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Structural Variation Governs Substrate Specificity for Organic Anion Transporter (OAT) Homologs

2007; Elsevier BV; Volume: 282; Issue: 33 Linguagem: Inglês

10.1074/jbc.m703467200

1083-351X

Gregory Kaler, David M. Truong, Akash Khandelwal, Megha Nagle, Satish A. Eraly, Peter W. Swaan, Sanjay K. Nigám,

Computational Drug Discovery Methods

Organic anion transporters (OATs, SLC22) interact with a remarkably diverse array of endogenous and exogenous organic anions. However, little is known about the structural features that determine their substrate selectivity. We examined the substrate binding preferences and transport function of olfactory organic anion transporter, Oat6, in comparison with the more broadly expressed transporter, Oat1 (first identified as NKT). In analyzing interactions of both transporters with over 40 structurally diverse organic anions, we find a correlation between organic anion potency (pKi) and hydrophobicity (logP) suggesting a hydrophobicity-driven association with transporter-binding sites, which appears particularly prominent for Oat6. On the other hand, organic anion binding selectivity between Oat6 and Oat1 is influenced by the anion mass and net charge. Smaller mono-anions manifest greater potency for Oat6 and di-anions for Oat1. Comparative molecular field analysis confirms these mechanistic insights and provides a model for predicting new OAT substrates. By comparative molecular field analysis, both hydrophobic and charged interactions contribute to Oat1 binding, although it is predominantly the former that contributes to Oat6 binding. Together, the data suggest that, although the three-dimensional structures of these two transporters may be very similar, the binding pockets exhibit crucial differences. Furthermore, for six radiolabeled substrates, we assessed transport efficacy (Vmax) for Oat6 and Oat1. Binding potency and transport efficacy had little correlation, suggesting that different molecular interactions are involved in substrate binding to the transporter and translocation across the membrane. Substrate specificity for a particular transporter may enable design of drugs for targeting to specific tissues (e.g. olfactory mucosa). We also discuss how these data suggest a possible mechanism for remote sensing between OATs in different tissue compartments (e.g. kidney, olfactory mucosa) via organic anions. Organic anion transporters (OATs, SLC22) interact with a remarkably diverse array of endogenous and exogenous organic anions. However, little is known about the structural features that determine their substrate selectivity. We examined the substrate binding preferences and transport function of olfactory organic anion transporter, Oat6, in comparison with the more broadly expressed transporter, Oat1 (first identified as NKT). In analyzing interactions of both transporters with over 40 structurally diverse organic anions, we find a correlation between organic anion potency (pKi) and hydrophobicity (logP) suggesting a hydrophobicity-driven association with transporter-binding sites, which appears particularly prominent for Oat6. On the other hand, organic anion binding selectivity between Oat6 and Oat1 is influenced by the anion mass and net charge. Smaller mono-anions manifest greater potency for Oat6 and di-anions for Oat1. Comparative molecular field analysis confirms these mechanistic insights and provides a model for predicting new OAT substrates. By comparative molecular field analysis, both hydrophobic and charged interactions contribute to Oat1 binding, although it is predominantly the former that contributes to Oat6 binding. Together, the data suggest that, although the three-dimensional structures of these two transporters may be very similar, the binding pockets exhibit crucial differences. Furthermore, for six radiolabeled substrates, we assessed transport efficacy (Vmax) for Oat6 and Oat1. Binding potency and transport efficacy had little correlation, suggesting that different molecular interactions are involved in substrate binding to the transporter and translocation across the membrane. Substrate