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A Selective and Slowly Reversible Inhibitor of L-Type Amino Acid Transporter 1 (LAT1) Potentiates Anti-Proliferative Drug Efficacy in Cancer Cells Kristiina Maria Huttunen, Mikko Gynther, Johanna Huttunen, Elena Puris, Julie A Spicer, and William A. Denny J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00190 • Publication Date (Web): 02 Jun 2016 Downloaded from http://pubs.acs.org on June 6, 2016

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Journal of Medicinal Chemistry

A Selective and Slowly Reversible Inhibitor of LType Amino Acid Transporter 1 (LAT1) Potentiates Anti-Proliferative Drug Efficacy in Cancer Cells

Kristiina M. Huttunen,†* Mikko Gynther, † Johanna Huttunen, † Elena Puris, † Julie A. Spicer, ‡ William A. Denny ‡

†

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland,

P.O. Box 1627, FI-70211 Kuopio, Finland ‡

Auckland Cancer Society Research Centre, The University of Auckland,

Private Bag 92019, Auckland 1142, New Zealand

ACS Paragon Plus Environment

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ABSTRACT

The L-type amino acid transporter 1 (LAT1) is a transmembrane protein carrying bulky and neutral amino acids into cells. LAT1 is over-expressed in several types of tumors and its inhibition can result in reduced cancer cell growth. However, known LAT1 inhibitors lack selectivity over other transporters. In the present study, we designed and synthesized a novel selective LAT1 inhibitor (1), which inhibited the uptake of LAT1 substrate, L-leucin as well as cell growth. It also significantly potentiated the efficacy of bestatin and cisplatin even at low concentrations (25 µM). Inhibition was slowly reversible, as the inhibitor was able to be detached from the cell surface and blood-brain barrier. Moreover, the inhibitor was metabolically stable and selective towards LAT1. Since the inhibitor was readily accumulated into the prostate after intraperitoneal injection to the healthy mice, this compound may be a promising agent or adjuvant especially for the treatment of prostate cancer.


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INTRODUCTION Amino acids are essential for protein synthesis, supporting cell growth and metabolism. System L is a family of sodium-independent transport proteins that can carry neutral amino acids across the cell membrane. It is comprised of four different transporters, LAT1-4.1-6 LAT1 and 2 are heterodimers that consist of a catalytic light chain (SLC7A5 or SLC7A8, respectively) linked via a disulfide bond to a heavy chain (4F2hc or CD98hc, SLC3A2). They belong to the solute carrier family 7 (SLC7),3-6 whereas their structurally and functionally distinct system L family members, LAT3 and LAT4, belong to the SLC family 43 (SCL43A1 and SCL43A2, respectively)1, 2. LAT1 transports branched or bulky neutral amino acids in the following order; Phe > Trp > Leu > Ile > Met > His > Tyr > Val, and it does not carry anionic or cationic amino acids.5 LAT1 has specific tissue distribution, different from those of other system L proteins. It is highly expressed in cells that require a constant supply of amino acids, such as neural, glial and placental cells as well as endothelial cells of the blood-brain barrier (BBB) and activated T cells.5, 7, 8 Furthermore, among the system L proteins it is the most extensively over-expressed in tumors and metastases.9-12 This increased expression of LAT1 has been associated with unfavorable prognosis and patient survival in many types of cancer, such as breast, prostate, lung, colorectal, head and neck cancer as well as in gliomas.13-18 Therefore, LAT1 has attained a growing interest as a diagnostic target, but more importantly, as a potential drug target by which the proliferation of cancer cells could be reduced.19

By depriving the cancer cells of essential amino acids, LAT1 inhibitors hinder protein synthesis and thus cell proliferation. However, amino acids, especially leucine, are also known to stimulate protein synthesis and cell growth via mammalian target of rapamycin (mTOR), and


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inhibition of LAT1 has been reported to suppress mTOR signaling and subsequently tumor growth.10,

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However, the detailed mechanisms how leucine activates mTOR in this nutrient

signaling pathway are still unclear. 2-Amino-2-norbornane-carboxylic acid (BCH) (Figure 1) has been referred to as a system L inhibitor in the literature and is reported to induce the suppression of cancer cell growth and apoptosis.21-23 However, BCH lacks selectivity for LAT1 over LAT2 and very high amounts (over 10 mM concentrations) are needed to produce antiproliferative effects.21-23 A novel selective LAT1 inhibitor, (S)-2-amino-3-(4-((5-amino-2phenylbenzo[d]oxazol-7-yl)methoxy)-3,5-dichlorophenyl)propanoic acid 2 (JPH203, previously known also as KYT-0353) (Figure 1), has been reported to have anti-tumor effects towards human colon cancer-derived HT-29 cells and human oral cancer cells (YD-38) at micromolar concentrations (IC50 4.1 and 69 µM, respectively, after 4 days).24, 25 Interestingly, inhibition of cell growth was 68-87-times greater than the inhibition of leucine uptake (IC50 0.06 and 0.79 µM, respectively) in HT-29 and YD-38 cells, implying that inhibition of LAT1-mediated amino acid uptake does not account alone for the anti-proliferative effects of inhibitor 2. In YD-38 cells, inhibitor 2 has also been found to induce apoptotic factors, including caspase-3, -7 and -9 as well as poly(ADP-ribose) polymerase (PARP), which may explain its overall efficacy.25 However, inhibitor 2 has been reported to be a substrate for organic anion transporting polypeptides (OATPs) 1B1, 1B3 and 2B1 and organic anion transporter 3 (OAT3), which increases its hepatic exposure.26 Since inhibitor 2 is also rapidly N-acetylated in hepatocytes,27 its systemic concentrations and availability to reach the target cancer cells is limited.

In the present study, we designed and synthesized a potent LAT1-inhibitor and evaluated its reversible and selective binding to LAT1 over LAT2, OATPs and monocarboxylate transporter 1


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(MCT1). The metabolic stability of this inhibitor was determined, as well as its potential to affect the viability of human breast cancer cells (MCF-7) alone and as a combination with other anticancer drugs, the aminopeptidase inhibitor bestatin and the DNA cross-linking agent cisplatin. The systemic pharmacokinetics of this inhibitor and its function at the blood-brain barrier (BBB) was also evaluated.


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RESULTS Synthesis and Properties of the LAT1-Inhibitor 1 (S)-2-Amino-3-(3-((2,4-dicyano-3-(4-(2-(methylamino)-2oxoethoxy)phenyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)carbamoyl)phenyl)propanoic

acid

1

(KMH-233) was designed from a 3-Dimensional Quantitative Structure Activity (3D-QSAR) model of the rat LAT1 binding site, which we have recently reported;28 the estimated inhibition of leucine uptake by docking the designed compound 1 was 76% (Scheme 1). Compound 3 was selected as a backbone of the inhibitor due to its large and bulky size, which was assumed to at least slow down the possibility of transportation across the cell membrane by LAT1. Compound 1 was then prepared by a previously described method, using 9-BBN to protect the amino acid group (compound 4), coupling the amino acid 4 and the compound 329 by the aid of EDC/DMAP and finally deprotecting the formed compound with ethylenediamine to obtain compound 1 in good overall yield.30 Compound 1 showed good aqueous solubility (1.16 ± 0.11 mg/mL).

