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: 10014333
• 负责人: SURESH AMBUDKAR
• 金额: $ 132.32万
• 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/10014333
• 关键词: ABCB1 gene; ABCG2 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Adenosine; Adenosine A3 Receptor; Affect; Affinity; Affinity Chromatography; Agonist; Anions; Antibodies; Antineoplastic Agents; Area; BODIPY; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biological Assay; Biological Availability; Biophysics; Bos taurus structural-GP protein; Cancer Patient; Candida; Carrier Proteins; Cell membrane; Cells; Chemicals; Chemotherapy-Oncologic Procedure; Chromatography; Chronic Disease; Clinic; Clinical; Collaborations; Complement; Computer Simulation; Cryoelectron Microscopy; Curcumin; Cyclosporine; Cystic Fibrosis Transmembrane Conductance Regulator; Derivation procedure; Detergents; Development; Docking; Drug Interactions; Drug Kinetics; Drug Transport; Environment; Exclusion; Exhibits; FDA approved; FLT3 inhibitor; Flow Cytometry; Fluorescence; Genetic Structures; Goals; Heating; High temperature of physical object; Human; Hydrophobicity; Immobilization; In Vitro; Insecta; Knowledge; Label; Libraries; Ligands; Link; Lipids; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Metals; Methods; Micelles; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Monoclonal Antibodies; Multi-Drug Resistance; Mus; Mutagenesis; Mutation; Names; National Heart, Lung, and Blood Institute; National Institute of Diabetes and Digestive and Kidney Diseases; Natural Products; Nickel; Nucleosides; Nucleotides; P-Glycoprotein; PDGFRB gene; Paclitaxel; Parents; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphorylcholine; Play; Primary carcinoma of the liver cells; Proteins; Psoriasis; Pump; Reporting; Resistance; Resolution; Rheumatoid Arthritis; Role; Site; Structure; Structure-Activity Relationship; Substrate Specificity; Suppressor Mutations; Temperature; Testing; Time; Transmembrane Domain; Tyrosine; Tyrosine Kinase Inhibitor; United States National Institutes of Health; Verapamil; Vesicle; Vinblastine; Work; Yeasts; analog; base; cancer type; chronic pain; clinical effect; design; dodecyl maltoside; imaging probe; in vivo; inhibitor/antagonist; insight; interdisciplinary approach; kinase inhibitor; member; molecular modeling; mutant; nanodisk; novel therapeutic intervention; particle; protein transport; proteoliposomes; reconstitution; screening; simulation; small molecule; three dimensional structure

We have designed a coordinated strategy using multidisciplinary approaches to understand the molecular basis of polyspecificity 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 their structure activity relationships, in silico molecular modeling and MD simulations 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: We previously have reported that the tyrosine rich 15Y P-gp mutant fails to transport large-size substrates. To understand how the 15Y mutant lost the ability to transport large size substrates, we decided first to make two mutants: one named 6Y with substitution of six residues in TMD1 (F72Y/F303Y/I306Y/F314Y/F336Y/L339Y) and another one termed 9Y with nine substitutions in TMD2 (F732Y/F759Y/F770Y/F938Y/F942Y/M949Y/L975Y/F983Y/F994Y). Out of six residues in TMD1 (6Y mutant), five are clustered in TMHs 5 and 6, whereas the nine residues in TMD2 (9Y mutant) are more widely dispersed throughout TMHs 7, 8, 11 and 12. Interestingly, the 6Y mutant partially transported BD-verapamil, but failed to transport all other tested substrates. On the other hand, the 9Y mutant transported all substrates, as did its parent 15Y mutant (including large-size substrates that are not transported by 15Y. This result indicated that certain 9Y residues were able to rescue the function of the 6Y mutant. To identify 9Y residues that can rescue the function of the 6Y mutant, we made two mutants in a 6Y background: one with 6Y plus three 9Y residues located in the upper leaflet (F732Y/F759Y/L975Y) and called it 6Y + ULY. The second mutant, termed 6Y + 3Y, contains 6Y plus three 9Y residues located in the lower leaflet of the membrane (F938Y/F942Y/F994). While the ULY mutant was able to transport eight out of ten substrates, the 6Y + 3Y mutant transported nine out of ten substrates. The 15Y, ULY and 6Y +3Y mutants failed to transport BD-vinblastine, showing that the mutation(s) in 6Y responsible for loss of BD-vinblastine transport are dominant mutations. The addition of ULY or 3Y substitutions from the 9Y mutant to WT P-gp did not have any effect on its expression or function. This is the first evidence of the presence of second site suppressor mutations in P-gp, although such mutations have been reported in other eukaryotic ABC transporters including CFTR (ABCC7), yeast PDR5 and Candida Cdr1. 