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: 7592629
• 负责人: SURESH AMBUDKAR
• 金额: $ 109.89万
• 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/7592629
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Adenine; Adjuvant; Affect; Affinity; Age related macular degeneration; Antihypertensive Agents; Antineoplastic Agents; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Availability; Biological Factors; Biological Models; Biological Process; Breast Cancer Cell; Carrier Proteins; Cells; Chronic Idiopathic Jaundice; Chronic-Phase Myeloid Leukemia; Class; Clinical; Collaborations; Complement component C1s; Condition; Curcumin; Cyclosporine; Cystic Fibrosis; Data; Detergents; Development; Developmental Therapeutics Program; Dihydropyridines; Disease; Doxorubicin; Drug Transport; Drug resistance; Exhibits; Gene Amplification; Gleevec; Goals; Haplotypes; Hematopoietic Stem Cell Transplantation; Hereditary Disease; Histones; Human; Human Genome; Hydrolysis; Imatinib; Immune; Immunosuppressive Agents; Joints; Kinetics; Link; MCF7 cell; Malignant Neoplasms; Mediating; Messenger RNA; Microfluidics; Molecular; Molecular Conformation; Molecular Profiling; Monoclonal Antibodies; Multi-Drug Resistance; Multidrug Resistance Associated Protein 1; Names; Nicardipine; Nifedipine; Nucleotides; Nutraceutical; Object Attachment; Oral; Organ; P-Glycoprotein; P-Glycoproteins; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Plants; Play; Porifera; Powder dose form; Preparation; Property; Protein Conformation; Protein Kinase Inhibitors; Protein Overexpression; Protein Tyrosine Kinase; Proteins; RNA Interference; Reaction; Reagent; Red Sea; Regulation; Resistance; Resistance development; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Scheme; Screening procedure; Single Nucleotide Polymorphism; Sirolimus; Site; Small Interfering RNA; Solid; Solutions; Source; Staging; Stargardt' s disease; Structure; Tacrolimus; Tangier Disease; Techniques; Technology; Thermodynamics; Treatment Protocols; Treatment Step; Tumeric; Tyrosine Kinase Inhibitor; Up-Regulation; Validation; Walker-A Motif; Walkers; Work; analog; base; bcr-abl Fusion Proteins; cancer cell; chemosensitizing agent; chemotherapeutic agent; chemotherapy; chromatin immunoprecipitation; cytotoxic; dihydropyridine; efflux pump; human ABCG2 protein; in vivo; inhibitor/antagonist; innovation; mRNA Expression; marine organism; member; mouse model; mutant; novel; novel strategies; novel therapeutics; protein kinase inhibitor; small molecule; small molecule libraries; tetrahydrocurcumin; three dimensional structure

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. Using this defined framework and the Walker B E556Q/E1201Q double mutant, we can precisely attribute the high-to-low affinity switch in the transport substrate binding site to the formation of the ES reaction intermediate. We have provided evidence that ATP-gamma-S, which is a non-hydrolyzable analog of ATP, can be used to generate the ES intermediate in wild-type protein. We have characterized a conserved subdomain 25 residues upstream of the Walker A motif of the ABC, which we named the A-loop (Aromatic residue interacting with the Adenine ring of ATP). In addition, we substituted the conserved H residue with Q, A, E, Y and K in the H-loop in both NBDs individually and together and these data suggest that the H residue in the H-loop most likely interacts with the gamma-P of ATP through H-bonding. 