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。使用这个定义的框架和 Walker B E556Q/E1201Q 双突变体，我们可以精确地将转运底物结合位点中的高到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（ATP 的不可水解类似物）可用于生成野生型蛋白质中的 ES 中间体。我们在 ABC 的 Walker A 基序上游 25 个残基处表征了一个保守的子结构域，我们将其命名为 A 环（与 ATP 腺嘌呤环相互作用的芳香残基）。此外，我们分别和一起用 Q、A、E、Y 和 K 取代了两个 NBD 中 H 环中的保守 H 残基，这些数据表明 H 环中的 H 残基最有可能与 γ- 相互作用。 ATP 通过 H 键合形成 P。 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：在小鼠模型系统中的筛选和验证：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。我们发现从姜黄粉中分离出的姜黄素是所有三种转运蛋白的有效调节剂。 有趣的是，姜黄素的细胞毒性不大，也不由 ABCB1、C1 或 G2 转运。 此外，我们发现四氢姜黄素（姜黄素的主要代谢物）也抑制 ABCB1、ABCC1 和 ABCG2 的活性，表明该代谢物在体内条件下也具有抑制潜力。因此，调节三种主要 ABC 药物转运蛋白功能的姜黄素似乎是开发作为有效化学增敏剂或至少作为增强化疗的营养佐剂的有希望的候选者。 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。我们发现临床上用作抗高血压药物的1, 4-二氢吡啶类、尼卡地平和硝苯地平是ABCG2的抑制剂和底物。在与 Maria R. Baer 博士的合作研究中，我们发现实体器官和造血干细胞移植中使用的免疫抑制剂（如环孢素 A、他克莫司和西罗莫司）通过 ABCB1、ABCC1 和 ABCG2 调节药物转运。 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。新开发的酪氨酸激酶抑制剂 AMN107（尼洛替尼）是伊马替尼（格列卫）的类似物，可抑制 BCR-ABL 蛋白的酪氨酸激酶活性，是治疗慢性期 CML 的有效一线疗法。我们与 Brendel 等人合作展示了这一点。它是 ABCG2 的高亲和力抑制剂。另一类蛋白激酶抑制剂包括吲哚并咔唑(ICZ)和双吲哚基马来酰亚胺(BIM)化合物。 在临床开发过程中，ICZ 和 BIM 均被证明可以与 ABC 转运蛋白相互作用。我们已经证明这些抑制剂可以阻断 ABCG2 介导的耐药性，从而可能增加 ABCG2 底物的口服生物利用度。开发治疗计划（DTP、NCI）化学库中的化合物已被分析用于抑制剂的开发。基于结构相似性命中，几种化合物被预测为潜在的抑制剂，我们的小组正在共同努力筛选 ABC 转运蛋白的潜在抑制剂。除了植物之外，海洋生物还提供了丰富的化合物来源，可以从中开发新的药物来克服多药耐药性。我们与陈哲生博士合作，表征了 sipholenol A 的调节作用，它是从红海海绵中分离出来的一种 sipholane 三萜类化合物。 Sipholenol A 选择性地克服 Pgp (ABCB1) 介导的抗癌药物耐药性。 3. 人类 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，已产生总蛋白 7.5-10.0 mg，纯度 > 99% Pgp 浓度为 10-12 mg/ml。我们观察到，只有在 NaCl 和 DHPC、DDM 等洗涤剂存在的情况下，较高浓度的均质纯 Pgp 才能保留在溶液中。在没有盐的情况下，即使存在较高浓度的 DHPC 或 DDM 洗涤剂，Pgp 也会聚集或沉淀。这种最新制备的均质纯 Pgp 蛋白保留了 ATP 酶活性的生物学功能。除了野生型蛋白外，还纯化了几种突变体，包括 E556Q/E1201Q 双突变体，该突变体在 ATP 存在下陷入 ES 预水解过渡状态。类似地，ES 预水解过渡态中核苷酸结合域的稳定应该有助于生成晶体。 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的多药耐药机制，我们开始研究治疗方案如何影响单步和多步选择中ABC药物转运蛋白的表达。 - 除了常规使用的生化和细胞生物学技术外，还通过采用 RT-PCR、siRNA 和染色质免疫沉淀 (ChIP) 对阿霉素等抗癌药物进行多步选择。我们发现，在 MCF-7 乳腺癌细胞中，ABC 转运蛋白 mRNA 表达模式随着阿霉素单步处理与多步处理的不同而变化。我们已经证明，多步选择的 MCF-7 细胞除了基因扩增之外，仅在 mRNA 和蛋白质水平过表达 ABCB1。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 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。与亲本 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 (ChIP) 测定观察到组蛋白过度乙酰化促进了 ABCG2 的上调。 5. ABCB1 中单核苷酸多态性和单倍型的表征：与 Drs 合作。 Michael Gottesman 和 Kimchi-Sarfaty 最近强调了同义 SNP 在确定蛋白质构象和功能中的重要性。我们的研究表明，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 (2677G>T) SNP，与改变的底物相关，并且在[摘要截断] 7800 个字符]