2. The mechanism of the molecular basis of polyspecificity of P-g: (i) We tested the interaction of P-gp with various A3 adenosine receptor agonists that are being developed for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain and hepatocellular carcinoma. Although compounds 3 and 8 displayed pronounced effects on P-gp function, we found that a BODIPY-conjugate of compound 8 (compound 24) was not transported by P-gp. The residues in the drug-binding pocket critical for interactions with adenosine analogs compound 3 and 8 were identified by in silico molecular docking. Molecular docking studies revealed that both compounds 3 and 8 bind in the same region of the drug-binding pocket as paclitaxel (Taxol). Collectively, these results indicate that nucleoside derivatives can exhibit varied modulatory effects on P-gp activity, depending on structural functionalization. This work was done in collaboration with Kenneth Jacobson, NIDDK. (ii) In addition, to study the transport function of P-gp, we synthesized a Bodipy-labeled fluorescent conjugate of cyclosporine A (BD-CsA). After synthesis and characterization of its chemical purity, BD-CsA was compared with the commonly used 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-CsA probe. In flow cytometry assays, the fluorescence intensity of BD-CsA was almost 10 times higher than that of NBD-CsA, enabling us to use significantly lower concentrations of BD-CsA to achieve the same fluorescence levels. We found that BD-CsA is recognized as a transport substrate by both human and mouse P-gp. In silico docking of BD-CsA and NBD-CsA to the human P-gp structure indicates that they both bind in the drug-binding pocket with similar docking scores and possibly interact with similar residues. Thus, we demonstrate that BD-CsA is a sensitive fluorescent substrate of P-gp that can be used to efficiently study the transporter's localization and function in vitro and in vivo. (This work was done in collaboration with Drs. Rolf Swenson and Raju Natarajan, Imaging Probe Development Center, NHLBI, NIH). (iii) Development of a thermal inactivation method for understanding the drug-substrate and ATP-dependent stability of P-gp. We are focusing on the thermal inactivation of ATPase activity and how it is affected by nucleotides, transported substrates and modulators. We developed a very simple assay consisting of heating insect cell membrane vesicles expressing P-gp to different temperatures ranging from 37C to 70C for 10 min and assessing the P-gp ATPase activity afterwards at 37C. Interestingly, in the absence of ATP, when the NBDs are separated from each other, the protein is highly susceptible to inactivation at higher temperature. In contrast, in the presence of ATP (under non-hydrolyzing condition), when the two NBDs are close together in an inward-closed conformation, the thermal stability increases over 20C. 3. Resolution of the three-dimensional structure of human Pgp: For structural studies it is important to obtain a large amount of purified functional protein. We compared three detergents (1,2-diheptanoyol-sn-glycero-3-phosphocholine, dodecyl maltoside and n-octyl-beta-D-glucopyranoside) used for solubilization and purification of human and mouse P-gp from insect High-Five cell membranes. P-gp purification was performed first using immobilized metal affinity chromatography, then followed by a second step of either anion exchange chromatography or size exclusion chromatography to yield protein in concentrations of 10 to 12 mg per ml. Size exclusion chromatography was the preferred method, as it allows separation of monomeric transporters from aggregates. We showed that the purified protein, when reconstituted in proteoliposomes and nanodiscs, exhibits both basal and substrate or inhibitor-modulated ATPase activity. We are currently using nickel-NTA followed by a size exclusion column for purification of P-gp and nanodiscs prepared with this protein are being used for cryo-EM studies. 4. Development of non-toxic natural product and small molecule modulators to overcome resistance mediated by P-gp and ABCG2: We continue to characterize the recently developed tyrosine kinase inhibitors, repurposed drugs, small molecules, natural products and synthetic derivations of curcumin for their effect on the function of P-gp and ABCG2. Our goal is to characterize the effect of these clinically important modulators to help us to understand the polyspecificity of these transporters. We found that the FLT3 inhibitor midostaurin selectively modulated the function of human P-gp. Similarly, glesatinib, a c-MET/SMO dual inhibitor also modulated the function of P-gp. On the other hand, the KIT and PDGFR-alfa inhibitor, avapritinib inhibited the function of both P-gp and ABCG2. Solensertib, an ASK1 inhibitor, sensitized P-gp- and ABCG2-expressing cells to anticancer drugs.