specificity for a particular transporter may enable design of drugs for targeting to specific tissues (e.g. olfactory mucosa). We also discuss how these data suggest a possible mechanism for remote sensing between OATs in different tissue compartments (e.g. kidney, olfactory mucosa) via organic anions. The proximal tubule of the mammalian kidney rapidly clears a very large number of structurally diverse small organic anions from the circulation (1Dantzler W.H. Wright S.H. Biochim. Biophys. Acta. 2003; 1618: 185-193Crossref PubMed Scopus (70) Google Scholar). Although several organic anion transporter proteins (OATs) 2The abbreviations used are: OAT, organic anion transporter; CoMFA, comparative molecular field analysis; ES, estrone sulfate; PAH, p-aminohippurate; QSAR, quantitative structure-activity relationship; PLS, partial least square; CoMFA, comparative molecular field analysis. are expressed in the kidney (2Eraly S.A. Bush K.T. Sampogna R.V. Bhatnagar V. Nigam S.K. Mol. Pharmacol. 2004; 65: 479-487Crossref PubMed Scopus (71) Google Scholar), it appears, on the basis of in vivo studies in knock-out mice, that among these, Oat1, is responsible for the bulk of classical renal secretion of organic anions (3Eraly S.A. Vallon V. Vaughn D.A. Gangoiti J.A. Richter K. Nagle M. Monte J.C. Rieg T. Truong D.M. Long J.M. Barshop B.A. Kaler G. Nigam S.K. J. Biol. Chem. 2006; 281: 5072-5083Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). There are numerous studies reporting the interaction of Oat1 with diverse groups of substrates, particularly various categories of drugs (e.g. nonsteroidal anti-inflammatory drugs, β-lactam antibiotics, and diuretics, reviewed in Ref. 4Burckhardt B.C. Burckhardt G. Rev. Physiol. Biochem. Pharmacol. 2003; 146: 95-158Crossref PubMed Scopus (263) Google Scholar); however, there has not been a comprehensive examination of substrate preferences within a single study or clear mechanistic insight into the structural requirements for transport by OATs. Oat6 is a close phylogenetic relation of Oat1, first identified as NKT (5Lopez-Nieto C.E. You G. Barros E.J.G. Beier D.R. Nigam S.K. J. Am. Soc. Nephrol. 1996; 7: 1301Google Scholar, 7Lopez-Nieto C.E. You G. Bush K.T. Barros E.J. Beier D.R. Nigam S.K. J. Biol. Chem. 1997; 272: 6471-6478Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 8Sweet D.H. Wolff N.A. Pritchard J.B. J. Biol. Chem. 1997; 272: 30088-30095Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). 3C. E. Lopez-Nieto, GenBank™ accession number MMU528. Unique among the OATs, it appears absent from the kidney but is instead expressed in olfactory mucosa (9Monte J.C. Nagle M.A. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2004; 323: 429-436Crossref PubMed Scopus (94) Google Scholar, 10Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (58) Google Scholar). In addition to different tissue localization, substrate discrimination between the two transporters (10Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (58) Google Scholar) may reflect their distinctive physiological functions. Both Oat1 and Oat6 interact (though with different affinity) with organic anions implicated in urinary odor-type specification (i.e. those contributing to the urinary odors and thus helping mice distinguish different individuals) (3Eraly S.A. Vallon V. Vaughn D.A. Gangoiti J.A. Richter K. Nagle M. Monte J.C. Rieg T. Truong D.M. Long J.M. Barshop B.A. Kaler G. Nigam S.K. J. Biol. Chem. 2006; 281: 5072-5083Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 10Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (58) Google Scholar). This raises the possibility that odorants secreted into the urine via one OAT might be absorbed and/or sensed in the olfactory mucosa via another OAT (10Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (58) Google Scholar). Furthermore, Oat6 or similar transporters might mediate nasal absorption of some of the organic anionic drugs that are also substrates for other SLC22 family transporters. This could allow for intranasal administration as an alternative route of drug delivery that might bypass the blood-brain barrier, suggesting the possibility of selective drug targeting for enhanced brain delivery (11Illum L. Eur. J. Pharm. Sci. 2000; 11: 1-18Crossref PubMed Scopus (778) Google Scholar, 12Bagger M.A. Bechgaard E. Eur. J. Pharm. Sci. 2004; 21: 235-242Crossref PubMed Scopus (52) Google Scholar, 13Vyas T.K. Shahiwala A. Marathe S. Misra A. Curr. Drug Deliv. 