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: 7732970
• 负责人: SURESH AMBUDKAR
• 金额: $ 115.14万
• 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/7732970
• 关键词: ABCA3 gene; ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Adrenoleukodystrophy; Affect; Affinity; Age related macular degeneration; Americas; Antineoplastic Agents; Binding; Binding Sites; Biochemical; Biological Assay; Biological Factors; Biological Models; Breast; Breast Cancer Cell; CCR; CD44 gene; Cancer cell line; Carrier Proteins; Cell Line; Cells; Cholesterol; Chronic Idiopathic Jaundice; Clinical; Collaborations; Colon; Condition; Crystallization; Cystic Fibrosis; Data; Defect; Detergents; Development; Dimerization; Disease; Dissociation; Dose; Doxorubicin; Drug Transport; Drug resistance; Energy-Generating Resources; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Epigenetic Process; Erlotinib; Exhibits; Exposure to; Generations; Genus Cola; Gleevec; Goals; Guanosine Triphosphate Phosphohydrolases; Haplotypes; Histones; Human; Human Genome; Hybridomas; Hydrolysis; Imatinib; Immune; Incubated; Kinetics; Length; Libraries; Link; MCF7 cell; Malignant Neoplasms; Malignant neoplasm of ovary; Mammary Neoplasms; Mediating; Messenger RNA; Miconazole; Microfluidics; Molecular; Molecular Conformation; Molecular Profiling; Molecular Target; Monoclonal Antibodies; Multi-Drug Resistance; Multidrug Resistance Associated Protein 1; Mus; Nucleotides; P-Glycoprotein; P-Glycoproteins; Parents; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Play; Population; Power stroke; Process; Program Development; Property; Protein Overexpression; Proteins; RNA Interference; Range; Reaction; Reagent; Regulation; Resistance; Resistance development; Resolution; Role; Sampling; Scheme; Screening procedure; Single Nucleotide Polymorphism; Site; Solutions; Specificity; Staging; Stargardt' s disease; Stem cells; Structure; Substrate Specificity; Tangier Disease; Technology; Testing; Therapeutic; Thermodynamics; Thiosemicarbazones; Treatment Protocols; Tyrosine Kinase Inhibitor; Up-Regulation; Validation; Vitamin K 3; Work; Xenobiotics; Yeasts; analog; base; cancer cell; cancer stem cell; cancer therapy; chemotherapeutic agent; chromatin immunoprecipitation; efflux pump; high throughput screening; human ABCG2 protein; improved; inhibitor/antagonist; innovation; killings; lapatinib; lipid transport; malignant breast neoplasm; member; mouse model; mutant; novel strategies; novel therapeutics; plumbagin; research study; response; small molecule; three dimensional structure; uridine triphosphatase; ABCA3 gene; ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Adrenoleukodystrophy; Affect; Affinity; Age related macular degeneration; Americas; Antineoplastic Agents; Binding; Binding Sites; Biochemical; Biological Assay; Biological Factors; Biological Models; Breast; Breast Cancer Cell; CCR; CD44 gene; Cancer cell line; Carrier Proteins; Cell Line; Cells; Cholesterol; Chronic Idiopathic Jaundice; Clinical; Collaborations; Colon; Condition; Crystallization; Cystic Fibrosis; Data; Defect; Detergents; Development; Dimerization; Disease; Dissociation; Dose; Doxorubicin; Drug Transport; Drug resistance; Energy-Generating Resources; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Epigenetic Process; Erlotinib; Exhibits; Exposure to; Generations; Genus Cola; Gleevec; Goals; Guanosine Triphosphate Phosphohydrolases; Haplotypes; Histones; Human; Human Genome; Hybridomas; Hydrolysis; Imatinib; Immune; Incubated; Kinetics; Length; Libraries; Link; MCF7 cell; Malignant Neoplasms; Malignant neoplasm