Identification of Novel Substrates and StructureâActivity Relationship

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Identification of novel substrates and structure activity relationship of cellular uptake mediated by the human organic cation transporters 1 and 2 (hOCT1 and hOCT2). Ramon Hendrickx, Jenny G Johansson, Christina Lohmann, RoseMarie Jenvert, Anders Blomgren, Lena Börjesson, and Lena Gustavsson J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400966v • Publication Date (Web): 28 Aug 2013 Downloaded from http://pubs.acs.org on September 3, 2013

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Journal of Medicinal Chemistry

Identification of novel substrates and structure activity relationship of cellular uptake mediated by the human organic cation transporters 1 and 2 (hOCT1 and hOCT2)

Ramon Hendrickx, Jenny G. Johansson1, Christina Lohmann2, Rose-Marie Jenvert3, Anders Blomgren4, Lena Börjesson and Lena Gustavsson5*

Respiratory, Inflammation and Autoimmunity Innovative Medicines Unit, AstraZeneca R&D Mölndal, SE-431 83 Mölndal, Sweden

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ABSTRACT

Recently the clinical importance of human organic cation transporter1 (hOCT1/SLC22A1) and 2 (hOCT2/SLC22A2) in drug disposition e.g. clearance, toxicity and drug-drug interactions have been highlighted1, 2. Consequently, there is an extensive need for experimental assessment of structure transport relationships as well as tools to predict drug uptake by these transporters in ADMET investigations. In the present study, we developed a robust assay for screening unlabeled compound uptake by hOCT1 and hOCT2 using HEK293 transfected cells. For the first time an extensive data set comprising uptake of 354 compounds is presented. As expected there was a large overlap in substrate specificity between the two organic cation transporters. However, several compounds selectively taken up by either hOCT1 or hOCT2 were identified. In particular a chemical series of phenyl thiophene carboxamide ureas was identified as selective hOCT1 substrates. Moreover, the drivers for transport differed, molecular volume being the most important determinant of hOCT1 substrates whereas H-bonding parameters like PSA dominated for hOCT2.

INTRODUCTION Drug transporters play a key role in drug disposition influencing absorption, distribution and excretion. Transporters of the solute carrier (SLC) family most commonly facilitates uptake of compounds into cells whereas transporters of the ATP binding cassette (ABC) family are pumping drugs out of cells. Together with passive diffusion, uptake and efflux transporters work in concert to modulate the intracellular concentration of drugs, thereby having impact on their


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pharmacological efficacy and toxicity1. By facilitating uptake into liver and kidney cells, transporters of the solute carrier families, e.g. organic cation transporters (OCTs) and organic anion transporters (OATPs/OATs), may be the first, and for some compounds the rate limiting step, in hepatobiliary and renal clearance3. Optimization to improve metabolic stability and oral bioavailability has increased the number of compounds with e.g. lower lipophilicity, for which non-metabolic, transporter-dependent clearance may be the dominating mechanism of elimination. The clinical importance of specific transporters in drug disposition and consequently the need to understand their impact in drug development were recently highlighted in a review by the International Transporter Consortium2. Organic cation transporters 1 and 2 (OCT1/SLC22A1) and OCT2/SLC22A2) are solute carriers that mediate uptake of organic cations into cells in an electrogenic fasion. In human, hOCT1 is highly expressed on the sinusoidal side of the hepatocytes4, 5 facilitating uptake of cations into the liver cells. In addition, this transporter protein is also expressed to a lower degree on the basolateral side of epithelial cells in the intestine, brain and lung. On the other hand hOCT2 is primarily expressed on the basolateral side of kidney proximal tubule epithelium4. In contrast, in the rat Oct1 and Oct2 are both extensively expressed in the kidney and consequently, the clearance route mediated by organic cation transporters in man may be poorly predicted if based on rodent in vivo studies. By mediating uptake of drugs into the liver (hOCT1) and kidney (hOCT2) respectively, organic cation transporters are involved in hepatic and renal clearance and consequently OCT substrates are subject to drug drug interactions. In addition, OCTs may be of importance to target drugs to their site of action as reported for metformin6 or to be a factor in cell toxicity as has been observed with cisplatin7. In drug discovery and development it is important to understand the transporter impact on clearance mechanisms in order to optimize and predict compound pharmacokinetics. 3 ACS Paragon Plus Environment


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Consequently, an enhanced knowledge of structure activity relationships (SAR) with regard to drug transport is needed as well as the development of predictive in silico models for calculation of substrate and inhibition potential of new chemical entities. Extensive sets of compounds were recently explored in terms of their inhibition of hOCT18 and hOCT29 in transporter transfected HEK293 cells and hOCT1 in hepatocytes10. High lipophilicity and a positive net charge were important drivers of OCT inhibition. In contrast to inhibition, only limited number of compounds has been assessed for their OCT substrate properties, main reason being the more laborious compound analysis of a variety of unlabeled compounds required. In the present paper, we have measured the uptake rate of compounds into HEK293 cells transfected with hOCT1 (343 compounds), hOCT2 (211 compounds) or empty vector (354 compounds). Based on the data we are presenting the structural requirements for the apparent transporter mediated uptake.

RESULTS Assessment of drug transport and method validation. A robust screening method for the assessment of transporter-mediated uptake of compounds was developed using HEK293 cell transfected with hOCT1 or empty vector. To quantify the amount of compound taken up by cells, a generic LC-MS/MS based protocol for analysis of compounds with a wide range of physicochemical properties was established. Definition of result parameters used in the SAR analysis is shown in Figure 1. The assay conditions were optimized using known hOCT1 substrates. The time course of compound uptake was close to linear for at least 4 minutes as exemplified by tetraethylammonium (TEA) uptake (Figure 2a). For screening at a single time point, 4 minutes was selected as this resulted in a robust measurement within a reasonably linear part of the time-


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course. The Km of TEA uptake was 140 µM (Figure 2b) which is within the range of 69-229 µM reported in the literature11, 12.

