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: 10925988
• 负责人: SURESH AMBUDKAR
• 金额: $ 176.23万
• 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/10925988
• 关键词: ABCB1 gene; ABCG2 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-binding cassette transport; Address; Adenosine A3 Receptor; Affinity; Alanine; Antineoplastic Agents; Area; Binding; Biochemical; Biological Assay; Biological Availability; Biophysics; Breast Cancer Cell; CCR; Cancer Patient; Carrier Proteins; Cells; Charge; Chemicals; Chemoresistance; Chemotherapy-Oncologic Procedure; Clinic; Collaborations; Cryoelectron Microscopy; Data; Development; Drug Efflux; Drug Interactions; Drug Kinetics; Drug Transport; Drug resistance; Energy Metabolism; Exhibits; Extramural Activities; FDA approved; Formulation; Genus Hippocampus; Goals; Hela Cells; Human; Hydrophobicity; Knowledge; Ligands; Light; Link; Lipids; Malignant Neoplasms; Maryland; Mediating; Membrane; Molecular; Molecular Conformation; Monoclonal Antibodies; Multi-Drug Resistance; Mus; Mutagenesis; Mutate; Mutation; Names; Natural Products; Nature; PUVA Photochemotherapy; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphatidylinositide 3-Kinase Inhibitor; Photochemistry; Photosensitizing Agents; Play; Poly(ADP-ribose) Polymerase Inhibitor; Production; Proteins; Pump; Reactive Oxygen Species; Regulation; Resistance; Resolution; Rhodamine 123; Role; Series; Site; Structure; Structure-Activity Relationship; Substrate Specificity; Tariquidar; Testing; Time; Transmembrane Domain; Tyrosine Kinase Inhibitor; Universities; Work; cancer cell; cancer drug resistance; cancer type; design; drug repurposing; flexibility; glycoprotein structure; improved; in silico; in vivo; inhibitor; insight; interdisciplinary approach; kinase inhibitor; member; molecular dynamics; molecular modeling; multidrug resistant cancer; mutant; nanodisk; nanomedicine; novel strategies; novel therapeutic intervention; programs; reconstitution; refractory cancer; screening; small molecule; small molecule libraries; tool; triple-negative invasive breast carcinoma; uptake