Ability of Compound 1 and BCH to Bind to LAT1 and Compound 1 to LAT2 The ability of compound 1 to bind to LAT1 and LAT2 was evaluated via a competitive inhibition assay with [14C]-L-leucine (LAT1 substrate) and [14C]-L-alanine (LAT2 substrate) in the MCF-7 (human breast cancer) cell line, in which the LAT1, LAT2 and 4F2hc mRNA expressions were determined (Figure 2A). Compound 1 inhibited the cellular uptake of [14C]-Lleucine (0.157 µM) significantly during 5 min incubation in a concentration-dependent manner (Figure 2B). The half maximal inhibitory concentration (IC50) value for compound 1 was 18.2 ± 1.2 µM, (evaluated by nonlinear regression analysis from results at 8 concentrations between 1 µM – 1 mM), indicating that it has good affinity for LAT1 as well as potency to inhibit LAT1 in


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MCF-7 cells. For comparison, the IC50 value of LAT1-mediated uptake was also determined for BCH in MCF-7 cells and it was over 6 -times higher (112 ± 12 µM) than corresponding IC50 value for compound 1 (Figure 2B). Contrarily, compound 1 was not able to inhibit [14C]-Lalanine (10.0 µM) significantly within the concentration range of 50-1000 µM (Figure 2C) and thus, the exact IC50 value of uptake mediated via LAT2 for compound 1 could not be calculated (> 1mM). However, comparing inhibition of the [14C]-L-leucine uptake by compound 1 at 25 µM concentration, close to its IC50 value of LAT1-mediated uptake (55.10 ± 2.81%), to inhibition of the [14C]-L-alanine uptake, which compound 1 did not inhibit at all, it can be concluded that compound 1 is able to bind and inhibit selectively to LAT1 over LAT2. Moreover, at 4-times higher concentration, (100 µM), when minor inhibition of the [14C]-L-alanine uptake was seen (3.93 ± 1.97%), inhibition of [14C]-L-leucine was already 77.75 ± 3.72%.

LAT1-Mediated Transport of Compound 1 and BCH into Cells To evaluate whether the compound 1 was transported into MFC-7 cells via LAT1 or only bound to it on the cell surface, we incubated compound 1 (100 µM) for 5 and 30 min at 37 ᵒC and washed the cells with cold (4 ᵒC; reduces activity of transporters) and warm (37 ᵒC, retains activity of transporters) buffer before lysing the cells. Thus, in the case of warm washing after 30 min, washing was done twice, after 5 min and after 30 min without lysing the cells in between time points. As seen in Figure 3A, the amount of compound 1 detected after 5 and 30 min with cold washing were equal; 2.80 ± 0.65 and 2.63 ± 0.11 nmol/mg of protein, respectively. The amount was significantly reduced after 5 and 5+30 min when the cells were washed with warm buffer (1.43 ± 0.49 and 0.21 ± 0.19 nmol/mg of protein, respectively), indicating that the compound 1 was reversibly bound to LAT1 and thus not transported to any meaningful extent


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into the MCF-7 cells. The binding of compound 1 to LAT1 was evaluated over the concentration range 25 - 400 µM during 30 min incubation and from the results in Figure 3B we can conclude that binding of compound 1 saturated at a concentration of 100 µM, equivalent to an amount of 2.81 ± 0.80 nmol/mg of protein. We also evaluated the amount of BCH transported into MCF-7 cells via LAT1, and for example, at 100 µM the amount detected was 11.95 ± 1.10 nmol/mg of protein; thus, over 4- times greater than the amount of compound 1 (Figure 3B). In this respect BCH behaves more as a substrate than an inhibitor of LAT1.

Selectivity of Compound 1 to Inhibit LAT1 over other Influx Transporters To clarify details of transporter-mediated binding of compound 1, its binding was evaluated in the presence (500 µM) of the known competing MCT1 inhibitor, phloretin,31 OATP1A2/2B1 inhibitor, naringin,32 OATP1B1/1B3 inhibitor, rifampicin,32, 33 or unselective OATP substrate, Lthyroxine (T4)34, as these transporters are reported to be expressed in MCF-7 cells.34-37 Figure 4 clearly illustrates that the binding of compound 1 was not changed by known transporter inhibitors, and is therefore, mediated primarily via LAT1.

Metabolic Stability of Compound 1 The chemical and metabolic stability of compound 1 was studied in isotonic Tris-HCl buffer (pH 7.4), with mouse liver S9 fraction (phase II hydrolytic reactions) and with mouse liver microsomes in the presence of NADPH (cytochrome P450 catalyzed phase I reactions) at 37 °C. Compound 1 was chemically stable in the buffer over 24 h and no change in UV-HPLC chromatograms was detected in S9 or microsome fractions during 2 h incubation. We also evaluated the stability of compound 1 in human plasma and showed, as expected, that it was


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stable for 2 h. Thus, compound 1 has the advantage over inhibitor 2 as a potent and selective LAT1 inhibitor of being more stable towards enzymatic metabolism.26, 27

The Ability of Compound 1 to Inhibit Cancer Cell Growth and Potentiate the Efficacy of Known Anti-Cancer Drugs The ability of compound 1 to affect cell growth was studied in MCF-7 cells, evaluating its ability to potentiate the anti-proliferative effects of the aminopeptidase inhibitor bestatin and the DNA cross-linking cisplatin. Over a 72 h incubation, compound 1 alone showed a significant reduction of cell growth with an IC50 value of 124 ± 24 µM (determined between concentration range 0.5-1000 µM) (Figure 5). At 100 µM concentration bestatin, cisplatin and BCH all showed very weak inhibition (0-24%) of the growth of MCF-7 cells over 72 h, whereas compound 1 reduced cell growth significantly (Figure 6, Table 1). Thus, the IC50 values for bestatin, cisplatin and BCH were 541 ± 86, 289 ± 47 and 362 ± 38 µM, respectively (Figure 5), with compound 1 being the most effective when tested alone. Under the same conditions, a combination of compound 1 close to its IC50 value (100 µM) with bestatin (100 µM) showed significantly increased anti-proliferative efficacy (69%) compared the latter compound alone (0%) (Table 1). Similarly with cisplatin (100 µM), inhibition of cell growth was increased from 0% (cisplatin alone) to 82% with combination of compound 1 (100 µM). Compared to BCH, which was able to inhibit the cell growth at 100 µM concentration only 24% and potentiate the anti-proliferative efficacy of 100 µM bestatin only 3% and 100 µM cisplatin 43%, the compound 1 proved to be more effective (Figure 6, Table 1). Moreover, compound 1 was effective and able to potentiate the anti-proliferative efficacy of bestatin (100 µM) and cisplatin (100 µM) at a


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lower concentration of 25 µM, inhibiting cell growth 53% and 50%, respectively (Figure 6, Table 1). In vivo Pharmacokinetics of Compound 1 in Mice In vivo pharmacokinetics and tissue distribution of compound 1 were determined after 23 µmol/kg i.p. injection at seven time points between 10 and 480 min. The time-concentration profiles of compound 1 in blood, brain, liver, prostate, heart and kidneys are presented in Figure 7, and the apparent pharmacokinetic parameters AUC0–480 min, Cmax , tmax ,and t½β calculated from the in vivo data are presented in Table 2. In addition, the compound 1 unbound fraction in tissues (fu, tissue) was determined and then used to calculate the unbound AUC0–480 min and Cmax from the pharmacokinetic data (Table 2.) As predicted, compound 1 was distributed efficiently into liver after i.p. injection, since from the injection site drugs are absorbed into the mesenteric vessels, which drain into the portal vein and pass through the liver.38 More surprising was the relatively high AUC value in prostate, which was not explained by high non-specific tissue binding (Table 2). Interestingly, the concentration of compound 1 in the brain was the lowest from the evaluated tissues, despite the high expression of LAT1 at the blood-brain barrier. This indicates that compound 1 is not a LAT1 substrate and transported across the BBB. However, we also determined blood-brain barrier permeation of compound 1 using an in situ mouse brain perfusion technique, which confirmed that compound 1 had extremely poor brain uptake. The unidirectional transfer constant (Kin) for compound 1 was only 8.2 ˟ 10-5 ± 0.5 ˟ 10-5 mL/s/g (mean ± SD, n = 3), whereas for endogenous LAT1 substrate, L-leucine, the Kin-value was 156times higher, 12.9 ˟ 10-3 ± 0.4 ˟ 10-3 mL/s/g (mean ± SD, n = 4). Thus, compound 1 was most probably only bound to LAT1 in the microvascular endothelial cells and not transported across


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the BBB, since in the present study a capillary depletion method was not used. Therefore, we concluded that compound 1 was a LAT1 inhibitor.