Based on their preferential uptake in hOCT1 or hOCT2 transfected cells respectively, ipratropium and cimetidine were selected as reference compounds that were included in all test occasions as quality controls. Concentration response experiments resulted in a Km of ipratropium uptake in hOCT1 transfected cells of 9 µM and a Km of cimetidine uptake in hOCT2 transfected cells of 60 µM (Figure 3). The precision of hOCT1 uptake was evaluated using ipratropium in HEK293-hOCT1 cells. The coefficient of variation was less than 5% within a plate (calculated from average and SD from 12 wells) and less than 10% between different assay occasions (n=12).

Physicochemical property space of compounds tested. The compounds tested in OCT assays were selected from marketed drugs (83 compounds) and AstraZeneca project compounds (271 compounds). The marketed drugs included 71 (86%) bases, 7 (8.4%) quaternary ammonium salts, 4 (4.8%) neutrals and 1 (1.2%) acid. The AstraZeneca compounds included 206 (76%) bases, 27 (10%) quaternary ammonium salts, 32 (12%) neutrals, 5 (1.8%) zwitterions and 1 (0.4%) acid. The majority of AstraZeneca compounds were from projects aiming at inhaled delivery.

To gain insight into the coverage of global drug space by the investigated compounds, principal component analysis (PCA) was carried out on 2358 marketed drugs based on calculated variables related to the compound lipophilicity, charge, polar/non-polar atom counts, size and ion class integers which depend on the compounds charge state at physiological pH (acidic, basic, neutral



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and zwitterionic). The resultant first 3 components described 70% of the variation in the global drug space based on the 2358 compounds which consisted of 39% neutrals, 34% bases, 18% acids, 6% zwitterions and 3% quaternary amines. Analysis of the loadings revealed p1 to be dominated by size related variables whereas p2 was mainly governed by charge and the number of hydrogen bond donors (Figure 4a). Lipophilic variables were found to be important in both p1 and p2.

Analyses of the resultant score plots (Figure 4b) with inclusion of the herein investigated compounds as test set demonstrated a good cover of global drug space (full grey circles) with the tested drug compounds (full black circles) in particularly for the bases, which constituted the majority of tested compounds. The positioning of the AstraZeneca compounds (open circles) in the lower right quadrant of the t1-t2 score plot indicated they are on average more polar than the collection of marketed drugs. This reflects the latter are by large optimized for oral drug delivery whereas the majority of tested project compounds were optimized for inhaled delivery with less tight polarity constraints. In all, both tested marketed drug and proprietary compounds provided a good cover of total drug space when both oral and inhaled routes are considered. In addition to the increased polarity of tested project compounds in comparison to the global drug space, the tested AstraZeneca quaternary amines were also found to be larger, on average, than their marketed counterparts.

Substrate identification and selectivity. A total of 354 compounds (83 marketed drugs and 271 AZ project compounds) were tested in HEK293 cells transfected with hOCT1 and empty vector and 211 compounds (77 marketed drugs and 134 AZ project compounds) in hOCT2 (all uptake data is presented in Supporting Information Table S1). In figure 5, uptake in hOCT1 and hOCT2 6 ACS Paragon Plus Environment

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transfected cells is plotted versus the uptake in empty vector transfected cells. At the line of unity, the uptake is being equal in transporter and empty vector transfected cells. Above a certain level of passive permeability (uptake > approximately 500 pmol/mg protein), no transporter dependent increase was observed. As expected, bases and quaternary amines were identified as substrates but also several neutral compounds were found to be transported by hOCT1 (Figure 5 and Supporting Information Table S1). The uptake of acids and zwitterions was similar in transporter and empty vector transfected cells. Compounds were defined as substrates when the ratio of uptake into OCT transfected/empty vector transfected cells was higher than 1.3. There was a large overlap in substrate specificity with most compounds being substrates of both hOCT1 and hOCT2. The overlap is exemplified in Table 1 with marketed drugs having the largest total OCT/passive ratios (>10). Some marketed drugs were found to be relatively selective substrates of either hOCT1 or hOCT2. Five and three marketed compounds were identified with a significantly higher uptake in hOCT1 and hOCT2 respectively (Table 2). One of the chemical series from the AstraZeneca projects, phenyl thiophene carboxamide ureas (phTCU), contained selective hOCT1 substrates (Table 3). In general, the phTCU compounds showed a low passive permeability and an extensive uptake into HEK293 cells transfected with hOCT1. Most of the phTCUs were poor or not substrates of hOCT2. In general, a pyrrolidine-3-ol substituent on phTCUs increased the uptake by hOCT1 while the hOCT2 mediated uptake was low (Table 3). Moreover, there may be a degree of stereoselectivity as indicated by the difference in uptake between stereoisomers (compound 43 and 128 in Table 3). However, the majority of compounds were tested as racemates and this has to be further explored to draw any conclusion.



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Structure transport relationships. Uptake into HEK293 transfected with empty vector reflects the passive permeability together with attachment to the cell membrane. The major determinant of uptake in the empty vector cells was lipophilicity as is illustrated in Figure 6. Molecular volume was the descriptor with highest impact on hOCT1 mediated uptake (Figure 7a). Increasing the molecular volume led to a decrease in uptake. Above 500 Å3, almost no substrates could be detected. This trend was not significant for hOCT2 (Figure 7a) when exploring all compounds. However, for quaternary amines a clear correlation of transporter mediated uptake to molecular volume was observed also for hOCT2 (Figure 7b). Interestingly, a substructure class of bases containing a β-amino alcohol moiety, showed a parabolic shaped curve for hOCT1 versus molecular volume with an optimum around 300 Å3 (Figure 8). This phenomenon could not be observed for hOCT2 (Figure 8). Polar surface area (PSA) was the most influential descriptor of compound uptake by hOCT2, in particular for the quaternary amines, showing a decreased uptake with increasing PSA (Fig. 9). Also there was a tendency of higher hOCT2-mediated uptake with increasing log D (Fig. 10). For hOCT1, there was no obvious correlation between uptake and either log D or PSA (Figs. 9 and 10).

