Biochemical Analysis of Multidrug Resistance-linked Tran

atp结合盒（ABC）转运体如p -糖蛋白（Pgp, ABCB1）、多药耐药相关蛋白（MRP1, ABCC1）和米托蒽酮耐药蛋白（MXR，也称为乳腺癌耐药蛋白、BCRP、ABCP或ABCG2）作为atp依赖的外排泵，在大多数癌症的多药耐药发展中起重要作用。人类有48种已知的ABC转运蛋白，其中至少14种转运蛋白参与多种两亲药物的运动，包括抗癌药物、核苷酸类似物和环核苷酸。因此，这些转运体中的一些也可能有助于恶性细胞中多药耐药的发展。我们的研究旨在了解与多药耐药相关的ABC转运蛋白的作用机制。通过杆状病毒-昆虫细胞表达系统，制备了大量（6- 7mg /ml）具有生物活性的Pgp，用于生物物理和结构研究，通过Pgp结构的解析将加速对这些转运体机制的进一步了解。在过去的几年里，我们一直致力于了解Pgp对ATP水解的催化循环，确定限速步骤以及底物和调节剂对ATP酶活性的调节。类似的研究已经开始于MRP1， MRP4和MRP8，以深入了解这两个ATP位点在这些转运体水解ATP中的作用。这些研究将有助于深入了解这些转运蛋白在癌症多药耐药发展中的作用，并有助于开发新的治疗策略。底物相互作用位点的表征以及底物与Pgp相互作用中涉及的区域和残基的鉴定：我们之前的研究表明，Pgp上至少存在两个不相同的底物相互作用位点。我们继续努力表征底物和抑制剂的相互作用，以阐明Pgp广泛底物特异性的生化基础。我们筛选了stipiamide（一种逆转pgp介导的耐药的合成多烯抗生素）的多种衍生物，以深入了解底物相互作用位点。我们还研究了用于治疗酒精中毒的药物双硫仑调节Pgp活性的机制。从临床角度来看，有趣的是，双硫仑还能调节人类MRP1、MRP4以及真菌ABC药物转运体Cdr1p的活性。二硫仑是一种相对无毒的二硫氨基甲酸酯（口服LD50 8.6 g/kg），半个多世纪以来一直用于酒精厌恶疗法。二硫代氨基甲酸酯与临界硫醇和复杂的金属离子反应。我们已经证明，在完整的细胞中，双硫仑可以逆转人MDR1或mrp1介导的荧光药物底物的外排。双硫仑抑制ATP水解和a- 32p8 -叠氮ATP与p -糖蛋白和MRP1的结合，其抑制曲线与半胱氨酸修饰剂n -乙基马来酰亚胺相似。然而，如果ATP位点受到过量ATP的保护，双硫仑会以浓度依赖的方式刺激两种转运体的ATP水解。因此，除了修饰atp位点的半胱氨酸外，双硫仑还可能与药物底物结合位点相互作用。我们证明，双硫仑，而不是n-乙基马来酰亚胺，以浓度依赖性的方式抑制多药转运体与125i -碘芳唑嗪和3h -叠氮嘧啶的光亲和标记。这表明双硫仑与药物结合位点的相互作用独立于其作为半胱氨酸修饰剂的作用。最后，我们利用MRP4 （ABCC4）证明了二硫仑可以通过在位于活性位点附近的半胱氨酸之间形成二硫键来抑制atp结合，尽管不是在活性位点本身。同样，我们已经证明双硫仑是白色念珠菌多药转运体Cdr1p的有效调节剂，它在多种抗真菌药物的耐药性发展中起着重要作用。抑制Cdr1p和人p糖蛋白的生化机制似乎是相似的。双硫仑以浓度依赖性的方式抑制ATP （a- 32p8 -叠氮ATP）和药物底物（125i - iodoaryazidoprazosin和3H-Azidopine）的光亲和类似物与Cdr1p的结合。与这些发现一致的是，低浓度（1mm）的无毒双硫仑使表达Cdr1p的酿酒酵母细胞对抗真菌药物如环己亚胺、氟康唑、咪康唑和制霉菌素更敏感。总之，我们的研究结果表明，双硫仑通过与ATP和底物结合位点的相互作用来调节药物转运体的功能，因此二硫代氨基甲酸酯可能为开发抗真菌和哺乳动物ABC药物转运体介导的耐药性的新干预措施提供了一个有用的分子支架。人MRP4 （ABCC4）和MRP8 （ABCC11）的特性：多药耐药蛋白4 (MRP4/ABCC4)，转运环单磷酸核苷、核苷类似物药物、化疗药物和前列腺素。我们已经通过在昆虫细胞中表达的人MRP4来表征ATP水解。MRP4水解ATP (Km, 0.62 mM)，这被原钒酸盐和氟化铍抑制。然而，与p糖蛋白的atp酶活性不同，p糖蛋白对两种抑制剂都同样敏感，mrp4 - atp酶对氟化铍比对正钒酸盐更敏感。a-32P8azidoATP与MRP4结合（半最大结合浓度为3mm），并被ATP或其不可水解的类似物AMPPNP（半最大抑制浓度为13.3 mM和308 mM）取代。MRP4底物，前列腺素E1和E2，刺激ATP水解2至3倍，但不影响ATP的Km。其他几种底物，如叠氮胸苷、9-（2-膦基-甲氧基乙基）腺嘌呤和甲氨蝶呤不刺激ATP水解，但抑制前列腺素e2刺激的ATP水解。虽然可以产生MRP4a-32P8azidoADPVi和MRP4a-32P8azidoADPBeFx两种水解后过渡态，但氟化铍的核苷酸捕获率高4倍。二价阳离子Mg2+和Mn2+支持相当水平的核苷酸结合、水解和捕获。然而，Co2+增加了a-32P8azidoADP结合和氟化铍诱导的a-32P8azidoADP捕获，但不支持稳态ATP水解。ADP抑制基础和前列腺素e2刺激的ATP水解（半最大抑制浓度分别为0.19和0.25 mM）和氟化铍诱导的a- 32p8azidoadp捕获，而Pi在20 mM以下没有影响。总的来说，我们的研究结果表明，MRP4表现出底物刺激的ATP水解，我们提出了一个动力学方案，表明ADP从水解后过渡态释放可能是催化循环中的限速步骤。最近，MRP亚家族的两个新成员被确定（MRP8和MRP9）。我们已经在杆状病毒感染的High Five昆虫细胞中表达了人MRP8。初步结果表明，与Pgp和MRP1类似，MRP8表现出底物刺激的atp酶活性。这些研究将有助于我们了解这些MRP亚家族成员的功能及其在癌细胞多药耐药发展中的作用。人类Pgp的二维和三维结构的分辨率：在催化循环的各个阶段的Pgp的高分辨率结构将是了解转运机制必不可少的。这是我们的主要兴趣之一，我们在过去投入了相当大的努力来开发获得大量纯活性Pgp的方法。

The ATP-binding cassette (ABC) transporters such as P-glycoprotein (Pgp, ABCB1), the multidrug resistance-associated protein (MRP1, ABCC1), and the mitoxantrone-resistance protein (MXR also known as breast cancer resistance protein, BCRP, ABCP or ABCG2), which function as ATP-dependent efflux pumps, play an important role in the development of multidrug resistance in most cancers. There are 48 known ABC transport proteins in the human and at least 14 of these transporters are involved in the movement of a variety of amphipathic agents including anticancer agents, nucleotide analogs and cyclic nucleotides. Thus, some of these transporters also may contribute to the development of multidrug resistance in malignant cells. Our studies are directed toward understanding the mechanism of action of the multidrug resistance-linked ABC transporters. By using a baculovirus-insect cell expression system, a