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: 9153530
• 负责人: SURESH AMBUDKAR
• 金额: $ 107.1万
• 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/9153530
• 关键词: ABCB1 gene; ABCG2 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Adopted; Affect; Affinity; Alanine; Baculoviruses; Binding; Binding Sites; Biochemical; Cancer Patient; Carrier Proteins; Cell surface; Cells; Chemicals; Clinic; Collaborations; Copper; Coupled; Crosslinker; Cryoelectron Microscopy; Cyclosporine; Cysteine; Cystic Fibrosis Transmembrane Conductance Regulator; Detergents; Development; Disease; Disulfides; Drug Binding Site; Drug resistance; Energy Transfer; Exhibits; Face; Fluorescence; Generations; Goals; HCT-15; Half-Life; Hela Cells; Human; Human Characteristics; Hydrogels; Hydrogen Bonding; Inhibitory Concentration 50; Investigation; Ions; Kinetics; Lead; Link; Liposomes; Location; Lysosomes; MG132; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Methods; Michigan; Microscopy; Molecular; Molecular Biology; Molecular Chaperones; Molecular Conformation; Molecular Models; Monitor; Monoclonal Antibodies; Multi-Drug Resistance; Mutagenesis; Mutate; Mutation; Optics; Oxidants; P-Glycoprotein; Paclitaxel; Pathway interactions; Pharmaceutical Preparations; Phenanthrolines; Phenylalanine; Photoaffinity Labels; Physiological; Play; Property; Proteins; Reagent; Research; Resolution; Rhodamine 123; Role; Scheme; Side; Site; Solutions; Structure; Structure-Activity Relationship; Substrate Specificity; Sulfhydryl Compounds; Tariquidar; Techniques; Transition Elements; Tyrosine; Tyrosine Kinase Inhibitor; Vanadates; Verapamil; Vesicle; Vinblastine; Walker-A Motif; X-Ray Crystallography; base; biophysical analysis; biophysical techniques; cancer type; chemotherapy; clinically relevant; colon cancer cell line; crosslink; drug mechanism; flexibility; high risk; inhibitor/antagonist; insight; ion mobility; lactacystin; luminescence; molecular dynamics; molecular modeling; molecular transporter; multi drug transporter; mutant; nanometer; neoplastic cell; novel; novel therapeutics; particle; pharmacophore; protein folding; reconstitution; screening; small molecule; therapeutic target; three dimensional structure; transport inhibitor; uptake

We have focused our efforts to elucidate: i) the catalytic cycle and transport pathway of human Pgp; ii) the molecular basis of the polyspecificity of Pgp; iii) the interaction of clinically relevant tyrosine kinase inhibitors (TKIs) with Pgp and ABCG2; iv) determination of the binding site of nilotinib, a second generation TKI, on Pgp; v) pharmacophore features required for binding of nilotinib to Pgp and ABCG2; and vi) the fate of cell surface Pgp, its folding and stability. We have been employing cell-based, biochemical, biophysical, pharmacological, and physiological techniques along with molecular biology and molecular modeling approaches to extend our understanding of the mechanistic aspects and the structure-function relationships of ABC drug transporters. In addition, we have devoted considerable efforts to the screening and development of TKIs and small molecule modulators for Pgp and ABCG2. We found that several tyrosine kinase inhibitors, which are used in the clinic for treatment of various types of cancers, are either transport substrates or inhibitors of Pgp and/or ABCG2. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of Pgp and the role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of Pgp. We are using molecular modeling and mutagenesis approaches to elucidate on a molecular level how this transporter recognizes and transports a wide variety of structurally dissimilar compounds. We have begun to use tmFRET, which is a novel biophysical method developed to determine short range (5 - 25 angstrom) 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 Pgp. With tmFRET, preliminary results show that there is a significant change in the distance of the two NBDs between the apo and closed conformations ( 20 angstrom). Similarly, results of disulfide crosslinking studies with the oxidant copper phenanthroline and bi-functional sulfhydryl group reagents indicate that human Pgp is a very flexible molecule and its NBDs are much closer to each other in the apo form. The distance between the C431 and C1074 residues in the Walker A motif of NBDs ranges from 5 to 25 angstrom in the apo (in the absence of ATP and drug-substrate) conformation. 