DISCUSSION AND CONCLUSIONS We report for the first time an extensive data set of uptake rates for hOCT1 and hOCT2 including the identification of compounds that are selectively taken up by either hOCT1 or hOCT2. In particular one series of new hOCT1 selective compounds are presented. Although there is a large overlap in substrate specificity, the drivers for compounds being transported by hOCT1 are notably different from those characteristic of hOCT2 substrates. Molecular volume


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was by far the most important driver of hOCT1-mediated uptake whereas PSA was one important factor correlating to uptake by hOCT2. The total cellular uptake of compounds is composed of the passive diffusion component and the transporter mediated component and their relative contribution depends on the compounds physicochemical properties13. In this study, uptake data were presented both as active uptake and as the ratio between the total uptake and passive. The passive component was defined as the uptake in empty vector transfected cells and includes diffusion across the cell membrane as well as association of compound to the membrane the latter which may be extensive in particular in the case of lipophilic bases. The active uptake represents the transporter dependent component of the total uptake and should, assuming the measurements are performed under linear conditions, reflect the intrinsic clearance i.e. the capacity of the transport. On the other hand, the total uptake into OCT transfected cells divided by the passive uptake reflects the impact of the transport on the disposition of the drug. In terms of substrate identification and selectivity we have therefore focused on compounds with a high ratio OCT/passive uptake (ratio >10). Compounds with a higher passive permeability may also be good substrates of the transporter protein but the impact of the carrier will be concealed by the dominating passive component. Also physiologically the solute carrier will most probably not have a large impact on the total uptake for compounds with high passive diffusion rate. As expected from the literature, there was a large overlap in substrate specificity between hOCT1 and hOCT24. Amiloride was by far the best substrate of both hOCT1 and hOCT2 followed by a set of quaternary amines including tiotropium. In accordance with the high uptake by hOCT1 and hOCT2, amiloride and tiotropium are extensively cleared non-metabolically with a large fraction, approximately 50%, of the dose being secreted as parent in urine14,

15

.

Metformin, cimetidine and albuterol were found to be mainly transported by hOCT2. This fits 9 ACS Paragon Plus Environment


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well with metformin and cimetidine being almost completely excreted as parent in urine16. Xameterol, ranitidine, procaterol, ipratropium and sumatriptan were found to be preferred substrates of hOCT1 rather than hOCT2. Ranitidine has previously been reported to be a good substrate for hOCT1 but only poorly transported by hOCT217. Ipratropium was recently demonstrated to be a substrate of both hOCT1 and hOCT218 but, in contrast to our study the uptake into hOCT2 transfected cells was much larger than uptake by hOCT1. To our knowledge there are no previous reports on xameterol, procaterol and sumatriptan uptake by organic cation transporters. All of the five drugs found to be more selective for hOCT1 still show a substantial component of renal clearance as parent in man ranging from 18% for procaterol19 to 77% for ranitidine16. The impact of hOCT1, which is highly expressed in the liver, is however difficult to assess from the reported in vivo pharmacokinetic studies since metabolism may occur following drug uptake and the interpretation of excretion data is more complex for the hepatobiliary pathway. Moreover, there is only a limited amount of clinical biliary clearance data available in the literature. Interestingly, a range of in-house AstraZeneca compounds from the phTCU class, were found to be extensively taken up by hOCT1 and only poorly by hOCT2. Since specific transporter tool compounds are rare, structural features from the phTCU class may be used to design an in vivo hOCT1 specific probe substrate. Although drugs from several disease areas have been demonstrated to interact with organic cation transporters4, most data originates from inhibition studies and there is only a limited number of compounds for which hOCT1 and hOCT2 uptake rates has been reported. Systematic studies of structure transport relationships have mainly been based on series of n-alkylammonium substances with different chain lengths. Using trans-stimulation as a means of investigating the actual translocation of a compound across the cell membrane, a decrease in transport by hOCT2 was indicated with increasing n-alkylammonium chain length20. Our data confirm the 10 ACS Paragon Plus Environment

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observations that only small organic cations are being transported by hOCT1 and hOCT2 with an upper limit around a molecular weight of 5004. Interestingly, we observed an optimum in hOCT1 but not hOCT2-mediated uptake for a specific subclass of compounds, β-amino alcohols, with a molecular volume of approximately 300 Å3. This has not previously been reported for organic cation transporters of the SLC22A family. Notably, in electrophysiological measurements on Xenopus Leavis oocytes transfected with rOct2, Schmitt and Koepsell21 demonstrated that permeation rate was optimal with alkali cations with a specific ion radius whereas smaller and larger ions were not transported at a substantial rate. They proposed a model of rOct2 as a selectivity filter with the pore size determining the optimal substrates. Our data would fit into such a hypothesis. There is a 70% homology in amino acid sequence of hOCT1 and hOCT2 and a large overlap in substrate specificity have previously been reported4. Still we observed a clear difference in the SAR between the transporters. Molecular volume was the dominant driver for hOCT1 whereas H-bonding e.g. PSA and charge/polarity were more important for transport mediated by hOCT2. Despite these interesting differences between the two cation transporters one should be wary to over-interpret these findings due to a larger range for the measured variable for the tested compounds in hOCT1 as compared to hOCT2. However, interestingly, a comparison of the interaction of n-alkylammonium substances with different chain lengths with hOCT1 and hOCT2 have previously indicated a difference in the substrate specificity between the two transporters22, hOCT2 preferring smaller, hydrophilic substrates compared to hOCT1. Most structure transport relationship on hOCT1 and hOCT2 in the literature is based on inhibition data. An extensive number of compounds have been screened for their inhibition of hOCT18,

