Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

我们采用多学科方法设计了一种协调策略，以了解atp结合盒（ABC）药物转运体p-糖蛋白（P-gp）所表现出的多特异性的分子基础以及P-gp介导的药物转运机制。我们的方法包括几种生化和生物物理分析，基于细胞的运输分析，在脂质纳米盘中使用冷冻电镜进行结构研究的纯化和重构，药物化学合成大量化合物以评估结构-活性关系，在硅分子模型和MD模拟中扩展我们对机制方面和结构-功能关系的理解。此外，我们采用了一种新的方法，在同源跨膜螺旋（TMHs）中用丙氨酸取代多个保守残基来阐明P-gp的转运机制。此外，我们正在投入大量精力筛选和开发酪氨酸激酶抑制剂（TKIs）和P-gp和ABCG2的小分子调节剂，用于临床治疗各种类型的癌症。1. 阐明ATP水解的催化循环和P-gp的转运途径：我们继续研究P-gp的催化循环，特别是将其结构构象与ATP水解过程中发生的各个步骤联系起来。Cryo-EM单颗粒研究揭示了两种主要的构象，一种是内向开放（IO）构象，其中nbd被分离，并且药物结合腔可与底物或抑制剂相互作用。在atp结合的E556Q/E1201Q （EQ）突变体中观察到第二种构象，内向封闭（IC），其中nbd二聚（封闭），药物结合腔坍塌。正钒酸盐是无机磷酸盐的过渡态类似物，可抑制P-gp和其他ABC转运体的atp酶活性。我们测试了几种多氧钒酸盐化合物对P-gp的药物转运和atp酶活性的影响。有趣的是，我们发现decavanadate抑制P-gp的药物转运和atp酶活性，其效力与原钒酸盐相同。分子对接研究表明，decavanadate似乎不会与药物结合袋结合，这表明它很可能与ATP位点相互作用。此外，decavanadate与P-gp的结合对构象敏感。我们计划利用decavanadate的这一特性来了解更多与催化循环相关的构象变化。2. P-gp介导的转运方向逆转：我们采用了一种新的方法，在同源跨膜螺旋中引入多个突变，生化和基于细胞的转运实验，以及分子模型来研究P-gp转运药物的机制。P-gp的14A变体在TMH 6和TMH 12中有14个突变，它们排列在药物结合袋的中心空腔中，失去了输出大多数被测试底物的能力，但获得了输入四种底物的能力，包括Rhodamine123和flutax1（紫杉醇衍生物）。通过在TMH 6和TMH 12中产生几个替换残基的变体，我们发现控制转运方向的开关位于中心区域。对一系列突变体的进一步表征表明，TMH 6中的3个残基（V334、F336和F336）和TMH 12中的6个残基（F978、S979、V981、V982、F983和M986）对4种底物的最大积累起作用。有趣的是，残基F978、S979、V981、V982和F983对药物转运方向的调节至关重要。为了评估TMH 4和TMH 10中残基的作用，我们构建了TM4,10- 14a突变体，其中TMH 4和TMH 10中的7个残基被Ala取代。该突变体P-gp没有介导任何测试底物的积累，也没有外排所有测试底物。这些数据表明，tmh4和tmh10中的残基对P-gp的输运功能至关重要。3. P-gp和ABCG2的光动力调控机制：我们已经开始阐明光动力疗法（PDT）介导的ABC药物转运体调控的分子机制。PDT是一种基于光化学的工具，它涉及光激活光敏剂来产生活性氧。在临床上，PDT已被用于治疗各种疾病，如光化性角化病、非小细胞肺癌、头颈癌等。atp酶活性和硅分子对接分析表明，光敏剂苯并卟啉衍生物（BPD）在无光条件下以微摩尔半最大抑制浓度与ABCB1和ABCG2结合。光激活BPD产生单线态氧，以光学剂量依赖性的方式进一步降低ABCB1和ABCG2的atp酶活性高达12倍。凝胶电泳和Western blotting显示，光激活BPD通过共价交联诱导这些转运蛋白聚集。因此，PDT通过调节ATPase活性和这些转运体的蛋白质完整性来影响ABCB1和ABCG2的功能。从这项研究中获得的关于ABC药物转运体光动力学操作的见解可以帮助开发和应用新的光学工具，以克服癌症化疗后经常出现的多药耐药。为了提高PDT治疗耐药癌症的效率，我们设计了一种结合疏水性BPD光敏剂和构象敏感的UIC2单克隆抗体的光免疫偶联制剂，用于鉴定三阴性乳腺癌（TNBC）细胞中P-gp的表达。我们发现UIC2-BPD偶联物可以在活TNBC细胞中荧光标记P-gp，这表明它可能用于表达这种多药物转运体的肿瘤的荧光成像。这些研究是与Huang- chiao (Joe) Huang合作进行的，是CCR， NCI和马里兰大学合作项目的一部分。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：我们继续表征最近开发的TKIs，包括SKLB610，恩沙替尼和OTS964，再用途药物，小分子（苯基呋烷香豆素衍生物和泛素激活酶抑制剂ak -243），以及天然产物对P-gp和ABCG2功能的影响。这些研究是与博士合作进行的。Glaucio Valdameri（巴西巴拉那联邦大学）、Wu Chung-Pu（台湾长工大学）、Chen zhesheng（纽约圣约翰大学）和Shinobu Ohnuma（日本东北大学医学院）。A3腺苷受体激动剂已被开发用于治疗慢性疾病，如类风湿关节炎、牛皮癣、慢性疼痛和肝细胞癌。以前我们证明了腺苷受体的各种激动剂和拮抗剂调节P-gp的功能。我们扩展了这些研究，以测试A3腺苷受体配体（腺嘌呤核苷和核碱基衍生物）是否与ABCG2相互作用，调节药物在肠道中的吸收。我们合成了63种化合物，并测试了它们对ABCG2 atp酶活性的影响。其中，化合物60，一个具有低腺苷受体亲和力的7-二氮杂-5'-酯衍生物，被确定为ABCG2的高亲和力配体（与Kenneth Jacobson合作）。我们发现A3腺苷受体配体可以表现为*TRUNCATED*

We have designed a coordinated strategy using multidisciplinary approaches to understand the molecular basis of the polyspecificity exhibited by the ATP-binding cassette (ABC) drug transporter P-glycoprotein (P-gp) and the mechanism of P-gp-mediated drug transport. Our approaches include several biochemical and biophysical assays, cell-based transport assays, purification and reconstitution in lipid nanodiscs for structural studies using cryo-EM, medicinal chemistry to synthesize a large number of compounds to assess structure-activity relationships, in silico molecular modeling and MD simulations to extend our understanding of the mechanistic aspects and structure-function relationships. In addition, we are employing a novel approach of substituting multiple conserved residues with alanine in homologous transmembrane