2005; 2: 165-175Crossref PubMed Scopus (211) Google Scholar). To characterize the multispecificity of OATs, two kinds of substrate-transporter interaction should be analyzed separately as follows: 1) interactions leading to the substrate association with the transporter-binding site, and 2) interactions of the bound substrate within the binding site that affect transporter conformational dynamics and substrate translocation. Previous studies by Ullrich (14Ullrich K.J. J. Membr. Biol. 1997; 158: 95-107Crossref PubMed Scopus (129) Google Scholar), performed on whole kidney proximal tubules prior to the cloning and identification of the key transporters expressed at renal as well as extrarenal sites, assumed specific interactions between hydrophobic moieties of the substrate with particular hydrophobic residues within the transporter channel; furthermore, optimal substrates contained two carboxylates spaced 6–7 Å apart. Aside from this work, which is focused on the whole kidney, no comprehensive studies have been carried out to clarify chemical space requirements for substrate specificity. Given the tremendous pharmacological relevance of the OATs, as well as their role in handling key metabolites in many different cells and tissues, it is important to define the structural basis for substrate preferences. To address this issue, we have performed a detailed wet lab and computational characterization of the substrate binding preferences and transport function of Oat6 in comparison to those of Oat1, testing over 40 compounds for their interaction with and/or transport by these OATs. We first analyzed correlations of the binding preferences of these compounds with their one-dimensional property activities (hydrophobicity, net charge, and molecular mass). Next, we extended these analyses by determining the three-dimensional quantitative structure-activity relationships (QSAR) of Oat1 and Oat6 binding using comparative molecular field analysis (CoMFA). In three-dimensional QSAR, differences in observed biological properties are correlated with determinations made by placing ligands in a three-dimensional field box and evaluating the electrostatic (Coulombic interactions) and steric (van der Waals interactions) fields at regularly spaced grid points. This approach has been used with much success on other transporters (15Suhre W.M. Ekins S. Chang C. Swaan P.W. Wright S.H. Mol. Pharmacol. 2005; 67: 1067-1077Crossref PubMed Scopus (91) Google Scholar) and in fact can be used to predict the affinity of a ligand for a transporter. Our experimental data together with computational analyses strongly support several novel conclusions regarding the structural determinants of OAT substrate selectivity, helping to explain the difference in Oat1 versus Oat6 substrate preferences and presumably reflecting specific differences in the transporter binding pockets. We find substrate potency (pKi) to be correlated with hydrophobicity (logP) mainly in the case of Oat6, suggesting a hydrophobicity-driven association of organic anions with the transporter-binding site. In contrast, substrate binding to Oat1 involves roughly equal contributions from hydrophobic and electrostatic interactions. Differences in substrate selectivity between Oat6 and Oat1 are influenced by ligand size and charge, rather than hydrophobicity, with smaller mono-anions manifesting a higher affinity for Oat6 and di-anions for Oat1. In addition, we determined the maximum uptake rates (Vmax) of six radiolabeled substrates for both transporters. Little correlation was found between Ki and Vmax, suggesting that for these transporters different substrate characteristics influence binding to the transporter versus actual translocation across the membrane. The data suggest ways of targeting drugs to specific OATs and also a mechanism for remote sensing, via