10

and hOCT29. The key drivers for inhibition of both hOCT1 and hOCT2 were

lipophilicity and positive charge at physiological pH8,

9, 10

. Lower PSA, lower number of 11

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hydrogen bond donors and acceptors correlated with a stronger inhibition of both transporter activities. In addition, higher molecular volume was characteristic of hOCT2 inhibitors9. Our study demonstrates that the structural drivers for substrates of hOCT1 and hOCT2 are different compared to that for inhibition. For both transporters and in particular for hOCT1, lipophilicity is not a significant driver of transport. The observation could be due to the difficulty to distinguish between active and passive uptake for lipophilic compounds. However, Zhang and coworkers showed that n-alkylammonium compounds with increasing chain length were accompanied with an increase in inhibition potency whereas the opposite was the case for trans-stimulation that is indicating the actual translocation20. Furthermore, one of the key determinants of hOCT1 substrate properties, molecular volume, did not show up as a driver of hOCT1 inhibition. Although inhibition studies are good indicators of whether a compound is interacting with organic cation transporters, experimentally assessing the transport as such is of importance for evaluating whether a compound is a good substrate. In conclusion, we have developed a robust assay for the quantification of cellular uptake of unlabelled compounds by organic cation transporters. Analysis of the relationship between structural/physicohemical properties and transport rates indicated that positive charge and molecular volume are most important drivers of transport by hOCT1 whereas positive charge and H-bonding properties are major determinants of hOCT2 mediated uptake. Compounds selective for either hOCT1 or hOCT2 were identified and may serve as useful probe substrates in drug disposition studies in vivo. hOCT1 and hOCT2 are important transporters regulating the disposition of cationic drugs. Thus, increasing our knowledge about structural determinants of drug transport are important in the assessment of drug disposition as well as investigating the risk of new drug candidates being subject to drug drug interactions.


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EXPERIMENTAL SECTION Materials. Dulbecco´s Modified Eagle Medium (DMEM), Foetal Bovine Serum, glutaMAXT1, Geneticin, Hank´s Balanced Salt Solution (HBSS) with CaCl2 and MgCl2 without phenol red, phosphate-buffered saline (Ca2+/Mg2+-free) and trypsin were obtained from Gibco. All other chemicals were from Sigma-Aldrich. Poly-D-Lysine coated 12-well dishes (BD BioCoat) were obtained from Becton–Dickinson and deep well plates (1 mL wells) from Greiner. T75 flasks were purchased from Corning Inc. [14C]-tetraethylammonium bromide (2.4 mCi/mmol) was purchased from Perkin Elmer Life Sciences. All test compounds were from the AstraZeneca compound bank and the purity >95% as analysed by HPLC. The structures of all test compounds were verified by NMR and MS.

Cell culture. Human embryonic kidney (HEK293; ATCC CRL 1573) cells stably transfected with hOCT1 or empty vector were produced as previously described23. HEK293 cells transfected with hOCT2 were obtained from professor Bönisch, Institute for Pharmacology and Toxicology, University of Bonn, Germany24. The cells were grown as adherent monolayer cultures in T75 flasks in DMEM supplemented with 10 % (v/v) Fetal Bovine Serum, 2 mM glutaMAXT-1 and 500 μg/ml Geneticin at 37 oC in a humidified 5% CO2 atmosphere. For subculturing, cells at 70– 80% confluence were washed with phosphate buffered saline (PBS) (Ca2+/Mg2+-free), and 3 ml trypsin/EDTA (0.05% trypsin, 0.53 mM EDTA) was added for 1–2 min. The cell suspension was collected in 5 ml growth medium, centrifuged for 4 min at 115 g, and resuspended in growth medium for seeding into new T75 flasks or into assay plates as described below. HEK293 cells transfected with hOCT1 and empty vector were used in passages ranging between 5 and 30. HEK293 cells transfected with hOCT2 were used in passages 18-40.



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For the uptake assay, cells were seeded in Poly-D-Lysine coated 12-well plates at a density of 3-4x105 cells per well. Cells were cultured in a humidified 5% CO2 atmosphere at 37oC for 3 days. Cell monolayers were confluent at the time for the assay. At 24 h before experiments, 10 mM sodium butyrate was added to the medium to stimulate expression of recombinant proteins25.

Uptake of compounds in transfected with hOCT1, hOCT2 and empty vector. Cells on 12-well plates were kept at 37 oC on a heating plate (PST-60HL-4, Biosan) during the experiments. The cells were carefully washed twice with 500 μl/well assay buffer (HBSS supplemented with 10 mM HEPES pH7.4) prewarmed to 37 oC. 500 μl assay buffer were added to each well and the cells preincubated for 5 min at 37°C. After removing the buffer, test compound diluted in assay buffer was added to the wells. For screening of compounds at one concentration and one time point, the cells were incubated with 2.5 μM test compound for 4 minutes. For time and concentration dependence experiment, time and concentrations are noted in the figure legends. All compounds were incubated in triplicates. In each experiment a reference compound was used as quality control which was ipratropium for hOCT1 and cimetidine for hOCT2. The incubation was stopped by removing the solution and adding 500 μl ice-cold assay buffer. The cells were washed twice with ice cold assay buffer. Removing the buffer as complete as possible during the washing procedure was critical for good results. After washing of cells, 500 μl lysing solution (methanol/1% acetic acid) containing internal standard was added. As internal standard for the LC/MS-MS analysis, 0.5 μM dexamethasone was used. For test compounds with a mass within 3 units of dexamethasone, 1 μM budesonide was used. The internal standard was used to compensate for evaporation of solvent during incubation and sample preparation. The cell plates were left with the lysing solution for at least 5 minutes with lid in room temperature. The cell lysates were then transferred to a 96-deep well plate which was centrifuged at 1500 g for 10 14 ACS Paragon Plus Environment