helices (TMHs) to elucidate the transport mechanism of P-gp. Furthermore, we are devoting considerable effort to the screening and development of tyrosine kinase inhibitors (TKIs) and small molecule modulators of both 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: We continue to study the catalytic cycle of P-gp, specifically correlating the structural conformations with the various steps that occur during ATP hydrolysis. Cryo-EM single particle studies have revealed two major conformations, one being the inward-open (IO) conformation, in which the NBDs are separated, and the drug-binding cavity is accessible for interaction with substrates or inhibitors. The second conformation, inward-closed (IC), is observed in the ATP-bound E556Q/E1201Q (EQ) mutant, in which the NBDs are dimerized (closed), and the drug-binding cavity is collapsed. Orthovanadate is a transition-state analog of inorganic phosphate that inhibits the ATPase activity of P-gp and other ABC transporters. We tested several polyoxyvanadate compounds for their effect on the drug transport and ATPase activity of P-gp. Interestingly, we found that decavanadate inhibits both the drug transport and ATPase activity of P-gp with the same potency as orthovanadate. Molecular docking studies indicate that decavanadate does not appear to bind to the drug-binding pocket, suggesting that most likely it interacts with the ATP sites. Moreover, decavanadate binding to P-gp is conformation-sensitive. We plan to exploit this property of decavanadate to learn more about conformational changes associated with the catalytic cycle. 2. Reversal of the direction of transport mediated by P-gp: We used a novel approach of introducing multiple mutations in homologous transmembrane helices, biochemical and cell-based transport assays, and molecular modeling to investigate the mechanism of drug transport by P-gp. A variant of P-gp termed 14A having 14 mutations in TMH 6 and TMH 12, which line the central cavity of the drug-binding pocket, lost the ability to export most of the substrates tested, but gained the ability to import four substrates, including Rhodamine123 and Flutax-1 (a Taxol derivative). By generating several variants with substitution of residues in both TMH 6 and 12, we found that the switch that controls the direction of transport resides in the center region. Further characterization of a series of mutants indicated that three residues (V334, F336 and F336) from TMH 6 and six residues (F978, S979, V981, V982, F983 and M986) from TMH 12 contribute to maximal accumulation of four substrates. Interestingly, residues F978, S979, V981, V982 and F983 are critical for modulation of the direction of drug transport. To assess the role of residues in TMH 4 and TMH 10, we generated a TM4,10-14A mutant in which seven residues from TMH 4 and seven from TMH 10 were substituted with Ala. This mutant P-gp did not mediate accumulation of any tested substrate and also failed to efflux all tested substrates. These data demonstrate that the residues in TMH 4 and 10 are critical for the transport function of P-gp. 3. Mechanism of photodynamic regulation of P-gp and ABCG2: We have begun to elucidate the molecular mechanism of photo dynamic therapy (PDT)-mediated regulation of ABC drug transporters. PDT is a