Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

我们设计了一个协调的策略，使用多学科的方法来了解多特异性的分子基础和p- gp介导的药物转运机制。我们的方法包括几种生化和生物物理分析，基于细胞的运输分析，在脂质纳米盘中使用冷冻电镜进行结构研究的纯化和重构，药物化学合成大量化合物以评估其结构-活性关系，在硅分子模型和MD模拟中扩展我们对ABC药物转运体的机制方面和结构-功能关系的理解。此外，我们采用了一种新的方法，用丙氨酸取代同源跨膜螺旋6和12中的多个保守残基，以阐明P-gp的转运机制。此外，我们正在投入大量精力筛选和开发酪氨酸激酶抑制剂（TKIs）和P-gp和ABCG2的小分子调节剂，用于临床治疗各种类型的癌症。1. 阐明ATP水解的催化循环和P-gp的转运途径：我们继续表征功能第二位点抑制基因突变的增益。我们之前发现TMD1中替换了6个残基的6Y突变体（F72Y/F303Y/I306Y/F314Y/F336Y/L339Y）部分运输了BD-verapamil，但未能运输所有其他被测试的底物。P-gp在atp结合构象中的原子结构表明，胞内螺旋4与NBD1的q环相互作用。为了验证用酪氨酸取代细胞内螺旋4中保守的F916残基是否可以挽救6Y突变体的功能，我们将该残基加入6Y中，产生6Y + F916Y突变体。令我们惊讶的是，我们发现F916Y确实挽救了6Y突变体的功能。与细胞内螺旋4的F916Y与NBD1 q环的F480Y之间的相互作用一致，我们发现，在6Y突变体中添加F480Y也至少在选定的底物中恢复了6Y的转运功能。我们还将NBD1和NBD2特征区的Q535Y或Q1180Y残基加入到6Y中，检查这些残基是否也挽救了6Y变体的功能。然而，nbd特征区的残基对6Y突变体的功能没有任何影响。这些第二位点抑制突变的结果证明了药物结合口袋与人类P-gp的NBD1之间的联系。这是P-gp中存在功能获得性第二位点抑制突变的第一个证据，尽管此类突变已在其他真核ABC转运体中报道，包括CFTR (ABCC7)，酵母PDR5和假丝酵母Cdr1。2. P-gp多特异性的分子基础机制：(i)开发了一种热失活方法，以了解P-gp的药物底物和atp依赖性稳定性。我们观察到P-gp的热稳定性取决于其nbd的构象。在蛋白质水平上，温度对P-gp的影响也可以通过SDS-PAGE量化转运体单体带的消失来跟踪。我们利用这种方法研究了P-gp突变体的热稳定性，这些突变体将ATP与NBDs （E556Q/E1201Q）或仅与NBD2 （Y401A）结合，但不能进行ATP水解，从而进一步支持ATP诱导的NBD二聚化在P-gp热稳定过程中的作用。与没有核苷酸的内向开放构象相比，atp结合nbd及其二聚化产生的内向封闭构象具有显著更高的热稳定性。我们还发现，在转运底物刺激ATP水解的情况下，P-gp的热稳定性与转运和ATP酶活性抑制剂存在显著差异。抑制剂通过稳定向内开放的构象来阻止ATP水解所需的nbd的ATP依赖性二聚化。这些发现表明，结合在TM区域的调节剂通过阻止P-gp的NBDs的ATP依赖性二聚化来抑制ATP水解和药物运输。目前，我们正在测试156种FDA批准的抗癌药物对P-gp和ABCG2热稳定性的影响。这将有助于我们确定P-gp和ABCG2的有效抑制剂。（ii）我们的合作者Kenneth Jacobson博士（NIDDK， NIH）正在开发各种A3腺苷受体激动剂，用于治疗慢性疾病，包括类风湿关节炎、牛皮癣、慢性疼痛和肝细胞癌。我们测试了40多种A3腺苷受体配体，发现这些配体对P-gp和ABCG2活性具有不同的调节作用，这取决于结构功能化。虽然MRS 7343（先导化合物）抑制ABCG2的转运功能，但这种腺苷受体配体与P-gp没有相互作用。分子模型研究表明，选定的A3腺苷受体类似物与P-gp和ABCG2的药物结合口袋中的残基相互作用。P-gp和ABCG2似乎都影响A3腺苷受体配体的药代动力学。MRS 7343似乎是开发P-gp和ABCG2高亲和调节剂的良好模板。3. 人类P-gp的三维结构解析：对于结构研究来说，获得大量纯化的功能蛋白是非常重要的。我们比较了用于从High-Five昆虫细胞膜中溶解和纯化人类和小鼠P-gp的三种洗涤剂（1,2-二庚醇- sng -3-甘油酸-3-磷酸胆碱、十二烷基麦芽糖苷和正辛基- β -d -葡萄糖吡喃苷）。P-gp首先使用固定化金属亲和层析进行纯化，然后使用阴离子交换层析或尺寸排除层析进行第二步纯化，得到浓度为10至12 mg / ml的蛋白质。尺寸排除层析是首选方法，因为它允许从聚集体中分离单体转运蛋白。我们发现纯化后的蛋白，当在蛋白脂质体和纳米圆盘中重组时，显示出基础和底物或抑制剂调节的atp酶活性。我们目前使用镍- nta，然后用大小排除柱纯化P-gp，用这种蛋白质制备的纳米片用于低温电镜研究。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：我们继续描述最近开发的酪氨酸激酶抑制剂，重新用途药物，小分子，天然产物和姜黄素的合成衍生物对P-gp和ABCG2功能的影响。我们的目标是描述这些临床上重要的调节剂的作用，以帮助我们了解这些转运蛋白的多特异性。我们发现选择性IIa类组蛋白去乙酰化酶抑制剂TMP195是P-gp和ABCG2的调节剂，而磷酸二酯酶5型抑制剂MY-5445、TKI抑制剂西特拉替尼、c-myc抑制剂tivatinib和licochalcone a仅调节ABCG2的功能。此外，PI3K/mTOR抑制剂samotolisib是P-gp和ABCG2的底物。这些研究是与博士合作进行的。吴忠普（台湾长工大学）和陈哲生（Jason）（纽约圣约翰大学）。

We have designed a coordinated strategy using multidisciplinary approaches to understand the molecular basis of polyspecificity and the mechanism of P-gp-mediated drug transport. Our approaches include several biochemical and biophysical assays, cell-based transport assays, purification and reconstitution in lipid nanodiscs for structural studies using cryo-EM, medicinal chemistry to synthesize a large number of compounds to assess their structure-activity relationships, in silico molecular modeling and MD simulations to extend our understanding of the mechanistic aspects and the structure-function relationships of ABC drug transporters. In addition, we are employing a novel approach of substituting multiple conserved residues with alanine in homologous transmembrane helices