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minutes. 100 μl of the supernatant was transferred to a new 96-deep well plate and each sample was diluted with 300 μl water. The sample plates were stored at -80°C until analysis. The amount of compound associated with the cells were quantified by LC-MS/MS (Sciex API3000 or API4000) using a standard curve. A matrix solution for preparation of standard curves was produced in the same way by adding lysing solution to cells not treated with compound. Aquasil, 5μm, 10x1mm, (Thermo Electron Corp.) was used as a pre-column and the analytical column was Aquasil, 5μm, 30x1mm, (Thermo Electron Corp). A linear gradient was used for the separation,

starting

with

100%

water/0.5%

acetic

acid

(v/v)

and

ending

with

acetonitrile/water/acetic acid (95/4.5/0.5) (v/v/v). For most compounds, the limit of quantification of the LC-MS/MS analysis was around 1 nM corresponding to a cellular uptake of 7 pmol/mg protein. For analysis of radiolabelled compound uptake ([14C]-tetraethylammonium only), cells were lysed with 1% triton X-100. Cell lysates were mixed with 10 ml scintillation cocktail (Ultima Gold, PerkinElmer) and the radioactivity analysed using a Packard Tri-Carb 2500 TR liquid scintillation counter (PerkinElmer).

Calculation of uptake. Total uptake by hOCT1, hOCT2 or empty vector transfected cells were calculated by dividing the amount of drug quantified in the drug lysate by the amount of cell protein present in one well. The active uptake is the total uptake in hOCT1 or hOCT2 transfected cells minus the uptake in the empty vector transfected cells. Data presented are an average of three determinations. Uptake data for all tested compounds are presented in Supporting Information Table S1. Physicochemical properties of compounds. Log D at pH 7.4 was experimentally determined by octanol water partitioning using a high throughput shake-flask method26. Other physicochemical parameters included in the SAR analysis were calculated using ACD/PhysChem 15 ACS Paragon Plus Environment


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Suite (Advanced Chemistry Development, Inc., Toronto, On, Canada, www.acdlabs.com). Physicochemical property data are presented in Supporting Information Table S1 (experimental) and S2 (calculated).

ASSOCIATED CONTENT Supporting Information Available. Experimental data for all test compounds including uptake in HEK293 cells transfected with hOCT1, hOCT2 and empty vector, and log D. Calculated physicochemical parameters used for SAR analysis. SMILES notation of test compounds. This material is available free of charge via the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION Corresponding Author *Lena Gustavsson, E-mail address: [email protected] , Phone no: +46 40 337756, present address, see below.

Present Addresses 1

Jenny Johansson, Lund University, Wallenberg Neuroscience Center, BMC A11, SE-221 84

Lund, Sweden 2

Christina Lohmann, University of Münster, 48149 Münster, Germany

3

Rose-Marie Jenvert, QA & Validation, ÅF Business Area South, Hallenborgs gata 4,

SE-211 74 Malmö, Sweden 16 ACS Paragon Plus Environment

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4


Anders Blomgren, Clinical Chemistry, University and Regional Laboratories, Skåne University

Hospital, SE-221 85 Lund, Sweden 5

Lena Gustavsson, Molecular Medicine, Department of Laboratory Medicine, Lund University,

Medicon Village B404, SE-223 81 Lund, Sweden. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ABBREVIATIONS HEK293, human embryonic kidney cells; hOCT1, human organic cation transporter 1; hOCT2, human organic cation transporter 2; HPLC, high performance liquid chromatography; MS, mass spectrometry; OCT, organic cation transporter; OAT, organic anion transporter; OATP, organic anion transporting polypeptide, PCA, principal component analysis; PSA, polar surface area; SLC, solute carrier; TEA, tetraethylammonium

REFERENCES 1. DeGorter, M. K.; Xia, C. Q.; Yang, J. J.; Kim, R. B., Drug transporters in drug efficacy and toxicity. Annual review of pharmacology and toxicology 2012, 52, 249-273. 2. International Transporter, C.; Giacomini, K. M.; Huang, S. M.; Tweedie, D. J.; Benet, L. Z.; Brouwer, K. L.; Chu, X.; Dahlin, A.; Evers, R.; Fischer, V.; Hillgren, K. M.; Hoffmaster, K. A.; Ishikawa, T.; Keppler, D.; Kim, R. B.; Lee, C. A.; Niemi, M.; Polli, J. W.; Sugiyama, Y.; Swaan, P. W.; Ware, J. A.; Wright, S. H.; Yee, S. W.; Zamek-Gliszczynski, M. J.; Zhang, L., Membrane transporters in drug development. Nature reviews. Drug discovery 2010, 9 (3), 215-236.



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3. Shitara, Y.; Horie, T.; Sugiyama, Y., Transporters as a determinant of drug clearance and tissue distribution. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences 2006, 27 (5), 425-446. 4. Koepsell, H.; Lips, K.; Volk, C., Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications. Pharmaceutical research 2007, 24 (7), 12271251. 5. Hilgendorf, C.; Ahlin, G.; Seithel, A.; Artursson, P.; Ungell, A. L.; Karlsson, J., Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines. Drug metabolism and disposition: the biological fate of chemicals 2007, 35 (8), 1333-1340. 6. Shu, Y.; Sheardown, S. A.; Brown, C.; Owen, R. P.; Zhang, S.; Castro, R. A.; Ianculescu, A. G.; Yue, L.; Lo, J. C.; Burchard, E. G.; Brett, C. M.; Giacomini, K. M., Effect of genetic variation in the organic cation transporter 1 (OCT1) on metformin action. J Clin Invest 2007, 117 (5), 1422-1431. 7. Ciarimboli, G.; Deuster, D.; Knief, A.; Sperling, M.; Holtkamp, M.; Edemir, B.; Pavenstadt, H.; Lanvers-Kaminsky, C.; am Zehnhoff-Dinnesen, A.; Schinkel, A. H.; Koepsell, H.; Jurgens, H.; Schlatter, E., Organic cation transporter 2 mediates cisplatin-induced oto- and nephrotoxicity and is a target for protective interventions. The American journal of pathology 2010, 176 (3), 1169-1180. 8. Ahlin, G.; Karlsson, J.; Pedersen, J. M.; Gustavsson, L.; Larsson, R.; Matsson, P.; Norinder, U.; Bergstrom, C. A.; Artursson, P., Structural requirements for drug inhibition of the liver specific human organic cation transport protein 1. Journal of medicinal chemistry 2008, 51 (19), 5932-5942. 9. Kido, Y.; Matsson, P.; Giacomini, K. M., Profiling of a prescription drug library for potential renal drug-drug interactions mediated by the organic cation transporter 2. Journal of medicinal chemistry 2011, 54 (13), 4548-4558. 10. Badolo, L.; Rasmussen, L. M.; Hansen, H. R.; Sveigaard, C., Screening of OATP1B1/3 and OCT1 inhibitors in cryopreserved hepatocytes in suspension. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences 2010, 40 (4), 282-288.