我们使用多学科方法设计了一个协调策略，以了解多特异性的分子基础和 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、基于细胞的转运测定、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其结构活性关系、在计算机分子建模和MD模拟中扩展我们对ABC药物转运蛋白的机械方面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ATP水解的催化循环和P-gp的转运途径：我们之前报道过富含酪氨酸的15Y P-gp突变体无法转运大尺寸底物。为了了解15Y突变体如何失去运输大尺寸底物的能力，我们决定首先制作两种突变体：一种称为6Y，在TMD1中替换了六个残基（F72Y/F303Y/I306Y/F314Y/F336Y/L339Y），另一种称为9Y，在TMD2中替换了九个残基 （F732Y/F759Y/F770Y/F938Y/F942Y/M949Y/L975Y/F983Y/F994Y）。 TMD1（6Y 突变体）中的 6 个残基中，5 个聚集在 TMH 5 和 6 中，而 TMD2（9Y 突变体）中的 9 个残基更广泛地分散在 TMH 7、8、11 和 12 中。有趣的是，6Y 突变体部分转运 BD-维拉帕米，但未能转运所有其他测试底物。另一方面，9Y突变体转运所有底物，与其亲本15Y突变体一样（包括15Y不转运的大尺寸底物）。这一结果表明某些9Y残基能够挽救6Y突变体的功能。为了鉴定可以挽救6Y突变体功能的9Y残基，我们在6Y背景下制备了两个突变体：一个具有6Y加上位于上部小叶的三个9Y残基 （F732Y/F759Y/L975Y）并将其称为 6Y + ULY。第二个突变体称为 6Y + 3Y，包含位于膜下部小叶的 6Y 和三个 9Y 残基 (F938Y/F942Y/F994)。 ULY 突变体能够转运十分之八的底物，而 6Y + 3Y 突变体则转运十分之九的底物。 15Y、ULY和6Y+3Y突变体未能转运BD-长春碱，表明6Y中导致BD-长春碱转运丧失的突变是显性突变。将 9Y 突变体的 ULY 或 3Y 取代添加到 WT P-gp 对其表达或功能没有任何影响。这是 P-gp 中存在第二位点抑制突变的第一个证据，尽管此类突变已被证实 在其他真核 ABC 转运蛋白中已有报道，包括 CFTR (ABCC7)、酵母 PDR5 和念珠菌 Cdr1。 2. P-g 多特异性的分子基础机制： (i) 我们测试了 P-gp 与各种 A3 腺苷受体激动剂的相互作用，这些激动剂正在开发用于治疗慢性疾病，包括类风湿关节炎、牛皮癣、慢性疼痛和 肝细胞癌。尽管化合物 3 和 8 对 P-gp 功能显示出显着影响，但我们发现化合物 8（化合物 24）的 BODIPY 缀合物不被 P-gp 转运。通过计算机分子对接鉴定了药物结合袋中对于与腺苷类似物化合物 3 和 8 相互作用至关重要的残基。分子对接研究表明，化合物 3 和 8 与紫杉醇 (Taxol) 结合在药物结合袋的同一区域。总的来说，这些结果表明核苷衍生物可以对 P-gp 活性表现出不同的调节作用，具体取决于结构功能化。