Immobilized Drug Transporters

膜转运蛋白可能在几种药物的药代动力学（当在正常组织中表达时）和/或对几种抗癌剂的耐药性（当在癌症中表达时）中起作用。这些蛋白质中的几种属于ABC（ATP结合盒）蛋白超家族，包括P-糖蛋白（Pgp，ABCB 1）、多药耐药蛋白（MRP 1，MRP 2，BCRP，ABCC 1）、米托蒽醌耐药蛋白（MXR，ABCG 2）、小管多特异性有机阴离子转运蛋白（cMOAT，ABCC 2）、胆盐输出泵（BSEP，ABCB 11）。其他转运蛋白，如有机阳离子转运蛋白（OCT 1和OCT 2）和有机阴离子转运蛋白（OAT）不属于ABC超家族。这些蛋白质似乎共享几种底物药物，可能包括一些抗癌药物。不同转运蛋白之间抗癌和非抗癌底物的部分共享可以解释用于抑制特定转运蛋白功能的药物的一些副作用。例如，环孢菌素A已被临床测试为Pgp引起的多药耐药性的抑制剂。其副作用之一是黄疸，这可能是由于其干扰cMOAT转运结合胆红素所致。因此，转运蛋白抑制剂的临床疗效（无论是用于逆转抗癌药物耐药性，还是用于调节药代动力学）可以通过优化其转运蛋白选择性来提高。Pgp是被研究得最透彻的转运蛋白。Pgp通过将其底物从细胞内膜转运到外叶或细胞外间隙而将其底物从细胞中排出。Pgp生物学的核心是它能够结合各种各样的底物和抑制剂，这表明可能存在多个结合位点。这些位点的定义的缺乏和Pgp的晶体结构的不可用性迄今阻碍了靶向Pgp功能的药物的合理设计。我们建议，细胞膜亲和层析（CMAC）可以被用来作为一种手段来表征结合位点和不同的转运蛋白的转运周期，并最终确定有效的和蛋白质选择性药效团的转运蛋白功能的药理学抑制。我们目前正在表征和优化Pgp的CMAC模型。这一过程还将为其他ABC和非ABC运输商的建模提供原型。从稳定转染的细胞系（MDA 435/LCC 6 MDR 1人乳腺癌细胞）获得含有Pgp的膜，并且从MDA 435/LCC 6细胞系获得不含Pgp的膜。 将膜固定在100微米i.d.玻璃管，形成开放的管状柱，Pgp（+）-OT和Pgp（-）-OT。 将色谱柱置于含有质谱仪的色谱系统中，并使用已知的Pgp底物和已知不与Pgp结合的化合物进行快速前沿色谱研究。 结果表明，两种色谱柱上的色谱保留差异（Δ时间）可用于将化合物分类为Pgp底物。 结果与使用细胞方法如Caco-2细胞渗透性获得的数据相关，并且该方法是筛选Pgp底物的快速、重复性和准确的方法。 来自用编码人多药耐药蛋白1（MRP 1）、多药耐药蛋白2（MRP 2）和乳腺癌耐药蛋白（BCRP）的cDNA转染的Sf 9昆虫细胞的细胞膜已被用于创建CMAC（MRP 1）、CMAC（MRP 2）和CMAC（BCPR）柱，并且来自未转染的Sf 9细胞系的膜已被用于创建CMAC（Sf 9）柱。 来自初始研究的数据表明，CMAC柱含有功能性转运蛋白，并且它们的柱可用于表征底物和抑制剂与固定化转运蛋白的相互作用。 将来自1321 N1星形细胞瘤细胞系的细胞膜固定化以产生CMAC（1321 N1）柱。 初步研究表明，该项目中开发的色谱方法可用于鉴定和区分该细胞系中表达的多种转运蛋白。 这些转运蛋白的性质和相互作用的全面表征正在进行中。 本研究的数据表明，人源性星形细胞瘤和胶质母细胞瘤细胞系表达Pgp、MRP 1和BCRP。 关键的观察结果是，Pgp和MRP 1在细胞膜以及核膜上表达。 后一个观察结果是第一次在核膜上鉴定出功能性ABC转运蛋白，并为克服这些肿瘤中的多药耐药性提供了新的关键靶点。使用胶质母细胞瘤组织的免疫组织化学证实了这些肿瘤中BCRP的存在。 已获得表达和不表达人有机转运蛋白（hOCT 1）的细胞系，并用于制备CMAC（hOCT 1（+））和CAMC（hOCT 1（-））色谱柱。通过将从各个细胞系获得的膜固定在固定化人工膜（IAM）液相色谱固定相的表面上来制备柱。 前沿亲和色谱已证明CMAC（hOCT 1（+））柱可用于鉴定hOCT 1的底物和抑制剂。 研究还表明，与hOCT 1的相互作用具有对映选择性，并已用于创建描述抑制剂与hOCT 1结合的药效团，并可用于通过计算机识别这些抑制剂。 已经创建了含有由单点突变产生的突变hOCT 1转运蛋白的CMAC柱，并用于表征这些突变对结合亲和力、选择性和转运的影响。已开发并表征了含有固定化有机阴离子转运体的初始柱。

Membrane transporter proteins may play a role both in the pharmacokinetics of several drugs (when expressed in normal tissues) and/or resistance to several anticancer agents (when expressed in cancers). Several of these proteins belong to the ABC (ATP Binding Cassette) protein superfamily, including, P-glycoprotein (Pgp, ABCB1), the Multidrug Resistance Protein (MRP1, MRP2, BCRP, ABCC1), the Mitoxantrone Resistance Protein (MXR, ABCG2), the canalicular Multispecific Organic Anion Transporter (cMOAT, ABCC2), the Bile Salt Export Pump (BSEP, ABCB11). Other transporter proteins, such the Organic Cation transporters (OCT1 and OCT2) and the Organic Anionic Transporter (OAT) do not belong to the ABC superfamily. These proteins seem to share several substrate drugs including, possibly, some anticancer agents. The partial sharing of anticancer and non-anticancer substrates between different transporters may explain some of the side effects of drugs used to inhibit the function of specific transporters. For example, Cyclosporin A has been tested clinically as an inhibitor of Pgp-caused multidrug resistance. One of its side effects is jaundice, which might be caused by its interference of the transport of conjugated bilirubin by cMOAT. So, the clinical efficacy of transporter inhibitors (whether used to reverse anticancer drug resistance, or to modulate pharmacokinetics) may be improved by optimizing their transporter selectivity. Pgp is the transporter that has been most thoroughly studied. Pgp effluxes its substrates from cells by transporting them from the inner cell membrane to the outer leaflet or to the extracellular space. Central to the biology of Pgp is its ability to bind a wide array of diverse substrates and inhibitors, suggesting the possible existence of multiple binding sites. The lack of definition of these sites and the unavailability of a crystal structure for Pgp have so far hindered a rational design of drugs targeting Pgp function. We propose that cellular membrane affinity chromatography (CMAC)can be used as a