photochemistry-based tool that involves light activation of photosensitizers to generate reactive oxygen species. In the clinic, PDT has been used to treat various diseases such as actinic keratosis, non-small cell lung cancer, and head and neck cancer. ATPase activity and in silico molecular docking analyses show that the photosensitizer benzoporphyrin derivative (BPD) binds to ABCB1 and ABCG2 with micromolar half-maximal inhibitory concentrations in the absence of light. Light activation of BPD generates singlet oxygen to further reduce the ATPase activity of ABCB1 and ABCG2 by up to 12-fold in an optical dose-dependent manner. Gel electrophoresis and Western blotting revealed that light-activated BPD induces aggregation of these transporters by covalent crosslinking. Thus, PDT affects the function of ABCB1 and ABCG2 by modulating the ATPase activity and protein integrity of these transporters. Insights gained from this study concerning the photodynamic manipulation of ABC drug transporters could aid in the development and application of new optical tools to overcome the multidrug resistance that often develops after cancer chemotherapy. To improve the efficiency of PDT for drug-resistant cancer, we devised a photoimmunoconjugate formulation combining hydrophobic BPD photosensitizers and a conformation-sensitive UIC2 monoclonal antibody to identify P-gp expression on triple negative breast cancer (TNBC) cells. We found that a UIC2-BPD conjugate can be used to fluorescently label P-gp in live TNBC cells, indicating its potential use for fluorescence imaging of tumors expressing this multidrug transporter. These studies were carried out in collaboration with Huang-Chiao (Joe) Huang as a part of partnership program between CCR, NCI and the University of Maryland. 4. Development of non-toxic natural product and small molecule modulators to overcome resistance mediated by P-gp and ABCG2: We continue to characterize recently developed TKIs including SKLB610, ensartinib, and OTS964, repurposed drugs, small molecules (phenylfurocoumarin derivative and ubiquitin-activating enzyme inhibitor TAK-243), and natural products for their effect on the function of P-gp and ABCG2. These studies were carried out in collaboration with Drs. Glaucio Valdameri (Federal University of Parana, Brazil), Chung-Pu Wu (Chang Gung University, Taiwan), Zhe-Sheng Chen (St. John's University, NY) and Shinobu Ohnuma (Tohoku University Graduate School of Medicine, Japan). A3 adenosine receptor agonists have been developed for the treatment of chronic diseases such as rheumatoid arthritis, psoriasis, chronic pain, and hepatocellular carcinoma. Previously we demonstrated that various agonists and antagonists of adenosine receptor modulate the function of P-gp. We expanded these studies to test whether A3 adenosine receptor ligands, both adenine nucleoside and nucleobase derivatives, interact with ABCG2, modulating the absorption of drugs in the intestine. We synthesized 63 compounds and tested them for their effect on the ATPase activity of ABCG2. Of these, compound 60, a 7-deaza-5'-ester derivative with low adenosine receptor affinity, was identified as a high-affinity ligand for ABCG2 (in collaboration with Kenneth Jacobson). We found that A3 adenosine receptor ligands can exhibit *TRUNCATED*