Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

多药耐药性相关转运蛋白的生化分析

• 批准号: 8763056
• 负责人: SURESH AMBUDKAR
• 金额: $ 98.9万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763056
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Affect; BRAF gene; Baculoviruses; Binding; Binding Sites; Biochemical; Biological Assay; Biology; CCR; Cancer Patient; Carrier Proteins; Cell surface; Cells; Chemicals; Chemistry; Chemosensitization; China; Clinical; Clinical Treatment; Collaborations; Combination Drug Therapy; Cryoelectron Microscopy; Crystallization; Cyclosporine; Cysteine; Data; Development; Disabled Persons; Docking; Drug Binding Site; Drug Interactions; Drug Transport; Drug resistance; Drug-sensitive; Electron Spin Resonance Spectroscopy; Electrons; Energy Transfer; Event; Exhibits; FK506; FLT3 gene; Fluorescence; Funding; Goals; Hela Cells; Homology Modeling; Human; Immunophilins; Insecta; Ions; Label; Life; Link; Location; Malignant Neoplasms; Maps; Maryland; Mediating; Melanoma Cell; Metabolic Pathway; Methods; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Models; Monitor; Monoclonal Antibodies; Multi-Drug Resistance; Multidrug Resistance Associated Protein 1; Mus; Mutagenesis; Mutation; Nucleotides; P-Glycoprotein; Pathway interactions; Pharmaceutical Preparations; Phase; Physiologic pulse; Play; Preparation; Proteins; Proto-Oncogene Proteins B-raf; Publishing; Regulation; Reporting; Resistance; Resolution; Role; Scheme; Site; Spin Labels; Staging; Structure; System; Taiwan; Tariquidar; Techniques; Testing; The Sun; Therapeutic; Three-Dimensional Image; Transition Elements; Transmembrane Domain; Tyrosine Kinase Inhibitor; United States National Institutes of Health; Universities; Valinomycin; Verapamil; Work; X-Ray Crystallography; base; cancer cell; cellular imaging; chemotherapy; cold temperature; crosslink; efflux pump; electron tomography; extracellular; flexibility; improved; inhibitor/antagonist; innovation; insight; kinase inhibitor; metabolomics; molecular modeling; mutant; neoplastic cell; novel; novel therapeutics; particle; programs; protein misfolding; protein misprocessing; single molecule; small molecule; three dimensional structure; trafficking

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.

