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: 10262054
• 负责人: SURESH AMBUDKAR
• 金额: $ 148.47万
• 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/10262054
• 关键词: ABCB1 gene; ABCG2 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Adenosine A3 Receptor; Affect; Affinity; Affinity Chromatography; Agonist; Alanine; Anions; Antibodies; Antineoplastic Agents; Area; Atomic Force Microscopy; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Availability; Biophysics; Cancer Patient; Candida; Carrier Proteins; Cell membrane; Cells; Chemicals; Chemotherapy-Oncologic Procedure; Chromatography; Chronic Disease; Clinic; Clinical; Collaborations; Complement; Cryoelectron Microscopy; Curcumin; Cystic Fibrosis Transmembrane Conductance Regulator; Derivation procedure; Detergents; Development; Dimerization; Docking; Drug Interactions; Drug Kinetics; Drug Transport; Environment; Exclusion; Exhibits; FDA approved; Genetic Structures; Goals; Histone Deacetylase Inhibitor; Human; Hydrophobicity; Immobilization; Insecta; Knowledge; Lead; 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; National Institute of Diabetes and Digestive and Kidney Diseases; Natural Products; Nickel; Nucleotides; P-Glycoprotein; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphorylcholine; Play; Primary carcinoma of the liver cells; Proteins; Psoriatic Arthritis; Pump; Purinergic P1 Receptors; Reporting; Resistance; Resolution; Rheumatoid Arthritis; Role; Site; Structure; Structure-Activity Relationship; Study models; Substrate Specificity; Suppressor Mutations; Taiwan; Temperature; Testing; Transmembrane Domain; Tyrosine; Tyrosine Kinase Inhibitor; United States National Institutes of Health; Universities; Variant; Verapamil; Work; Yeasts; analog; base; c-myc Genes; cancer type; chronic pain; clinical effect; design; dodecyl maltoside; gain of function; in silico; inhibitor/antagonist; insight; interdisciplinary approach; kinase inhibitor; mTOR Inhibitor; member; molecular modeling; mutant; nanodisk; novel strategies; novel therapeutic intervention; particle; phosphoric diester hydrolase; prevent; proteoliposomes; reconstitution; screening; simulation; small molecule; structural glycoprotein; thermostability; 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 are employing a novel approach of substituting multiple conserved residues with alanine in homologous transmembrane helices six and twelve 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 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 characterize the gain of function second-site suppressor mutations. We previously found that the 6Y mutant with substitution of six residues in TMD1 (F72Y/F303Y/I306Y/F314Y/F336Y/L339Y) partially transported BD-verapamil but failed to transport all other tested substrates. The atomic structure of P-gp in the ATP-bound conformation showed that intracellular helix four interacts with the Q-loop of NBD1. To test whether the substitution of the conserved F916 residue in intracellular helix four with tyrosine can rescue the function of the 6Y mutant, we added this residue to 6Y to produce the 6Y + F916Y mutant. To our surprise, we found that F916Y indeed rescues