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。其中一些属于ABC(三磷酸腺苷结合盒)蛋白超家族，包括P-糖蛋白(PGP，ABCB1)，多药耐药蛋白(MRP1，ABCC1)，米托蒽醌耐药蛋白(MXR，ABCG2)，小管多特异性有机阴离子转运体(cMOAT，ABCC2)，胆盐输出泵(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能解释了用于抑制特定转运体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引起的多药耐药的抑制剂。其副作用之一是黄疸，可能是由于干扰了cMOAT对结合胆红素的转运所致。因此，通过优化转运蛋白的选择性可以提高转运蛋白抑制剂的临床疗效(无论是用于逆转抗癌耐药性，还是用于调节药代动力学)。PGP是研究最深入的转运蛋白。PGP通过将其底物从细胞膜内部输送到外部小叶或细胞外空间而将其从细胞中排出。PGP生物学的核心是它能够结合多种不同的底物和抑制剂，这表明可能存在多个结合位点。到目前为止，缺乏对这些位点的定义和Pgp的晶体结构的缺乏，阻碍了针对Pgp功能的药物的合理设计。我们认为，亲和层析可以作为一种手段来表征不同转运蛋白的结合部位和转运周期，最终确定有效的、蛋白质选择性的药效团来抑制转运蛋白功能。目前，我们正在研究和优化Pgp的亲和层析模型。这一过程也将为其他ABC和非ABC运输者的建模提供一个原型。从Stable ey转基因细胞系(MDA435/LCC6MDR1人乳腺癌细胞)获得含有Pgp的膜，从MDA435/LCC6细胞系获得不含Pgp的膜)。这些膜被固定在100微米的直径表面。玻璃管形成开放的管状柱，PGP()-OT和PGP(-)-OT。这些色谱柱被放置在包含质谱仪的色谱系统中，并对已知的PGP亚状态和已知不与PGP结合的化合物进行快速正面同色谱研究。结果表明，两种柱色谱保留值的差值(增量时间)可以用来区分化合物为PGP底物。结果与Caco-2细胞易感性等细胞学方法获得的数据相关联，该方法是一种快速、重复性和准确性较高的筛选PGP底物的方法。已经获得了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柱。通过将从各自的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来制备柱。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的相互作用是对映体选择性的，并已被用于创建一个药效团，该药效团描述了与hOCT1结合的抑制剂，并可用于在硅胶中鉴定这些抑制剂。含有由单点突变产生的突变hOCT1转运体的柱已经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。已开发了一种含有固定化有机阴离子转运体的初始柱，并正在对该柱进行表征。

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 create a 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.