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Binding of Compound 1 to LAT1 Expressed at the Blood-Brain Barrier To clarify if compound 1 is reversibly bound to LAT1 at the blood-brain barrier, inhibition of the brain uptake of [14C]-L-leucine (0.157 µM) was determined using an in situ mouse brain perfusion technique. Figure 8 illustrates that compound 1 significantly inhibits the brain uptake of [14C]-L-leucine, when 100 µM compound 1 is co-perfused with [14C]-L-leucine (79% inhibition). The uptake inhibition was also significant but decreased when the brain perfusion was performed 10 min after an i.p. injection of compound 1 (27% inhibition). The plasma concentration of compound 1 at the time of perfusion was ca. 20 µM. As the perfusion buffer did not contain the compound 1, and thus the brain vasculature was cleared of the compound, it was confirmed that compound 1 was bound to LAT1 in a slow but reversible manner. When brain perfusion was performed 180 min after the i.p. injection of compound 1, the brain uptake of [14C]-L-leucine was regained (only 14% inhibition). At 180 min compound 1 was nearly completely eliminated from plasma (0.23 µM), and as the uptake of [14C]-L-leucine was not inhibited, it was concluded that LAT1 binding of compound 1 was reversible.


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DISCUSSION AND CONCLUSIONS In the present study we report a novel, potent and selective LAT1 inhibitor. This compound (1) inhibited the uptake of [14C]-L-leucine with an IC50 value of 18 µM in human breast cancer (MCF-7) cells, being 6.2-times more potent than BCH (IC50 value of 112 µM) (Figure 2B). However, in MCF-7 cells, the compound 1 did not show as significant inhibition of [14C]-Lleucine uptake as inhibitor 2 in human colon cancer-derived HT-29 cells (IC50 value of 0.06 µM) and in human YD-38 oral cancer cells (IC50 value of 0.79 µM)24, 25 In the MCF-7 cells, the mean normalized mRNA expression levels of LAT1 and 4F2hc were also lower than in HD-29 and YD-38 cells (Figure 2A). However, the mRNA levels poorly correlate with protein levels on the cell surface or the function of the protein.39 Therefore, the inhibitions of [14C]-L-leucine uptake should be compared with extreme caution between laboratories, even if the mRNA expression levels of the studied transporters are known.

Inhibition of LAT1 by compound 1 was reversible, as the compound was able to be removed from the surface of the MCF-7 cells (Figure 3A), and it was not transported into the cells (Figure 3B). Thus, to our best knowledge, compound 1 is the first reported compound that is truly behaving as an inhibitor and not as a substrate of LAT1. A known system L inhibitor, BCH was transported at very high rate into the MCF-7 cells (Figure 3B) and therefore in our opinion, it is a substrate of LAT1. BCH is a small compound (Mw. 155.19 g/mol) and thus readily transported into the cells, which also explains why in many studies the L-leucine uptake and cell growth have been reduced only at higher BCH concentrations.21-23 In comparison, compound 1 is much larger and bulkier (Mw. 587.60 g/mol), and with negligible LAT1-mediated cellular uptake (Figure 3B), it has more potential than BCH to act as an inhibitor of LAT1. No data has


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been reported to date to what extent the novel reported LAT1-inhibitor, inhibitor 2 is transported into the cells via LAT1. However, the inhibition of L-leucine uptake does not explain all of the anti-proliferative efficacy of inhibitor 2 and as it is a substrate for OATP1B1, 1B3 and 2B1 and OAT3, it is likely that it may be transported into the cancer cells.25,

26, 34, 40, 41

Inhibitor 2 is

slightly smaller (Mw. 472.32 g/mol) than compound 1 and it is structurally related to thyroid hormones, triiodothyronine (T3) and thyroxine (T4). According to Oda et al.24 inhibitor 2 was originally designed by a reference to the amino acid backbone of T3, a substrate of LAT1, as it was thought to have high affinity for LAT1 over LAT2 but low transportability via LAT1 into the cells. However, thyroid hormones are known to be transported by OATP1C1 across the BBB and they also utilize monocarboxylate transporters 8 and 10 (MCT8 and MCT10) for their cellular uptake.42 Therefore, there is a risk of unselective transporter-mediated uptake if the inhibitor is designed based on thyroid hormones, which may eventually impair the clinical usefulness of the inhibitor.20, 26

Compound 1 was designed by our 3D-QSAR model of the rat LAT1 binding site and therefore, it was expected that this compound had high selective affinity for human LAT1.28 Indeed, the inhibition of L-leucine uptake (76%) via rat LAT1 estimated by the 3D-QSAR model correlated extremely well with the value gained in MCF-7 cells expressing human LAT1 (78%). In this study we also evaluated whether the compound 1 had affinity for LAT2, MCT1, as well as OATP1A2, 2B1, 1B1 and 1B3. According to our results, compound 1 had no affinity for LAT2 as it was not significantly able to inhibit the uptake of [14C]-L-alanine at concentration level of 50-1000 µM (Figure 2C). Therefore, our interpretation is that compound 1 is not a LAT2 substrate. LAT2 transports also smaller amino acids than LAT13-6 and therefore, it carries


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smaller non-natural compounds, such as BCH. However, although LAT1 and LAT2 have similar substrate selectivity of natural amino acids, they are not identical. Moreover, transport capacity of non-natural compounds, such as drugs, via LAT2 has not been thoroughly studied to date and therefore, it is yet unknown, if LAT2 is able to interact and carry larger compounds, such as inhibitor 2, which uses other transporters for the cellular uptake. Binding of compound 1 was also studied in the presence of the known competing MCT1 inhibitor, phloretin, OATP1A2/2B1 inhibitor, naringin, OATP1B1/1B3 inhibitor, rifampicin, and unselective OATP substrate, Lthyroxine (T4). According to our results (Figure 4), none of these compounds were able to change the detected amount of compound 1 bound mainly to LAT1. However, at this point we relied on the literature data for which of these transporters are reported to be expressed in MCF734-37 and thus, we did not quantify their expression or function. Therefore, detailed selectivity studies need to be carried out in future, with specific transporters transfected or using knockdown cell-lines. Nonetheless, at this point it was concluded that compound 1 is a selective inhibitor of LAT1 and maybe thus superior to inhibitor 2 in terms of potency to inhibit L-leucine uptake but also prospect of having less off-target adverse effects. Moreover, compound 1 was found to be both chemically stable (at pH 7.4 for 24 h) as well as metabolically stable (in mouse liver S9 and microsomes as well as human plasma) for 2 h. Therefore, compared to inhibitor 2, which undergoes relatively fast N-acetylation in liver, intestine and kidney of various species,27 compound 1 seems also to be metabolically superior.