我们的工作重点是阐明 ATP 结合盒 (ABC) 药物转运蛋白在癌症多药耐药性 (MDR) 发展中的作用，并开发新的治疗策略以提高癌症患者化疗的效率。在这些研究中，我们正在研究人类 P-糖蛋白（Pgp、ABCB1）和 ABCG2，并采用了创新方法，包括生物物理技术，例如连续波和脉冲双电子共振 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转运的分子机制，使用单克隆抗体的 Fab 和在不同步骤中捕获的各种突变蛋白催化循环使我们能够将转运蛋白固定在特定的构象中，以便通过 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 3D 图像分析。最近，我们通过X射线晶体学获得了从昆虫细胞中纯化的小鼠Pgp的7至8埃分辨率的结构。 1. 阐明ATP水解的催化循环和Pgp的运输途径以及ATP结合盒中保守基序的作用：我们正在继续对Pgp的催化循环和运输途径的研究。为了监测 ATP 水解和药物转运过程中发生的构象变化，我们使用 EPR 光谱和自旋标记方法。基于同源模型，我们在 cys-less Pgp 的不同位置引入了单个 cys 残基或两个 cys 残基，包括细胞外环、跨膜结构域、细胞内环和核苷酸结合结构域 (NBD) 的区域。我们已经开始与 NIH 资助的设施（康奈尔大学化学与化学生物学系）的 Jack Freed 博士合作，使用连续波和脉冲双电子电子共振 (DEER) ESR 光谱来监测存在和存在的构象变化。缺乏药物底物和 ATP。双 cys 突变体的 DEER ESR 光谱研究也将使我们能够验证人类 Pgp 的同源模型。此外，过渡金属离子福斯特共振能量转移 (tmFRET) 是一种新颖的生物物理方法，用于测定极低浓度下蛋白质不同位置内的短程（5 - 20 埃）和小尺度距离。使用这种灵敏的基于荧光的方法，我们已经开始确定与 apo 和 P-gp 的闭合（ATP/Vi 捕获）构象相关的距离变化。通过tmFRET，初步结果表明，apo和闭合构象之间的两个NBD的距离有很小的变化（小于20埃）。 DEER 和化学交联研究的初步结果表明，人 Pgp 是一种非常灵活的分子，其 NBD 比已发表的小鼠 Pgp 结构中的 NBD 彼此更接近。我们使用 QZ59RRR 结合形式的小鼠 Pgp 结构作为模板，将环孢素 A、塔利奎达、维拉帕米、缬氨霉素和 FSBA 对接在人 Pgp 的药物结合结构域中。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。我们发现环孢菌素A、塔利奎达和缬氨霉素都不能抑制Y307C/Q725C和V982C三重突变体中IAAP的标记，表明这些药物已经失去了与主要药物结合位点结合的能力。然而，这些药物仍然通过在替代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方面表现出卓越的化学灵活性。 2.开发有效的无毒ABC转运蛋白小分子调节剂/抑制剂：我们继续研究临床上重要的酪氨酸激酶抑制剂（TKI）与ABC药物转运蛋白的相互作用。我们与 Maria Baer 博士（马里兰大学）合作，证明 PIM 激酶抑制剂 SGI-1776 和 FLT3 激酶抑制剂 quizartinib 都能在药理学相关浓度下调节 ABCG2 的功能，并对化疗增敏和不良药物相互作用产生影响。我们发现最近开发的 TKI saracatinib 和 Tandutinib 与 Pgp 和 ABCG2 有效相互作用，影响肿瘤细胞的化疗敏感性（与 Zhe-Sheng Chen 博士和 Tanaji Talele 博士 [圣约翰大学] 以及 Li-wu Fu [Sun Yet] 合作）森大学，中国广州]）。我们表征了维莫非尼与 Pgp 和 ABCG2 的相互作用，后者用于治疗含有 V600E 突变 BRAF 激酶的黑色素瘤细胞，并发现在功能性 ABCG2 存在的情况下，维莫非尼对 BRAF (V600E) 突变 A375 细胞中 BRAF 激酶的抑制作用减弱。这些发现表明 ABCG2 赋予 A375 细胞对维罗非尼的耐药性，这表明针对多种途径的联合化疗可能是一种有效的治疗策略，可以克服携带 BRAF(V600E) 突变的癌症对维罗非尼的获得性耐药性（与 Chug-Pu 博士合作）吴，长庚大学，台湾）。我们与 Stuart Yuspa 博士（LG、CCR、NCI）合作，使用荧光和相差活细胞成像系统 IncuCyteTMFLR（Essen BioScience）开发了一种高通量 Pgp 介导的外排测定。该测定对于评估药物间相互作用和预测临床治疗中的 MDR 非常有用。 3. 人类Pgp三维结构的解析：Pgp三维结构的解析是一个正在进行的项目，为此我们开发了一种纯化方案，获得了7.5-10.0 mg的99%均质的总蛋白浓度为 10-12 mg/ml 的纯 Pgp。我们使用为纯化人类 Pgp 开发的条件，从昆虫细胞中纯化了大量（10-12 毫克蛋白质/毫升）小鼠 Pgp (mdr1a)。使用该制剂，我们通过 X 射线晶体学获得了小鼠 Pgp apo 构象的 7 至 8 埃分辨率结构。在这种低分辨率下，apo 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，以提高晶体的质量，以获得高分辨率（小于 3 埃）的结构分辨率并获得 apo 和闭合构象的高分辨率结构。我们已加入 NIH-FEI 生活实验室计划，通过单粒子冷冻电子显微镜研究获得人类和小鼠 Pgp 的高分辨率结构。 4.细胞内环1和3在人Pgp折叠和稳定性中的作用：我们研究了细胞内环1和3中的残基在人Pgp折叠和成熟中的作用。在不含半胱氨酸的背景中，ICL1 中的残基 D164 和 ICL3 中的残基 D805 被半胱氨酸取代。据观察，D164C/D805C 突变体在 HeLa 细胞中表达时，会导致 Pgp 错误加工，从而无法转运药物底物。通过在较低温度 (27°C) 下培养细胞或通过在不依赖于亲免素的途径中用底物（环孢素 A、FK506）、调节剂（tariquidar）或小校正分子进行处理，可以将错误折叠的蛋白质拯救到细胞表面。细胞内捕获的错误加工蛋白与分子伴侣 Hsp70 的结合更多，环孢菌素 A 的处理减少了突变型 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 残基对于 Pgp 的正确折叠至关重要。