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, 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的方式将底物从细胞外排。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。我们建议亲和层析可以作为表征不同转运体的结合位点和转运周期的手段，并最终定义有效的和蛋白质选择性的药物载体，以药理抑制转运体的功能。我们目前正在表征和优化亲和层析模型的Pgp。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。色谱柱放置在含有质谱仪的色谱系统中，用已知的Pgp基态和已知不与Pgp结合的化合物进行快速正面层析研究。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。结果与细胞方法（如Caco-2细胞premeability）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。已获得表达和不表达人有机转运蛋白（hOCT1）的细胞系，并用于制备hOCT1(+)- iam和hOCT1(-)- oct色谱柱。用固定人工膜（IAM）液相色谱固定相表面固定不同细胞系的膜制备色谱柱。正面亲和层析表明，hOCT1(+)-IAM柱可用于鉴定hOCT1的底物和抑制剂。研究还表明，与hOCT1的相互作用具有对映选择性，并已被用于创建描述与hOCT1结合的抑制剂的药效团，并可用于在计算机上识别这些抑制剂。包含由单点突变产生的突变hOCT1转运蛋白的列已被创建，并用于表征这些突变对结合亲和力、选择性和运输的影响。开发了一种含有固定化有机阴离子转运体的初始柱，并对该柱进行了表征。

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, 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 cell membrane inner 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 affinity chromatography 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 affinity chromatography 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 chromatopgraphic system containing a mass spectrometer and rapid frontal corhomatographic studies were performed with known Pgp substates 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 correlatable with data obtained using cellular methods such as Caco-2 cell premeability and the method is a rapid, reproducible and accurate appraoch to screen for Pgp substrates. Cell lines which express and do not express the human organic transporter (hOCT1)have been obtained and used to prepare hOCT1(+)-IAM and hOCT1(-)-OCT 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 hOCT1(+)-IAM 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 createa pharmacophore that describes inhibitor binding to the hOCT1 and can be used to indentify these inhibitors in silico. 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 the column is being characterized.