We have designed a coordinated strategy using multidisciplinary approaches to understand the molecular basis of the polyspecificity exhibited by the ATP-binding cassette (ABC) drug transporter P-glycoprotein (P-gp) 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 structure-activity relationships, in silico molecular modeling and MD simulations to extend our understanding of the mechanistic aspects and structure-function relationships. In addition, we are employing a novel approach of substituting multiple conserved residues with alanine in homologous transmembrane helices (TMHs) to elucidate the transport mechanism of P-gp. Furthermore, we are devoting considerable effort to the screening of repurposed drugs, dual inhibitors, tyrosine kinase inhibitors (TKIs) and small molecule modulators of both 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 study the catalytic cycle of P-gp, specifically the mechanism of ATP hydrolysis by inhibitors. Cryo-EM analysis of P-gp structures has revealed that two molecules of inhibitors such as zosuquidar, tariquidar, elacridar and encequidar are bound in the transmembrane region. One of the molecules of the inhibitor is bound in the substrate-binding pocket (SBP) and another in a cavity referred to as an "access tunnel" extending from the SBP to the gate formed by TMHs 4 and 10. It was hypothesized that inhibitor occupancy of the access tunnel would impede substrate transport. To test this hypothesis, residues lining the access tunnel that interact with four inhibitors were substituted with alanine to generate mutants named L-site-5A, L-site-8A, and L-site-9A. These mutants, along with the wild-type (WT) P-gp, were expressed in HeLa cells. The mutants showed expression levels similar to those of WT P-gp. In addition, the L-site-5A mutant showed normal transport activity for eight of the ten fluorescent substrates tested and partial transport for two of them, whereas the L-site-8A and L-site- 9A mutants exhibited progressive loss of transport function. When compared to the L-site-5A mutant, L-site-8A and L-site-9A have residues N296/I299/F770/V991 substituted by Ala. These residues, or at least some of them, may be essential for transport of the tested fluorescent substrates, even though they are located at some distance from the SBP. Surprisingly, all four inhibitors partially inhibited or completely inhibited (70%) the drug efflux activity of the L-site mutants. The inhibition of Rhod-2, AM efflux was further characterized, obtaining IC50 values for tariquidar inhibition for the three L-site mutants not significantly different from WT (13 nM). However, there were significant differences observed in the case of inhibition by zosuquidar, with the mutants displaying values up to 200-fold higher compared to WT-P-gp, indicating decreased affinity. 2. Mechanism of the reversal of the direction of P-gp-mediated drug transport from efflux to uptake: Previously, we generated the 14A mutant of human P-gp in which seven conserved residues each from TMH6 and TMH12 were mutated to alanine and found that this mutant could not efflux most of the substrates tested. But surprisingly, it was able to import four of the tested substrates including Rh123 and Flutax-1. We continue to study the mechanism of how the direction of transport from efflux to uptake is changed. We focused on these three sub-aims. (a) Determination of the minimum number of mutations in TMHs 6 and 12 necessary for the uptake function: We generated a series of mutants, with mutations ranging from 4 to 16 in TMH6 and TMH12 based on their possible interaction with substrates. We found that a minimum of 7 residues (3 from TMH 6 and 4 from TMH 12, a mutant named 7AII) are required for complete loss of efflux of tested substrates and to gain the ability to mediate uptake of 5 substrates. These findings are consistent with the presence of a switch region comprised of residues present in the upper halves of both TMH6 and TMH12 which determines the direction of substrate transport. We plan to test whether the substitution of 2 to 4 residues in this region of TMH6 and 12 with positively (Arg) or negatively (Asp) charged residues changes the direction of transport. (b) Conversion of mouse P-gp into drug uptake pumps for structural and in vivo functional studies: Human P-gp is a highly conserved transporter and shares 87% identity with mouse P-gp Abcb1a. We substituted the same residues in TMH6 and 12 of mouse P-gp, forming the human P-gp 7A-II mutant to determine whether the murine transporter can be converted to a drug uptake pump. We found that indeed mouse P-gp 7A mutant also lost efflux function but could mediate uptake of 5 substrates. Furthermore, rhodamine 123 uptake by the mouse P-gp-7A mutant was inhibited by substrates and inhibitors similar to the human 7A-II mutant. 3. The molecular basis of the polyspecificity of human P-gp: To understand the molecular basis of the broad substrate specificity of P-gp, we substituted seven residues with Ala in both homologous transmembrane helices (TMHs) 4 and 10 which undergo significant conformational changes during the catalytic cycle of P-gp. We found that the transport function of the mutants containing seven Ala substitutions either in TMH4 or 10 alone is almost the same as that of WT P-gp. However, when the same seven residues are mutated in both TMHs 4 and 10, the TMH4,10-14A mutant lost the ability to transport most tested substrates. These data, consistent with the flexible nature of the TMDs, indicate that multiple mutations in a single TMH are well tolerated. Additional data including molecular dynamics (MD) simulations suggest that residues in TMHs 4 and 10 function cooperatively to generate conformational changes necessary for the translocation of substrate drugs out of cells. Thus, our findings provide a functional correlation with the observed structural changes in TMHs 4 and 10 in the inward-open and -closed states of P-gp. 4. Mechanism of photodynamic regulation of P-gp and ABCG2: We have begun to elucidate the molecular mechanism of photo dynamic therapy (PDT)-mediated regulation of ABC drug transporters. These studies are carried out in collaboration with Dr. Huang-Chiao Huang (partnership program between CCR, NCI and the University of Maryland). PDT is a photochemistry-based tool that involves light activation of photosensitizers to generate reactive oxygen species. PDT using the photosensitizer benzoporphyrin (BPD, which is a substrate of P-gp and ABCG2), inhibits both transporters by modulating their ATPase activity and protein integrity. To improve the efficiency of PDT for drug-resistant cancer, we devised a photoimmunoconjugate formulation combining hydrophobic BPD photosensitizers and a conformation-sensitive UIC2 monoclonal antibody to identify P-gp expression on triple negative breast cancer (TNBC) cells. We plan to use Seahorse-based assays to assess the effect of PDT on the energy metabolism and ATP production in P-gp or ABCG2 expressing drug-resistant cancer cells. 5. Screening of non-toxic natural products, small molecules, and repurposed drugs as modulators to overcome resistance mediated by P-gp and ABCG2: We continue to characterize natural products, recently developed tyrosine kinase inhibitors, repurposed drugs, and small molecules for their effect on the function of P-gp and ABCG2. These studies are carried out in collaboration with intramural and extramural collaborators.