endogenous organic anions, between OAT-expressing tissue compartments. Organic Anions (OAT Substrates and Inhibitors)—3H-Labeled OAT substrates, p-[3H]aminohippurate (PAH, specific activity 4.2 Ci/mmol) and [3H]estrone 3-sulfate (ES, 57 Ci/mmol) were purchased from PerkinElmer Life Sciences; [3H]ochratoxin A (15 Ci/mmol), [3H]methotrexate (20 Ci/mmol), [3H]ibuprofen (0.5 Ci/mmol), and [3H]prostaglandin E2 (193.5 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). Unlabeled organic anions (potential substrates/inhibitors) were obtained from Sigma as free acids or sodium salts, and their stock solutions (100 mm) were prepared and adjusted to pH 7.4 as described previously (10Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (58) Google Scholar). LogP values (octanol/water partition coefficients) of the organic anions were calculated using Molinspiration software. Uptake in Xenopus Oocytes—Capped RNA was synthesized from linearized plasmid DNA (mOat6, Image clone ID 6309674; mOat1, Image clone ID 4163278) using mMessage mMachine in vitro transcription kit (Ambion, Inc., Austin, TX). Xenopus oocyte uptake assays were performed as described previously (10Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (58) Google Scholar). Briefly, oocytes were isolated and maintained in Barth's buffer (88 mm NaCl, 1 mm KCl, 0.33 mm Ca(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4, 2.4 mm NaHCO3, and 10 mm HEPES, pH 7.4) supplemented with 5% fetal horse serum, 0.05 mg/ml gentamycin sulfate, and 2.5 mm sodium pyruvate. The day after oocytes were harvested, cRNA solution (mOat6 or mOat1, 1 μg/μl) was injected into oocytes (23 nl/oocyte) using the Nanoliter 2000 nanoinjector (World Precision Instruments, Sarasota, FL). Two days after injection, oocytes were washed in serum-free Barth's buffer, and experimental groups of 20–30 oocytes each were placed in wells of a 24-well plate with 1 ml of Barth's buffer containing 1 μCi of a 3H-labeled tracer ion and an unlabeled organic anion (tracer uptake inhibitor), with no inhibitor in a control group. After 1 h of incubation at room temperature, oocytes were washed three times with ice-cold Barth's buffer, and each experimental group was divided into four samples of 5–8 oocytes, and radioactivity was measured by scintillation counting. Transport activity was calculated for [3H]-labeled tracers as tracer clearance from the incubation medium (CL = Vtransport/S), by dividing the amount of tracer absorbed per oocyte per unit time (Vtransport, cpm/oocyte/h) by the tracer concentration in the incubation medium (S, cpm/μl). In all experiments, the background (non-OAT-mediated) tracer uptake was measured in uninjected oocytes (in preliminary experiments, uptake in water-injected and uninjected oocytes was not found to be significantly different). This background uptake (probably combining nonsaturable tracer diffusion and endogenous transport expressed by the oocytes) was subtracted from the uptake measured in injected oocytes to calculate the OAT-mediated component of uptake. In a typical experiment, the control (uninhibited) transporter-mediated tracer clearance was 0.35 to 0.9 μl/oocyte/h for [3H]ES uptake in Oat6-injected oocytes and 0.7–2.0 μl/oocyte/h for [3H]PAH uptake in Oat1-injected oocytes. This indicates that the percentage of tracer that is transported in 1 h did not exceed 6% (assuming maximum experimental group size of 30 oocytes and maximum tracer clearance of 2.0 μl/oocyte/h), so that the tracer concentration was practically constant during the incubation time. In all inhibition experiments, the uninhibited OAT-mediated clearance exceeded the background clearance of the same tracer in uninjected oocytes by at least 10-fold. Calculations and Statistics—In each experimental group, tracer clearance was calculated as mean ± S.E. of quadruplicate samples (with exception of the control group that included eight samples). In uptake inhibition experiments, the OAT-mediated clearance (i.e. difference between cRNA-injected and uninjected oocytes) in the presence of an inhibiting organic anion was expressed as a percentage of the mean OAT-mediated clearance in the control