Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

我们的工作重点是阐明：i)人类P-gp的催化循环和转运途径；ii) P-gp多特异性的分子基础；iii)临床相关酪氨酸激酶抑制剂（TKIs）和其他天然产物调节剂与P-gp和ABCG2的相互作用；iv)第三代环肽抑制剂衍生物（BTT化合物）与P-gp和ABCG2结合所需的药效团特征；(6)利用单粒子低温电镜分析人P-gp在催化循环过程中的构象景观。我们一直在使用基于细胞的、生物化学的、生物物理的、药理学的和生理学的技术，以及分子生物学和分子建模方法来扩展我们对ABC药物转运体的机制方面和结构-功能关系的理解。此外，我们还投入了大量的精力来筛选和开发用于临床治疗各种类型癌症的TKIs和P-gp和ABCG2的小分子调节剂。1. 阐明ATP水解的催化循环和P-gp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P-gp的催化循环和转运途径进行研究。我们正在使用分子模型和诱变方法在分子水平上阐明这种转运体如何识别和运输各种结构不同的化合物。我们最近在P-gp的药物结合口袋中发现了一个苯丙氨酸-酪氨酸结构基序，这对于抑制zosuquidar和其他高亲和力调节剂水解ATP至关重要。由于酪氨酸既是氢键供体又是受体，非共价相互作用是药物转运的关键，我们利用基因合成技术对药物结合口袋进行了全局改变，在药物结合口袋中引入了15个酪氨酸残基，研究其对P-gp药物结合和转运功能的影响。通过计算分析，在人P-gp药物结合口袋中鉴定出15个与底物相互作用的保守残基，并将其替换为酪氨酸，包括11个苯丙氨酸、2个亮氨酸、1个异亮氨酸和1个蛋氨酸。对HeLa细胞中富含酪氨酸的P-gp突变体的表征表明，通过引入15个额外的酪氨酸残基来改变药物结合袋是耐受良好的，并且对该突变体的总表达或细胞表面表达没有可测量的影响。虽然富含酪氨酸的突变体P-gp可以运输小到中等大小的荧光底物，但其运输大型（1000道尔顿以上）底物如nbd -环孢素A、bodipy紫杉醇和bodipyvinblastine的能力显著降低，从而揭示了富含酪氨酸的P-gp突变体的药物运输与分子大小呈负相关。据我们所知，这是第一个报告表明，药物结合口袋的整体改变与替代15个残基是良好的耐受性。15Y突变体在TMD1中有6个突变残基，在TMD2中有9个。在TMD1中有6个取代或在TMD2中有9个取代的突变体的产生将有助于评估转运体的每个TMD的作用，并缩小有助于确定底物大小的残基的数量。2. 多特异性的分子基础机制是多药转运体的重要特性，通过分子建模和诱变方法：为了了解P-gp广泛底物特异性的分子基础，我们表征了洗涤剂胶束环境对药物介导的P-gp atp酶活性抑制的影响。P-gp的大多数底物或调节剂刺激其基础atp酶活性，只有少数药物被发现抑制它。对纯化P-gp的一些结构研究已经被报道，但在十二烷基麦芽糖苷洗涤剂环境中，配体与分离转运体相互作用的信息非常有限，在某些情况下相互矛盾。基于这些原因，我们比较了人类和小鼠P-gp在天然膜、洗涤剂胶束和人工膜中重组后的生化特性。我们发现调节剂zosuquidar， tariquidar和elacridar在洗涤剂胶束环境中刺激纯化的人或小鼠P-gp的atp酶活性，而这些药物抑制转运蛋白在天然膜中或在蛋白脂质体中重组时的atp酶活性。同样，与天然或人工膜相比，洗涤剂胶束对维拉帕米和环肽抑制剂QZ59-SSS的表观亲和力降低了30至150倍。综上所述，这些发现表明，由于构象变化或洗涤剂在洗涤剂胶束环境中的结合位点结合，高亲和位点无法进入。配体与低亲和力位点结合，导致P-gp atp酶活性的改变。因此，配体与纯化P-gp相互作用的结构和功能方面需要在无洗涤剂的天然或人工膜环境中进行研究。3. 人类Pgp三维结构的分辨率：P-gp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，产生了7.5-10.0 mg的99%均质纯P-gp总蛋白。由于人类P-gp的灵活性，我们也使用低温电子显微镜技术进行单粒子分析。为了将分辨率提高到原子水平，我们已经开始使用在纳米片中重组的纯化P-gp。我们优化了在纳米圆盘中以功能形式重组纯化P-gp的条件。膜支架蛋白（MSP） 1与纯化P-gp的比例以及纳米圆盘的脂质组成是保持纯化蛋白在纳米圆盘中功能的关键。P-gp在纳米圆盘中的性质与在天然膜中观察到的性质相似。在不含ATP-Mg、adp -钒酸盐和UIC2抗体Fab的情况下，对P-gp-纳米片进行阴性染色，电镜分析表明，每个纳米片含有一个P-gp分子。单粒子低温电镜研究是与Sriram Subramaniam博士（LCB）合作进行的。4. 开发无毒天然产物和小分子调节剂以克服P-gp和ABCG2介导的耐药性：我们与Tanaji Talele博士（纽约圣约翰大学）合作，通过合成超过100种环肽抑制剂TTT28衍生物，扩展了这些研究。我们发现至少有两种衍生物抑制P-gp的atp酶活性，并使表达P-gp和ABCB2的细胞对抗癌药物敏感。对接研究表明，这些衍生物与P-gp的药物结合口袋结合。为了验证其中一种抑制衍生物（化合物109）的对接姿势，我们在药物结合口袋中使用了单或双突变。此外，我们继续表征最近开发的酪氨酸激酶抑制剂，这些抑制剂用于临床治疗癌症患者，因为它们对P-gp和ABCG2功能的影响。磷脂酰肌醇3-激酶抑制剂PF-498216可调节ABCG2的功能，而regorafenib仅调节P-gp的功能。我们还发现BTK抑制剂ibrutinib （PCI-32765）可以克服P-gp和ABCC10 （MRP7）介导的紫杉醇耐药小鼠异种移植模型。这些研究是与博士合作进行的。吴忠普（台湾长工大学）、陈哲生（美国纽约圣约翰大学）。

We have focused our efforts to elucidate: i) the catalytic cycle and transport pathway of human P-gp; ii) the molecular basis of the polyspecificity of P-gp; iii) the interaction of clinically relevant tyrosine kinase inhibitors (TKIs) and other natural product modulators with P-gp and ABCG2; iv) pharmacophore features required for binding of third generation cyclic peptide inhibitor derivatives (BTT compounds) to P-gp and ABCG2; and vi) the use of single particle cryo-EM for the analysis of the conformational landscape of human P-gp during its catalytic cycle. We have been employing