of ovary; Mammary Neoplasms; Mediating; Messenger RNA; Miconazole; Microfluidics; Molecular; Molecular Conformation; Molecular Profiling; Molecular Target; Monoclonal Antibodies; Multi-Drug Resistance; Multidrug Resistance Associated Protein 1; Mus; Nucleotides; P-Glycoprotein; P-Glycoproteins; Parents; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Play; Population; Power stroke; Process; Program Development; Property; Protein Overexpression; Proteins; RNA Interference; Range; Reaction; Reagent; Regulation; Resistance; Resistance development; Resolution; Role; Sampling; Scheme; Screening procedure; Single Nucleotide Polymorphism; Site; Solutions; Specificity; Staging; Stargardt' s disease; Stem cells; Structure; Substrate Specificity; Tangier Disease; Technology; Testing; Therapeutic; Thermodynamics; Thiosemicarbazones; Treatment Protocols; Tyrosine Kinase Inhibitor; Up-Regulation; Validation; Vitamin K 3; Work; Xenobiotics; Yeasts; analog; base; cancer cell; cancer stem cell; cancer therapy; chemotherapeutic agent; chromatin immunoprecipitation; efflux pump; high throughput screening; human ABCG2 protein; improved; inhibitor/antagonist; innovation; killings; lapatinib; lipid transport; malignant breast neoplasm; member; mouse model; mutant; novel strategies; novel therapeutics; plumbagin; research study; response; small molecule; three dimensional structure; uridine triphosphatase

1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of Pgp and role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of Pgp. Based on the thermodynamic and kinetic properties, we have identified the ES and EP stable reaction intermediates of the Pgp-mediated ATPase reaction. Recently, we observed that the ES conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analog ATP-g-S. ATP-g-S, similar to ATP in E556Q/E1201Q mutant, is occluded into the wild-type Pgp NBDs at 34-37C but not at 4C. When Pgp is occluded with ATP-g-S, it exhibits reduced affinity for transport substrates. Collectively, these data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs and although, two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. It appears that in a full-length ABC transporter like Pgp, the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site. In collaboration with John Golin (The Catholic Univ. of America), we characterized the ATPase activity of the yeast Pdr5p transporter that effluxes a variety of xenobiotic compounds. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is known to be a potent transport substrate, however both GTPase and UTPase activities are relatively resistant to this drug. Our results indicate that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. We propose that Pdr5p increases its transport substrate specificity by using more than one nucleotide as an energy source. 2. Development of potent natural product and other non-toxic modulators/inhibitors of ABC transporters: To develop modulator(s) that will inhibit multiple transporters we screened synthetic compounds as well as natural products. The ABCG2 transporter confers resistance to multiple chemotherapeutic agents. One approach to combat MDR mediated by this transporter is the development of inhibitors/modulators that block its function at non-toxic concentrations. We found that napthoquinones, vitamin K3 and its structural analogue plumbagin are substrates of ABCG2. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, a thiosemicarbazone, NSC73306, which kills specifically Pgp expressing cells, was found to be a potent modulator of ABCG2. Thus, NSC73306 exhibits due mode of action that can be exploited to overcome drug resistance by eliminating Pgp-overexpressing cells and by acting as a potent modulator to resensitize ABCG2 expressing cancer cells to chemotherapeutics. In collaboration with Drs. Susan Bates, Curtis Henrich, Michael Dean and James McMahon (CCR and Molecular Targets Development Program, NCI) we have screened with a high-throughput assay a library of 7400 natural products and synthetic compounds to identify potent new inhibitors of ABCG2. We are also studying tyrosine kinase inhibitors for their potential use as inhibitors of ABC drug transporters. We found that the newly developed tyrosine kinase inhibitor AMN107 (nilotinib), and its parent drug imatinib (Gleevec) modulates activity of Pgp and ABCG2 by interacting at the drug-substrate binding sites instead of ATP sites as is the case with tyrosine kinase inhibitors. In collaboration with Dr. Zhe-Sheng Chen (St. Johns Univ.), we have demonstrated that Erlotinib (Tarceva), which is an EGFR tyrosine kinase inhibitor, reverses Pgp and ABCG2-mediated resistance by directly inhibiting the efflux function of these transporters. 3. Resolution of three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of Pgp is an ongoing project and for this we have developed a purification scheme that has yielded total protein 7.5-10 mg of more than 99% homogeneously pure Pgp at 10-12 mg/ml concentration. For improving the crystallization of Pgp, we have initiated another approach where in the Fab of the conformation-sensitive monoclonal antibody, UIC2 is incubated along with the purified Pgp during crystallization. We have optimized the conditions to generate Fab of UIC2 with protein concentration in the range of 5-7 mg/ml. Additional experiments demonstrate that Fab of UIC2 binds to Pgp in detergent solution under similar conditions that are used for generations of crystals indicating that it is feasible to generate co-crystals of Pgp and UIC2-Fab. At present we are in the process of generating 400-500 mg of UIC2 from the hybridoma cell line HB1287. This will allow us to prepare 150-200 mg of Fab for testing new crystallization conditions. 4. Molecular mechanism of drug resistance in single- and multi-step selection with anticancer agents in cancer cells: To understand the mechanism of multidrug resistance (MDR) under clinical conditions, we have begun to examine how treatment regimens affect the expression of ABC drug transporters in single- and multi-step selection with anticancer drugs such as doxorubicin. We established single-step doxorubicin-selected MCF-7 sublines using very low concentrations, 14 or 21 nM. We have found that ABCC2, ABCC4 and ABCG2 were overexpressed at the mRNA level in these single-step selected sublines. Yet, only ABCC4 and ABCG2 were overexpressed at the protein level. Both 14 and 21 nM single-step doxorubicin-selected sublines exhibit nearly 5-fold resistance to doxorubicin compared to parental MCF-7 cells. However, as ABCC4 does not confer resistance to doxorubicin it is most likely that ABCG2 is the major transporter responsible for the development of resistance. We also observed by using chromatin immunoprecipitation assay that the upregulation of ABCG2 is facilitated by histone hyperacetylation. Interestingly, stem cell markers including CD44 and CD24 are not enriched in single-step clones of breast cancer cell line MCF-7 selected with low concentration (21 nM) of doxorubicin. These results indicate that the MDR phenotype arises following lose-dose, single-step exposure to doxorubicin, and further suggest that overexpression of ABCG2 by epigenetic changes may mediate early stages of MDR development. In another related study in collaboration with Lyuba Varticovski (CCR, NCI), we sought to determine whether cancer stem cells occur in BRCA1-associated breast cancer and contribute to therapeutic response. We observed that Brca-1-deficient mouse mammary tumors harbor heterogeneous cancer stem cell populations, and CD44+/CD24- cells represent a population that correlates with human breast cancer stem cells. 