group. To determine potencies of organic anions (tested as tracer uptake inhibitors), tracer uptake was measured in the presence of increasing inhibitor concentration. For each organic anion, a series of 3–4 concentrations in successive 10-fold increments was tested. The inhibition data were considered sufficient for curve-fitting when the inhibitor concentration points spanned the interval comprising 50% inhibition. The tracer uptake versus log[inhibitor] data were fit in the one-site competition equation incorporated in Prism 4.0 software (GraphPad Inc., San Diego, CA), and log(IC50) was calculated as mean ± S.E. The IC50 value was calculated as 10mean and standard error for IC50 as S.E.IC50 = 10mean – 10mean – S.E.. Analysis of the uptake inhibition of 3H-labeled tracer substrate (237 nm [3H]PAH in Oat1-injected oocytes or 17 nm [3H]ES in Oat6-injected oocytes) by "cold" PAH and ES yielded Kd values of 9.4 ± 1.1 and 58 ± 10 μm, respectively (10Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (58) Google Scholar). These results indicate that the concentrations of both tracers in our uptake inhibition experiments were far below their Kd values, so that analyzing inhibition data using the Cheng-Prusoff equation (Ki = IC50/(1 + [substrate]/Kd)) for all organic anions yielded Ki values not distinguishable from respective IC50 values (the difference being always significantly less than the standard error for IC50; data not shown). Based on these results, we used the calculated values of IC50 as inhibition constant (Ki) values and calculated organic anion potencies as pKi =–log(Ki). The maximum transporter-mediated uptake rate was determined for six 3H-labeled substrates in Oat6- and Oat1-injected oocytes as Vmax = CL × (S + Ki) (see under "Results" for details). Standard error for Vmax was calculated as S.E.Vmax = (S.E.CL2 × (S + Ki)2 + S.E.Ki2 × CL2)1/2. Molecular Modeling and Alignment—Three-dimensional structure building was performed using SYBYL 7.1 (Sybyl). Energy minimizations were performed using the Tripos force field (17Clark M. Cramer R.D. OpdenBosch N. J. Comput. Chem. 1989; 10: 982-1012Crossref Scopus (2872) Google Scholar) and Gasteiger-Huöckel charges with distance-dependent dielectrics and the conjugate gradient method with a convergence criterion of 0.001 kcal/mol. The most important requirement for CoMFA (18Cramer R.D. Patterson D.E. Bunce J.D. J. Am. Chem. Soc. 1988; 110: 5959-5967Crossref PubMed Scopus (4148) Google Scholar) is the structural overlap of the molecules to be analyzed to a suitable template, which is assumed to be a "bioactive conformation." The three-dimensional coordinates of methotrexate were obtained from the Protein Data Bank (access code 1RX3) (19Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (28983) Google Scholar) and used as a template for the superimposition of all other molecules using the "Flexible Superposition" option in the FlexS (20Lemmen C. Lengauer T. Klebe G. J. Med. Chem. 1998; 41: 4502-4520Crossref PubMed Scopus (222) Google Scholar) module of Sybyl. CoMFA Three-dimensional QSAR Models—CoMFA explains the gradual changes in observed biological properties by evaluating the electrostatic (Coulombic interactions) and steric (van der Waals interactions) fields at regularly spaced grid points surrounding a set of mutually aligned ligands for a specific target protein. A statistical algorithm, partial least square (PLS), was used to correlate the field descriptors with biological activities (i.e. pKi). The standard CoMFA settings were applied for developing Oat1, Oat6, and selectivity models (i.e. pKi(Oat6) – pKi(Oat1); see Table 3). The global model contains 28 molecules in training set (19 mono-anions and 9 di-anions) and 7 molecules in test set (5 mono-anions and 2 di-anions). The pKi values of 2-hydroxy-3-methyl butyrate, 2-methyl butyrate, 3-hydroxy butyrate, and 2-ethyl hexanoate were tested using racemic mixtures; hence, their activities were predicted as separate enantiomers. The aligned molecules were placed inside a three-dimensional cubic lattice box with grid spacing of 2.0 Å in x, y, and z directions. The CoMFA steric and electrostatic descriptors