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，在脂质纳米圆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. ATP水解的催化循环和P-gp转运途径的阐明：我们之前报道过富含酪氨酸的15Y P-gp突变体不能转运大尺寸底物。为了了解15Y突变体如何失去转运大尺寸底物的能力，我们决定首先制造两个突变体：一个突变体命名为6Y，在TMD1中替换了6个残基（F72Y/F303Y/I306Y/F314Y/F336Y/L339Y），另一个突变体命名为9Y，在TMD2中替换了9个残基（F732Y/F759Y/F770Y/F938Y/F942Y/M949Y/L975Y/F983Y/F994Y）。在TMD1 （6Y突变体）的6个残基中，5个聚集在TMHs 5和6中，而TMD2 （9Y突变体）的9个残基更广泛地分布在TMHs 7、8、11和12中。有趣的是，6Y突变体部分运输了bd -维拉帕米，但未能运输所有其他测试底物。另一方面，9Y突变体运输了所有底物，其亲本15Y突变体也是如此（包括不被15Y运输的大尺寸底物）。这一结果表明，某些9Y残基能够挽救6Y突变体的功能。为了寻找能够挽救6Y突变体功能的9Y残基，我们在6Y背景下制造了两个突变体：一个是6Y加位于上部小叶的三个9Y残基（F732Y/F759Y/L975Y），称为6Y + ULY。第二个突变体称为6Y + 3Y，包含6Y +三个9Y残基，位于膜的下叶（F938Y/F942Y/F994）。而ULY突变体能够运输10个底物中的8个，6Y + 3Y突变体能够运输10个底物中的9个。15Y、ULY和6Y +3Y突变体不能转运bd -长春碱，表明6Y中导致bd -长春碱转运丧失的突变是显性突变。将9Y突变体中的ULY或3Y替换到WT P-gp中，对其表达和功能没有任何影响。这是P-gp中存在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-g多特异性的分子基础机制：(i)我们测试了P-gp与各种A3腺苷受体激动剂的相互作用，这些激动剂正在开发用于治疗慢性疾病，包括类风湿关节炎、牛皮癣、慢性疼痛和肝细胞癌。虽然化合物3和8对P-gp的功能有明显的影响，但我们发现化合物8的bodipy共轭物（化合物24）并没有被P-gp转运。通过硅分子对接，鉴定了与腺苷类似物化合物3和8相互作用至关重要的药物结合口袋中的残基。分子对接研究表明，化合物3和8与紫杉醇（Taxol）结合在药物结合口袋的同一区域。总之，这些结果表明核苷衍生物对P-gp活性的调节作用取决于结构功能化。这项工作是与NIDDK的Kenneth Jacobson合作完成的。（ii）此外，为了研究P-gp的转运功能，我们合成了一种bodipy标记的环孢素a荧光偶联物（BD-CsA）。在合成并对其化学纯度进行表征后，将BD-CsA与常用的7-硝基苯-2-氧-1,3-二唑-4-基(NBD)-CsA探针进行了比较。在流式细胞术中，BD-CsA的荧光强度几乎是NBD-CsA的10倍，这使得我们可以使用较低浓度的BD-CsA来达到相同的荧光水平。我们发现BD-CsA被人和小鼠P-gp识别为转运底物。BD-CsA和NBD-CsA与人类P-gp结构的硅对接表明，它们都以相似的对接分数结合在药物结合口袋中，并可能与相似的残基相互作用。因此，我们证明了BD-CsA是P-gp的敏感荧光底物，可以有效地研究P-gp在体内和体外的定位和功能。(这项工作是与博士合作完成的。Rolf Swenson和Raju Natarajan，成像探针开发中心，NHLBI， NIH)。（iii）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。我们专注于atp酶活性的热失活，以及它如何受到核苷酸、转运底物和调节剂的影响。我们开发了一个非常简单的实验，包括将表达P-gp的昆虫细胞膜囊泡加热到37℃至70℃的不同温度10分钟，然后在37℃下评估P-gp atp酶活性。有趣的是，在没有ATP的情况下，当nbd相互分离时，蛋白质在较高温度下极易失活。相反，在ATP存在的情况下（非水解条件下），当两个nbd以内向封闭的构象靠近时，热稳定性在20℃以上增加。3. 人类Pgp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。我们比较了三种用于从昆虫High-Five细胞膜中溶解和纯化人类和小鼠P-gp的洗涤剂（1,2-二庚醇- n-甘油-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FLT3抑制剂midoin选择性地调节人类P-gp的功能。同样，c-MET/SMO双抑制剂glesatinib也能调节P-gp的功能。另一方面，KIT和PDGFR-alfa抑制剂avapritinib抑制P-gp和ABCG2的功能。Solensertib是一种ASK1抑制剂，可使表达P-gp和abcg2的细胞对抗癌药物敏感。

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 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: We previously have reported that the tyrosine rich 15Y P-gp mutant fails to transport large-size substrates. To understand how the 15Y mutant lost the ability to transport large size substrates, we decided first to make two mutants: one named 6Y with substitution of six residues in TMD1 (F72Y/F303Y/I306Y/F314Y/F336Y/L339Y) and another one termed 9Y with nine substitutions in TMD2 (F732Y/F759Y/F770Y/F938Y/F942Y/M949Y/L975Y/F983Y/F994Y). Out of six residues in TMD1 (6Y mutant), five are clustered in TMHs 5 and 6, whereas the nine residues in TMD2 (9Y mutant) are more widely dispersed throughout TMHs 7, 8, 11 and 12. Interestingly, the 6Y mutant partially transported BD-verapamil, but failed to transport all other tested substrates. On the other hand, the 9Y mutant transported all substrates, as did its parent 15Y mutant (including large-size substrates that are not transported by 15Y. This result indicated that certain 9Y residues were able to rescue the function of the 6Y mutant. To identify 9Y residues that can rescue the function of the 6Y mutant, we made two mutants in a 6Y background: one with 6Y plus three 9Y residues located in the upper leaflet (F732Y/F759Y/L975Y) and called it 6Y + ULY. The second mutant, termed 6Y + 3Y, contains 6Y plus three 9Y residues located in the lower leaflet of the membrane (F938Y/F942Y/F994). While the ULY mutant was able to transport eight out of ten substrates, the 6Y + 3Y mutant transported nine out of ten substrates. The 15Y, ULY and 6Y +3Y mutants failed