the function of the 6Y mutant. Consistent with the interaction between F916Y of intracellular helix four and F480Y from the Q-loop of NBD1, we found that addition of F480Y to the 6Y mutant also rescued the transport function of 6Y at least for selected substrates. We also added the residues Q535Y or Q1180Y from the signature region of NBD1 and NBD2 to 6Y to check whether these residues also rescue the function of the 6Y variant. However, the residues from the signature region of the NBDs did not have any effect on the function of the 6Y mutant. These results with second-site suppressor mutations demonstrate the connection between the drug-binding pocket and NBD1 of human P-gp. This is the first evidence of the presence of gain-of-function 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) Development of a thermal inactivation method for understanding the drug-substrate and ATP-dependent stability of P-gp. We observed that the thermostability of P-gp depends on the conformation of its NBDs. The effect of temperature on P-gp at the protein level can also be followed by quantifying the disappearance of the monomeric band of the transporter by SDS-PAGE. We used this approach to study the thermostability of P-gp mutants, which bind ATP to both NBDs (E556Q/E1201Q) or only to NBD2 (Y401A), but are unable to carry out ATP hydrolysis, to provide further support for the role of ATP-induced NBD dimerization during thermo-stabilization of P-gp. The inward-closed conformation that results from ATP-binding to both NBDs and their dimerization has remarkably higher thermostability when compared with the inward-open conformation in the absence of nucleotides. We also find significant differences in the thermostability of P-gp in the presence of transport substrates that stimulate ATP hydrolysis vs. inhibitors of both transport and ATPase activity. The inhibitors prevent the ATP-dependent dimerization of the NBDs, which is required for ATP hydrolysis, by stabilizing the inward-open conformation. These findings suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing ATP-dependent dimerization of the NBDs of P-gp. Currently, we are testing 156 FDA approved cancer drugs for their effect on the thermal stabilization of P-gp and ABCG2. This will help us to identify potent inhibitors of P-gp and ABCG2. (ii) Various A3 adenosine receptor agonists are being developed by our collaborator Dr. Kenneth Jacobson (NIDDK, NIH) for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain and hepatocellular carcinoma. We tested more than forty A3 adenosine receptor ligands and found that these ligands can have different modulatory effects on P-gp and ABCG2 activity, depending on structural functionalization. Although MRS 7343 (lead compound) inhibits the transport function of ABCG2, this adenosine receptor ligand showed no interaction with P-gp. Molecular modeling studies revealed that selected A3 adenosine receptor analogs interact with residues in the drug-binding pockets of both P-gp and ABCG2. Both P-gp and ABCG2 appear to affect the pharmacokinetics of A3 adenosine receptor ligands. MRS 7343 appears to be a good template to develop high affinity modulators for P-gp and ABCG2. 