初步结果表明，载子构象和闭合构象之间的两个nbd距离变化很小（小于20埃）。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。我们以qz59rrr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、valinomycin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：我们继续研究临床上重要的酪氨酸激酶抑制剂（TKIs）与ABC药物转运蛋白的相互作用。与马里兰大学的Maria Baer博士合作，我们证明PIM激酶抑制剂SGI-1776和FLT3激酶抑制剂quizartinib在药理学上相关浓度下调节ABCG2的功能，这对化学致敏和药物不良相互作用有影响。我们发现最近开发的TKIs saracatinib和Tandutinib与Pgp和ABCG2有效相互作用，影响肿瘤细胞的化学致敏。陈哲生，Tanaji Talele[美国圣约翰大学]，傅立武[中山大学，中国广州])。我们描述了vemurafenib与Pgp和ABCG2（用于治疗含有V600E突变BRAF激酶的黑色素瘤细胞）的相互作用，并发现在功能性ABCG2存在的情况下，vemurafenib对BRAF （V600E）突变A375细胞的BRAF激酶抑制作用降低。这些发现表明，ABCG2在A375细胞中赋予vemurafenib耐药性，这表明针对多种途径的联合化疗可能是克服BRAF（V600E）突变癌症对vemurafenib获得性耐药的有效治疗策略（与台湾长公大学吴忠普博士合作）。我们与Stuart Yuspa博士（LG， CCR， NCI）合作，利用荧光和相衬活细胞成像系统IncuCyteTMFLR （Essen BioScience）开发了一种高通量pgp介导的外排试验。该分析将非常有用的评估药物-药物相互作用和预测耐多药临床治疗。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，在10-12 mg/ml浓度下，获得了7.5-10.0 mg 99%均质纯度的Pgp总蛋白。我们使用纯化人类Pgp的条件，从昆虫细胞中大量纯化小鼠Pgp (mdr1a) （10-12 mg蛋白/ml）。利用这种制备方法，我们通过x射线晶体学获得了载脂蛋白构象的小鼠Pgp的7 ~ 8埃分辨率结构。在这种低分辨率下，载脂蛋白构象的结构与Aller等人在2009年报道的略有不同。目前，我们正在测试各种结晶条件，以提高晶体质量，以获得高分辨率（小于3埃）的结构，并获得载脂蛋白和封闭构象的高分辨率结构。我们加入了NIH-FEI活体实验室项目，利用单粒子冷冻电镜研究获得了人和小鼠Pgp的高分辨率结构。4. 细胞内环1和3在人类Pgp折叠和稳定性中的作用：我们研究了细胞内环1和3残基在人类Pgp折叠和成熟中的作用。在无半胱氨酸背景下，ICL1中的残基D164和ICL3中的残基D805被半胱氨酸取代。我们观察到，当D164C/D805C突变体在HeLa细胞中表达时，会导致Pgp的错误加工，从而无法运输药物底物。通过在较低温度（27℃）下培养细胞，或用底物（环孢素A， FK506）、调节剂（tariquidar）或不依赖于亲免疫蛋白途径的小校正分子处理，错误折叠的蛋白可以被拯救到细胞表面。细胞内捕获的错误加工蛋白更多地与伴侣蛋白Hsp70结合，而环孢素A的处理减少了突变体Pgp与Hsp70的结合，从而使其能够被运输到细胞表面。这些数据表明，D164和D805残基对Pgp的正常折叠至关重要。

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 the 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, transition metal ion Forster resonance energy transfer (tmFRET), chemical crosslinking, directed mutagenesis, and 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, we have been able to obtain by X-ray crystallography a 7- to 8-angstrom resolution structure of mouse Pgp purified from insect cells. 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 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 (Department of 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. In addition, transition metal ion Forster resonance energy transfer (tmFRET) is a novel biophysical method developed to determine short range (5 - 20 angstrom) and small-scale distances within different locations of the protein at very low concentrations. Using this sensitive fluorescence-based method, we have begun to determine the changes in distance associated with the apo and the closed (ATP/Vi trapped) conformations of P-gp. With tmFRET, preliminary results show that there is a small change in the distance of the two NBDs between the apo and closed conformations (less than 20 angstrom). 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. We have docked cyclosporine A, tariquidar, verapamil, valinomycin and FSBA in the drug-binding domain of human Pgp using the structure of mouse Pgp in QZ59RRR-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 clinically important tyrosine kinase inhibitors (TKIs) for their interactions with ABC drug transporters. In collaboration with Dr. Maria Baer (University of Maryland), we demonstrated that both PIM kinase inhibitor SGI-1776 and the FLT3 kinase inhibitor quizartinib modulate the function of ABCG2 at pharmacologically relevant concentrations with implications for chemosensitization and adverse drug interactions. We found that the recently developed TKIs saracatinib and Tandutinib interact potently with Pgp and ABCG2 affecting the chemosensitization of tumor cells (in collaboration with Drs. Zhe-Sheng Chen and Tanaji Talele [St. Johns University], and Li-wu Fu [Sun Yet Sen University, Guangzhou, China]). We have characterized the interaction of vemurafenib with Pgp and ABCG2, which is used for treatment of melanoma cells harboring V600E mutant BRAF kinase and found that in the presence of functional ABCG2, BRAF kinase inhibition by vemurafenib is reduced in BRAF (V600E) mutant A375 cells. These findings indicate that ABCG2 confers resistance to vemurafenib in A375 cells, suggesting that combination chemotherapy targeting multiple pathways could be an effective therapeutic strategy to overcome acquired resistance to vemurafenib for cancers harboring the BRAF(V600E) mutation (in collaboration with Dr. Chug-Pu Wu, Chang Gung University, Taiwan). In collaboration with Dr. Stuart Yuspa (LG, CCR, NCI), we have developed a high throughput Pgp-mediated efflux assay using the fluorescent and phase-contrast live cell imaging system, the IncuCyteTMFLR (Essen BioScience). This assay will be very useful for assessing drug-drug interactions and for predicting MDR in clinical treatment. 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. 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. Using this preparation, we have obtained by X-ray crystallography a 7- to 8-angstrom resolution structure of mouse Pgp in apo conformation. At this low resolution, the structure in apo conformation is slightly different than previously reported by Aller et al., in 2009. Currently, we are testing various crystallization conditions to improve the quality of crystals to obtain structure at high (less than 3 angstrom) resolution and to obtain a high-resolution structure in apo and closed conformations. We have joined the NIH-FEI living lab program to obtain the high-resolution structure of both human and mouse Pgp by using single particle cryo-electron microscopy studies. 4. Role of intracellular loops 1 and 3 in folding and stability of human Pgp: We investigated the role of residues in intracellular loops 1 and 3 in folding and maturation of human Pgp. The residue D164 in ICL1 and the residue D805 in ICL3 were replaced with cysteine in a cysteine-less background. It was observed that the D164C/D805C mutant, when expressed in HeLa cells, led to misprocessing of Pgp, which thus failed to transport the drug substrates. The misfolded protein could be rescued to the cell surface by growing the cells at lower temperature (27C) or by treatment with substrates (cyclosporine A, FK506), modulators (tariquidar) or small corrector molecules in an immunophilin-independent pathway. The intracellularly trapped misprocessed protein associates more with chaperone Hsp70 and the treatment with cyclosporine A reduces association of mutant Pgp with Hsp70, thus allowing it to be trafficked to the cell surface. These data demonstrate that the D164 and D805 residues are critical for proper folding of Pgp.