2. Mechanism of the drug-mediated inhibition of Pgp ATPase activity. Most of the substrates or modulators of Pgp stimulate its basal ATPase activity, and only a few drugs have been found to inhibit it. Zosuquidar, tariquidar and elacridar, high affinity inhibitors of transport function, also inhibit Pgp ATPase activity, while a variety of substrates including verapamil, paclitaxel and vinblastine stimulate ATP hydrolysis. The molecular mechanisms that are in play, in either case (stimulation or inhibition), remain elusive. The development of an effective Pgp inhibitor certainly would benefit from the understanding of drug-mediated inhibition of ATP hydrolysis. Using directed mutagenesis we identified a pair of phenylalanine-tyrosine structural motifs of Pgp that are critical for the inhibition of ATP hydrolysis by high-affinity modulators. These structural motifs are located at the drug-binding pocket of Pgp. We found that drugs that inhibit the ATPase activity switch to stimulating the ATPase activity when any of these residues are mutated. For instance, zosuquidar inhibits the basal ATP hydrolysis of cysless WT Pgp with high affinity (IC50 = 10 nM). The inhibition is completely lost upon mutation of Y953 to alanine and is switched to stimulation when three polar residues are mutated (Y307A/Q725A/Y953A). Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with the tyrosines Y953 and Y310, respectively, in an edge-to-face conformation, helping thetyrosines to adopt the proper orientation to effectively establish hydrogen-bond contact with the inhibitors. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport. Upon mutation, they bind at lower affinity sites that lead to stimulation of ATP hydrolysis and a poor inhibition of transport. 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. Due to the flexible nature of human Pgp and the difficulty of generating crystals of good diffraction quality, we are also using single particle analysis by the cryo-electron microscopy technique. The current studies indicate that the structural features of human Pgp in the presence and absence of a Fab of conformation-sensitive monoclonal antibody can be observed at 15- to 20-angstrom resolution. We are optimizing conditions to reach a sub-nanometer resolution to obtain the structure of human Pgp in at least three different (apo, ADP-vanadate trapped and Fab-bound) conformations. The structural studies are carried out in collaboration with Drs. Di Xia and Sriram Subramanian. In addition, we have reconstituted human Pgp in giant unilamellar liposomes. Giant unilamellar liposomes (vesicles) are very useful to study the biophysical and kinetic properties of transport by Pgp, as these liposomes can be observed individually by optical microscopy. In collaboration with Dr. Michael Mayer (Univ. of Michigan), we reconstituted purified human Pgp into giant liposomes using a hydrogel method. Pgp in the giant liposomes was reconstituted with inside-out and right side out (same as in intact cells) orientations at equal levels, and it exhibited substrate-stimulated ATPase activity and ATP -dependent rhodamine 123 uptake. 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. To gain insight into the stability of cell surface Pgp, we assessed the degradation of cell surface Pgp. We characterized the pathway involved in degradation of Pgp following its internalization. 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 26-27 h and treatment with the lysosomal inhibitor bafilomycin results in prolonged retention at the cell surface (half-life 35-36 h), indicating that the cell surface Pgp is degraded in lysosomes. Consistent with these results, internalized Pgp was found localized to the lysosomes. When cells were treated with the proteasomal inhibitors MG132 or lactacystin, 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. To further explore the role of ICL 3 residues in folding, we mutated conserved R798, D800, D805, D806 and K808 to alanine and the mutant Pgps were expressed in HeLa cells using bac-mam baculovirus. We found that the cell surface expression of R798A, D800A, D805A and D806A was greatly reduced, and these mutants were retained in the ER. As expected, the mutant proteins were rescued to the cell surface by treatment with cyclosporine A and they were fully functional. However, the expression of K808A was not affected, and the mutation had no effect on transport function. Thus, the interaction of ICL3 and NBD2 is critical for folding of Pgp, but not for its function.