six and twelve to elucidate the transport mechanism of P-gp. Furthermore, we are devoting considerable effort to the screening and development of tyrosine kinase inhibitors (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: We continue to characterize the gain of function second-site suppressor mutations. We previously found that the 6Y mutant with substitution of six residues in TMD1 (F72Y/F303Y/I306Y/F314Y/F336Y/L339Y) partially transported BD-verapamil but failed to transport all other tested substrates. The atomic structure of P-gp in the ATP-bound conformation showed that intracellular helix four interacts with the Q-loop of NBD1. To test whether the substitution of the conserved F916 residue in intracellular helix four with tyrosine can rescue the function of the 6Y mutant, we added this residue to 6Y to produce the 6Y + F916Y mutant. To our surprise, we found that F916Y indeed rescues the function of the 6Y mutant. Consistent with the interaction between F916Y of intracellular helix four and F480Y from the Q-loop of NBD1, we found that addition of F480Y to the 6Y mutant also rescued the transport function of 6Y at least for selected substrates. We also added the residues Q535Y or Q1180Y from the signature region of NBD1 and NBD2 to 6Y to check whether these residues also rescue the function of the 6Y variant. However, the residues from the signature region of the NBDs did not have any effect on the function of the 6Y mutant. These results with second-site suppressor mutations demonstrate the connection between the drug-binding pocket and NBD1 of human P-gp. This is the first evidence of the presence of gain-of-function second-site suppressor mutations in P-gp, although such mutations have been reported in other eukaryotic ABC transporters including CFTR (ABCC7), yeast PDR5 and Candida Cdr1. 2. The mechanism of the molecular basis of polyspecificity of P-g: (i) Development of a thermal inactivation method for understanding the drug-substrate and ATP-dependent stability of P-gp. We observed that the thermostability of P-gp depends on the conformation of its NBDs. The effect of temperature on P-gp at the protein level can also be followed by quantifying the disappearance of the monomeric band of the transporter by SDS-PAGE. We used this approach to study the thermostability of P-gp mutants, which bind ATP to both NBDs (E556Q/E1201Q) or only to NBD2 (Y401A), but are unable to carry out ATP hydrolysis, to provide further support for the role of ATP-induced NBD dimerization during thermo-stabilization of P-gp. The inward-closed conformation that results from ATP-binding to both NBDs and their dimerization has remarkably higher thermostability when compared with the inward-open conformation in the absence of nucleotides. We also find significant differences in the thermostability of P-gp in the presence of transport substrates that stimulate ATP hydrolysis vs. inhibitors of both transport and ATPase activity. The inhibitors prevent the ATP-dependent dimerization of the NBDs, which is required for ATP hydrolysis, by stabilizing the inward-open conformation. These findings suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing ATP-dependent dimerization