Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

1. 阐明ATP水解的催化循环和Pgp转运途径以及保守基序在ATP结合盒中的作用：我们正在继续对Pgp的催化循环和转运途径进行研究。基于热力学和动力学性质，我们确定了pgp介导的atp酶反应的ES和EP稳定反应中间体。利用这一定义框架和Walker B E556Q/E1201Q双突变体，我们可以精确地将转运底物结合位点的高低亲和性切换归因于ES反应中间体的形成。我们提供的证据表明，ATP- γ - s是一种ATP的不可水解类似物，可以用来在野生型蛋白中产生ES中间体。我们在ABC的Walker a基序上游发现了一个保守的亚结构域25个残基，我们将其命名为a环（与ATP的腺嘌呤环相互作用的芳香残基）。此外，我们分别用两个nbd中H环中的Q、A、E、Y和K代替了保守的H残基，这些数据表明H环中的H残基很可能通过H键与ATP的γ - p相互作用。2. 开发有效的天然产物和其他无毒的ABC转运蛋白调节剂/抑制剂：在小鼠模型系统中的筛选和验证：为了开发能够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三种转运蛋白的有效调节剂。有趣的是，姜黄素没有很强的细胞毒性，也不是由ABCB1、C1或G2运输的。此外，我们发现姜黄素的主要代谢物四氢姜黄素也能抑制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，姜黄素调节三种主要ABC药物转运体的功能，似乎是一个很有前途的候选物，作为一种有效的化疗增敏剂，或至少作为一种营养辅助剂来增强化疗。ABCG2转运体对多种化疗药物具有耐药性。对抗这种转运体介导的耐多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降压药的1,4 -二氢吡啶、尼卡地平和硝苯地平是ABCG2的抑制剂和底物。在与Maria R. Baer博士的一项合作研究中，我们发现用于实体器官和造血干细胞移植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。我们也在研究酪氨酸激酶抑制剂作为ABC药物转运体抑制剂的潜在用途。新开发的酪氨酸激酶抑制剂AMN107 （nilotinib）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，是一种有效的慢性粒细胞白血病（CML）的一线治疗药物。我们与Brendel等人合作表明，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咔唑（ICZ）和双吲哚聚马来酰胺（BIM）化合物。在临床开发过程中，icz和BIMs都被证明与ABC转运蛋白相互作用。我们已经证明这些抑制剂阻断ABCG2介导的耐药性，从而可能增加ABCG2底物的口服生物利用度。从发育治疗计划（DTP， NCI）化学文库中提取的化合物已被分析用于开发抑制剂。基于结构相似点，一些化合物被预测为潜在的抑制剂，我们的团队正在共同努力筛选ABC转运蛋白的潜在抑制剂。除了植物，海洋生物也提供了丰富的化合物来源，从中可以开发出新的药物来克服多药耐药性。与陈哲生博士合作，我们表征了虹吸酚A的调节作用，这是一种从红海海绵中分离出来的虹吸烷三萜。Sipholenol A选择性克服Pgp （ABCB1）介导的抗癌药物耐药。3. 人类Pgp三维结构的分辨率：Pgp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，以10-12 mg/ml的浓度获得了总蛋白7.5-10.0 mg > 99%均质纯度的Pgp。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才能留在溶液中。在没有盐的情况下，即使存在高浓度的DHPC或DDM洗涤剂，Pgp也会聚集或沉淀。最近制备的均质纯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，这些突变体在ATP存在下处于ES预水解过渡态。同样，ES预水解过渡态中核苷酸结合结构域的稳定也有助于晶体的生成。4. 肿瘤细胞单步和多步选择抗癌药耐药的分子机制为了了解临床条件下多药耐药的机制，除了常规的生化和细胞生物学技术外，我们已经开始通过RT-PCR、siRNA和染色质免疫沉淀（ChIP）技术，研究在单步和多步选择抗癌药物（如阿霉素）时，治疗方案如何影响ABC药物转运体的表达。我们发现，在MCF-7乳腺癌细胞中，ABC转运蛋白mRNA的表达模式随阿霉素单步治疗和多步治疗而变化。我们已经证明，多步骤选择的MCF-7细胞仅在mRNA和蛋白质水平上过表达ABCB1，此外还有基因扩增。我们还建立了单步多柔比星选择MCF-7亚组，使用非常低的浓度，14或21 nM。我们发现ABCC2， ABCC4和ABCG2在mRNA水平上在这些单步选择的亚系中过表达。然而，只有ABCC4和ABCG2在蛋白水平上过表达。与亲本MCF-7细胞相比，14和21 nM单步阿霉素选择亚群对阿霉素的耐药性均接近5倍。然而，由于ABCC4不会产生对阿霉素的耐药性，因此ABCG2很可能是导致耐药性产生的主要转运蛋白。我们还通过染色质免疫沉淀（ChIP）实验观察到，组蛋白超乙酰化促进了ABCG2的上调。5. ABCB1单核苷酸多态性和单倍型的表征：与博士合作。Michael Gottesman和Kimchi-Sarfaty最近强调了同义snp在决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，两个同义SNP （3435C b> T和1236C>T）和一个非同义SNP （2677G>T）与底物改变有关，并且在[摘要截断为7800个字符]

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]