5. Characterization of Single nucleotide polymorphisms and haplotypes in ABCB1: In collaboration with Dr. Michael Gottesman we showed that in MDR1, synonymous SNPs in the context of a haplotype, with two synonymous (3435C>T & 1236C>T) and one non-synonymous (2677G>T) SNP, were associated with altered substrate and inhibitor specificity. Further recent work in collaboration with Drs. Gottesman and Nussinov (SAIC, Frederick Inc., and CCR, NCI) suggests that long- [summary truncated at 7800 characters]

1。阐明PGP的ATP水解和运输途径的催化循环以及保守基序在ATP结合盒中的作用：我们正在继续研究PGP的催化循环和转运途径。基于热力学和动力学特性，我们已经确定了PGP介导的ATPase反应的ES和EP稳定反应中间体。最近，我们观察到，可以通过使用不可水解的ATP ATP ATP-G-S来获得PGP（以前在E556Q/E1201Q突变体中证明的ES构象）。 E556Q/E1201Q突变体中类似于ATP的ATP-G-S在34-37C中被遮住到野生型PGP NBDS中，但在4C中却不在4C中。当PGP被ATP-G-S遮挡时，它表现出对运输底物的亲和力降低。总的来说，这些数据为NBD的ATP驱动的二聚化和ADP驱动的解离提供了证据，尽管两个ATP分子可能启动二聚化，但只有一个被驱动到闭塞前的水解前中间状态。看来，在像PGP这样的全长ABC转运蛋白中，NBDS之一的遮挡核苷酸构象在传输底物位置提供了动力冲程。我们与约翰·戈林（John Golin）（美国天主教大学）合作，表征了酵母PDR5P转运蛋白的ATPase活性，该活性消失了多种异生元化合物。 PDR5P特异性ATPase活性表明，克洛司酰亚唑（已知是有效的运输底物）完全依赖浓度的抑制作用，但是GTPase和UTPase活性都对此药物具有相对抗性。我们的结果表明，这种抑制作用是非竞争的，并且是由于氯咪唑与转运蛋白在与ATP结合域不同的位点上的相互作用引起的。我们建议PDR5P通过使用多个核苷酸作为能源来增加其转运底物的特异性。 2。ABC转运蛋白的有效天然产物和其他无毒调节剂/抑制剂的开发：为了开发调节剂，为了抑制多个转运蛋白，我们筛选了合成化合物以及天然产物。 ABCG2转运蛋白赋予对多种化学治疗剂的耐药性。打击该转运蛋白介导的MDR的一种方法是开发抑制剂/调节剂，这些抑制剂/调节剂以无毒浓度阻断其功能。 我们发现Napthoquinones，Vitamin K3及其结构模拟铅质是ABCG2的底物。因此，ABCG2可能在体内维生素K3水平的调节中起作用。此外，发现杀死特异性PGP细胞的NSC73306是ABCG2的有效调节剂。因此，NSC73306表现出适当的作用方式，可以通过消除过表达PGP的细胞并充当有效的调节剂来使ABCG2对化学治疗药的表达ABCG2敏感，从而可以利用该模式来克服耐药性。与Drs合作。 Susan Bates，Curtis Henrich，Michael Dean和James McMahon（CCR和Molecular Targets Development计划，NCI）我们已经使用高通量测定法进行了一个7400天然产品和合成化合物的库，以识别ABCG2的有效新抑制剂。我们还正在研究酪氨酸激酶抑制剂，以潜在用作ABC药物转运蛋白的抑制剂。我们发现，新开发的酪氨酸激酶抑制剂AMN107（Nilotinib）及其母体药物伊马替尼（Gleevec）通过在药物覆盖的结合位点相互作用而不是与酪氨酸激酶抑制剂的情况下，通过在药物覆盖的结合位点上进行相互作用来调节PGP和ABCG2的活性。我们与Zhe-Sheng Chen博士（圣约翰大学）合作，我们证明了Erlotinib（Tarceva）是EGFR酪氨酸激酶抑制剂，可以通过直接抑制这些转运蛋白的外排功能来逆转PGP和ABCG2介导的耐药性。 3。人PGP的三维结构的分辨率：PGP的三维结构的分辨率是一个正在进行的项目，为此，我们开发了一种纯化方案，该方案在10-12 mg/mL浓度下产生了总蛋白7.5-10 mg，均质纯PGP超过99％。为了改善PGP的结晶，我们启动了另一种方法，其中在构象敏感的单克隆抗体中，UIC2在结晶过程中与纯化的PGP一起孵育。我们优化了以5-7 mg/ml范围内的蛋白质浓度生成UIC2 Fab的条件。 其他实验表明，UIC2的Fab在与几代晶体的相似条件下在洗涤剂溶液中与PGP结合，表明可以生成PGP和UIC2-FAB的共结晶是可行的。目前，我们正在从杂交瘤细胞系HB1287产生400-500 mg的UIC2。这将使我们能够准备150-200毫克的FAB来测试新的结晶条件。 4。在癌细胞中使用抗癌剂的单步选择中耐药性的分子机制：在临床条件下了解多药耐药性（MDR）的机制，我们已经开始研究治疗方案如何影响ABC药物转运蛋白在单步选择中使用抗癌药物在诸如doxorubiubin等抗癌药物中选择的ABC药物转运蛋白的表达。我们使用非常低的浓度（14或21 nm）建立了单步阿霉素选择的MCF-7 subline。我们发现，在这些单步选择的sublines中，ABCC2，ABCC4和ABCG2在mRNA水平上过表达。然而，在蛋白质水平上，仅ABCC4和ABCG2过表达。与亲本MCF-7细胞相比，14和21 nm单步阿霉素选择的subline对阿霉素的耐药性近5倍。但是，由于ABCC4不赋予阿霉素的抵抗力，因此ABCG2很可能是负责耐药性发展的主要转运蛋白。我们还通过使用染色质免疫沉淀测定法观察到，ABCG2的上调通过组蛋白高乙酰化促进。 有趣的是，包括CD44和CD24在内的干细胞标记物不富集于乳腺癌细胞系MCF-7的单步克隆中，选择了浓度低（21 nm）阿霉素。这些结果表明，MDR表型是在损失剂量，单步暴露于阿霉素后产生的，进一步表明，表观遗传变化对ABCG2的过表达可能介导MDR发育的早期阶段。在另一项与Lyuba Varticovski（CCR，NCI）合作的相关研究中，我们试图确定癌症干细胞是否发生在BRCA1相关的乳腺癌中，并有助于治疗反应。我们观察到BRCA-1缺陷型小鼠乳腺肿瘤含有异质性癌症干细胞群体，CD44+/CD24-细胞代表与人类乳腺癌干细胞相关的群体。 5。在ABCB1中的单核苷酸多态性和单倍型的表征：与迈克尔·戈特斯曼（Michael Gottesman）博士合作，我们表明，在MDR1中，在单型单型中的同义SNP中，具有两个同义词（3435c> t＆1236c> t），并且与一个非单词式（2677g> 2677g> t）相关联。与博士合作的进一步最近工作。 Gottesman and Nussinov（SAIC，Frederick Inc.和CCR，NCI）提出长期[摘要以7800个字符截断]