The liver and kidney excrete from the body a wide array of positively charged organic molecules of physiological, pharmacological and toxicological significance. The first step in the transepithelial secretion of the """"""""organic cations"""""""" (OCs) by tissues in the kidney and liver involves mediated OC uptake from the blood into cells, across the basolateral membrane. This process, the entry step in OC secretion, is mediated by members of the SLC22A family of transport proteins: OCT2 (in the kidney), and OCT1 (in the liver). OCTs are sites of clinically important drug-drug interactions, and genetic polymorphisms of these transporters have been shown to influence both the efficacy and pharmacokinetics of selected drugs. Development of programs for rational drug design will require an understanding of the structure of these proteins. During the course of the current grant cycle of this continuing research program we developed a homology model of OCT2 structure, based upon crystal structures of several related transporters from the Major Facilitator Superfamily of transport proteins. This model, along with models of other SLC22A transport proteins, has provided novel insight into relationships between transporter structure and function. However, confidence in the accuracy of these models will remain modest, at best, until structural and functional predictions based upon these models receive rigorous testing and validation. This proposal describes four sets of related studies that extend and expand our ongoing examination of the structure and function of the human ortholog of the organic cation transporter, OCT2. (1) Using site-directed mutagenesis and the substituted cysteine accessibility scanning method (SCAM), we will test the hypothesis that changes orientation of the transporter (from inward- to outward-facing) changes the profile of amino acid residues within the translocation pathway. (2) Proteomic methods (photoaffinity labeling and mass spectrometry) will identify regions in the cleft to which OC substrates bind. (2) The results of these experiments will be integrated into parallel studies employing computational methods, including the application of 3D-QSAR methods and molecular dynamics simulations, to refine the OCT2 model, predict conformational changes associated with the transport process, and characterize ligand interactions with putative binding surfaces. (4) Fluorescence Resonance Energy Transfer (FRET) will be used to test the hypothesis that oligomerization of OCT2 in the plasma membrane changes the affinity of the transporter for substrates. These studies will be essential for development of models that accurately predict and, ideally, preempt unwanted interactions of cationic drugs in both the kidney and liver.

Public Health Relevance

The kidney and liver actively secrete many drugs from the body, and unwanted drug-drug interactions at the sites of secretion in these organs are the source of substantial morbidity and mortality. The organic cation transporters, OCT1 and OCT2, mediate the first step in secretion of cationic drugs in the human liver and kidney, respectively. During the current cycle of the research program, we generated a model of the 3D structure of OCT2 that has been used to help identify specific sites in this protein that influence how drug molecules bind. In this proposal we outline experiments to validate and refine this model, and probe the structural changes of this protein associated with the transport process itself. The results of these studies will help predict and, ideally, preempt unwanted drug-drug interactions in both the kidney and liver, and can be expected to assist in development of programs of rational drug design.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK058251-10
Application #
7900580
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Ketchum, Christian J
Project Start
2000-08-15
Project End
2013-07-31
Budget Start
2010-08-01
Budget End
2011-07-31
Support Year
10
Fiscal Year
2010
Total Cost
$359,608
Indirect Cost
Name
University of Arizona
Department
Physiology
Type
Schools of Medicine
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Sandoval, Philip J; Zorn, Kimberley M; Clark, Alex M et al. (2018) Assessment of Substrate-Dependent Ligand Interactions at the Organic Cation Transporter OCT2 Using Six Model Substrates. Mol Pharmacol 94:1057-1068
Severance, Alyscia Cory; Sandoval, Philip J; Wright, Stephen H (2017) Correlation between Apparent Substrate Affinity and OCT2 Transport Turnover. J Pharmacol Exp Ther 362:405-412
He, Xiao; Garza, Denisse; Nigam, Sanjay K et al. (2016) Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2. PLoS One 11:e0152969
Martínez-Guerrero, L J; Evans, K K; Dantzler, W H et al. (2016) The multidrug transporter MATE1 sequesters OCs within an intracellular compartment that has no influence on OC secretion in renal proximal tubules. Am J Physiol Renal Physiol 310:F57-67
Shibayama, Takahiro; Morales, Mark; Zhang, Xiaohong et al. (2015) Unstirred Water Layers and the Kinetics of Organic Cation Transport. Pharm Res 32:2937-49
Ekins, Sean; Clark, Alex M; Wright, Stephen H (2015) Making Transporter Models for Drug-Drug Interaction Prediction Mobile. Drug Metab Dispos 43:1642-5
Pelis, Ryan M; Wright, Stephen H (2014) SLC22, SLC44, and SLC47 transporters--organic anion and cation transporters: molecular and cellular properties. Curr Top Membr 73:233-61
Belzer, Mathew; Morales, Mark; Jagadish, Bhumasamudram et al. (2013) Substrate-dependent ligand inhibition of the human organic cation transporter OCT2. J Pharmacol Exp Ther 346:300-10
Harper, Jaclyn N; Wright, Stephen H (2013) Multiple mechanisms of ligand interaction with the human organic cation transporter, OCT2. Am J Physiol Renal Physiol 304:F56-67
Pelis, Ryan M; Dangprapai, Yodying; Cheng, Yaofeng et al. (2012) Functional significance of conserved cysteines in the human organic cation transporter 2. Am J Physiol Renal Physiol 303:F313-20

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