2. Development of potent natural product and other non-toxic modulators/inhibitors of ABC transporters: screening and validation in mouse model systems: To develop modulator(s) that will inhibit multiple transporters, we screened synthetic compounds as well as natural products. We found that curcumin isolated from turmeric powder, is a potent modulator of all three transporters. Interestingly, curcumin is not very cytotoxic nor is it transported by ABCB1, C1 or G2. Additionally, we showed that tetrahydrocurcumin, which is a major metabolite of curcumin, also inhibits the activity of ABCB1, ABCC1 and ABCG2, suggesting that this metabolite also has inhibitory potential under in vivo conditions. Thus, curcumin, which modulates the function of three major ABC drug transporters, appears to be a promising candidate for development as an effective chemosensitizer, or at least as a nutraceutical adjuvant to enhance chemotherapy. 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 1, 4-dihydropyridines, nicardipine and nifedipine, which are used clinically as antihypertensive agents, are inhibitors as well as substrates of ABCG2. In a collaborative study with Dr. Maria R. Baer, we showed that the immunosuppressive agents used in solid organ and hematopoietic stem cell transplantation such as cyclosporine A, tacrolimus and sirolimus modulate drug transport by ABCB1, ABCC1 and ABCG2. We are also studying tyrosine kinase inhibitors for their potential use as inhibitors of ABC drug transporters. The newly developed tyrosine kinase inhibitor AMN107 (nilotinib), which is an analog of imatinib (Gleevec) inhibits the tyrosine kinase activity of the BCR-ABL protein and is an effective, frontline therapy for chronic-phase CML. We have shown in collaboration with Brendel et al. that it is a high affinity inhibitor of ABCG2. Another class of protein kinase inhibitors includes indolocarbazole (ICZ) and bisindolylmalemide (BIM) compounds. During their clinical development, both ICZs and BIMs were demonstrated to interact with ABC transporters. We have demonstrated that these inhibitors block ABCG2-mediated drug resistance and thus may increase oral bioavailability of ABCG2 substrates. Compounds from the Developmental Therapeutics Programs (DTP, NCI) chemical libraries have been analyzed for the development of inhibitors. Based on structural similarity hits, several compounds were projected to be potential inhibitors, and our group is involved in a joint effort to screen the potential inhibitors of ABC transporters. In addition to plants, marine organisms also provide a rich source of compounds from which novel agents can be developed to overcome multidrug resistance. In collaboration with Dr. Zhe-Sheng Chen, we have characterized the modulatory effect of sipholenol A, which is a sipholane triterpenoid isolated from the Red Sea sponge. Sipholenol A selectively overcomes the resistance to anticancer drugs mediated by Pgp (ABCB1). 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 7.5-10.0 mg of > 99% homogeneously pure Pgp at 10-12 mg/ml concentration. We have observed that homogeneously pure Pgp at higher concentration stays in solution only in the presence of NaCl, and detergents such as DHPC, DDM. In the absence of salt, Pgp either aggregates or precipitates even in the presence of higher concentrations of DHPC or DDM detergents. This most recent preparation of homogeneously pure Pgp protein has retained the biological function of ATPase activity. In addition to wild-type protein, several mutants including the E556Q/E1201Q double mutant, which is trapped in an ES pre-hydrolysis transition-like state in the presence of ATP, have also been purified. Similarly, the stabilization of nucleotide-binding domains in the ES pre-hydrolysis transition state should help to generate crystals. 