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: 10702323
• 负责人: SURESH AMBUDKAR
• 金额: $ 150.33万
• 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/10702323
• 关键词: ABCB1 gene; ABCG2 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; ATP-binding cassette transport; Actinic keratosis; Address; Adenine; Adenosine A3 Receptor; Affect; Affinity; Agonist; Alanine; Antibodies; Antineoplastic Agents; Area; Binding; Biochemical; Biological Assay; Biological Availability; Biophysics; Brazil; Breast Cancer Cell; CCR; Cancer Patient; Carrier Proteins; Cells; Chemicals; Chemoresistance; Chemotherapy-Oncologic Procedure; Chronic Disease; Clinic; Clinical; Collaborations; Complement; Cryoelectron Microscopy; Curcumin; Data; Derivation procedure; Development; Disease; Docking; Dose; Drug Interactions; Drug Kinetics; Drug Transport; Drug resistance; Enzyme Inhibitor Drugs; Esters; Exhibits; FDA approved; Formulation; Goals; Head and Neck Cancer; Hydrophobicity; Intestines; Japan; Knowledge; Label; Learning; Libraries; Ligands; Light; Link; Lipids; Malignant Neoplasms; Maryland; Mediating; Molecular; Molecular Conformation; Monoclonal Antibodies; Multi-Drug Resistance; Mutation; Natural Products; Non-Small-Cell Lung Carcinoma; Nucleosides; Optics; Orthovanadate; PUVA Photochemotherapy; Paclitaxel; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphotransferases; Photochemistry; Photosensitizing Agents; Play; Primary carcinoma of the liver cells; Property; Proteins; Psoriatic Arthritis; Pump; Purinergic P1 Receptors; Reactive Oxygen Species; Regulation; Resistance; Resolution; Rheumatoid Arthritis; Role; Series; Singlet Oxygen; Site; Structure; Structure-Activity Relationship; Substrate Specificity; Taiwan; Testing; Time; Transmembrane Domain; Tyrosine Kinase Inhibitor; Ubiquitin-Activating Enzymes; Universities; Variant; Western Blotting; Work; absorption; analog; antagonist; base; cancer imaging; cancer type; chronic pain; crosslink; design; dimer; drug repurposing; fluorescence imaging; gel electrophoresis; improved; in silico; inhibitor; inhibitor therapy; inorganic phosphate; insight; interdisciplinary approach; medical schools; member; molecular dynamics; molecular modeling; multi drug transporter; multidrug resistant cancer; mutant; nanodisk; nanomedicine; novel strategies; novel therapeutic intervention; nucleobase; particle; programs; reconstitution; refractory cancer; screening; small molecule; tool; triple-negative invasive breast carcinoma

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*

