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: 9556248
• 负责人: SURESH AMBUDKAR
• 金额: $ 105.23万
• 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/9556248
• 关键词: 1-Phosphatidylinositol 3-Kinase; ABCB1 gene; ABCG2 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Affinity; Antibodies; Antineoplastic Agents; Artificial Membranes; Binding; Binding Sites; Biochemical; Biophysics; Cancer Patient; Carrier Proteins; Cell surface; Cells; Chemicals; Chemotherapy-Oncologic Procedure; Clinic; Collaborations; Computer Analysis; Conflict (Psychology); Coupled; Cryoelectron Microscopy; Cyclic Peptides; Cyclosporine; Detergents; Development; Disease; Docking; Drug Binding Site; Drug Transport; Environment; Exhibits; Generations; Goals; Hela Cells; Human; Human Characteristics; Hydrogen Bonding; Isoleucine; Leucine; Ligand Binding; Ligands; Link; Lipids; Malignant Neoplasms; Mass Spectrum Analysis; Measurable; Mediating; Membrane; Methionine; Micelles; Molecular; Molecular Biology; Molecular Conformation; Molecular Models; Monoclonal Antibodies; Multi-Drug Resistance; Mus; Mutagenesis; Mutate; Mutation; Natural Products; Negative Staining; P-Glycoprotein; Paclitaxel; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phenylalanine; Photoaffinity Labels; Physiological; Property; Proteins; Reporting; Research; Resistance; Resolution; Role; Sampling; Scaffolding Protein; Scheme; Site; Structure; Structure-Activity Relationship; Substrate Specificity; TAP1 gene; Taiwan; Tariquidar; Techniques; Tyrosine; Tyrosine Kinase Inhibitor; Universities; Vanadates; Verapamil; Vinblastine; Xenograft Model; base; cancer type; clinically relevant; dalton; dodecyl maltoside; flexibility; gene synthesis; high risk; improved; inhibitor/antagonist; ion mobility; kinase inhibitor; molecular modeling; molecular size; multi drug transporter; mutant; nanodisk; neoplastic cell; novel therapeutic intervention; particle; pharmacophore; protein transport; proteoliposomes; reconstitution; screening; single molecule; small molecule; three dimensional structure

We have focused our efforts to elucidate: i) the catalytic cycle and transport pathway of human P-gp; ii) the molecular basis of the polyspecificity of P-gp; iii) the interaction of clinically relevant tyrosine kinase inhibitors (TKIs) and other natural product modulators with P-gp and ABCG2; iv) pharmacophore features required for binding of third generation cyclic peptide inhibitor derivatives (BTT compounds) to P-gp and ABCG2; and vi) the use of single particle cryo-EM for the analysis of the conformational landscape of human P-gp during its catalytic cycle. 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 effort to the screening and development of TKIs and small molecule modulators of P-gp and ABCG2 that are used in the clinic for treatment of various types of cancers. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of P-gp and the role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of P-gp. 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 recently identified a phenylalanine-tyrosine structural motif in the drug-binding pocket of P-gp that is critical for the inhibition of ATP hydrolysis by zosuquidar and other high-affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, we made a global alteration of the drug-binding pocket using gene synthesis to introduce fifteen tyrosine residues in the drug-binding pocket to study its effect on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine, two leucine, one isoleucine, and one methionine. Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size fluorescent substrates, its ability to transport large ( above 1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased, thus revealing a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. To our knowledge, this is the first report demonstrating that the global alteration of the drug-binding pocket with substitution of fifteen residues is well tolerated. The 15Y mutant has six mutated residues located in TMD1 and nine in TMD2. The generation of mutants with either six substitutions in TMD1 or nine in TMD2 will be useful to assess the role of each TMD of the transporter and to narrow the number of residues contributing to substrate size determination. 2. The mechanism of the molecular basis of polyspecificity, which is an important property of multidrug transporters, by using molecular modeling and mutagenesis approaches: Towards the goal of understanding the molecular basis of broad substrate specificity of P-gp, we characterized the effect of a detergent micelle environment on the drug-mediated inhibition of P-gp ATPase activity. Most of the substrates or modulators of P-gp stimulate its basal ATPase activity, and only a few drugs have been found to inhibit it. Several structural studies on purified P-gp have been reported and there is very limited and in some cases conflicting information available on ligand interactions with isolated transporters in a dodecyl maltoside detergent environment. For these reasons, we compared the biochemical properties of human and mouse P-gp in native membranes, detergent micelles, and after reconstitution in artificial membranes. We found that the modulators zosuquidar, tariquidar and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment, whereas these drugs inhibited the ATPase activity of the transporter in native membranes or when it was reconstituted in proteoliposomes. Similarly, a 30- to 150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared to native or artificial membranes. These findings in aggregate demonstrated that the high-affinity site is inaccessible either due to a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. Thus, the structural and functional aspects of ligand interactions with purified P-gp need to be studied in a detergent-free native or artificial membrane environment. 3. Resolution of the three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of P-gp 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 P-gp. Due to the flexible nature of human P-gp, we are also using single particle analysis by the cryo-electron microscopy technique. To improve the resolution to an atomic level, we have begun to use the purified P-gp reconstituted in nanodiscs. We have optimized the conditions for reconstituting purified P-gp in nanodiscs in a functional form. The ratio of membrane scaffold protein (MSP) 1 to purified P-gp and the lipid composition of nanodiscs is critical to retain the function of purified protein in nanodiscs. The properties of P-gp in nanodiscs are similar to those observed in native membranes. The EM analysis of negatively stained P-gp-nanodisc samples in the absence and presence of ATP-Mg, ADP-vanadate and Fab of UIC2 antibody suggests that there is a single molecule of P-gp per nanodiscs. The single particle cryo-EM studies are carried out in collaboration with Dr. Sriram Subramaniam (LCB). 