我们设计了一种采用多学科方法的协调策略，以了解 ATP 结合盒 (ABC) 药物转运蛋白 P-糖蛋白 (P-gp) 所表现出的多特异性的分子基础以及 P-gp 介导的药物转运机制。我们的方法包括多种生物化学和生物物理测定、基于细胞的运输测定、脂质纳米盘的纯化和重构，以使用冷冻电镜进行结构研究、药物化学合成大量化合物以评估结构-活性关系、在计算机分子建模和MD 模拟可扩展我们对机械方面和结构-功能关系的理解。此外，我们正在采用一种新方法，用丙氨酸取代同源跨膜螺旋（TMH）中的多个保守残基来阐明 P-gp 的转运机制。此外，我们还投入大量精力筛选临床上用于治疗各种癌症的再利用药物、双重抑制剂、酪氨酸激酶抑制剂（TKI）以及P-gp和ABCG2的小分子调节剂。 1.阐明P-gp的ATP水解催化循环和转运途径：我们继续研究P-gp的催化循环，特别是抑制剂水解ATP的机制。 P-gp 结构的冷冻电镜分析表明，两个抑制剂分子（例如 zosuquidar、tariquidar、elacridar 和 encequidar）结合在跨膜区域。抑制剂的一个分子结合在底物结合袋 (SBP) 中，另一个分子结合在从 SBP 延伸到 TMH 4 和 10 形成的门的被称为“通道隧道”的空腔中。据推测，抑制剂占用通道会阻碍基材的运输。为了检验这一假设，与四种抑制剂相互作用的通道内的残基被丙氨酸取代，产生名为 L-site-5A、L-site-8A 和 L-site-9A 的突变体。这些突变体与野生型 (WT) P-gp 一起在 HeLa 细胞中表达。突变体表现出与WT P-gp相似的表达水平。此外，L-site-5A 突变体对所测试的 10 种荧光底物中的 8 种表现出正常的转运活性，并对其中的两种表现出部分转运，而 L-site-8A 和 L-site-9A 突变体则表现出转运功能逐渐丧失。与 L-site-5A 突变体相比，L-site-8A 和 L-site-9A 的残基 N296/I299/F770/V991 被 Ala 取代。这些残基或至少其中一些残基可能对于运输至关重要测试的荧光底物，即使它们距离 SBP 有一定距离。令人惊讶的是，所有四种抑制剂都部分抑制或完全抑制（70%）L位点突变体的药物流出活性。进一步表征了 Rhod-2、AM 外流的抑制，获得了三种 L 位点突变体的 tariquidar 抑制的 IC50 值，与 WT 没有显着差异 (13 nM)。然而，在 zosuquidar 抑制的情况下观察到显着差异，与 WT-P-gp 相比，突变体显示的值高出 200 倍，表明亲和力降低。 2. P-gp介导的药物转运从流出到摄取的方向逆转的机制：之前，我们生成了人P-gp的14A突变体，其中TMH6和TMH12各7个保守残基突变为丙氨酸，并发现该突变体不能流出大多数测试的底物。但令人惊讶的是，它能够导入四种测试底物，包括 Rh123 和 Flutax-1。我们继续研究从流出到摄取的运输方向如何改变的机制。我们专注于这三个子目标。 (a) 确定摄取功能所需的 TMH 6 和 12 中突变的最小数量：我们生成了一系列突变体，根据它们可能与底物的相互作用，TMH6 和 TMH12 中的突变范围为 4 至 16 个。我们发现，要完全丧失测试底物的外排并获得介导 5 种底物摄取的能力，至少需要 7 个残基（3 个来自 TMH 6，4 个来自 TMH 12，一种名为 7AII 的突变体）。这些发现与 TMH6 和 TMH12 上半部分存在的残基组成的开关区域的存在一致，该区域决定底物传输的方向。我们计划测试 TMH6 和 12 的该区域中的 2 至 4 个残基用带正电 (Arg) 或带负电 (Asp) 的残基取代是否会改变运输方向。 (b) 将小鼠 P-gp 转化为药物摄取泵，用于结构和体内功能研究：人 P-gp 是一种高度保守的转运蛋白，与小鼠 P-gp Abcb1a 具有 87% 的同一性。我们替换了小鼠P-gp的TMH6和12中相同的残基，形成人P-gp 7A-II突变体，以确定小鼠转运蛋白是否可以转化为药物摄取泵。我们发现小鼠 P-gp 7A 突变体确实也失去了外排功能，但可以介导 5 种底物的摄取。此外，与人 7A-II 突变体类似，小鼠 P-gp-7A 突变体对罗丹明 123 的摄取受到底物和抑制剂的抑制。 3. 人 P-gp 多特异性的分子基础：为了了解 P-gp 广泛底物特异性的分子基础，我们用 Ala 取代了同源跨膜螺旋 (TMH) 4 和 10 中的 7 个残基，这两个螺旋经历了显着的构象变化。 P-gp催化循环过程中的变化。我们发现，TMH4 或单独的 10 中含有 7 个 Ala 取代的突变体的转运功能几乎与 WT P-gp 相同。然而，当 TMH 4 和 10 中相同的七个残基发生突变时，TMH4,10-14A 突变体失去了转运大多数测试底物的能力。这些数据与 TMD 的灵活性一致，表明单个 TMH 中的多个突变具有良好的耐受性。包括分子动力学 (MD) 模拟在内的其他数据表明，TMH 4 和 10 中的残基协同作用，产生底物药物从细胞中转位所需的构象变化。因此，我们的研究结果提供了与 P-gp 向内开放和封闭状态下 TMH 4 和 10 中观察到的结构变化的功能相关性。 4. P-gp和ABCG2的光动力调节机制：我们已经开始阐明光动力疗法（PDT）介导的ABC药物转运蛋白调节的分子机制。这些研究是与 Huang-Chiao Huang 博士（CCR、NCI 和马里兰大学之间的合作项目）合作进行的。 PDT 是一种基于光化学的工具，涉及光敏剂的光激活以产生活性氧。使用光敏剂苯并卟啉（BPD，是 P-gp 和 ABCG2 的底物）的 PDT 通过调节 ATP 酶活性和蛋白质完整性来抑制这两种转运蛋白。为了提高PDT治疗耐药癌症的效率，我们设计了一种结合疏水性BPD光敏剂和构象敏感的UIC2单克隆抗体的光免疫缀合物制剂，以鉴定三阴性乳腺癌（TNBC）细胞上的P-gp表达。我们计划使用基于 Seahorse 的检测来评估 PDT 对表达 P-gp 或 ABCG2 的耐药癌细胞中能量代谢和 ATP 产生的影响。 5. 筛选无毒天然产物、小分子和再利用药物作为调节剂以克服 P-gp 和 ABCG2 介导的耐药性：我们继续表征天然产物、最近开发的酪氨酸激酶抑制剂、再利用药物和小分子的特性对 P-gp 和 ABCG2 功能的影响。这些研究是与校内和校外合作者合作进行的。