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11. Zhang, L.; Schaner, M. E.; Giacomini, K. M., Functional characterization of an organic cation transporter (hOCT1) in a transiently transfected human cell line (HeLa). J Pharmacol Exp Ther 1998, 286 (1), 354-361. 12. Umehara, K. I.; Iwatsubo, T.; Noguchi, K.; Kamimura, H., Functional involvement of organic cation transporter1 (OCT1/Oct1) in the hepatic uptake of organic cations in humans and rats. Xenobiotica; the fate of foreign compounds in biological systems 2007, 37 (8), 818-831. 13. Sugano, K.; Kansy, M.; Artursson, P.; Avdeef, A.; Bendels, S.; Di, L.; Ecker, G. F.; Faller, B.; Fischer, H.; Gerebtzoff, G.; Lennernaes, H.; Senner, F., Coexistence of passive and carrier-mediated processes in drug transport. Nature reviews. Drug discovery 2010, 9 (8), 597-614. 14. Spahn, H.; Reuter, K.; Mutschler, E.; Gerok, W.; Knauf, H., Pharmacokinetics of amiloride in renal and hepatic disease. European journal of clinical pharmacology 1987, 33 (5), 493-498. 15. Turck, D.; Weber, W.; Sigmund, R.; Budde, K.; Neumayer, H. H.; Fritsche, L.; Rominger, K. L.; Feifel, U.; Slowinski, T., Pharmacokinetics of intravenous, single-dose tiotropium in subjects with different degrees of renal impairment. Journal of clinical pharmacology 2004, 44 (2), 163-172. 16. Varma, M. V.; Feng, B.; Obach, R. S.; Troutman, M. D.; Chupka, J.; Miller, H. R.; El-Kattan, A., Physicochemical determinants of human renal clearance. Journal of medicinal chemistry 2009, 52 (15), 4844-4852. 17. Bourdet, D. L.; Pritchard, J. B.; Thakker, D. R., Differential substrate and inhibitory activities of ranitidine and famotidine toward human organic cation transporter 1 (hOCT1; SLC22A1), hOCT2 (SLC22A2), and hOCT3 (SLC22A3). J Pharmacol Exp Ther 2005, 315 (3), 1288-1297. 18. Nakanishi, T.; Haruta, T.; Shirasaka, Y.; Tamai, I., Organic cation transporter-mediated renal secretion of ipratropium and tiotropium in rats and humans. Drug metabolism and disposition: the biological fate of chemicals 2011, 39 (1), 117-122. 19. Eldon, M. A.; Battle, M. M.; Coon, M. J.; Nordblom, G. D.; Sedman, A. J.; Colburn, W. A., Clinical pharmacokinetics and relative bioavailability of oral procaterol. Pharmaceutical research 1993, 10 (4), 603-605. 19 ACS Paragon Plus Environment


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20. Zhang, L.; Gorset, W.; Dresser, M. J.; Giacomini, K. M., The interaction of n-tetraalkylammonium compounds with a human organic cation transporter, hOCT1. J Pharmacol Exp Ther 1999, 288 (3), 11921198. 21. Schmitt, B. M.; Koepsell, H., Alkali cation binding and permeation in the rat organic cation transporter rOCT2. The Journal of biological chemistry 2005, 280 (26), 24481-24490. 22. Dresser, M. J.; Xiao, G.; Leabman, M. K.; Gray, A. T.; Giacomini, K. M., Interactions of ntetraalkylammonium compounds and biguanides with a human renal organic cation transporter (hOCT2). Pharmaceutical research 2002, 19 (8), 1244-1247. 23. Lohmann, C.; Gelius, B.; Danielsson, J.; Skoging-Nyberg, U.; Hollnack, E.; Dudley, A.; Wahlberg, J.; Hoogstraate, J.; Gustavsson, L., Scintillation proximity assay for measuring uptake by the human drug transporters hOCT1, hOAT3, and hOATP1B1. Analytical biochemistry 2007, 366 (2), 117-125. 24. Hayer-Zillgen, M.; Bruss, M.; Bonisch, H., Expression and pharmacological profile of the human organic cation transporters hOCT1, hOCT2 and hOCT3. British journal of pharmacology 2002, 136 (6), 829-836. 25. Grunberg, J.; Knogler, K.; Waibel, R.; Novak-Hofer, I., High-yield production of recombinant antibody fragments in HEK-293 cells using sodium butyrate. BioTechniques 2003, 34 (5), 968-972. 26. Alelyunas, Y. W.; Pelosi-Kilby, L.; Turcotte, P.; Kary, M. B.; Spreen, R. C., A high throughput dried DMSO LogD lipophilicity measurement based on 96-well shake-flask and atmospheric pressure photoionization mass spectrometry detection. Journal of chromatography. A 2010, 1217 (12), 1950-1955.