cell-based, biochemical, biophysical, pharmacological, and physiological techniques along with molecular biology and molecular modeling approaches to extend our understanding of the mechanistic aspects and the structure-function relationships of ABC drug transporters. In addition, we have devoted considerable effort to the screening and development of TKIs and small molecule modulators of P-gp and ABCG2 that are used in the clinic for treatment of various types of cancers. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of P-gp and the role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of P-gp. We are using molecular modeling and mutagenesis approaches to elucidate on a molecular level how this transporter recognizes and transports a wide variety of structurally dissimilar compounds. We recently identified a phenylalanine-tyrosine structural motif in the drug-binding pocket of P-gp that is critical for the inhibition of ATP hydrolysis by zosuquidar and other high-affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, we made a global alteration of the drug-binding pocket using gene synthesis to introduce fifteen tyrosine residues in the drug-binding pocket to study its effect on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine, two leucine, one isoleucine, and one methionine. Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size fluorescent substrates, its ability to transport large ( above 1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased, thus revealing a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. To our knowledge, this is the first report demonstrating that the global alteration of the drug-binding pocket with substitution of fifteen residues is well tolerated. The 15Y mutant has six mutated residues located in TMD1 and nine in TMD2. The generation of mutants with either six substitutions in TMD1 or nine in TMD2 will be useful to assess the role of each TMD of the transporter and to narrow the number of residues contributing to substrate size determination. 2. The mechanism of the molecular basis of polyspecificity, which is an important property of multidrug transporters, by using molecular modeling and mutagenesis approaches: Towards the goal of understanding the molecular basis of broad substrate specificity of P-gp, we characterized the effect of a detergent micelle environment on the drug-mediated inhibition of P-gp ATPase activity. Most of the substrates or modulators of P-gp stimulate its basal ATPase activity, and only a few drugs have been found to inhibit it. Several structural studies on purified P-gp have been reported and there is very limited and in some cases conflicting information available on ligand interactions with isolated transporters in a dodecyl maltoside detergent environment. For these reasons, we compared the biochemical properties of human and mouse P-gp in native membranes, detergent micelles, and after reconstitution in artificial membranes. We found that the modulators zosuquidar, tariquidar and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment, whereas