Compound 1 had good anti-proliferative efficacy, alone with an IC50 value of 124 µM in MCF7 cells after 72 h incubation (Figure 5).Thus, compound 1 showed only 6.9-times less inhibition of L-leucine uptake than cell growth, indicating that inhibition of LAT1 could be the primary


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mechanism of its anti-proliferative efficacy. In contrast, inhibitor 2 had 68-87-fold difference between the L-leucine uptake and cell growth inhibition in YD-38 and HT-29 cells and therefore, there must be some other apoptic mechanisms involved to its anti-proliferative efficacy other than L-leucine uptake inhibition. However, it is likely that when cancer cells are deprived of essential amino acids, they develop other compensating routes for amino acid supply, such as via autophagy and ubiquitin-proteasome pathway, by which peptides are degraded into amino acids by intracellular aminopeptidases. Therefore, a LAT1 inhibitor would be beneficial when administered in combination with aminopeptidase inhibitor, such as bestatin.43 In our studies, bestatin (100 µM) alone was not as effective as compound 1 in inhibiting the cell growth in MCF-7 cells, and its IC50 value was therefore rather high (541 µM). However, in combination with compound 1, inhibition was increased from 0% to 53% already with 25 µM concentration of compound 1 during 72 h incubation (Figure 6, Table 1). For comparison, we studied whether compound 1 can also potentiate the anti-proliferative efficacy of the DNA cross-linking agent cisplatin, which has been used against breast cancer. Similarly, cisplatin (100 µM) alone inhibited the cell growth with relatively high IC50 value (289 µM), but in combination with compound 1, the anti-proliferative efficacy was improved significantly from 0% to 50% inhibition with 25 µM concentration of compound 1 (Figure 5, Table 1). Moreover, compound 1 was more potent as an anti-proliferative agent than BCH (IC50 of 362 µM), which did not inhibit the cell growth at 100 µM concentration significantly alone or in combination with bestatin or cisplatin. Therefore, it can be concluded that compound 1 may have great potential alone and especially in combination with other anti-cancer or immunomodulating drugs, such as aminopeptidase inhibitors, but also with other DNA alkylating agents. Moreover, the potentiating efficacy can be achieved already at lower concentration of compound 1.


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The pharmacokinetic evaluation of compound 1 revealed that the brain uptake was low even though LAT1 expression at the BBB is known to be high44 and compound 1 showed high affinity for LAT1 (Figure 7). This is clear evidence that compound 1 is a LAT1 inhibitor and not a substrate. In accordance with the in vitro data, the in situ brain perfusion results indicated that compound 1 is a slowly reversible inhibitor, since the uptake inhibition of LAT1 substrate [14C]L-leucine

was time-dependent (Figures 3A and 8). Thus, the reversibility of LAT1 inhibition at

the BBB ensures that the source of amino acids for the brain or other healthy cells is recovered over time. This is a desired property for a LAT1 inhibitor, since the function of LAT1 is necessary for the uptake of essential amino acids into neurons and astrocytes as well as activated T cells.7, 8 Moreover, the low brain uptake in vivo suggests that compound 1 is not able to utilize other influx transporters present at the BBB. However, it has to be remembered that there can be substrate recognition, tissue distribution as well as protein levels differences between mouse and human LAT1 and in different diseases or disease stages the expression and function of LAT1 may be altered.

As can be expected after an i.p. injection, the highest Cmax and AUC values were reached in the liver. However, the first pass metabolism did not eliminate the compound 1 entirely and it distributed into other tissues as well (Figure 7, Table 2). Perhaps the most notable result of the in vivo pharmacokinetic evaluation was the accumulation of compound 1 into prostate. The AUC value in prostate (1275 nmol/g×min) was nearly as high as AUC value in kidney (1426 nmol/g×min) and over 5-times higher than in heart (249 nmol/g×min), despite the latter tissues are more highly perfused by blood. The prostate accumulation was not due to higher non-specific


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binding compared to other tissues (Table 2). Interestingly, the Cmax value in prostate (24.1 µM) was above the in vitro IC50-value of LAT1-mediated uptake of compound 1 (18.15 µM), and at 25 µM concentration, compound 1 significantly potentiated the in vitro anti-proliferative effect of bestatin and cisplatin. Therefore, accumulation into prostate makes compound 1 a promising candidate for the treatment of prostate cancer, in which high LAT1 expression has been reported and associated with a poor prognosis for patients.17 However, the mechanism, which leads to accumulation of compound 1 into prostate should be studied more thoroughly in future.


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In conclusion, we have designed and synthesized a potent and selective LAT1 inhibitor (1) that is able to inhibit binding and transport of essential neutral amino acids and thus, inhibit the cell growth of cancer cells. The inhibitor reduced the cell growth effectively alone, but also in combination with aminopeptidase inhibitor, bestatin, and DNA cross-linking agent, cisplatin, even at low concentrations. The inhibitor was not transported into the cells, like BCH and thus, it is not a substrate of LAT1. In contrast, it was classified as a slowly reversible LAT1 inhibitor, as it was able to be detached from the cell surface as well as from the BBB over the time course. As the inhibitor was not metabolized and it showed high selectivity for LAT1 over LAT2, OATPs and MCT1 in our primary screening, it may be superior to a novel LAT1 inhibitor 2. Moreover, according to the pharmacokinetic and tissue distribution analysis, the inhibitor was able to readily accumulate in the prostate. Due to the fact that LAT1 is over-expressed in prostate cancer and associated with poor prognosis and patient survival, the inhibitor reported herein may be a promising drug candidate as such or as an adjuvant for the treatment of prostate cancer in future.


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EXPERIMENTAL SECTION General Procedures All general procedures and experimental information are given in Supporting Information. Synthesis and characterization of the compound 1, analytical methods (HPLC-UV and LS-MS), characterization of MCF-7 cells and the binding and uptake of compounds in MCF-7 cells, antiproliferative efficacy of compounds in MCF-7 cells, in situ mouse brain perfusion, pharmacokinetics of compound 1 in mice and plasma and tissue sample preparation and binding are given below.

(S)-2-Amino-3-(3-((2,4-dicyano-3-(4-(2-(methylamino)-2oxoethoxy)phenyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)carbamoyl)phenyl)propanoic acid (1) 2-(4-(1-Amino-2,4-dicyanobenzo[4,5]imidazo[1,2-a]pyridin-3-yl)phenoxy)-Nmethylacetamide

3

carboxyethyl)benzoic

(0.28 acid

g,

0.71 4

mmol) (0.35

and

BBN-protected

g,

1.066

mmol),

(R)-3-(2-amino-21-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (0.27 g, 1.41 mmol) and 4-dimethylaminopyridine (0.17 g, 1.41 mmol) were refluxed in anhydrous CH2Cl2/dimethylformamide (10:1, 10 mL) solution under Ar-atm overnight. The solvent volume was reduced by evaporation and the compound was purified by flash column chromatography eluting with 1-10% MeOH/CH2Cl2 solution to yield yellow solid (0.39 g, 77%). 1H NMR ((CD3)2SO): δ ppm 8.75 (d, J = 8.3 Hz, 1H), 8.15 (s, 1H), 8.04 (d, J = 7.7 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.55-7.49 (m, 2H), 7.48-7.41 (m, 2H), 7.287.24 (m, 1H), 7.12 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 7.4 Hz, 1H), 6.58-6.49 (m, 1H), 6.00-5.91 (m, 1H), 4.56 (s, 2 H), 3.91-3.83 (m, 1H), 3.30-3.24 (m, 1H), 3.19-3.14 (m, 1H), 2.69 (d, J = 4.6 Hz,


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3H), 1.89-1.14 (m, 12H), 0.54-0.45 (m, 2H). MS (ESI-) for C40H37BN7O5 (M-H)-: Calcd 707.60, Found 706.37.