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 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 by employing RT-PCR, siRNA and chromatin immunoprecipitation (ChIP) in addition to regularly used biochemical and cell biological techniques. We have found that ABC transporter mRNA expression patterns vary with single- vs. multi-step treatment with doxorubicin in MCF-7 breast cancer cells. We have shown that multi-step selected MCF-7 cells overexpress only ABCB1 at the mRNA and protein levels and that in addition to gene amplification. We also 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 (ChIP) assay that the upregulation of ABCG2 is facilitated by histone hyperacetylation. 5. Characterization of Single nucleotide polymorphisms and haplotypes in ABCB1: In collaboration with Drs. Michael Gottesman and Kimchi-Sarfaty we have recently highlighted the importance of synonymous SNPs in determining protein conformation and function. Our study 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 in [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。使用此定义的框架和Walker B E556Q/E1201Q双重突变体，我们可以精确地将运输底物结合位点中的高低亲和力开关归因于ES反应中间体的形成。我们提供的证据表明，ATP-GAMMA-S是ATP的不可溶解的类似物，可用于在野生型蛋白质中产生ES中间体。我们已经表征了助行器上游的一个保守子域25个残基是ABC的图案，我们将其命名为A-Loop（与ATP的腺嘌呤环相互作用的芳族残基）。此外，我们在NBD的H-LOOP中分别和一起将保守的H残基用Q，A，E，Y和K代替，这些数据表明，H环中的H残基很可能通过H键与ATP的Gamma-P相互作用。 2。开发有效的天然产品和其他无毒调节剂/ABC转运蛋白的抑制剂：小鼠模型系统中的筛选和验证：开发将抑制多个转运蛋白的调节剂，我们筛选了合成化合物以及天然产物。我们发现从姜黄粉中分离出的姜黄素是所有三个转运蛋白的有效调节剂。 有趣的是，姜黄素不是很细胞毒性，也不是由ABCB1，C1或G2运输的。 此外，我们表明四氢瓜蛋白是姜黄素的主要代谢产物，也抑制了ABCB1，ABCC1和ABCG2的活性，这表明该代谢产物在体内条件下也具有抑制潜力。因此，调节三个主要ABC药物转运蛋白的功能的姜黄素似乎是有效发展作为有效化学敏化剂的有前途的候选者，或者至少是营养辅助剂以增强化学疗法。 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，临床上用作抗高血压剂的1、4-二氢吡啶，尼古丁和硝苯地平是抑制剂以及ABCG2的底物。在与玛丽亚·R·巴尔（Maria R. Baer）博士的一项合作研究中，我们表明，用于固体器官和造血干细胞移植的免疫抑制剂，例如环孢素A，他克莫司和西洛略木斯通过ABCB1，ABCC1，ABCC1和ABCG2调节药物转运。 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。新开发的酪氨酸激酶抑制剂AMN107（Nilotinib）是伊马替尼（Gleevec）的类似物（Gleevec）抑制了BCR-ABL蛋白的酪氨酸激酶活性，并且是一种有效的前线治疗，用于慢性相CML。我们已经与Brendel等人合作展示了。它是ABCG2的高亲和力抑制剂。另一种类型的蛋白激酶抑制剂包括吲哚核巴唑（ICZ）和双丁丁甲乳甲酰胺（BIM）化合物。 在其临床发育过程中，ICZ和BIMS均证明与ABC转运蛋白相互作用。我们已经证明，这些抑制剂阻断了ABCG2介导的耐药性，因此可能会增加ABCG2底物的口服生物利用度。已经对开发治疗计划（DTP，NCI）化学库的化合物进行了分析，以开发抑制剂。基于结构相似性，预计几种化合物是潜在的抑制剂，我们的小组参与了筛选ABC转运蛋白的潜在抑制剂的共同努力。除植物外，海洋生物还提供了丰富的化合物来源，可以从中开发出新的药物来克服多药耐药性。我们与Zhe-Sheng Chen博士合作，表征了西哌啶酚A的调节作用，Sipholenol A是一种从红海海绵中分离出来的Sipholane三萜。 Sipholenol A有选择地克服了对PGP介导的抗癌药物的抗性（ABCB1）。 3.人类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7.5-10.0 mg，同一件纯PGP均质> 99％。我们已经观察到，在NaCl存在下仅在溶液中均匀地纯PGP和DHPC，DDM等洗涤剂。在没有盐的情况下，即使存在较高浓度的DHPC或DDM洗涤剂，PGP即使存在聚集体或沉淀。最近的纯PGP蛋白的最新制备保留了ATPase活性的生物学功能。除野生型蛋白外，还纯化了几种在ATP存在下被困在ES中的液化溶解过渡状态的包括E556Q/E1201Q双突变体在内的几种突变体。同样，在Hydrolyssy跃迁态中，核苷酸结合结构域的稳定应有助于产生晶体。 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：为了了解临床条件下的多药耐药性的机制，我们开始研究治疗方案如何通过使用毒药和sirna inip rt-pcr（siraptrapt）在单一和多步中选择的ABC药物转运蛋白的表达（通过使用sirna rt-rt-rt-rt-rt-rt-rt-rt-prat）（定期使用的生化和细胞生物学技术。我们发现，在MCF-7乳腺癌细胞中，ABC转运蛋白mRNA表达模式因单步与多步骤处理而变化。我们已经表明，多步选择了在mRNA和蛋白水平上仅过表达ABCB1的MCF-7细胞，除了基因扩增外。我们还使用非常低的浓度（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（CHIP）测定法观察到，ABCG2的上调通过组蛋白高乙酰化促进。 5。ABCB1中的单核苷酸多态性和单倍型的表征：与DRS合作。迈克尔·戈特斯曼（Michael Gottesman）和泡菜 - 萨法蒂（Kimchi-Sarfaty）我们最近强调了同义SNP在确定蛋白质构象和功能方面的重要性。我们的研究表明，在MDR1中，在单倍型中的同义SNP中，具有两个同义词（3435c> t＆1236c> t）和一个非同义词（2677G> t）SNP，与底物的变化和[以7800个字符的摘要中的摘要有关