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Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

多药耐药性相关转运蛋白的生化分析

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

我们采用多学科方法设计了一种协调策略，以了解atp结合盒（ABC）药物转运体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结构-活性关系，在硅分子模型和MD模拟中扩展我们对机制方面和结构-功能关系的理解。此外，我们采用了一种新的方法，在同源跨膜螺旋（TMHs）中用丙氨酸取代多个保守残基来阐明P-gp的转运机制。此外，我们正在投入大量精力筛选用于临床治疗各种类型癌症的重用途药物、双重抑制剂、酪氨酸激酶抑制剂（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的转运途径：我们继续研究P-gp的催化循环，特别是抑制剂对ATP水解的机理。P-gp结构的低温电镜分析表明，两种抑制剂zosuquidar、tariquidar、elacridar和encequidar在跨膜区结合。抑制剂的一个分子结合在底物结合袋（SBP）中，另一个分子结合在称为“通道”的空腔中，从SBP延伸到由TMHs 4和10形成的门。假设抑制剂占据通道会阻碍底物的运输。为了验证这一假设，研究人员用丙氨酸取代了通道内与四种抑制剂相互作用的残基，产生了名为l - 5a、l - 8a和l - 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。突变体的表达水平与WT P-gp相似。此外，L-site- 5a突变体对10种荧光底物中的8种表现出正常的转运活性，其中2种表现出部分转运活性，而L-site- 8a和L-site- 9A突变体表现出转运功能的逐渐丧失。与l - 5a突变体相比，l - 8a和l - 9a的残基N296/I299/F770/V991被Ala取代。这些残基，或至少其中的一些残基，可能对被测荧光底物的运输是必需的，即使它们位于离SBP有一定距离的地方。令人惊讶的是，所有四种抑制剂都部分或完全抑制了l位点突变体的药物外排活性（70%）。进一步表征了对Rhod-2、AM外排的抑制作用，得到了三种l位点突变体对tariquar抑制的IC50值，与WT （13 nM）无显著差异。然而，在zosuquidar抑制的情况下，观察到显著差异，突变体的值比WT-P-gp高200倍，表明亲和力降低。2. P-gp介导的药物转运从外排到摄取方向逆转的机制：之前，我们产生了人P-gp的14A突变体，其中TMH6和TMH12各有7个保守残基突变为丙氨酸，并发现该突变体不能外排大多数被测试的底物。但令人惊讶的是，它能够进口四种测试底物，包括Rh123和flutax1。我们将继续研究从外排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TMH6和TMH12中摄取功能所需的最小突变数：我们生成了一系列突变，根据TMH6和TMH12可能与底物的相互作用，突变数从4到16不等。我们发现，至少需要7个残基（3个来自TMH 6, 4个来自TMH 12，一个名为7AII的突变体）才能完全失去被测试底物的外排，并获得介导5个底物摄取的能力。这些发现与TMH6和TMH12上半部分的残基组成的开关区域的存在一致，该开关区域决定了底物运输的方向。我们计划测试在TMH6和12的这个区域用带正电（Arg）或带负电（Asp）的残基取代2到4个残基是否会改变转运的方向。(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：人类P-gp是一种高度保守的转运蛋白，与小鼠P-gp Abcb1a具有87%的同源性。我们在小鼠P-gp的TMH6和12中替换了相同的残基，形成了人类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，小鼠P-gp-7A突变体对罗丹明123的摄取被类似于人类7A-II突变体的底物和抑制剂抑制。3. 人类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，我们在同源跨膜螺旋（TMHs） 4和10上用Ala取代了7个残基，这些残基在P-gp的催化循环中发生了显著的构象变化。我们发现含有7个Ala取代的突变体，无论是在TMH4还是在10中，其转运功能与WT P-gp几乎相同。然而，当TMH4和tmh10中相同的7个残基发生突变时，TMH4,10- 14a突变体失去了运输大多数被测底物的能力。这些数据与TMDs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，TMHs 4和10中的残基协同作用，产生底物药物转运出细胞所必需的构象变化。因此，我们的研究结果提供了在P-gp的内向开放和封闭状态下观察到的TMHs 4和10结构变化的功能相关性。4. P-gp和ABCG2的光动力调控机制：我们已经开始阐明光动力疗法（PDT）介导的ABC药物转运体调控的分子机制。这些研究是与Huang- chiao Huang博士（CCR、NCI和马里兰大学的合作项目）合作进行的。PDT是一种基于光化学的工具，它涉及光激活光敏剂来产生活性氧。PDT使用光敏剂苯并卟啉（BPD， P-gp和ABCG2的底物），通过调节它们的atp酶活性和蛋白质完整性来抑制这两种转运体。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偶联制剂，用于鉴定三阴性乳腺癌（TNBC）细胞中P-gp的表达。我们计划利用海马为基础的实验来评估PDT对表达P-gp或ABCG2的耐药癌细胞的能量代谢和ATP产生的影响。5. 筛选无毒天然产物、小分子和再用途药物作为克服P-gp和ABCG2介导的耐药性的调节剂：我们继续表征天然产物、最近开发的酪氨酸激酶抑制剂、再用途药物和小分子对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

期刊论文数量（92）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Selonsertib (GS-4997), an ASK1 inhibitor, antagonizes multidrug resistance in ABCB1- and ABCG2-overexpressing cancer cells.

Selonsertib (GS-4997) 是一种 ASK1 抑制剂，可拮抗 ABCB1 和 ABCG2 过表达癌细胞的多药耐药性。

DOI：
10.1016/j.canlet.2018.10.007
发表时间：
2019-01
期刊：
Cancer letters
影响因子：
9.7
作者：
Ji N;Yang Y;Cai CY;Lei ZN;Wang JQ;Gupta P;Shukla S;Ambudkar SV;Kong D;Chen ZS
通讯作者：
Chen ZS

Molecular basis of the polyspecificity of P-glycoprotein (ABCB1): recent biochemical and structural studies.

P-糖蛋白多蛋白多蛋白多蛋白的分子基础（ABCB1）：最近的生化和结构研究。