The compound prepared above (0.39 g, 0.55 mmol) and ethylenediamine (0.18 mL, 2.76 mmol) were refluxed in anhydrous tetrahydrofurane/dimethylformamide (10:1, 10 mL) for 10 min. The solvent was removed by evaporation and the compound was purified by flash column chromatography eluting with 5-75% MeOH/CH2Cl2 solution and triturated with Et2O/CH2Cl2 solution to yield the compound 1 as a yellow solid (0.16 g, 70%). 1H NMR ((CD3)2SO): δ ppm 8.73 (d, J = 8.0 Hz, 1H), 8.06 (s, 1H), 8.02 (d, J = 7.4 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.51 (d, J = 8.1 Hz, 2H) 7.49-7.37 (m, 3H), 7.27 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 8.1 Hz, 2H), 4.56 (s, 2 H), 3.42-3.37 (m, 1H), 3.33-3.23 (m, 1H), 2.92-2.83 (m, 1H), 2.69 (d, J = 4.2 Hz, 3H); 13C NMR ((CD3)2SO): δ ppm 173.44, 169.73, 167.80, 158.54, 154.33, 153.82, 148.52, 144.74, 142.51, 137.99, 131.71, 130.40 (2C), 129.89, 128.76, 127.97, 127.21, 125.12, 120.92, 117.71, 117.57, 117.15, 117.09, 114.48 (2C), 107.63, 82.43, 82.02, 67.06, 55.87, 37.47, 25.36. MS (ESI-) for C32H24N7O5 (M-H)-: Calcd 587.60, Found 586.22. Elemental analysis for C32H25N7O5 * 1.30 Et2O * 0.5 CH2Cl2, Clcd. C, 62.34%,; H, 5.34%; N, 13.50%, Found C, 62.43%; H, 5.73%, N, 13.34%. UV-HPLC purity 97.39%

High-Performance Liquid Chromatography (HPLC) Analyses The amount of compound 1 and BCH were determined by the HPLC system (Agilent 1100 series, Agilent Technologies Inc., Wilmington, DE, USA), at the wavelength of 285 nm and 200 nm for compound 1 and BCH, respectively. The compounds were separated on a Agilent Zorbax SB-C18 analytical column (4.6mm x 250 mm, 5 µm; Agilent Technologies Inc., Wilmington,


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DE, USA) running with 32 % of acetonitrile and 68% of 0.1% formic acid buffer (pH ca. 3.0) for compound 1 and with a 10:90 (v/v) ratio for BCH (1.0 mL/min). The lower limit of quantification for both compounds was 0.10 pmol/mg of protein. These HPLC methods were also selective, accurate and precise over the range 0.25-50 µM.

Liquid Chromatographic and Mass Spectrometric (LC-MS) Method Concentrations of compound 1 in the tissue samples were analyzed by Agilent 1200 Series Rapid Resolution LC System, together with Agilent 6410 Triple Quadrupole Mass Spectrometer equipped with an electrospray ionization source (Agilent Technologies Inc., Wilmington, DE, USA) LC-MS. Poroshell 120 EC-C-18 column (50 mm x 2.1 mm, 2.7 µm) (Agilent Technologies Inc., Wilmington, DE, USA) was used for chromatography. The high-performance liquid chromatography eluents were water (A) and acetonitrile (B), both containing 0.1% formic acid. A gradient elution of 20–90% B was applied in 1–3 min, followed by 3 min column equilibration. The mobile phase flow rate was 0.3 mL/min, the column temperature 60 °C and injection volume 5 µL. The following mass spectrometry conditions were used for acquiring the data data: electrospray ionization with positive ion mode; nitrogen drying gas at 300 °C and flow rate of 10 L/min; nebulizer pressure, 40 psi; and capillary voltage, 4000 V. Analyte detection was performed using multiple reaction monitoring, the transitions being 588 → 191.6, for compound 1. The transition for the internal standard, repaglinide, was 453.5 → 229.9. Fragmentor voltages and collision energies used for compound 1 were 130 V and 18 V, respectively, and for repaglinide the parameters were 75V and 8V, respectively. The lower limit or quantification of compound 1 in the tissue samples was 10 pmol/g.


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Expression and Function of LAT1 and LAT2 MCF-7 human breast adenocarcinoma cells (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Total RNA extraction from MCF-7 cells by using RNeasy Micro Kit (50) (Qiajen, Hilden, Germany), according to manufacturer’s instructions. The extracted RNA was treated with DNase (DNA fee, AMbion, TX, USA), and the amount of RNA was quantified by using the RiboGreen assay (Molecular Probes, Leiden, Netherlands). The RNA (0.5 µg) was converted into cDNA by using M-MuLV reverse transcriptase (400 U), random hexamers (20 µg) and dNTPs (10 mM) (Fermentas, Hanover, MD, USA). Quantification of the LAT1 and LAT2 genes was performed by employing Prism 7500 sequence detection system (Applied Biosystems, Inc., Foster City, CA, USA). Briefly, 6 µL of each sample was mixed with 10 µL of PCR reagent mixture containing 0.5 µL of primer probe mix (TaqMan Gene Expression assay, Applied Biosystems), 0.5 µL of sterile water and 5 µL of TaqMan master mix (Applied Biosystems). The used primer probe mixes were Hs01001183_m1 (LAT1, SLC7A5) and Hs00794796_m1 (LAT2, SLC7A8).

The function of LAT1 and LAT2 were determined known radiolabelled substrates, [14C]-Lleucine (PerkinElmer, Waltham, MA, USA) for LAT1 or [14C]-L-alanine (PerkinElmer, Waltham, MA, USA) for LAT2. MCF-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with L-glutamine (2 mM), heat-inactivated fetal bovine serum (10%), penicillin (50 U/mL) and streptomycin (50 µg/mL). MCF-7 cells were seeded at the density of 1 × 105 cells/well onto 24-well plates. The cells were used for the uptake experiments one day after seeding. After removal of the culture medium, the cells were carefully washed with pre-warmed HBSS (Hank’s balance salt solution) containing 125 mM choline chloride, 4.8 mM


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KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.3 mM CaCl2, 5.6 mM glucose, and 25 mM HEPES (pH 7.4). The cells were then pre-incubated in 500 µL of pre-warmed HBSS at 37 °C for 10 min before adding substrates (250 µL in HBSS) for the uptake experiment. The uptake time of radiolabeled substrates (min 0.157-1000 µM of [14C]-L-leucine or [14C]-L-alanine) was 5 min (concentration dependent uptake) or 0.5-30 min of 0.157 µM of [14C]-L-leucine or 10.0 µM [14C]-L-alanine (time-dependent uptake). The uptake of [14C]-L-leucine and [14C]-L-alanine was also studied at 4 °C to confirm that the transport was active and not passively mediated. The uptake of [14C]-L-leucine was evaluated in the presence and absence of Na+ to confirm that Lleucine was not transported via Na+-dependent transporters. The cells were washed three times with ice-cold HBSS. The cells were then lysed with 500 µL of 0.1 M NaOH on the ice-bath and the lysate was mixed with 3.5 mL of Emulsifier safe cocktail (PerkinElmer, Waltham, MA, USA). The radioactivity was measured by liquid scintillation counting (Wallac 1450 MicroBeta; Wallac Oy, Finland).