means to characterize the binding sites and transport cycle of the different transporters and, ultimately, to define effective and protein-selective pharmacophores for the pharmacological inhibition of transporter function. We are presently characterizing and optimizing CMAC models of Pgp. This process will also provide a prototype for the modeling of other ABC and non-ABC transporters. Membranes containing Pgp were obtained from a stabley trnasfected cell line (MDA435/LCC6MDR1 human breast cancer cells)and membranes which do not contain Pgp were obtained from the MDA435/LCC6 cell line). The membranes were immobilized on the surface of a 100 micron i.d. glass tube to form open tubnular columns, Pgp(+)-OT and Pgp(-)-OT. The columns were placed in a chromatographic system containing a mass spectrometer and rapid frontal chromatographic studies were performed with known Pgp substrates and compounds known not to bind to Pgp. The results indicate that the difference in the chromatographic retention on the two columns (the delta-time)can be used to classify compounds as Pgp substrates. The results are correlated with data obtained using cellular methods such as Caco-2 cell permeability and the method is a rapid, reproducible and accurate appraoch to screen for Pgp substrates. Cellular membranes from Sf9 insect cells transfected with cDNA coding the human multidrug resistance protein 1 (MRP1), multidrug resistance protein 2 (MRP2) and the breast cancer resistance protein (BCRP) have been utilized to create CMAC(MRP1), CMAC(MRP2) and CMAC(BCPR)columns and membranes from the non-transfected Sf9 cell line have been used to create CMAC(Sf9) columns. The data from the initial studies indicate that the CMAC columns contain functioning transporters and that they columns can be used to characterize the interactions of substrates and inhibitors with the immobilized transporters. Cellular membranes from a 1321N1 astrocytoma cell line have been immobilized to create a CMAC(1321N1) column. The initial studies demonstrate that the chromatographic approach developed in this project can be used to identify and distinguish between the multiple transporters expressed in this cell line. Full characterization of the properties and interactions of these transporters in underway. The data from this study indicate that human-derived astrocytoma and glioblastoma cell lines express Pgp, MRP1 and BCRP. The key observation is that Pgp and MRP1 are expressed on both the cellular membrane as well as the nuclear membrane. The latter observation is the first time that functional ABC transporters have been identified on the nuclear membrane and provide a new and key target for over coming multiple drug resistance in these tumors. The presence of BCRP in these tumors was confirmed using immunohistochemistry on tissues from glioblastoma tumors. Cell lines which express and do not express the human organic transporter (hOCT1)have been obtained and used to prepare CMAC(hOCT1(+))and CAMC(hOCT1(-))columns. The columns were prepared by immobilizing membranes obtained from the respective cell lines on the surface of immobilized artificial membrane (IAM) liquid chromatography stationary phases. Frontal affinity chromatography has demonstrated that the CMAC(hOCT1(+))column can be used to identify substrates and inhibitors of the hOCT1. The studies have also demonstrated that interactions with the hOCT1 are enantioselective and has been used to create a pharmacophore that describes inhibitor binding to the hOCT1 and can be used to identify these inhibitors in silico. CMAC columns containing mutated hOCT1 transporters created by single point mutations have been created and used to characterize the effects of these mutations on binding affintiy, selectivity and transport. An initial column containing an immobilized organic anion transporter has been developed and characterized.