Immobilized Drug Transporters

膜转运蛋白可能在几种药物的药代动力学（在正常组织中表达时）和/或对几种抗癌药物的耐药性（在癌症中表达时）中发挥作用。这些蛋白中有几个属于ABC （ATP结合盒）蛋白超家族，包括p -糖蛋白（Pgp, ABCB1）、多药耐药蛋白（MRP1, MRP2， BCRP, ABCC1）、米托沙酮耐药蛋白（MXR, ABCG2）、小管多特异性有机阴离子转运蛋白（cMOAT, ABCC2）、胆汁盐输出泵（BSEP, ABCB11）。其他转运蛋白，如有机阳离子转运蛋白（OCT1和OCT2）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。不同转运体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体功能的药物的一些副作用。例如，环孢素A已被临床测试为pgp引起的多药耐药抑制剂。它的副作用之一是黄疸，这可能是由于它干扰了cMOAT对共轭胆红素的转运。因此，转运蛋白抑制剂的临床疗效（无论是用于逆转抗癌耐药，还是用于调节药代动力学）都可以通过优化其转运蛋白选择性来提高。Pgp是目前研究最彻底的转运蛋白。Pgp通过将底物从细胞膜内转运到外小叶或细胞外空间，将底物从细胞外排。Pgp生物学的核心是其结合多种底物和抑制剂的能力，这表明可能存在多个结合位点。由于缺乏这些位点的定义和Pgp晶体结构的不可获得性，迄今为止阻碍了针对Pgp功能的药物的合理设计。我们提出细胞膜亲和层析（CMAC）可以作为表征不同转运体的结合位点和转运周期的手段，并最终确定有效的和蛋白质选择性的药物载体，用于药理抑制转运体功能。我们目前正在对Pgp的CMAC模型进行表征和优化。这一过程也将为其他ABC和非ABC转运体的建模提供一个原型。从稳定转染的细胞系（MDA435/LCC6MDR1人乳腺癌细胞）中获得含Pgp的膜，从MDA435/LCC6细胞系中获得不含Pgp的膜。将膜固定在100微米玻璃管表面，形成开放的管状柱，Pgp(+)- ot和Pgp(-)- ot。色谱柱放置在包含质谱仪的色谱系统中，使用已知的Pgp底物和已知不与Pgp结合的化合物进行快速正面色谱研究。结果表明，两柱色谱保留量（δ -时间）的差异可用于将化合物分类为Pgp底物。结果与Caco-2细胞渗透性等细胞方法获得的数据相关，该方法是一种快速，可重复性和准确的筛选Pgp底物的方法。

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. We have recently developed a series of column containing immobilized membranes obtained from mitochodria expressed in the U87 glioblastoma cell line. These columns are being characterized for the expression, content and function of ion and drug transporters contained within these membranes. These columns will then be used to study the binding of drugs known to affect mitochondrial function and the data compared to functional data. The goal is to develop a series of columns based upon human lymphocytes for use in the determination of disease status on mitochondrial function.