Ability of Compound 1 and BCH to Bind to LAT1 and/or LAT2 and Half of Maximum Inhibitory Concentration (IC50) Value The ability of compound 1 and BCH to inhibit the uptake of a known LAT1 substrate, [14C]-Lleucine, and ability of compound 1 to inhibit the uptake of a known LAT2 substrate, [14C]-Lalanine was carried out as described above. MCF-7 cells were incubated at 37 °C for 5 min in 250 µL of uptake medium containing 0.157 µM of [14C]-L-leucine or 10.0 µM of [14C]-L-alanine and 1-1000 µM of the compound 1 or BCH (or DMSO as blank). Subsequently, the cells were washed three times with ice-cold HBSS and then lysed and analyzed as described above.


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Transporter-Mediated Binding/Transport of Compound 1 or BCH in MCF-7 Cells The binding/transport of compound 1 and BCH were studied by adding 25-400 µM of compound in pre-warmed HBSS buffer (250 µL) on the top of the cell layer and incubated the mixture at 37 °C for 30 min. The cells were then washed three times with ice-cold HBSS and lysed with 500 µl of 0.1 M NaOH. The supernatants were analyzed by the HPLC method described above and the concentration of each experiment was calculated from the standard curve that was prepared by spiking known amounts of compound 1 of BCH to cell lysate. The protein concentrations on each plate were determined as mean of 3 samples by Bio-Rad Protein Assay (EnVision, Perkin Elmer, Inc., Waltham, MA, USA). The reversible binding was studied by incubating the compound 1 (100 µM) 5 and 30 min and then washing the cells three times with warm (37 ᵒC) HBSS buffer before lysing the cells. In the case of 30 min experiment the cells were washed two times, after 5 min and 30 min incubation before the cells were lysed.

The competitive binding in the presence of 500 µM phloretin (MCT1 inhibitor), naringin (OATP1A2/2B1 inhibitor), rifampicin (OATP 1B1/1B3 inhibitor) or L-thyroxine (T4, unselective OATP substrate) was carried out as described above by HBSS buffer solution that contained 100 µM of compound 1. The amounts of compound 1 were analyzed by the HPLC method described above and calculated from the spiked standard curve.

Anti-proliferative Activity in Vitro MCF-7 cells were seeded at the density of 2 × 104 cells/well onto collagen-coated 96-well plates. The cells were used for the proliferation experiments one day after seeding. A concentration of 0.5 - 1000 µM of test compounds; compound 1, BCH, bestatin and cisplatin, as


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well as, combinations of compound 1 (25 and 100 µM) or BCH (100 µM) with bestatin (100 µM) or cisplatin (100 µM) were added into the growth medium and incubated for 3 days. Each day the cell viability was determined by resazurin cell proliferation kit (Sigma, St. Louis, MO, USA), which is directly proportional to aerobic respiration and cellular metabolism of cells. The samples were measured fluorometrically by monitoring the increase in fluorescence at a wavelength of 590 nm using an excitation wavelength of 560 nm (EnVision, Perkin Elmer, Inc., Waltham, MA, USA). The cell death was confirmed in the decrease of cell amount by visualizing the wells with microscopy.

In Situ Mouse Brain Perfusion Adult male mice weighing 25 ± 5 g, supplied by Envigo (Venray, Netherlands) were used for the in vivo pharmacokinetic and in situ brain perfusion experiments. Mice were anesthetized with intraperitoneal (i.p.) bolus injections of ketamine (120 mg/kg) and xylazine (8 mg/kg). The right external carotid artery was ligated, and the right common carotid artery was cannulated with a PE-10 catheter (i.d. 0.28 mm; o.d. 0.61 mm) filled with saline including 100 IU/mL of heparin. Investigated compounds were perfused at 37 °C with a flow rate of 2.5 mL/min. The presence of functional LAT1-transporters in the mouse blood-brain barrier was confirmed with [14C]-L-leucine, which is a known substrate for LAT1. The 100 % Kin of 0.157 µM [14C]-Lleucine was determined by perfusing the mouse brain for 30 s, followed by perfusing the brain with analyte free perfusion buffer for 2 s at 4 °C. Kin was calculated using the equation 1.

=

×

(1)

where Qtot is total brain concentration, Cpf is the analyte concentration in the perfusion fluid and T is the perfusion time.


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The ability of compound 1 to bind to LAT1 was determined by co-perfusing 100 µM of compound 1 with [14C]-L-leucine and calculating the decrease of [14C]-L-leucine Kin compared to the 100 % control. The reversibility of LAT1 inhibition by compound 1 was determined by injecting 23 µmol/kg of compound 1 i.p. and then performing the brain perfusion with [14C]-Lleucine 10 or 180 min after the i.p. injection. The Kin of compound 1 was determined by perfusing the mouse brain with 100 µM of compound 1 for 60 s, followed by 2 s perfusion with drug free buffer at 4 °C.

In Vivo Pharmacokinetics of Compound 1 A concentration of 1.36 mM of compound 1 was dissolved in a vehicle containing 10 % (v/v) of DMSO, 20 % (w/v) of hydroxypropyl-β-cyclodextrin and 0.9 % (w/v) NaCl in water. A dose of 23 µmol/kg of compound 1 was administered as a bolus injection (i.p.) to mice. The mice were sacrificed by decapitation at selected time points between (10–480 min) and plasma, brain, liver, prostate, kidney and heart were collected for analysis.

Plasma and Tissue Sample Preparation Plasma samples were prepared by precipitating of 100 µL of plasma with 200 µL of acetonitrile containing the internal standard, repaglinide. Samples were vortexed and centrifuged for 10 min at 14,000 x g at 4 °C. Then 100 µL of supernatant was mixed with 100 µL of ultrapure water. Tissue samples were weighed and homogenized with ultrapure water (1:3). An aliquot of 100 µL from the homogenates was taken, and the analyte was isolated from the samples by protein precipitation with 300 µL of acetonitrile containing the internal standard.


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Samples were vortexed and centrifuged for 10 min at 14,000 x g at 4 °C. Prior to LC-MS analysis, 200 µL of supernatant was mixed with 100 µL of ultrapure water.

Plasma and Tissue Protein Binding of Compound 1 Non-specific tissue binding of the compound 1 was determined at 100 µM in mouse plasma, brain, liver, kidney, heart and prostate homogenates using equilibrium dialysis. The experiment protocol has been previously described by Gynther et al. 2015 45 and can be found in Supporting Information.

Data analysis All statistical analyses were performed using GraphPad Prism v. 5.03 software (GraphPad Software, San Diego, CA, USA). Statistical differences between groups were tested using oneway ANOVA, followed by a two-tailed Dunnett’s or Tukey’s test. Half of maximum inhibitory concentration (IC50) values were calculated by nonlinear regression analysis and presented as mean ± SD, (n=3). The pharmacokinetic parameters, area under the concentration-time curve from time zero to 360 min (AUC0-480), the maximum concentration after dosing (Cmax), time to reach Cmax (tmax), elimination half-lives (t½β) in plasma, brain, liver, prostate, kidneys and heart were obtained from the pharmacokinetic data. All in vivo data is presented as mean ± SEM.