of the NBDs of P-gp. Currently, we are testing 156 FDA approved cancer drugs for their effect on the thermal stabilization of P-gp and ABCG2. This will help us to identify potent inhibitors of P-gp and ABCG2. (ii) Various A3 adenosine receptor agonists are being developed by our collaborator Dr. Kenneth Jacobson (NIDDK, NIH) for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain and hepatocellular carcinoma. We tested more than forty A3 adenosine receptor ligands and found that these ligands can have different modulatory effects on P-gp and ABCG2 activity, depending on structural functionalization. Although MRS 7343 (lead compound) inhibits the transport function of ABCG2, this adenosine receptor ligand showed no interaction with P-gp. Molecular modeling studies revealed that selected A3 adenosine receptor analogs interact with residues in the drug-binding pockets of both P-gp and ABCG2. Both P-gp and ABCG2 appear to affect the pharmacokinetics of A3 adenosine receptor ligands. MRS 7343 appears to be a good template to develop high affinity modulators for P-gp and ABCG2. 3. Resolution of the three-dimensional structure of human P-gp: For structural studies it is important to obtain a large amount of purified functional protein. We compared three detergents (1,2-diheptanoyol-sn-glycero-3-phosphocholine, dodecyl maltoside and n-octyl-beta-D-glucopyranoside) used for solubilization and purification of human and mouse P-gp from High-Five insect cell membranes. P-gp purification was performed first using immobilized metal affinity chromatography, then followed by a second step of either anion exchange chromatography or size exclusion chromatography to yield protein in concentrations of 10 to 12 mg per ml. Size exclusion chromatography was the preferred method, as it allows separation of monomeric transporters from aggregates. We showed that the purified protein, when reconstituted in proteoliposomes and nanodiscs, exhibits both basal and substrate or inhibitor-modulated ATPase activity. We are currently using nickel-NTA followed by a size exclusion column for purification of P-gp and nanodiscs prepared with this protein are being used for cryo-EM studies. 4. Development of non-toxic natural product and small molecule modulators to overcome resistance mediated by P-gp and ABCG2: We continue to characterize recently developed tyrosine kinase inhibitors, repurposed drugs, small molecules, natural products and synthetic derivations of curcumin for their effect on the function of P-gp and ABCG2. Our goal is to characterize the effect of these clinically important modulators to help us to understand the polyspecificity of these transporters. We found that the selective class IIa histone deacetylase inhibitor TMP195 is a modulator of both P-gp and ABCG2, whereas the phosphodiesterase type 5 inhibitor MY-5445, the TKI inhibitor sitravatinib, the c-myc inhibitor tivatinib and licochalcone A modulate only the function of ABCG2. In addition, the PI3K/mTOR inhibitor samotolisib is a substrate for both P-gp and ABCG2. These studies were carried out in collaboration with Drs. Chung-Pu Wu (Chang Gung University, Taiwan) and Zhe-Sheng (Jason) Chen (St. John's University, NY).