这项工作是与 NIDDK 的 Kenneth Jacobson 合作完成的。 (ii)此外，为了研究P-gp的转运功能，我们合成了Bodipy标记的荧光蛋白 环孢菌素 A (BD-CsA) 缀合物。经过合成和化学纯度表征后，BD-CsA 与常用的 7-硝基苯甲-2-氧杂-1,3-二唑-4-基 (NBD)-CsA 探针进行了比较。在流式细胞术检测中，BD-CsA 的荧光强度几乎比 NBD-CsA 高 10 倍，使我们能够 使用明显较低浓度的 BD-CsA 来达到相同的荧光水平。我们发现 BD-CsA 被人和小鼠 P-gp 识别为转运底物。 BD-CsA 和 NBD-CsA 与人 P-gp 结构的计算机对接表明，它们都以相似的对接分数结合在药物结合袋中，并且可能与相似的残基相互作用。因此，我们证明 BD-CsA 是一种 P-gp 的敏感荧光底物，可用于有效研究转运蛋白的体外和体内定位和功能。 （这项工作是与 NHLBI、NIH 成像探针开发中心的 Rolf Swenson 和 Raju Natarajan 博士合作完成的）。 (iii) 开发热灭活方法来了解 P-gp 的药物底物和 ATP 依赖性稳定性。 我们重点研究 ATP 酶活性的热失活以及它如何受到核苷酸、转运底物和调节剂的影响。我们开发了一种非常简单的测定方法，包括将表达 P-gp 的昆虫细胞膜囊泡加热到 37°C 至 70°C 的不同温度，持续 10 分钟，然后在 37°C 下评估 P-gp ATP 酶活性。有趣的是，在没有 ATP 的情况下，当 NBD 被 由于蛋白质彼此分离，因此在较高温度下极易失活。相比之下，在ATP存在下（非水解条件下），当两个NBD以内闭构象靠近在一起时，热稳定性增加超过20℃。 3. 人Pgp三维结构的解析：对于结构研究来说，获得大量纯化的功能蛋白非常重要。我们比较了三个 去垢剂（1,2-二庚酰基-sn-甘油-3-磷酸胆碱、十二烷基麦芽糖苷和正辛基-β-D-吡喃葡萄糖苷）用于从昆虫 High-5 细胞膜中溶解和纯化人和小鼠 P-gp。首先使用固定金属亲和层析进行 P-gp 纯化，然后进行第二步阴离子交换层析或大小 排阻色谱法产生浓度为 10 至 12 mg/ml 的蛋白质。尺寸排阻色谱法是优选的方法，因为它允许将单体转运蛋白与聚集体分离。我们表明，纯化的蛋白质在蛋白脂质体和纳米盘中重建时，表现出基础和底物或抑制剂调节的 ATP 酶活性。我们目前使用镍-NTA，然后使用尺寸排阻柱 P-gp 的纯化和用该蛋白质制备的纳米圆盘正用于冷冻电镜研究。 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酪氨酸激酶抑制剂、再利用药物、小分子、天然产物和姜黄素的合成衍生物对 P-gp 功能的影响 和ABCG2。我们的目标是表征这些临床上重要的调节剂的作用，以帮助我们了解这些转运蛋白的多特异性。我们发现FLT3抑制剂midostaurin选择性调节人P-gp的功能。类似地，c-MET/SMO双重抑制剂glesatinib也调节P-gp的功能。另一方面，KIT和PDGFR-α抑制剂， avapritinib 抑制 P-gp 和 ABCG2 的功能。 Solenertib 是一种 ASK1 抑制剂，可使表达 P-gp 和 ABCG2 的细胞对抗癌药物敏感。