3. Resolution of the three-dimensional structure of human P-gp: 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 High-Five insect 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 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 selective class IIa histone deacetylase inhibitor TMP195 is a modulator of both P-gp and ABCG2, whereas the phosphodiesterase type 5 inhibitor MY-5445, the TKI inhibitor sitravatinib, the c-myc inhibitor tivatinib and licochalcone A modulate only the function of ABCG2. In addition, the PI3K/mTOR inhibitor samotolisib is a substrate for both P-gp and ABCG2. These studies were carried out in collaboration with Drs. Chung-Pu Wu (Chang Gung University, Taiwan) and Zhe-Sheng (Jason) Chen (St. John's University, NY).

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物化学和生物物理测定、基于细胞的运输测定、脂质纳米盘的纯化和重构，以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，在计算机分子建模中和 MD 模拟，以扩展我们对 ABC 药物转运蛋白的机制和结构功能关系的理解。此外，我们采用了一种新方法，用丙氨酸取代同源跨膜螺旋 6 和 12 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：我们继续表征第二位点抑制突变的功能获得。我们之前发现，TMD1 中六个残基被取代的 6Y 突变体（F72Y/F303Y/I306Y/F314Y/F336Y/L339Y）部分转运 BD-维拉帕米，但未能转运所有其他测试底物。 ATP 结合构象中 P-gp 的原子结构表明，细胞内螺旋四与 NBD1 的 Q 环相互作用。为了测试用酪氨酸取代细胞内螺旋四中保守的F916残基是否可以挽救6Y突变体的功能，我们将该残基添加到6Y中以产生6Y + F916Y突变体。令我们惊讶的是，我们发现F916Y确实挽救了6Y突变体的功能。与细胞内螺旋四的 F916Y 和 NBD1 Q 环的 F480Y 之间的相互作用一致，我们发现向 6Y 突变体添加 F480Y 至少对于选定的底物也挽救了 6Y 的转运功能。我们还将NBD1和NBD2的特征区域中的残基Q535Y或Q1180Y添加到6Y中，以检查这些残基是否也挽救了6Y变体的功能。然而，NBD 特征区域的残基对 6Y 突变体的功能没有任何影响。这些第二位点抑制突变的结果证明了药物结合袋和人 P-gp 的 NBD1 之间的联系。这是 P-gp 中存在功能获得性第二位点抑制突变的第一个证据，尽管此类突变已在其他真核 ABC 转运蛋白中报道，包括 CFTR (ABCC7)、酵母 PDR5 和念珠菌 Cdr1。 2. P-gp 多特异性的分子基础机制： (i) 开发热灭活方法以了解 P-gp 的药物底物和 ATP 依赖性稳定性。我们观察到 P-gp 的热稳定性取决于其 NBD 的构象。温度对蛋白质水平上的 P-gp 的影响也可以通过 SDS-PAGE 量化转运蛋白单体带的消失来跟踪。我们利用这种方法研究了P-gp突变体的热稳定性，该突变体将ATP与NBD（E556Q/E1201Q）或仅与NBD2（Y401A）结合，但无法进行ATP水解，为P-gp突变体的作用提供进一步支持。 P-gp 热稳定过程中 ATP 诱导的 NBD 二聚化。与不存在核苷酸的情况下的向内开放构象相比，由 ATP 与 NBD 结合及其二聚化产生的向内闭合构象具有显着更高的热稳定性。我们还发现，在刺激 ATP 水解的转运底物存在下，P-gp 的热稳定性与转运和 ATP 酶活性抑制剂存在显着差异。抑制剂通过稳定向内开放的构象来防止 NBD 的 ATP 依赖性二聚化，这是 ATP 水解所必需的。这些发现表明，结合在 TM 区域的调节剂通过阻止 P-gp NBD 的 ATP 依赖性二聚化来抑制 ATP 水解和药物转运。目前，我们正在测试 156 种 FDA 批准的癌症药物对 P-gp 和 ABCG2 热稳定性的影响。这将帮助我们鉴定 P-gp 和 ABCG2 的有效抑制剂。 (ii) 我们的合作者 Kenneth Jacobson 博士（NIDDK、NIH）正在开发各种 A3 腺苷受体激动剂，用于治疗慢性疾病，包括类风湿性关节炎、牛皮癣、慢性疼痛和肝细胞癌。我们测试了四十多种 A3 腺苷受体配体，发现这些配体可以根据结构功能化对 P-gp 和 ABCG2 活性产生不同的调节作用。尽管 MRS 7343（先导化合物）抑制 ABCG2 的转运功能，但该腺苷受体配体未显示与 P-gp 的相互作用。分子模型研究表明，选定的 A3 腺苷受体类似物与 P-gp 和 ABCG2 药物结合袋中的残基相互作用。 P-gp 和 ABCG2 似乎都会影响 A3 腺苷受体配体的药代动力学。 MRS 7343 似乎是开发 P-gp 和 ABCG2 高亲和力调节剂的良好模板。 3. 人P-gp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。我们比较了三种用于从 High-5 昆虫细胞中溶解和纯化人和小鼠 P-gp 的去污剂（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）膜。首先使用固定金属亲和层析进行 P-gp 纯化，然后进行阴离子交换层析或尺寸排阻层析的第二步，以产生浓度为 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来纯化 P-gp，并且用这种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、天然产物和姜黄素合成衍生物的作用P-gp 和 ABCG2 的功能。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。我们发现选择性IIa类组蛋白脱乙酰酶抑制剂TMP195是P-gp和ABCG2的调节剂，而5型磷酸二酯酶抑制剂MY-5445、TKI抑制剂sitravatinib、c-myc抑制剂tivatinib和甘草查尔酮A仅调节功能ABCG2 的。此外，PI3K/mTOR 抑制剂 samotolisib 是 P-gp 和 ABCG2 的底物。这些研究是与博士合作进行的。 Chung-Pu Wu（台湾长庚大学）和 Zhe-Sheng (Jason) Chen（纽约圣约翰大学）。