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TABLES Table 1. Drugs with overlapping substrate specificities for hOCT1 and hOCT2. The table presents compounds with the highest active uptake and a ratio of total to passive uptake >10. Trivial name

Ion Class

hOCT1 active (pmol/mg)

hOCT1 total/passive


hOCT2 total/passive

Amiloride Tiotropium* Glycopyrrolate Oxyphenonium Phenformin Fenoterol

B Q Q Q B B

1430 820 640 640 570 140

76 75 59 25 >39** 18

1660 460 830 230 450 110

74 39 93 14 >35** 12

*Mean of two batches, **Passive uptake into empty vector cells below limit of quantification, B=base, Q=quaternary amine salt



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Table 2. Drugs found to be most selective for either a) hOCT1 or b) hOCT2. Compounds classified as good substrates (ratio of total hOCT uptake/passive uptake > 10) for either hOCT1 (a) or hOCT2 (b) are presented. a) Compounds with higher uptake in hOCT1 than hOCT2 Trivial name Ion hOCT1 active hOCT1 (pmol/mg) Class total/passive Xamoterol * Ranitidine Procaterol Ipratropium Sumatriptan

B B B Q B

230 600 140 370 620


hOCT2 total/passive

5.2 22 14 56 94

1.6 1.6 1.9 8.3 3.3

43 18 11 42 15

*average of R and S form b) Compounds with a higher uptake in hOCT2 than hOCT1. Trivial name Ion hOCT1 active hOCT1 hOCT2 active (pmol/mg) (pmol/mg) Class total/passive Metformin Cimetidine Albuterol

B B B

5.4 65 58

>1.7** 6.5 11

110 270 270

hOCT2 total/passive >15** 15 15

** Passive uptake (empty vector cells) below limit of quantification 22 ACS Paragon Plus Environment

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Table 3. AstraZeneca project compounds selective for hOCT1. Several compounds from the phTCU series were found to be highly selective for hOCT1 over hOCT2. ID

X

Substituent in R1position

43

C

128 129 50* 51 54* 134* 64*

C C C C C C C

70 76

N N

Substituent in R2, R3, R4 (default=H)


hOCT1 total/ passive


hOCT2 total/ passive

(3S)-pyrrolidine-3-ol

1047

26

37

1.9

(3R)-pyrrolidine-3-ol piperidine-4-ol pyrrolidine-3-ol azetidine-3-ol H H 3-hydroxy-1,1dimethylpyrrolidinium 2-aminoethanol 3-aminopropanol

586 821 1653 641 1352 1493 903

15 17 39 11 68 73 53

6.6 35 22 63 21 8.5 46

1.1 1.8 1.6 2.2 1.6 1.4 4.3

796 739

13 14

40 23

1.3 1.5

R4=CN R2=pyrrolidine-3-ol R3=pyrrolidine-3-ol

*Racemates.



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LEGENDS TO THE FIGURES

Figure 1. Definition of result parameters used for the SAR analysis. Figure 2. Time-course (a) and concentration dependency (b) of [14C]tetraethylammonium uptake in HEK293 cells transfected with hOCT1 and empty vector. For the time course, cells were incubated with 2.5 M [14C]-tetraethylammonium. In the concentration dependency experiments, cells were incubated for 1 minute with different concentrations of [14C]tetraethylammonium. n=3 in each data point. Figure 3. Concentration dependency of reference compound uptake in HEK293 cells transfected with organic cation transporters and empty vector, a) ipratropium uptake in hOCT1 and empty vector and b) cimetidine uptake in hOCT2 and empty vector cells. Cells were incubated with different concentrations of reference compound for 1 minute and the amount of compound taken up by the cells was analysed by LC-MS/MS (n=3 in each data point). Figure 4. PCA analysis of tested compounds as compared to the general drug space; a) loadings of all compounds and b) score plots of compounds based on total, bases, quaternary amines, neutrals and acids/zwitterions. Figure 5. Total uptake of compounds in organic cation transporter versus empty vector transfected cells, a) hOCT1 and b) hOCT2. HEK293 cells transfected with hOCT1, hOCT2 or empty vector were incubated with test compounds (2.5 µM) for 4 minutes and the amount of compound taken up into the cells quantified by LC-MS/MS. For further details, see Experimental section and Supporting Information Table S1.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Uptake of compounds in HEK293 cells transfected with empty vector versus a) log D, b) PSA and c) molecular volume. HEK293 cells transfected with empty vector were incubated with test compounds (2.5 µM) for 4 minutes and the amount of compound taken up into the cells quantified by LC-MS/MS. Log D at pH7.4 was measured as described in the Experimental section. PSA and molecular volume were calculated. For data, see Supporting Information Tables S1 and S2. Figure 7. Active uptake of compounds by hOCT1 and hOCT2 versus molecular volume; a) all compounds, b) quaternary amines. HEK293 cells transfected with hOCT1, hOCT2 or empty vector were incubated with test compounds (2.5 µM) for 4 minutes and the amount of compound taken up into the cells quantified by LC-MS/MS. Molecular volume was calculated. For data, see Supporting Information Tables S1 and S2. Figure 8. Active uptake by hOCT1 and hOCT2 versus molecular volume for a subseries of bases containing β-amino alcohols. HEK293 cells transfected with hOCT1, hOCT2 or empty vector were incubated with test compounds (2.5 µM) for 4 minutes and the amount of compound taken up into the cells quantified by LC-MS/MS. Molecular volume was calculated. For data, see Supporting Information Tables S1 and S2. Figure 9. Active uptake of compounds by hOCT1 and hOCT2 versus polar surface area (PSA). HEK293 cells transfected with hOCT1, hOCT2 or empty vector were incubated with test compounds (2.5 µM) for 4 minutes and the amount of compound taken up into the cells quantified by LC-MS/MS. PSA was calculated. For data, see Supporting Information Tables S1 and S2.



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Page 26 of 49

Figure 10. Active uptake of compounds by hOCT1 and hOCT2 versus measured log D at pH 7.4. HEK293 cells transfected with hOCT1, hOCT2 or empty vector were incubated with test compounds (2.5 µM) for 4 minutes and the amount of compound taken up into the cells quantified by LC-MS/MS. Log D was measured by octanol water partitioning. For further details, see Experimental section and Supporting Information Table S1.


