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.

我们已经使用多学科方法设计了一种协调的策略，以了解ATP结合盒（ABC）药物转运蛋白P-糖蛋白（P-GP）和P-GP介导的药物运输的机制所表现出的多孕科特异性的分子基础。我们的方法包括几种生物化学和生物物理测定，基于细胞的运输测定，脂质纳米散发的纯化和重建，用于使用冷冻EM，药物化学，在硅基分子建模模拟中评估结构性关系的大量化合物，以扩展我们对机械性方面的理解，以评估结构 - 分子建模模拟和MD模拟的理解。此外，我们正在采用一种新型的方法，可以在同源跨膜螺旋（TMHS）中取代多个保守的残基来阐明P-gp的转运机理。此外，我们正在努力筛查重新利用的药物，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，这些分子调节剂均用于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：我们继续研究P-gp的催化循环，特别是抑制剂ATP水解的机理。 P-gp结构的冷冻EM分析表明，两个抑制剂的分子，例如Zosuquidar，Tariquidar，Elacridar和Chencequidar都结合在跨膜区域。抑制剂的分子之一与底物结合口袋（SBP）结合，另一个在称为“接入隧道”的空腔中结合，该漏洞从SBP延伸到TMHS 4和10形成的栅极。可以假设，抑制剂的抑制剂占用率会影响访问隧道的运输。为了检验该假设，将与四个抑制剂相互作用的接口隧道内衬的残基用丙氨酸取代，以产生称为L-Site-5a，L-Site-8A和L-Site-9A的突变体。这些突变体以及野生型（WT）P-gp在HeLa细胞中表达。突变体的表达水平与WT P-gp相似。此外，L位点-5a突变体在测试的十个荧光底物中的八个中表现出正常的转运活性，其中两个荧光底物和部分转运的运输活性，而L-Site-8a和L-Site-8a和L-Site-9a 9A突变体表现出逐渐逐渐失去运输功能。与L-Site-5a突变体相比，L-Site-8a和L-Site-9a具有残基N296/I299/F770/V991，由ALA代替。这些残基，或至少其中一些对于测试的荧光底物的运输可能至关重要，即使它们位于SBP的某些距离。令人惊讶的是，所有四个抑制剂部分抑制或完全抑制了L位点突变体的药物外排活性。 RHOD-2的抑制作用，AM外排是进一步表征的，对于三种L位点突变体的IC50值与WT（13 nm）无显着差异（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，突变体的值高达200倍，表明亲和力降低。 2. P-gp介导的药物从外排向摄取的逆转机制：以前，我们产生了人类P-gp的14A突变体，其中七个来自TMH6和TMH12的保守残基突变为丙氨酸，发现该突变体无法在测试的大多数底物中排出。但令人惊讶的是，它能够导入包括RH123和Flutax-1在内的四个经过测试的底物。我们继续研究从外排向吸收方向的方向的机制。我们专注于这三个子艾姆。（a）确定摄取功能所需的TMHS 6和12中最小突变数：我们产生了一系列突变体，突变在TMH6和TMH12中的突变范围为4至16，并且基于其可能与底物的相互作用。我们发现，至少有7个残基（来自TMH 12的TMH 6和4的3个残基，一个名为7AII的突变体）才能完全损失测试底物的排出，并获得介导5个底物摄取的能力。这些发现与由TMH6和TMH12上半部的残基组成的开关区域的存在一致，该残基决定了底物转运的方向。我们计划用正面（ARG）或负（ASP）带电的残留物在TMH6和12区域中取代2至4个残基会改变运输方向。（b）将小鼠P-gp转化为用于结构和体内功能研究的药物摄取泵：人P-gp是一种高度保守的转运蛋白，与小鼠P-gp ABCB1A共享87％的身份。我们在小鼠P-gp的TMH6和12中取代了相同的残基，形成了人类P-gp 7a-II突变体，以确定是否可以将鼠转运蛋白转换为药物摄取泵。我们发现确实，小鼠P-gp 7a突变体也失去了排出功能，但可以介导5个底物的摄取。此外，小鼠p-gp-7a突变体的摄取的若丹明123被类似于人类7A-II突变体的底物和抑制剂抑制。 3。人P-gp的多性特异性的分子基础：为了了解P-gp的广泛底物特异性的分子基础，我们在两个同源跨膜螺旋（TMHS）4和10中用ALA取代了七个残基，在P-gp的催化周期中均经历了显着构象变化。我们发现，单独使用TMH4或10中包含七个ALA取代的突变体的传输功能几乎与WT P-gp的转运函数相同。但是，当TMHS 4和10中相同的七个残基被突变时，TMH4,10-14A突变体失去了运输大多数测试底物的能力。这些数据与TMD的柔性性质一致，表明单个TMH中的多个突变良好。包括分子动力学（MD）模拟在内的其他数据表明，TMHS 4和10中的残基合作起作用，以产生将底物药物从细胞中移动所必需的构象变化。因此，我们的发现与P-gp的内向开幕式和截断的状态中观察到的结构变化提供了功能相关性。 4。P-gp和ABCG2的光动力调节机制：我们开始阐明光动力学治疗（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计划）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。 PDT使用光敏剂苯并核磷脂（BPD，是P-gp和ABCG2的底物），通过调节其ATPase活性和蛋白质完整性来抑制这两种转运蛋白。为了提高PDT对药物抗药性癌症的效率，我们设计了一种将疏水性BPD光敏剂和对构象敏感的UIC2单克隆抗体结合的摄影偶联物配方，以鉴定在三重阴性乳腺癌（TNBC）细胞上识别P-gp的表达。我们计划使用基于海马的测定法评估PDT对表达耐药性癌细胞的P-gp或ABCG2中PDT对能源代谢和ATP产生的影响。 5。筛选无毒天然产物，小分子和重新利用的药物作为调节剂，以克服由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，重复的药物以及小分子对P-GP和ABCG2功能的作用。这些研究是与壁内和壁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

Elucidation of the structural basis of interaction of the BCR-ABL kinase inhibitor, nilotinib (Tasigna) with the human ABC drug transporter P-glycoprotein.