Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

多药耐药性相关转运蛋白的生化分析

基本信息

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
项目类别：
财政年份：
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&apos 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

项目摘要

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ATP水解的催化循环和Pgp的运输途径以及ATP结合盒中保守基序的作用：我们正在继续对Pgp的催化循环和运输途径的研究。基于热力学和动力学性质，我们鉴定了 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 Pgp 的 ES 构象（之前在 E556Q/E1201Q 突变体中得到证实）可以通过使用不可水解的 ATP 类似物 ATP-g-S 与野生型蛋白获得。 ATP-g-S 与 E556Q/E1201Q 突变体中的 ATP 类似，在 34-37°C 时被封闭到野生型 Pgp NBD 中，但在 4°C 时则不然。当 Pgp 被 ATP-g-S 封闭时，它对转运底物的亲和力降低。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 分子可能引发二聚化，但只有一个被驱动到封闭的预水解中间状态。看来，在像 Pgp 这样的全长 ABC 转运蛋白中，其中一个 NBD 处的封闭核苷酸构象在转运底物位点提供了动力冲程。我们与 John Golin（美国天主教大学）合作，表征了流出各种外源化合物的酵母 Pdr5p 转运蛋白的 ATP 酶活性。 Pdr5p 特异性 ATP 酶活性显示出克霉唑的完全浓度依赖性抑制，克霉唑是一种有效的转运底物，但 GTP 酶和 UTP 酶活性对该药物相对具有抵抗力。我们的结果表明，这种抑制是非竞争性的，是由克霉唑与转运蛋白在不同于 ATP 结合域的位点相互作用引起的。我们建议 Pdr5p 通过使用一种以上的核苷酸作为能源来提高其转运底物特异性。 2.开发ABC转运蛋白的有效天然产物和其他无毒调节剂/抑制剂：为了开发抑制多种转运蛋白的调节剂，我们筛选了合成化合物和天然产物。 ABCG2 转运蛋白赋予对多种化疗药物的耐药性。对抗这种转运蛋白介导的多药耐药的一种方法是开发抑制剂/调节剂，在无毒浓度下阻断其功能。我们发现萘醌、维生素 K3 及其结构类似物白花丹素是 ABCG2 的底物。因此，ABCG2 可能在体内维生素 K3 水平的调节中发挥作用。此外，缩氨基硫脲 NSC73306 可特异性杀死 Pgp 表达细胞，被发现是 ABCG2 的有效调节剂。因此，NSC73306 表现出适当的作用模式，可通过消除 Pgp 过度表达的细胞并作为有效的调节剂使表达 ABCG2 的癌细胞对化疗重新敏感来克服耐药性。与博士合作。 Susan Bates、Curtis Henrich、Michael Dean 和 James McMahon（CCR 和分子靶点开发项目，NCI）我们通过高通量分析筛选了 7400 种天然产物和合成化合物库，以鉴定 ABCG2 的有效新抑制剂。我们还在研究酪氨酸激酶抑制剂作为 ABC 药物转运蛋白抑制剂的潜在用途。我们发现新开发的酪氨酸激酶抑制剂 AMN107（尼洛替尼）及其母体药物伊马替尼（格列卫）通过药物-底物结合位点而不是酪氨酸激酶抑制剂的 ATP 位点相互作用来调节 Pgp 和 ABCG2 的活性。我们与陈哲生博士（圣约翰大学）合作，证明厄洛替尼（特罗凯）是一种 EGFR 酪氨酸激酶抑制剂，可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三维结构的解析：Pgp三维结构的解析是一个正在进行的项目，为此我们开发了一种纯化方案，已获得总蛋白7.5-10 mg，均匀性超过99%浓度为 10-12 mg/ml 的纯 Pgp。为了改善Pgp的结晶，我们启动了另一种方法，在构象敏感单克隆抗体的Fab中，UIC2在结晶过程中与纯化的Pgp一起孵育。我们优化了生成 UIC2 Fab 的条件，其蛋白质浓度在 5-7 mg/ml 范围内。其他实验表明，在用于晶体生成的类似条件下，UIC2 的 Fab 在洗涤剂溶液中与 Pgp 结合，表明生成 Pgp 和 UIC2-Fab 的共晶是可行的。目前，我们正在从杂交瘤细胞系 HB1287 中产生 400-500 mg UIC2。这将使我们能够制备 150-200 毫克的 Fab 来测试新的结晶条件。 4. 癌细胞单步和多步选择抗癌药物耐药的分子机制：为了了解临床条件下多药耐药（MDR）的机制，我们开始研究治疗方案如何影响ABC药物的表达转运蛋白与抗癌药物（如阿霉素）的单步和多步选择。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 亚系。我们发现ABCC2、ABCC4和ABCG2在这些单步选择的亚系中在mRNA水平上过表达。然而，只有 ABCC4 和 ABCG2 在蛋白质水平上过表达。与亲代 MCF-7 细胞相比，14 和 21 nM 单步阿霉素选择的亚系对阿霉素的耐药性几乎是亲本 MCF-7 细胞的 5 倍。然而，由于 ABCC4 不赋予阿霉素耐药性，因此 ABCG2 很可能是导致耐药性产生的主要转运蛋白。我们还通过使用染色质免疫沉淀测定观察到组蛋白过度乙酰化促进了 ABCG2 的上调。有趣的是，包括 CD44 和 CD24 在内的干细胞标记物在用低浓度 (21 nM) 多柔比星选择的乳腺癌细胞系 MCF-7 的一步克隆中并未富集。这些结果表明，MDR 表型是在低剂量、单步暴露于阿霉素后出现的，并进一步表明表观遗传变化引起的 ABCG2 过度表达可能介导 MDR 发展的早期阶段。在与 Lyuba Varticovski（CCR、NCI）合作的另一项相关研究中，我们试图确定癌症干细胞是否出现在 BRCA1 相关乳腺癌中并有助于治疗反应。我们观察到 Brca-1 缺陷的小鼠乳腺肿瘤具有异质性癌症干细胞群，而 CD44+/CD24- 细胞代表与人类乳腺癌干细胞相关的群体。 5. ABCB1 中单核苷酸多态性和单倍型的表征：与 Michael Gottesman 博士合作，我们表明在 MDR1 中，单倍型背景下的同义 SNP，具有两个同义（3435C>T 和 1236C>T）和一个非同义 SNP。同义 (2677G>T) SNP 与改变的底物和抑制剂特异性相关。最近与博士合作的进一步工作。 Gottesman 和 Nussinov（SAIC、Frederick Inc. 和 CCR、NCI）建议长-[摘要被截断为 7800 个字符]