were calculated at each grid point using an sp3 hybridized carbon atom probe with +1 charge at 1.52 Å van der Waals radius. The cutoff value for steric and electrostatic fields was set to +30 kcal/mol. To derive CoMFA models, pKi values were used as the dependent variables, and CoMFA (steric and electrostatics) and logP values were used as the independent variables in PLS analysis. The predictive ability of the model was evaluated using leave-one out (LOO) cross-validation studies (r2cv) and the predictive residual sum of squares (PRESS) of the test set. The optimum number of components from r2cv was used for the final PLS model and calculating the conventional r2 of the model. The r2cv was calculated using Equation 1, rcv2=1−Σ(Yobserved−Ypredictedjbc2/Σ(Yobserved−Ymeanjbc21 where Ypredicted, Yobserved, and Ymean are the predicted, observed, and mean pKi values, respectively. The numerator in Equation 1 indicates PRESS, which is the difference of squared deviations between predicted and observed bioactivity values for molecules in the test set.TABLE 3Statistical analysis of global modelsModel no.r2cvaCross-validated correlation coefficient is shown.NbOptimum number of components was obtained from cross-validated PLS analysis.r2cNoncross-validated correlation coefficient is shown.SEEdStandard error of estimate is shown.PRESSePredictive residual sum of squares of test set is shown.FfF test value is shown.ContributionsgField contributions are as follows: steric, electrostatic, and logP contributions from CoMFA.StericElectrostaticsLogP%%%10.64340.9690.2437.766177.50650.549.520.62230.9090.3023.78880.01940.159.930.56030.9000.45517.74272.7449.051.040.68950.9720.2348.219153.25447.144.98.050.63830.9010.3154.76372.91930.248.121.760.51640.9230.40816.16969.20847.149.63.3a Cross-validated correlation coefficient is shown.b Optimum number of components was obtained from cross-validated PLS analysis.c Noncross-validated correlation coefficient is shown.d Standard error of estimate is shown.e Predictive residual sum of squares of test set is shown.f F test value is shown.g Field contributions are as follows: steric, electrostatic, and logP contributions from CoMFA. Open table in a new tab Inhibition of Tracer Uptake in Oat6- and Oat1-injected Oocytes—The ability of different organic anions to interact with Oat6 and Oat1 was assessed by inhibiting the transporter-mediated uptake of a radiolabeled tracer, [3H]ES or [3H]PAH, respectively, in Oat6-injected or Oat1-injected oocytes. A diverse collection of 41 organic anions (mono- and di-anionic) was examined, including endogenous metabolites previously identified as potential Oat1 substrates (4-hydroxyphenyl-pyruvate, 4-hydroxyphenyl-lactate, N-acetyl aspartate, and 3-hydroxybutyrate) (3Eraly S.A. Vallon V. Vaughn D.A. Gangoiti J.A. Richter K. Nagle M. Monte J.C. Rieg T. Truong D.M. Long J.M. Barshop B.A. Kaler G. Nigam S.K. J. Biol. Chem. 2006; 281: 5072-5083Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), anions of volatile organic acids described as odor-type components (propionate, 2- and 3-methyl butyrates, benzoate, heptanoate, and 2-ethyl hexanoate) (21Singer A.G. Beauchamp G.K. Yamazaki K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2210-2214Crossref PubMed Scopus (219) Google Scholar, 22Willse A. Belcher A.M. Preti G. Wahl J.H. Thresher M. Yang P. Yamazaki K. Beauchamp G.K. Anal. Chem. 2005; 77: 2348-2361Crossref PubMed Scopus (56) Google Scholar) and tested in our previous work (10Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (58) Google Scholar), as well as a number of anionic drugs and model compounds that are either described elsewhere as OAT substrates/inhibitors (4Burckhardt B.C. Burckhardt G. Rev. Physiol. Biochem. Pharmacol. 