title: Multi-level analysis of organic anion transporters 1, 3, and 6 reveals major differences in structural determinants of antiviral discrimination.
abstract: Long-term exposure to antivirals is associated with serious cellular toxicity to the kidney and other tissues. Organic anion transporters (OATs) are believed to mediate the cellular uptake, and hence cytotoxicity, of many antivirals. However, a systematic in vitro and ex vivo analysis of interactions between these compounds with various OAT isoforms has been lacking. To characterize substrate interactions with mOat1, mOat3, and mOat6, a fluorescence-based competition assay in Xenopus oocytes as well as wild-type and knock-out whole embryonic kidney (WEK) organ culture systems was developed using 6-carboxyfluorescein, 5-carboxyfluorescein, and fluorescein. Of nine common antiviral drugs assessed in oocytes, many manifested higher affinity for SLC22a6 (mOat1), originally identified as NKT (e.g. adefovir and cidofovir), two (ddC and ddI) manifested significantly higher affinity for mOat3, while mOat6 had comparatively low but measurable affinity for certain antivirals. A live organ staining approach combined with fluorescent uptake in WEK cultures allowed the visualization of OAT-mediated uptake ex vivo into developing proximal tubule-like structures, as well as quantification of substrate interactions of individual OAT isoforms. In general, antiviral specificity of SLC22a6 (Oat1) (in Oat3(-/-) WEK culture) and SLC22a8 (Oat3) (in Oat1(-/-) WEK culture) was consistent with the Xenopus oocyte data. The combined observations suggest SLC22a8 (Oat3) is the major transporter interacting with ddC and ddI. Finally, quantitative structure-activity relationship analysis of the nine antivirals' physicochemical descriptors with their OAT affinity indicates that antiviral preferences of mOat1 are explained by high polar surface areas (e.g. phosphate groups), whereas mOat3 prefers hydrogen bond acceptors (e.g. amines, ketones) and low rotatable bond numbers. In contrast, hydrogen bond donors (e.g. amides, alcohols) diminish binding to mOat6. This suggests that, despite sharing close overall sequence homology, Oat1, Oat3, and Oat6 have signficantly different binding pockets. Taken together, the data provide a basis for understanding potential drug interactions in combination antiviral therapy, as well as suggesting structural mdifications for drug design, especially in the context of targeting toward or away from specific tissues.
authors.affiliation: Department of Pediatrics, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Truong
authors.forename: Gregory
authors.initials: G
authors.lastname: Kaler
authors.forename: Akash
authors.initials: A
authors.lastname: Khandelwal
authors.forename: Peter W
authors.initials: PW
authors.lastname: Swaan
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Nigam
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry* / Q000494:pharmacology*; D002478:Cells, Cultured; D005260:Female; D006678:HIV / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000196:embryology / Q000378:metabolism; D007700:Kinetics; D027381:Liver-Specific Organic Anion Transporter 1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D015394:Molecular Structure; D009865:Oocytes; D027362:Organic Anion Transport Protein 1 / Q000235:genetics / Q000378:metabolism*; D029362:Organic Anion Transporters, Sodium-Independent / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D000998:Antiviral Agents; D027381:Liver-Specific Organic Anion Transporter 1; D027362:Organic Anion Transport Protein 1; D029362:Organic Anion Transporters, Sodium-Independent; C114338:organic anion transport protein 3