large amount (6-7 mg/ml) of biologically active Pgp has been prepared for biophysical and structural studies, as further understanding of the mechanism of these transporters would be accelerated by resolution of the structure of Pgp. In last couple of years we have directed our efforts towards understanding the catalytic cycle of ATP hydrolysis by Pgp, identification of rate-limiting step(s) and modulation of the ATPase activity by substrates and modulators. Similar studies have been initiated with MRP1, MRP4 and MRP8 to gain insight into the role of the two ATP sites in ATP hydrolysis by these transporters. Such studies will provide an insight into the role of these transporters in the development of multidrug resistance in cancers and aid in the development of new therapeutic strategies.1.Characterization of substrate interaction sites and identification of regions and residues involved in interaction of substrates with Pgp: Our previous studies demonstrated the presence of at least two non-identical substrate interaction sites on Pgp. We have continued our efforts to characterize the interactions of substrates and inhibitors to elucidate the biochemical basis for the broad substrate specificity of Pgp. We have screened a large variety of derivatives of stipiamide, a synthetic polyene antibiotic that reverses Pgp-mediated drug resistance, to gain insight into the substrate interaction sites. We have also studied the mechanism by which disulfiram, a drug used to treat alcoholism, modulates Pgp activity. From a clinical perspective it is interesting that disulfiram also modulates activities of human MRP1, MRP4 as well as fungal ABC drug transporter Cdr1p. Disulfiram, or tetraethylthiuram disulfide, is a relatively nontoxic (oral LD50 8.6 g/kg) dithiocarbamate, which has been used for over half a century for alcohol aversion therapy. Dithiocarbamates react with critical thiols and also complex metal ions. We have demonstrated that in intact cells disulfiram reverses either human MDR1- or MRP1-mediated efflux of fluorescent drug-substrates. Disulfiram inhibits ATP hydrolysis and the binding of a-32P8-azidoATP to P-glycoprotein and MRP1, with inhibition curves comparable to N-ethylmaleimide, a cysteine-modifying agent. However, if the ATP sites are protected with excess ATP, disulfiram stimulates ATP hydrolysis by both transporters in a concentration-dependent manner. Thus, in addition to modifying cysteines at the ATP-sites, disulfiram may interact with the drug-substrate binding site. We demonstrate that disulfiram, but not N-ethylmaleimide, inhibits in a concentration-dependent manner the photoaffinity labeling of the multidrug transporter with 125I-Iodoarylazidoprazosin and 3H-Azidopine. This suggests that the interaction of disulfiram with the drug-binding site is independent of its role as a cysteine-modifying agent. Finally, we have exploited MRP4 (ABCC4) to demonstrate that disulfiram can inhibit ATP-binding by forming disulfide bonds between cysteines located in the vicinity of, though not in the active site per se. Similarly, we have shown that disulfiram is an effective modulator of the multidrug transporter Cdr1p from Candida albicans, which plays an important role in the development of resistance to a variety of antifungal agents. The biochemical mechanisms for the inhibition of Cdr1p and human P-glycoprotein appear to be similar. Disulfiram inhibits the binding of photoaffinity analogs of both