我们的工作重点是阐明：i)人类Pgp的催化循环和转运途径；ii) Pgp多特异性的分子基础；iii)临床相关酪氨酸激酶抑制剂（TKIs）与Pgp和ABCG2的相互作用；iv)测定第二代TKI尼罗替尼在Pgp上的结合位点；v)尼罗替尼与Pgp和ABCG2结合所需的药效团特征；vi)细胞表面Pgp的命运、折叠和稳定性。我们一直在使用基于细胞的、生物化学的、生物物理的、药理学的和生理学的技术，以及分子生物学和分子建模方法来扩展我们对ABC药物转运体的机制方面和结构-功能关系的理解。此外，我们还在Pgp和ABCG2的TKIs和小分子调节剂的筛选和开发方面投入了大量的精力。我们发现临床上用于治疗各种类型癌症的几种酪氨酸激酶抑制剂是Pgp和/或ABCG2的转运底物或抑制剂。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。我们正在使用分子模型和诱变方法在分子水平上阐明这种转运体如何识别和运输各种结构不同的化合物。我们已经开始使用tmFRET，这是一种新的生物物理方法，用于在非常低的浓度下确定蛋白质不同位置的短距离（5 - 25埃）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Pgp的封闭（ATP/Vi捕获）构象相关的距离变化。利用tmFRET，初步结果表明两个nbd在载子构象和闭合构象（20埃）之间的距离有显著变化。同样，与氧化剂菲罗啉铜和双功能巯基试剂的二硫交联研究结果表明，人Pgp是一种非常灵活的分子，其ndb以载脂蛋白形式彼此更接近。NBDs的Walker A基序中C431和C1074残基之间的距离在载脂蛋白构象（在没有ATP和药物底物的情况下）为5 - 25埃。2. 药物介导的Pgp atp酶活性抑制机制。Pgp的大多数底物或调节剂刺激其基础atp酶活性，只有少数药物被发现抑制它。Zosuquidar， tariquidar和elacridar是高亲和力的转运功能抑制剂，也抑制Pgp ATP酶活性，而各种底物包括维拉帕米，紫杉醇和长春花碱刺激ATP水解。在这两种情况下（刺激或抑制）起作用的分子机制仍然难以捉摸。开发有效的Pgp抑制剂肯定会受益于对药物介导的ATP水解抑制的理解。通过定向诱变，我们确定了Pgp的一对苯丙氨酸-酪氨酸结构基序，它们对高亲和调节剂抑制ATP水解至关重要。这些结构基序位于Pgp的药物结合口袋。我们发现，当这些残基中的任何一个发生突变时，抑制atp酶活性的药物会转变为刺激atp酶活性。例如，zosuquidar具有高亲和力（IC50 = 10 nM）抑制无cyysless WT Pgp的基础ATP水解。当Y953突变为丙氨酸时，抑制作用完全消失，当三个极性残基突变（Y307A/Q725A/Y953A）时，抑制作用转变为刺激作用。分子模型显示，苯丙氨酸残基F978和F728分别与酪氨酸Y953和Y310形成边对面相互作用，帮助酪氨酸选择合适的取向，有效地与抑制剂建立氢键接触。生化研究和完整细胞的转运研究表明，抑制剂在高亲和力位点结合，产生ATP水解和转运的抑制。突变后，它们结合在较低的亲和力位点，导致ATP水解刺激和运输抑制不良。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，产生了7.5-10.0 mg的99%均质纯Pgp总蛋白。由于人类Pgp的柔韧性和产生良好衍射质量的晶体的困难，我们也使用低温电子显微镜技术进行单粒子分析。目前的研究表明，在构象敏感单克隆抗体Fab存在和不存在的情况下，人类Pgp的结构特征可以在15至20埃的分辨率下观察到。我们正在优化条件以达到亚纳米分辨率，以获得至少三种不同构象（载脂蛋白，adp -钒酸盐捕获和fab结合）的人类Pgp结构。结构研究是与博士合作进行的。迪夏和斯里拉姆·萨勃拉曼尼亚。此外，我们还在巨大的单层脂质体中重组了人Pgp。巨型单层脂质体（囊泡）是研究Pgp运输的生物物理和动力学性质非常有用的，因为这些脂质体可以通过光学显微镜单独观察到。与Michael Mayer博士（密歇根大学）合作，我们使用水凝胶法将纯化的人Pgp重组为巨大的脂质体。巨大脂质体中的Pgp以相同水平的内向外和右向外（与完整细胞相同）取向重组，并表现出底物刺激的ATP酶活性和ATP依赖性罗丹明123摄取。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。为了深入了解细胞表面Pgp的稳定性，我们评估了细胞表面Pgp的降解。我们描述了Pgp内化后降解的途径。我们发现Pgp在结肠癌细胞系HCT-15细胞表面的半衰期在26-27小时之间，用溶酶体抑制剂巴菲霉素处理后，Pgp在细胞表面的停留时间延长（半衰期为35-36小时），表明细胞表面的Pgp在溶酶体中被降解。与这些结果一致，内化Pgp被发现定位于溶酶体。当用蛋白酶体抑制剂MG132或lactacystin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。为了进一步探索icl3残基在折叠中的作用，我们将保守的R798、D800、D805、D806和K808突变为丙氨酸，并利用bacman杆状病毒在HeLa细胞中表达了突变的Pgps。我们发现R798A、D800A、D805A和D806A的细胞表面表达量大大降低，并且这些突变体保留在内质网中。正如预期的那样，突变蛋白通过环孢素A处理被拯救到细胞表面，并且它们具有完全的功能。然而，K808A的表达不受影响，突变对转运功能没有影响。因此，ICL3和NBD2的相互作用对Pgp的折叠至关重要，但对其功能并不重要。