项目成果

期刊论文数量（40）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx.

VAMP2 依赖性胞吐作用调节 TRPC3 通道的质膜插入，并有助于激动剂刺激的 Ca2 流入。

DOI：
发表时间：
2004-08-27
期刊：
影响因子：
16
作者：
Singh, Brij B;Lockwich, Timothy P;Bandyopadhyay, Bidhan C;Liu, Xibao;Bollimuntha, Sunitha;Brazer, So;Combs, Christian;Das, Sunit;Leenders, A G Miriam;Sheng, Zu;Knepper, Mark A;Ambudkar, Suresh V;Ambudkar, Indu S
通讯作者：
Ambudkar, Indu S

The role of hydrogen bond acceptor groups in the interaction of substrates with Pdr5p, a major yeast drug transporter.

氢键受体基团在底物与 Pdr5p（主要酵母药物转运蛋白）相互作用中的作用。

DOI：
发表时间：
2005-07-19
期刊：
影响因子：
2.9
作者：
Hanson, Leanne;May, Leopold;Tuma, Pamela;Keeven, James;Mehl, Patrick;Ferenz, Michelle;Ambudkar, Suresh V;Golin, John
通讯作者：
Golin, John

Imatinib mesylate and nilotinib (AMN107) exhibit high-affinity interaction with ABCG2 on primitive hematopoietic stem cells.

甲磺酸伊马替尼和尼罗替尼 (AMN107) 与原始造血干细胞上的 ABCG2 表现出高亲和力相互作用。

DOI：
发表时间：
2007-06
期刊：
影响因子：
0
作者：
Brendel, C;Scharenberg, C;Dohse, M;Robey, R W;Bates, S E;Shukla, S;Ambudkar, S V;Wang, Y;Wennemuth, G;Burchert, A;Boudriot, U;Neubauer, A
通讯作者：
Neubauer, A

Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein.

人类 P-糖蛋白催化循环的单周转期间需要在两个不同步骤中进行 ATP 水解的证据。

DOI：
10.1073/pnas.97.6.2515
发表时间：
2000-03-14
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Z. Sauna;S. Ambudkar
通讯作者：
S. Ambudkar

Functional characterization of Candida albicans ABC transporter Cdr1p.

白色念珠菌 ABC 转运蛋白 Cdr1p 的功能表征。