ATP (a-32P8-azidoATP) and drug substrates (125I-Iodoarylazidoprazosin and 3H-Azidopine) to Cdr1p in a concentration-dependent manner. Consistent with these findings, a non-toxic low concentration (1 mM) of disulfiram makes the Cdr1p expressing S. cerevisiae cells more susceptible to antifungal agents such as cycloheximide, fluconazole, miconazole and nystatin. Collectively, our results demonstrate that disulfiram modulates function of drug transporters by interaction with both ATP and substrate-binding sites and thus dithiocarbamates may provide a useful molecular scaffold for developing novel interventions against resistance-mediated by both fungal and mammalian ABC drug transporters.2.Characterization of human MRP4 (ABCC4) and MRP8 (ABCC11): Multidrug Resistance Protein 4 (MRP4/ABCC4), transports cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents and prostaglandins. We have characterized ATP hydrolysis by human MRP4 expressed in insect cells. MRP4 hydrolyzes ATP (Km, 0.62 mM), which is inhibited by orthovanadate and beryllium fluoride. However, unlike ATPase activity of P-glycoprotein, which is equally sensitive to both inhibitors, MRP4-ATPase is more sensitive to beryllium fluoride than to orthovanadate. a-32P8azidoATP binds to MRP4 (concentration for half-maximal binding 3 mM) and is displaced by ATP or by its non-hydrolysable analog AMPPNP (concentrations for half-maximal inhibition 13.3 mM and 308 mM). MRP4 substrates, the prostaglandins E1 and E2, stimulate ATP hydrolysis 2 to 3-fold but do not affect the Km for ATP. Several other substrates, azidothymidine, 9-(2-phosphonyl-methoxyethyl) adenine and methotrexate do not stimulate ATP hydrolysis but inhibit prostaglandin E2-stimulated ATP hydrolysis. Although both post-hydrolysis transition states MRP4a-32P8azidoADPVi and MRP4a-32P8azidoADPBeFx can be generated, nucleotide trapping is 4-fold higher with beryllium fluoride. The divalent cations Mg2+ and Mn2+ support comparable levels of nucleotide binding, hydrolysis and trapping. However, Co2+ increases a-32P8azidoATP binding and beryllium fluoride-induced a-32P8azidoADP trapping but does not support steady state ATP hydrolysis. ADP inhibits basal and prostaglandin E2-stimulated ATP hydrolysis (concentrations for half-maximal inhibition 0.19 and 0.25 mM, respectively) and beryllium fluoride-induced a-32P8azidoADP trapping while Pi has no effect up to 20 mM. In aggregate, our results demonstrate that MRP4 exhibits substrate-stimulated ATP hydrolysis and we propose a kinetic scheme suggesting that ADP release from the post-hydrolysis transition state may be the rate-limiting step during the catalytic cycle. Recently, two new members of the MRP subfamily have been identified (MRP8 and MRP9). We have expressed human MRP8 in baculovirus-infected High Five insect cells. The initial results indicate that MRP8, similar to Pgp and MRP1, exhibits substrate-stimulated ATPase activity. Such studies will help us to understand the function of these MRP subfamily members and their role in the development of multidrug resistance in cancer cells.3.Resolution of two- and three-dimensional structure of human Pgp: The high-resolution structure of Pgp at various stages during the catalytic cycle will be essential to understand the transport mechanism. This is one of our major interests and we have invested considerable effort in the past to develop methods for obtaining pure and active Pgp in large amounts.