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ANCILLARY INFORMATION Supporting Information More detailed information of materials (chemicals and animals) and experimental procedures of aqueous solubility, chemical and enzymatic stabilities and plasma and tissue protein binding of compound 1. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author Information * Phone: (358) 40 3553 684; E-mail: [email protected]

Funding Sources The work was financially supported by the Academy of Finland (#256837), Sigrid Juselius Foundation, Emil Aaltonen Foundation and Orion Research Foundation.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors would like to thank Ms. Helly Rissanen for invaluable technical assistance with in vitro bioconversion studies, Henna Ylikangas, M.Sc., for predicting LAT1 inhibition from 3D-


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QSAR, Dr. Tarja Kokkola, Ph.D. for help with MCF-7 cells and Dr. Kati-Sisko Vellonen for help with RT-qPCR.

ABBREVIATIONS BBB, blood-brain barrier; BCH, 2-amino-2-norbornane-carboxylic acid; 3D-QSAR, 3dimensional quantitative structure activity; LAT1, L-type amino acid transporter 1; MCF-7, human breast cancer cells; MCT1, monocarboxylate transporter 1; mTOR, mammalian target of rapamycin; OAT3, organic anion transporter 3; OATPs organic anions transporting polypeptides; PARP, poly(ADP-ribose) polymerase.


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Wang, Q.; Tiffen, J.; Bailey, C. G.; Lehman, M. L.; Ritchie, W.; Fazli, L.; Metierre, C.;

Feng, Y. J.; Li, E.; Gleave, M.; Buchanan, G.; Nelson, C. C.; Rasko, J. E.; Holst, J., Targeting amino acid transport in metastatic castration-resistant prostate cancer: effects on cell cycle, cell growth, and tumor development. J. Natl. Cancer Inst. 2013, 105, (19), 1463-1473. 21.

Imai, H.; Kaira, K.; Oriuchi, N.; Shimizu, K.; Tominaga, H.; Yanagitani, N.; Sunaga, N.;

Ishizuka, T.; Nagamori, S.; Promchan, K.; Nakajima, T.; Yamamoto, N.; Mori, M.; Kanai, Y., Inhibition of L-type amino acid transporter 1 has antitumor activity in non-small cell lung cancer. Anticancer Res. 2010, 30, (12), 4819-4828. 22.

Kim, C. S.; Cho, S. H.; Chun, H. S.; Lee, S. Y.; Endou, H.; Kanai, Y.; Kim do, K., BCH,

an inhibitor of system L amino acid transporters, induces apoptosis in cancer cells. Biol. Pharm. Bull. 2008, 31, (6), 1096-1100. 23.

Shennan, D. B.; Thomson, J., Inhibition of system L (LAT1/CD98hc) reduces the growth

of cultured human breast cancer cells. Oncol. Rep. 2008, 20, (4), 885-889. 24.

Oda, K.; Hosoda, N.; Endo, H.; Saito, K.; Tsujihara, K.; Yamamura, M.; Sakata, T.;

Anzai, N.; Wempe, M. F.; Kanai, Y.; Endou, H., L-type amino acid transporter 1 inhibitors inhibit tumor cell growth. Cancer Sci. 2010, 101, (1), 173-179. 25.

Yun, D. W.; Lee, S. A.; Park, M. G.; Kim, J. S.; Yu, S. K.; Park, M. R.; Kim, S. G.; Oh,

J. S.; Kim, C. S.; Kim, H. J.; Kim, J. S.; Chun, H. S.; Kanai, Y.; Endou, H.; Wempe, M. F.; Kim do, K., JPH203, an L-type amino acid transporter 1-selective compound, induces apoptosis of YD-38 human oral cancer cells. J. Pharmacol. Sci. 2014, 124, (2), 208-217.


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Toyoshima, J.; Kusuhara, H.; Wempe, M. F.; Endou, H.; Sugiyama, Y., Investigation of

the role of transporters on the hepatic elimination of an LAT1 selective inhibitor JPH203. J. Pharm. Sci. 2013, 102, (9), 3228-3238. 27.

Wempe, M. F.; Rice, P. J.; Lightner, J. W.; Jutabha, P.; Hayashi, M.; Anzai, N.; Wakui,

S.; Kusuhara, H.; Sugiyama, Y.; Endou, H., Metabolism and pharmacokinetic studies of JPH203, an L-amino acid transporter 1 (LAT1) selective compound. Drug Metab. Pharmacokinet. 2012, 27, (1), 155-161. 28.

Ylikangas, H.; Malmioja, K.; Peura, L.; Gynther, M.; Nwachukwu, E. O.; Leppanen, J.;

Laine, K.; Rautio, J.; Lahtela-Kakkonen, M.; Huttunen, K. M.; Poso, A., Quantitative insight into the design of compounds recognized by the L-type amino acid transporter 1 (LAT1). ChemMedChem. 2014, 9, (12), 2699-2707. 29.

Lyons, D. M.; Huttunen, K. M.; Browne, K. A.; Ciccone, A.; Trapani, J. A.; Denny, W.

A.; Spicer, J. A., Inhibition of the cellular function of perforin by 1-amino-2,4dicyanopyrido[1,2-a]benzimidazoles. Bioorg. Med. Chem. 2011, 19, (13), 4091-4100. 30.

Peura, L.; Malmioja, K.; Huttunen, K.; Leppanen, J.; Hamalainen, M.; Forsberg, M. M.;

Gynther, M.; Rautio, J.; Laine, K., Design, synthesis and brain uptake of LAT1-targeted amino acid prodrugs of dopamine. Pharm. Res. 2013, 30, (10), 2523-2537. 31.

Wang, Q.; Morris, M. E., Flavonoids modulate monocarboxylate transporter-1-mediated

transport of gamma-hydroxybutyrate in vitro and in vivo. Drug Metab. Dispos. 2007, 35, (2), 201-208. 32.

Kalliokoski, A.; Niemi, M., Impact of OATP transporters on pharmacokinetics. Br. J.

Pharmacol. 2009, 158, (3), 693-705.


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Roth, M.; Obaidat, A.; Hagenbuch, B., OATPs, OATs and OCTs: the organic anion and

cation transporters of the SLCO and SLC22A gene superfamilies. Br. J. Pharmacol. 2012, 165, (5), 1260-1287. 34.

Buxhofer-Ausch, V.; Secky, L.; Wlcek, K.; Svoboda, M.; Kounnis, V.; Briasoulis, E.;

Tzakos, A. G.; Jaeger, W.; Thalhammer, T., Tumor-specific expression of organic aniontransporting polypeptides: transporters as novel targets for cancer therapy. J. Drug Deliv. 2013, 2013, 863539-863550. 35.

Hussien, R.; Brooks, G. A., Mitochondrial and plasma membrane lactate transporter and

lactate dehydrogenase isoform expression in breast cancer cell lines. Physiol. Genomics. 2011, 43, (5), 255-264. 36.

Stute, P.; Reichenbach, A.; Szuwart, T.; Kiesel, L.; Gotte, M., Impact of testosterone on

the expression of organic anion transporting polypeptides (OATP-1A2, OATP-2B1, OATP-3A1) in malignant and non-malignant human breast cells in vitro. Maturitas. 2012, 71, (4), 376-384. 37.