4. Development of non-toxic natural product and small molecule modulators to overcome resistance mediated by P-gp and ABCG2: We have extended these studies by synthesizing over 100 derivatives of the cyclic peptide inhibitor TTT28 in collaboration with Dr. Tanaji Talele (St. John's University, NY). We found that at least two derivatives inhibited the ATPase activity of P-gp and sensitized cells expressing P-gp and ABCB2 to anticancer drugs. The docking studies indicated that these derivatives bind to the drug-binding pocket of P-gp. To validate the docking poses of one of the inhibitory derivatives (compound 109), we used single or double mutations in the drug-binding pocket. In addition, we continue to characterize the recently developed tyrosine kinase inhibitors, which are used in the clinic to treat cancer patients, for their effect on the function of P-gp and ABCG2. The phosphatidylinositol 3-kinase inhibitor PF-498216 was found to modulate the function of ABCG2, whereas regorafenib was found to modulate only the function of P-gp. We also found that the BTK inhibitor, ibrutinib (PCI-32765) can overcome the resistance to paclitaxel-mediated by P-gp and ABCC10 (MRP7) in a mouse xenograft model. These studies were carried out in collaboration with Drs. Chug-Pu Wu (Chang Gung University, Taiwan) and Zhe-Sheng Chen (St. John's University, NY).

我们致力于阐明：i) 人类 P-gp 的催化循环和运输途径； ii) P-gp 多特异性的分子基础； iii) 临床相关酪氨酸激酶抑制剂 (TKI) 和其他天然产物调节剂与 P-gp 和 ABCG2 的相互作用； iv) 第三代环肽抑制剂衍生物（BTT化合物）与P-gp和ABCG2结合所需的药效团特征； vi) 使用单粒子冷冻电镜分析人 P-gp 在其催化循环过程中的构象景观。我们一直采用基于细胞的生物化学、生物物理、药理学和生理学技术以及分子生物学和分子建模方法来扩展我们对 ABC 药物转运蛋白的机制和结构功能关系的理解。此外，我们还投入了大量精力筛选和开发用于临床治疗各类癌症的TKI以及P-gp和ABCG2小分子调节剂。 1. 阐明P-gp的ATP水解催化循环和运输途径以及ATP结合盒中保守基序的作用：我们正在继续对P-gp的催化循环和运输途径进行研究。我们正在使用分子建模和诱变方法在分子水平上阐明这种转运蛋白如何识别和转运各种结构不同的化合物。我们最近在 P-gp 的药物结合袋中发现了一个苯丙氨酸-酪氨酸结构基序，它对于 zosuquidar 和其他高亲和力调节剂抑制 ATP 水解至关重要。由于酪氨酸既是氢键供体又是受体，非共价相互作用是药物转运的关键，因此我们利用基因合成对药物结合口袋进行了整体改变，在药物结合口袋中引入了15个酪氨酸残基，以研究其对P-gp药物结合和转运功能的影响。通过计算分析，鉴定出人P-gp药物结合口袋中与底物相互作用的15个保守残基，然后用酪氨酸取代，其中包括11个苯丙氨酸、2个亮氨酸、1个异亮氨酸和1个蛋氨酸。 HeLa 细胞中富含酪氨酸的 P-gp 突变体的表征表明，通过引入 15 个额外的酪氨酸残基对药物结合袋的这种重大改变具有良好的耐受性，并且对该突变体的总表达或细胞表面表达没有可测量的影响。虽然富含酪氨酸的突变体 P-gp 可以转运小至中等尺寸的荧光底物，但其转运大尺寸（超过 1000 道尔顿）底物（如 NBD-环孢素 A、Bodipy-紫杉醇和 Bodipy-长春花碱）的能力显着下降，从而揭示了富含酪氨酸的 P-gp 突变体的药物转运和分子大小之间呈负相关。据我们所知，这是第一份证明用十五个残基取代的药物结合口袋的整体改变具有良好耐受性的报告。 15Y 突变体有 6 个突变残基位于 TMD1 中，9 个突变残基位于 TMD2 中。 TMD1 中具有 6 个取代或 TMD2 中具有 9 个取代的突变体的产生将有助于评估转运蛋白的每个 TMD 的作用，并缩小有助于底物大小测定的残基数量。 2.多特异性的分子基础机制，这是多药转运蛋白的一个重要特性，通过使用分子建模和诱变方法：为了了解P-gp广泛底物特异性的分子基础，我们表征了去垢剂胶束环境对药物介导的 P-gp ATP 酶活性抑制的影响。大多数 P-gp 的底物或调节剂都会刺激其基础 ATP 酶活性，只有少数药物被发现可以抑制它。已经报道了一些关于纯化的 P-gp 的结构研究，并且关于十二烷基麦芽糖苷洗涤剂环境中配体与分离的转运蛋白的相互作用的信息非常有限，并且在某些情况下是相互矛盾的。出于这些原因，我们比较了人和小鼠 P-gp 在天然膜、洗涤剂胶束中以及在人工膜中重构后的生化特性。我们发现调节剂 zosuquidar、tariquidar 和 elacridar 在洗涤剂胶束环境中刺激纯化的人或小鼠 P-gp 的 ATP 酶活性，而这些药物抑制天然膜中或在蛋白脂质体中重构时转运蛋白的 ATP 酶活性。类似地，与天然膜或人造膜相比，在洗涤剂胶束中观察到维拉帕米和环肽抑制剂 QZ59-SSS 的表观亲和力降低了 30 至 150 倍。这些发现总的来说表明，由于去污剂胶束环境中的构象变化或去污剂在结合位点的结合，高亲和力位点是不可接近的。配体与低亲和力位点结合，导致 P-gp ATP 酶活性调节发生改变。因此，配体与纯化 P-gp 相互作用的结构和功能方面需要在无去污剂的天然或人造膜环境中进行研究。 3. 人类 Pgp 三维结构的解析：P-gp 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，已产生 7.5-10.0 mg 的 99 总蛋白％均质纯P-gp。由于人类 P-gp 的灵活性，我们还使用冷冻电子显微镜技术进行单颗粒分析。为了将分辨率提高到原子水平，我们已经开始使用在纳米圆盘中重构的纯化 P-gp。我们优化了在纳米圆盘中以功能形式重构纯化 P-gp 的条件。膜支架蛋白 (MSP) 1 与纯化 P-gp 的比例以及纳米圆盘的脂质组成对于保留纳米圆盘中纯化蛋白的功能至关重要。纳米圆盘中 P-gp 的特性与天然膜中观察到的特性相似。在不存在和存在 ATP-Mg、ADP-钒酸盐和 UIC2 抗体 Fab 的情况下，对阴性染色的 P-gp-纳米圆盘样品进行 EM 分析，表明每个纳米圆盘有一个 P-gp 分子。单粒子冷冻电镜研究是与 Sriram Subramaniam 博士 (LCB) 合作进行的。 4. 开发无毒天然产物和小分子调节剂以克服 P-gp 和 ABCG2 介导的耐药性：我们与 Tanaji Talele 博士（圣路易斯）合作，合成了 100 多种环肽抑制剂 TTT28 的衍生物，从而扩展了这些研究。纽约约翰大学）。我们发现至少两种衍生物抑制 P-gp 的 ATP 酶活性，并使表达 P-gp 和 ABCB2 的细胞对抗癌药物敏感。对接研究表明这些衍生物与 P-gp 的药物结合口袋结合。为了验证一种抑制性衍生物（化合物 109）的对接姿势，我们在药物结合口袋中使用了单突变或双突变。此外，我们继续表征最近开发的酪氨酸激酶抑制剂对 P-gp 和 ABCG2 功能的影响，这些抑制剂在临床上用于治疗癌症患者。磷脂酰肌醇 3 激酶抑制剂 PF-498216 被发现可调节 ABCG2 的功能，而瑞戈非尼仅调节 P-gp 的功能。我们还发现，在小鼠异种移植模型中，BTK 抑制剂依鲁替尼 (PCI-32765) 可以克服 P-gp 和 ABCC10 (MRP7) 介导的紫杉醇耐药性。这些研究是与博士合作进行的。 Chug-Pu Wu（台湾长庚大学）和 Zhe-Sheng Chen（纽约圣约翰大学）。