2003; 146: 95-158Crossref PubMed Scopus (263) Google Scholar, 14Ullrich K.J. J. Membr. Biol. 1997; 158: 95-107Crossref PubMed Scopus (129) Google Scholar) or are presumed to be OAT ligands based on their structural similarity to known OAT substrates or inhibitors. Fig. 1 shows the side-by-side comparison of the inhibition of Oat6- and Oat1-mediated uptake by different organic anions tested at a single concentration (in the range of 1 μm to 1 mm, depending on the organic anion potency). The majority of the organic anions in the study significantly inhibited radioactive tracer uptake through Oat6 and/or Oat1, when tested at up to 1 mm concentration. Of all organic anions tested, 16 displayed significantly stronger inhibition of Oat6, 15 inhibited Oat1 to a greater extent, and 10 showed no apparent preference for either transporter. To determine the potency of organic anions that exhibited submillimolar activity upon at least one of the two transporters, we examined the concentration-dependent inhibition of tracer uptake. Organic anions producing <50% inhibition of both Oat1- and Oat6-mediated transport at 1 mm (ascorbate, maleate, tartrate, taurocholate, cholate, and gluconate) (Fig. 1) were excluded from the potency assessment experiments. With the remaining 35 anions, concentration-dependent inhibition of tracer uptake was measured, and the potencies (apparent pKi values, see under "Experimental Procedures" for details) were calculated for both transporters. Fig. 2 shows representative examples of the concentration-dependent inhibition of tracer uptake by organic anions selective for Oat6 (benzoate and pyruvate) or for Oat1 (PAH and fluorescein), and by those displaying similar potency for the two transporters (probenecid and penicillin G). The summarized results, along with the structures of the organic anions, their molecular masses, and logP values (octanol/water partition coefficient, indicating hydrophobicity), are presented in Table 1.TABLE 1Molecular characteristics of the organic anions tested as inhibitors of OAT-mediated uptake, and the Ki values calculated from the concentration dependences of the tracer uptake inhibition in Oat6- and Oat1-injected oocytes(1) Chemical structures, MW and logP values are presented for neutral (protonated) form.(2) Estimated logP values are calculated using Molinspiration software (see "Materials and Methods").(3) Data from (9Monte J.C. Nagle M.A. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2004; 323: 429-436Crossref PubMed Scopus (94) Google Scholar). Open table in a new tab (1) Chemical structures, MW and logP values are presented for neutral (protonated) form. (2) Estimated logP values are calculated using Molinspiration software (see "Materials and Methods"). (3) Data from (9Monte J.C. Nagle M.A. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2004; 323: 429-436Crossref PubMed Scopus (94) Google Scholar). Plotting the pKi values of Oat6 against those for Oat1 (Fig. 3) demonstrates a correlation between the organic anion potencies for the two transporters. This correlation was found to be significant for both single-charged and double-charged anions. In addition, Fig. 3 reveals the generally higher potency of monoanions for Oat6 and of di-anions for Oat1. Physicochemical and Structural Determinants of the Oat6/Oat1 Affinity and Selectivity of Organic Anions—The data in Table 1 were analyzed to determine the one-dimensional quantitative property-activity relationship between the potency of organic anions for the OATs and the most common molecular features that impact the interaction of a small molecule with protein-binding sites (molecular mass, hydrophobicity, and net charge). The potencies of the organic anions (pKi values) plotted versus their molecular masses are presented in Fig. 4, A and B, as determined for Oat6 and Oat1, respectively. Although no correlation between organic anion potency and molecular mass was detected for mono-anions on Oat6 or for dianions with Oat1, a weak correlation was observed for di-anions on Oat6 (Fig. 4A) and a strong correlation for mono-anions with Oat1 (Fig. 4B) (in both cases, smaller anions tend to manifest lower potency). To verify the influence of the molecular mass of organic anions on their selectivity between the two transporters, we also analyzed the potency difference between the transporters, pKi(Oat6) – pKi(Oat1). This approach compensates for the structural features of organic anions that contribute equally to their interaction with both Oat1 and Oat6 and reveals the features distinguishing between the two transporte