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FIGURES

Figure 1



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 2


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Uptake (pmol/min/mg protein)

Figure 3

1600

a)

1400 1200 1000

hOCT1

800

Empty vector

600

Active uptake

400 200 0 0

100

200

300

400

500

Concentration (µM)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3500

b)

3000 2500 2000 1500 1000 500 0 0

100

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Concentration (µM)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 4 a)


Page 31 of 49

Figure 5 Uptake hOCT1 (pmol/mg protein)

a)

10000

1000

100

Base

Quaternary Neutral

10

Acid Zwitterion 1

1

10

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1000

10000

Uptake empty vector (pmol/mg protein)

b) 10000

Uptake hOCT2 (pmol/mg protein)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1000

100

Base Quaternary Neutral Acid Zwitterion

10

1 1

10

100

1000

10000




Figure 6 a) Uptake empty vector (pmol/mg protein)

10000

1000

100 Base Quaternary Neutral Acid Zwitterion

10

1 -3

-1

1

3

5

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400

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800

Log D (7.4)

10000


b)

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1 0

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PSA c) Uptake empty vector (pmol/mg protein)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Molecular Volume

(Å3)


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Figure 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 8.


Page 35 of 49

10000


hOCT1 1000

100

10

1

0 0

100

200

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Active uptake hOCT2 (pmol/mg protein)

Figure 9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10000

hOCT2 1000

100

10

PSA

1

0 0

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PSA



Figure 10

-4

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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hOCT1 1000

100

10


1

0 -2

0

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6

Log D (7.4)

-4

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hOCT2 1000

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0 -2

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TABLE OF CONTENTS GRAPHICS



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

UptakeOCT

UptakeEmpty vector

Active uptake = UptakeOCT – UptakeEmpty vector Passive uptake = UptakeEmpty vector Ratio OCT/Passive = UptakeOCT/UptakeEmpty vector


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120

a)

100 80 60

hOCT1 Empty vector

40

Active uptake

20 0 0

2

4

6

8

10

Time (minutes) 2500


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


Uptake (pmol/mg protein)

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b) 2000 1500 1000 500 0 0

500

1000

Concentration (µM)


1600

a)

1400 1200

1000

hOCT1

800

Empty vector

600

Active uptake

400 200 0 0

100

200

300

400

500

Concentration (µM)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58



3500

b)

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Page 41 of 49

a)

Polarity/Charge

0.4

-0.1

ACD LogD7.4 ACD LogD6.5 ClogP ACD LogP

0.3

0.2 Neutral 0.1 0.0

P2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Log fraction neutral pKa (Acid 1)

Size

pKa (Acid 2)

n Rings

Base

pKa 0.0 pKa 0.1 (Base 1) (Base 2) -0.1 Acid Zwitterion Log fraction -0.2 ionised -0.3

n Donors

0.2

NPSA (Å²) 0.3

Lipinski n Rotatable Bonds

PSA (Å²)

n Acceptors

-0.4 P1


0.4 Volume (Å³) MW

n Heavy Atoms


b) 10

10

All compounds

t2

5

Bases

5

0

0

-5

-5

Global drug space (2368) Test cmpds - marketed drugs (83) Test cmpds – AZ cmpds (271)

-10

-10 -10

10

-5

0

5

10

-10

-5

10

Quaternary amines

0

5

10

10

Neutrals

5

5

5

0

0

0

-5

-5

-5

-10

-10

t2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

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-10

-5

0 t1

5

10

Acids

-10 -10

-5

0

5

10

t1


-10

-5

0 t1

5

10

Page 43 of 49

a)


10000

1000

100

Base Quaternary Neutral

10

Acid Zwitterion 1 1

10

100

1000

10000

Uptake empty vector (pmol/mg protein) b) 10000


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


1000

100


10

1 1

10

100

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10000




a) Uptake empty vector (pmol/mg protein)

10000

1000

100 Base Quaternary Neutral Acid Zwitterion

10

1 -3

-1

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3

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b)

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PSA c) Uptake empty vector (pmol/mg protein)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

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1000

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1 0

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Molecular Volume

(Å3)


Page 45 of 49


a) Active uptake hOCT1 (pmol/mg protein)




1 2 10000 10000 Base 3 hOCT2 hOCT1 4 Quaternary 5 Neutral 6 1000 1000 Acid 7 8 Zwitterion 9 100 100 10 11 12 10 10 13 14 15 16 1 1 17 18 19 0 0 20 0 200 400 600 0 200 400 600 21 3 Molecular volume (Å3 ) 22 Molecular volume (Å ) 23 24 25 b) 26 27 10000 10000 28 hOCT1 hOCT2 29 30 31 1000 32 1000 33 34 35 36 100 100 37 38 39 40 10 10 41 42 43 44 1 1 45 46 250 300 350 400 450 500 250 300 350 400 450 47 3 Molecular volume (Å3 ) Molecular volume (Å ) 48 49 50 51 52 53 54 55 ACS Paragon Plus Environment 56 57 58

500

10000


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58



hOCT1 1000

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1 0

200

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Molecular volume

(Å3

Page 46 of 49

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hOCT2 1000

100

10

1 0

)


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Molecular volume (Å3 )


1 2 3 4 5 6 7 8 9 10 10000 11 hOCT1 12 13 14 1000 15 16 17 100 18 19 20 10 21 22 23 1 24 25 26 27 0 28 0 100 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58



200

300


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hOCT2 1000

100

10

1

0 0

PSA

100

200

PSA


300

-4

10000


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58



hOCT1 1000

100

10


1

0 -2

0

2

4

6

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10000

hOCT2

-4

Log D (7.4)

1000

100

10

1

0 -2

0

2

Log D (7.4)


4

6


Uptake empty vector


Active uptake hOCT1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Uptake hOCT1

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Molecular volume

Identification of Novel Substrates and StructureâActivity Relationship

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