to transport BD-vinblastine, showing that the mutation(s) in 6Y responsible for loss of BD-vinblastine transport are dominant mutations. The addition of ULY or 3Y substitutions from the 9Y mutant to WT P-gp did not have any effect on its expression or function. This is the first evidence of the presence of 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) We tested the interaction of P-gp with various A3 adenosine receptor agonists that are being developed for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain and hepatocellular carcinoma. Although compounds 3 and 8 displayed pronounced effects on P-gp function, we found that a BODIPY-conjugate of compound 8 (compound 24) was not transported by P-gp. The residues in the drug-binding pocket critical for interactions with adenosine analogs compound 3 and 8 were identified by in silico molecular docking. Molecular docking studies revealed that both compounds 3 and 8 bind in the same region of the drug-binding pocket as paclitaxel (Taxol). Collectively, these results indicate that nucleoside derivatives can exhibit varied modulatory effects on P-gp activity, depending on structural functionalization. This work was done in collaboration with Kenneth Jacobson, NIDDK. (ii) In addition, to study the transport function of P-gp, we synthesized a Bodipy-labeled fluorescent conjugate of cyclosporine A (BD-CsA). After synthesis and characterization of its chemical purity, BD-CsA was compared with the commonly used 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-CsA probe. In flow cytometry assays, the fluorescence intensity of BD-CsA was almost 10 times higher than that of NBD-CsA, enabling us to use significantly lower concentrations of BD-CsA to achieve the same fluorescence levels. We found that BD-CsA is recognized as a transport substrate by both human and mouse P-gp. In silico docking of BD-CsA and NBD-CsA to the human P-gp structure indicates that they both bind in the drug-binding pocket with similar docking scores and possibly interact with similar residues. Thus, we demonstrate that BD-CsA is a sensitive fluorescent substrate of P-gp that can be used to efficiently study the transporter's localization and function in vitro and in vivo. (This work was done in collaboration with Drs. Rolf Swenson and Raju Natarajan, Imaging Probe Development Center, NHLBI, NIH). (iii) Development of a thermal inactivation method for understanding the drug-substrate and ATP-dependent stability of P-gp. We are focusing on the thermal inactivation of ATPase activity and how it is affected by nucleotides, transported substrates and modulators. We developed a very simple assay consisting of heating insect cell membrane vesicles expressing P-gp to different temperatures ranging from 37C to 70C for 10 min and assessing the P-gp ATPase activity afterwards at 37C. Interestingly, in the absence of ATP, when the NBDs are separated from each other, the protein is highly susceptible to inactivation at higher temperature. In contrast, in the presence of ATP (under non-hydrolyzing condition), when the two NBDs are close together in an inward-closed conformation, the thermal stability increases over 20C. 3. Resolution of the three-dimensional structure of human Pgp: 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 insect High-Five 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 the 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 FLT3 inhibitor midostaurin selectively modulated the function of human P-gp. Similarly, glesatinib, a c-MET/SMO dual inhibitor also modulated the function of P-gp. On the other hand, the KIT and PDGFR-alfa inhibitor, avapritinib inhibited the function of both P-gp and ABCG2. Solensertib, an ASK1 inhibitor, sensitized P-gp- and ABCG2-expressing cells to anticancer drugs.