these drugs inhibited the ATPase activity of the transporter in native membranes or when it was reconstituted in proteoliposomes. Similarly, a 30- to 150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared to native or artificial membranes. These findings in aggregate demonstrated that the high-affinity site is inaccessible either due to a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. Thus, the structural and functional aspects of ligand interactions with purified P-gp need to be studied in a detergent-free native or artificial membrane environment. 3. Resolution of the three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of P-gp is an ongoing project and for this we have developed a purification scheme that has yielded total protein of 7.5-10.0 mg of 99% homogeneously pure P-gp. Due to the flexible nature of human P-gp, we are also using single particle analysis by the cryo-electron microscopy technique. To improve the resolution to an atomic level, we have begun to use the purified P-gp reconstituted in nanodiscs. We have optimized the conditions for reconstituting purified P-gp in nanodiscs in a functional form. The ratio of membrane scaffold protein (MSP) 1 to purified P-gp and the lipid composition of nanodiscs is critical to retain the function of purified protein in nanodiscs. The properties of P-gp in nanodiscs are similar to those observed in native membranes. The EM analysis of negatively stained P-gp-nanodisc samples in the absence and presence of ATP-Mg, ADP-vanadate and Fab of UIC2 antibody suggests that there is a single molecule of P-gp per nanodiscs. The single particle cryo-EM studies are carried out in collaboration with Dr. Sriram Subramaniam (LCB). 4. Development of non-toxic natural product and small molecule modulators to overcome resistance mediated by P-gp and ABCG2: We have extended these studies by synthesizing over 100 derivatives of the cyclic peptide inhibitor TTT28 in collaboration with Dr. Tanaji Talele (St. John's University, NY). We found that at least two derivatives inhibited the ATPase activity of P-gp and sensitized cells expressing P-gp and ABCB2 to anticancer drugs. The docking studies indicated that these derivatives bind to the drug-binding pocket of P-gp. To validate the docking poses of one of the inhibitory derivatives (compound 109), we used single or double mutations in the drug-binding pocket. In addition, we continue to characterize the recently developed tyrosine kinase inhibitors, which are used in the clinic to treat cancer patients, for their effect on the function of P-gp and ABCG2. The phosphatidylinositol 3-kinase inhibitor PF-498216 was found to modulate the function of ABCG2, whereas regorafenib was found to modulate only the function of P-gp. We also found that the BTK inhibitor, ibrutinib (PCI-32765) can overcome the resistance to paclitaxel-mediated by P-gp and ABCC10 (MRP7) in a mouse xenograft model. These studies were carried out in collaboration with Drs. Chug-Pu Wu (Chang Gung University, Taiwan) and Zhe-Sheng Chen (St. John's University, NY).