Cloning and characterization of a novel functional organic anion transporting polypeptide 3A1 isoform highly expressed in the human brain and testis.

Organic anion transporting polypeptide 3A1 (OATP3A1, encoded by the SLCO3A1 gene) is a prostaglandin, oligopeptide, and steroid/thyroid hormone transporter with wide tissue distribution, expressed, e.g., in the human brain and testis. Although the physiological importance of OATP3A1 has not yet been clarified, based on its expression pattern, substrate recognition, and evolutionary conservation, OATP3A1 is a potential pharmacological target. Previously, two isoforms of OATP3A1, termed as V1 and V2, have been characterized. Here, we describe the cloning and functional characterization of a third isoform, OATP3A1_V3. The mRNA of isoform V3 is formed by alternative splicing and results in an OATP3A1 protein with an altered C-terminus compared to isoforms V1 and V2. Based on quantitative PCR, we demonstrate the widespread expression of SLCO3A1_V3 mRNA in human organs, with the highest expression in the brain and testis. By generation of an isoform V3-specific antibody and immunostaining, we show that the encoded protein is expressed in the human choroid plexus, neurons, and both germ and Sertoli cells of the testis. Moreover, we demonstrate that in contrast to isoform V1, OATP3A1_V3 localizes to the apical membrane of polarized MDCKII cells. Using HEK-293 cells engineered to overexpress OATP3A1_V3, we verify the protein's functionality and identify dehydroepiandrosterone sulfate as a novel OATP3A1 substrate. Based on their distinct expression patterns but overlapping functions, OATP3A1 isoforms may contribute to transcellular (neuro)steroid transport in the central nervous system.

Two common polymorphic variants of OATP4A1 as potential risk factors for colorectal cancer.

Genetic variations in the organic-anion-transporting polypeptide (OATP)-encoding solute carrier of organic anions (SLCO) genes can promote cancer development and progression. The overexpression of solute carrier organic anion transporter family member 4A1 (OATP4A1), a transporter for steroid hormones, prostaglandins, and bile acids, has been previously associated with tumor recurrence and progression in colorectal cancer (CRC). Therefore, the present study aimed to investigate the association between 2 frequent single nucleotide polymorphisms (SNPs) in SLCO4A1 (rs34419428, R70Q; rs1047099G, V78I) and CRC predisposition. Following restriction fragment length polymorphism-PCR analysis in 178 patients with CRC [Union for International Cancer Control (UICC) stage I/II] and 65 healthy controls, no significant difference was observed in allele frequency and the number of heterozygous/homozygous individuals between the groups. Notably, the R70Q minor allele was identified to be associated with the V78I minor allele in the genome. Comparing of the individual genotypes of CRC patients to clinical data, including sex, UICC-stage and relapse revealed no increased risk for CRC. In addition, the OATP4A1 immunoreactivity assay in paraffin-embedded CRC and adjacent non-tumorous mucosa sections, examined using quantitative microscopy image analysis, did not reveal any association with these polymorphisms. No significant differences were observed in the expression levels, localization, and sodium fluorescein transport capacity among the OATP4A1 variants, which was studied using functional assays in Sf9-insect and A431 tumor cells overexpressing the 2 single and a double mutant OATP4A1 SNP variants. These results suggested that the 2 most frequent polymorphisms located in the first intracellular loop of OATP4A1 do not associate with CRC predisposition and tumor recurrence. They are unlikely to affect the outcome of CRC in patients.

Synergistic transport of a fluorescent coumarin probe marks coumarins as pharmacological modulators of Organic anion-transporting polypeptide, OATP3A1.

Organic anion-transporting polypeptide 3A1 (OATP3A1) is a membrane transporter mediating the cellular uptake of various hormones such as estrone-3-sulfate, prostaglandins E1 and E2 and thyroxine. OATP3A1 is widely expressed in the human body and its presence in tissue-blood barriers, neurons and muscle cells marks it as a potential pharmacological target. Herein we demonstrate that an otherwise membrane impermeant, zwitterionic fluorescent coumarin probe, bearing a sulfonate function is a potent substrate of human OATP3A1, thus readily transported into HEK-293-OATP3A1 cells allowing functional investigation and the screen of drug interactions of the OATP3A1 transporter. At the same time, dyes lacking either the sulfonate motif or the coumarin scaffold showed a dramatic decrease in affinity or even a complete loss of transport. Furthermore, we observed a distinct inhibition/activation pattern in the OATP3A1-mediated uptake of closely related fluorescent coumarin derivatives differing only in the presence of the sulfonate moiety. Additionally, we detected a synergistic effect between one of the probes tested and the endogenous OATP substrate estrone-3-sulfate. These data, together with docking results indicate the presence of at least two cooperative substrate binding sites in OATP3A1. Besides providing the first sensitive probe for testing OATP3A1 substrate/inhibitor interactions, our results also help to understand substrate recognition and transport mechanism of the poorly characterized OATP3A1. Moreover, coumarins are good candidates for OATP3A1-targeted drug delivery and as pharmacological modulators of OATP3A1.