我们设计了一种采用多学科方法的协调策略，以了解 ATP 结合盒 (ABC) 药物转运蛋白 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。我们的方法包括多种生化和生物物理测定、基于细胞的运输测定、脂质纳米盘的纯化和重构，用于使用冷冻电镜进行结构研究、药物化学合成大量化合物以评估结构-活性关系、在计算机分子建模和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，我们正在采用一种新方法，用丙氨酸取代同源跨膜螺旋（TMH）中的多个保守残基来阐明 P-gp 的转运机制。此外，我们还投入大量精力筛选和开发酪氨酸激酶抑制剂（TKI）以及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的运输途径：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。冷冻电镜单粒子研究揭示了两种主要构象，一种是向内开放（IO）构象，其中 NBD 是分离的，并且药物结合腔易于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，即向内闭合 (IC)，其中 NBD 被二聚化（闭合），并且药物结合腔塌陷。 Orthovanadate 是无机磷酸盐的过渡态类似物，可抑制 P-gp 和其他 ABC 转运蛋白的 ATP 酶活性。我们测试了几种聚氧钒酸盐化合物对 P-gp 药物转运和 ATP 酶活性的影响。 有趣的是，我们发现十钒酸盐抑制 P-gp 的药物转运和 ATP 酶活性，其效力与原钒酸盐相同。分子对接研究表明十钒酸盐似乎没有与药物结合袋结合，这表明它很可能与 ATP 位点相互作用。此外，十钒酸盐与 P-gp 的结合是构象敏感的。我们计划利用十钒酸盐的这一特性来了解更多关于与催化循环相关的构象变化。 2. P-gp 介导的转运方向逆转：我们采用在同源跨膜螺旋中引入多个突变的新方法、生化和基于细胞的转运测定以及分子建模来研究 P-gp 的药物转运机制。 P-gp 的一种变体，称为 14A，在 TMH 6 和 TMH 12 中具有 14 个突变，位于药物结合袋的中央空腔，失去了输出大部分测试底物的能力，但获得了输入四种底物的能力，包括罗丹明 123 和 Flutax-1（一种紫杉醇衍生物）。通过生成几个替换 TMH 6 和 12 中残基的变体，我们发现控制运输方向的开关位于中心区域。一系列突变体的进一步表征表明，来自TMH 6的三个残基（V334、F336和F336）和来自TMH 12的六个残基（F978、S979、V981、V982、F983和M986）有助于四种底物的最大积累。有趣的是，残基 F978、S979、V981、V982 和 F983 对于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，我们生成了TM4,10-14A突变体，其中来自TMH 4的七个残基和来自TMH 10的七个残基被丙氨酸取代。该突变体P-gp不介导任何测试底物的积累，也未能流出所有测试底物。这些数据表明 TMH 4 和 10 中的残基对于 P-gp 的转运功能至关重要。 3. P-gp和ABCG2的光动力调节机制：我们已经开始阐明光动力疗法（PDT）介导的ABC药物转运蛋白调节的分子机制。 PDT 是一种基于光化学的工具，涉及光敏剂的光激活以产生活性氧。在临床上，PDT已被用于治疗光化性角化病、非小细胞肺癌、头颈癌等多种疾病。 ATP 酶活性和计算机分子对接分析表明，光敏剂苯并卟啉衍生物 (BPD) 在无光情况下以微摩尔半最大抑制浓度与 ABCB1 和 ABCG2 结合。 BPD 的光激活产生单线态氧，以光学剂量依赖性方式进一步降低 ABCB1 和 ABCG2 的 ATPase 活性高达 12 倍。凝胶电泳和蛋白质印迹表明，光激活的 BPD 通过共价交联诱导这些转运蛋白聚集。 因此，PDT 通过调节 ATP 酶活性和这些转运蛋白的蛋白质完整性来影响 ABCB1 和 ABCG2 的功能。从这项关于 ABC 药物转运蛋白光动力操纵的研究中获得的见解可能有助于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性。为了提高PDT治疗耐药癌症的效率，我们设计了一种光免疫缀合物制剂，结合疏水性BPD光敏剂和构象敏感的UIC2单克隆抗体来识别三阴性乳腺癌（TNBC）细胞上的P-gp表达。我们发现 UIC2-BPD 缀合物可用于荧光标记活 TNBC 细胞中的 P-gp，表明其可用于表达这种多药转运蛋白的肿瘤的荧光成像。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。 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：我们继续表征最近开发的 TKI，包括 SKLB610、ensartinib 和 OTS964、再利用药物、小分子（苯基呋喃香豆素衍生物和泛素激活酶抑制剂 TAK-243）以及天然产物的作用 P-gp 和 ABCG2 的功能。这些研究是与博士合作进行的。 Glaucio Valdameri（巴西巴拉那联邦大学）、Chung-Pu Wu（台湾长庚大学）、Zhe-Sheng Chen（纽约圣约翰大学）和 Shinobu Ohnuma（日本东北大学医学研究生院）。 A3腺苷受体激动剂已被开发用于治疗慢性疾病，如类风湿性关节炎、牛皮癣、慢性疼痛和肝细胞癌。之前我们证明了多种腺苷受体激动剂和拮抗剂可以调节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，从而调节药物在肠道的吸收。 We synthesized 63 compounds and tested them for their effect on the ATPase activity of ABCG2.其中，化合物 60（一种具有低腺苷受体亲和力的 7-脱氮杂-5'-酯衍生物）被鉴定为 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出*截断*