项目成果

Comparison of ATP-binding cassette transporter interactions with the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib.

ATP 结合盒转运蛋白与酪氨酸激酶抑制剂伊马替尼、尼罗替尼和达沙替尼相互作用的比较。

• 发表时间: 2010-08
• 作者: Dohse, Marius;Scharenberg, Christian;Shukla, Suneet;Robey, Robert W;Volkmann, Thorsten;Deeken, John F;Brendel, Cornelia;Ambudkar, Suresh V;Neubauer, Andreas;Bates, Susan E
• 通讯作者: Bates, Susan E

The phosphodiesterase-5 inhibitor vardenafil is a potent inhibitor of ABCB1/P-glycoprotein transporter.

磷酸二酯酶 5 抑制剂伐地那非是 ABCB1/P-糖蛋白转运蛋白的有效抑制剂。

• 发表时间: 2011
• 影响因子: 3.7
• 作者: Ding, Pei;Tiwari, Amit K;Ohnuma, Shinobu;Lee, Jeferson W K K;An, Xin;Dai, Chun;Lu, Qi;Singh, Satyakam;Yang, Dong;Talele, Tanaji T;Ambudkar, Suresh V;Chen, Zhe
• 通讯作者: Chen, Zhe

Biochemical mechanism of modulation of human P-glycoprotein by stemofoline.

Stefoline 调节人 P-糖蛋白的生化机制。

• 发表时间: 2011-12
• 影响因子: 2.7
• 作者: Chanmahasathien, Wisinee;Ohnuma, Shinobu;Ambudkar, Suresh V;Limtrakul, Pornngarm
• 通讯作者: Limtrakul, Pornngarm

The transmission interface of the Saccharomyces cerevisiae multidrug transporter Pdr5: Val-656 located in intracellular loop 2 plays a major role in drug resistance.

位于细胞内环2的酿酒酵母多药转运蛋白Pdr5:Val-656的传输界面在耐药性中起主要作用。

• 发表时间: 2013-02
• 影响因子: 4.9
• 作者: Downes, Marianne T;Mehla, Jitender;Ananthaswamy, Neeti;Wakschlag, Adina;Lamonde, Micheala;Dine, Elliot;Ambudkar, Suresh V;Golin, John
• 通讯作者: Golin, John

Human ABCB1 (P-glycoprotein) and ABCG2 mediate resistance to BI 2536, a potent and selective inhibitor of Polo-like kinase 1.

人 ABCB1（P-糖蛋白）和 ABCG2 介导对 BI 2536 的耐药性，BI 2536 是 Polo 样激酶 1 的有效选择性抑制剂。

• 发表时间: 2013-10-01
• 影响因子: 5.8
• 作者: Wu, Chung;Hsiao, Sung;Sim, Hong;Luo, Shi;Tuo, Wei;Cheng, Hsing;Li, Yan;Huang, Yang;Ambudkar, Suresh V
• 通讯作者: Ambudkar, Suresh V