A novel fluorescence-based functional assay for human OATP1A2 and OATP1C1 identifies interaction between third-generation P-gp inhibitors and OATP1A2.

Organic anion-transporting polypeptide 1A2 (OATP1A2), expressed in the human blood-brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system-targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell-impermeable fluorescent dyes was screened in HEK-293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live-or-Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third-generation P-glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2-expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence-based assays developed in the current study are a good alternative of radioligand-based tests and pave the way toward high-throughput screens for OATP1A2/1C1 drug interaction studies.

Identification of novel cell-impermeant fluorescent substrates for testing the function and drug interaction of Organic Anion-Transporting Polypeptides, OATP1B1/1B3 and 2B1.

Organic Anion-Transporting Polypeptides are multispecific membrane proteins that regulate the passage of crucial endobiotics and drugs across pharmacological barriers. OATP1B1 and OATP1B3 have been described to play a major role in the hepatic uptake of statins, antivirals and various chemotherapeutics; whereas the pharmacological role of the ubiquitously expressed OATP2B1 is less well characterized. According to current industry standards, in vitro testing for susceptibility to OATP1B1 and 1B3 mediated transport is recommended for drug candidates that are eliminated in part via the liver. Here we show that human OATP1B1, 1B3 and 2B1 transport a series of commercially available viability dyes that are generally believed to be impermeable to intact cells. We demonstrate that the intracellular accumulation of Zombie Violet, Live/Dead Green, Cascade Blue and Alexa Fluor 405 is specifically increased by OATPs. Inhibition of Cascade Blue or Alexa Fluor 405 uptake by known OATP substrates/inhibitors yielded IC50 values in agreement with gold-standard radioligand assays. The fluorescence-based assays described in this study provide a new tool for testing OATP1B/2B1 drug interactions.

Functional expression of the 11 human Organic Anion Transporting Polypeptides in insect cells reveals that sodium fluorescein is a general OATP substrate.

Organic Anion Transporting Polypeptides (OATPs), encoded by genes of the Solute Carrier Organic Anion (SLCO) family, are transmembrane proteins involved in the uptake of various compounds of endogenous or exogenous origin. In addition to their physiological roles, OATPs influence the pharmacokinetics and drug-drug interactions of several clinically relevant compounds. To examine the function and molecular interactions of human OATPs, including several poorly characterized family members, we expressed all 11 human OATPs at high levels in the baculovirus-Sf9 cell system. We measured the temperature- and inhibitor-sensitive cellular accumulation of sodium fluorescein and fluorescein-methotrexate, two fluorescent substrates of the OATPs, OATP1B1 and 1B3. OATP1B1 and 1B3 were functional in Sf9 cells, showing rapid uptake (t1/2(fluorescein-methotrexate) 2.64 and 4.16 min, and t1/2(fluorescein) 6.71 and 5.58 min for OATP1B1 and 1B3, respectively) and high-affinity transport (Km(fluorescein-methotrexate) 0.23 and 0.53 µM, and Km(fluorescein) 25.73 and 38.55 µM for OATP1B1 and 1B3, respectively) of both substrates. We found that sodium fluorescein is a general substrate of all human OATPs: 1A2, 1B1, 1B3, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1, while fluorescein-methotrexate is only transported by 1B1, 1B3, 1A2 and 2B1. Acidic extracellular pH greatly facilitated fluorescein uptake by all OATPs, and new molecular interactions were detected (between OATP2B1 and Imatinib, OATP3A1, 5A1 and 6A1 and estradiol 17-ß-d-glucuronide, and OATP1C1 and 4C1 and prostaglandin E2). These studies demonstrate, for the first time, that the insect cell system is suitable for the functional analysis of the entire human OATP family, and for drug-OATP interaction screening.