Wlcek, K.; Svoboda, M.; Thalhammer, T.; Sellner, F.; Krupitza, G.; Jaeger, W., Altered

expression of organic anion transporter polypeptide (OATP) genes in human breast carcinoma. Cancer Biol. Ther. 2008, 7, (9), 1450-1455. 38.

Lukas, G.; Brindle, S. D.; Greengard, P., The route of absorption of intraperitoneally

administered compounds. J. Pharmacol. Exp. Ther. 1971, 178, (3), 562-564. 39.

Maier, T.; Guell, M.; Serrano, L., Correlation of mRNA and protein in complex

biological samples. FEBS Lett. 2009, 583, (24), 3966-3973. 40.

Li, Q.; Shu, Y., Role of solute carriers in response to anticancer drugs. Mol. Cell Ther.

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Thakkar, N.; Lockhart, A. C.; Lee, W., Role of Organic Anion-Transporting Polypeptides

(OATPs) in Cancer Therapy. AAPS J. 2015, 17, (3), 535-545. 42.

Kinne, A.; Schulein, R.; Krause, G., Primary and secondary thyroid hormone

transporters. Thyroid Res. 2011, 4 Suppl 1, S7-S16. 43.

Fan, X.; Ross, D. D.; Arakawa, H.; Ganapathy, V.; Tamai, I.; Nakanishi, T., Impact of

system L amino acid transporter 1 (LAT1) on proliferation of human ovarian cancer cells: a possible target for combination therapy with anti-proliferative aminopeptidase inhibitors. Biochem. Pharmacol. 2010, 80, (6), 811-818. 44.

Boado, R. J.; Li, J. Y.; Nagaya, M.; Zhang, C.; Pardridge, W. M., Selective expression of

the large neutral amino acid transporter at the blood-brain barrier. Proc. Natl. Acad. Sci. U S A. 1999, 96, (21), 12079-12084. 45.

Gynther, M.; Kaariainen, T. M.; Hakkarainen, J. J.; Jalkanen, A. J.; Petsalo, A.;

Lehtonen, M.; Peura, L.; Kurkipuro, J.; Samaranayake, H.; Yla-Herttuala, S.; Rautio, J.; Forsberg, M. M., Brain pharmacokinetics of ganciclovir in rats with orthotopic BT4C glioma. Drug Metab. Dispos. 2015, 43, (1), 140-146.


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Page 38 of 51

FIGURES

Figure 1. Chemical structures of 2-amino-2-norbornane-carboxylic acid (BCH) and inhibitor 2.


38

Page 39 of 51

0.15

0.10

0.05

T2

ch ai 4F 2

he

av

y

LA

LA

n

0.00 T1

Mean normalized expression

A

B Remaining 0.157 µ M [14C]-Leucine uptake (%)

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


100 90 80 70 60 50 40 30 20 10 0 10 - 6 . 5

10 - 6 . 0

10 - 5 . 5

10 - 5 . 0

10 - 4 . 5

10 - 4 . 0

10 - 3 . 5

10 - 3 . 0

Concentration (log M)


39


C 130

Remaining 10.0 µM [14C]-Alanine uptake (%)

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

Page 40 of 51

120 110 100 90 80 70 60 50 40 30 20 10 0 10 - 4 . 5 0

10 - 4 . 2 5

10 - 4 . 0 0

10 - 3 . 7 5

10 - 3 . 5 0

10 - 3 . 2 5

10 - 3 . 0 0


Figure 2. A) Quantitative expression of LAT1, LAT2 and 4F2hc mRNA in MCF-7 cells (mean ± S.E.M., n=3). B) Inhibition of 0.157 µM [14C]-L-leucine uptake by compound 1 (filled circles ●) and BCH (open circles ○) in MCF-7 cells. IC50 values were 18.2 ± 1.2 µM and 112 ± 12 µM, for compound 1 and BCH, respectively (mean ± SD, n=3). C) Inhibition of 10.0 µM [14C]-Lalanine uptake by compound 1 in MCF-7 cells (mean ± SD, n=3). Predicted IC50 value was over 1 mM for compound 1.


40

Page 41 of 51

A **

nmol/mg protein

3

***

2

1

x2 as h w m

w ar

in ,

in ,

m

m

(3 0

(3 0

1 p. C

om

p.

1

om C

)

) w

ar m in ,w m (5

1 p. om C

co ld

w

w ol d in ,c m (5 1 p. om C

as h

as h)

as h)

0

B 20

nmol/mg of 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


15 10 5 0 0

100

200

300

400

µM

Figure 3. A) The total transporter binding of compound 1 (100 µM) in MFC-7 cells at 37 ᵒC after 5 and 30 min with cold wash and after 5 and 5+30 min with warm wash before cell lysing. B) Binding of compound 1 (filled circles ●) and transport of BCH (open circles ○) into the MCF7 cells over a concentration range of 25 - 400 µM. The data is presented as mean ± SD (n=3). An asterisk denotes a statistically significant difference from the respective control (*** P < 0.001, one-way ANOVA, followed by Tukey’s test).


41


nmol/mg of protein

5 4 3 2 1

Co m

C om

p. 1

p. 1

(to ta l)

(P hl or et in p. ) 1 (N C om ar in p. gi 1 n) (R C i fa om m p. pi ci 1 n) (L -T hy ro xi ne )

0

C om

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

Page 42 of 51

Figure 4. Selectivity of compound 1 (100 µM) towards LAT1 over MCT1 (500 µM phloretin), OATP1A2/2B1 (500 µM naringin), OATP1B1/1B3 (500 µM rifampicin) and other possible OATPs (500 µM L-thyroxine) expressed in MCF-7 cells. The data is presented as mean ± SD (n=3).


42

Page 43 of 51

120 110

Remaining viability (%)

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


100 90 80 70 60 50 40 30 20 10 0 -5.5

-5.0

-4.5

-4.0

-3.5

-3.0

-2.5


Figure 5. Concentration-dependent anti-proliferative efficacy of compound 1 (filled circles ●), BCH (open circles ○), bestatin (filled triangles▲) and cisplatin (open squares □) in MCF-7 cells after 72 h. IC50 value were 124 ± 14, 541 ± 86, 289 ± 47 and 362 ± 38 µM for compound 1, bestatin, cisplatin and BCH, respectively (mean ± SD, n=3).


43


*** *** 120

Remaining viability (%)

*** *

100 80 60 40 20

om C p. om 1 B es p. 25 µM 1 B tati es n B 100 C ta 1 0 µM tin 0 Be H µM sta 10 1 B +C t i n 0 µ es 00 ta µM om 10 M tin +C p 0 10 o m . 1 µM C is 2 0 p µM p. 1 5 µ C lati + 10 M is pl n 1 0 C B µM at 00 is CH pl in 1 µ 0 a M C is 100 +C tin 0 µ M pl 1 at µM om 00 in +C p µ 10 o . 1 M 0 mp 25 µM .1 µ +B 10 M 0 C µ H 10 M 0 µM

0

C

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

Page 44 of 51

Figure 6. Effects of compound 1, bestatin and cisplatin alone on the viability of MCF-7 cells and the potentiating effect of 25 and 100 µM compound 1 with 100 µM bestatin and cisplatin antiproliferative efficacy after 72 h. The percentage cell viability was calculated as a ratio of the drug treated cells and untreated control cells. The data is presented as mean ± SD, (n=3). An asterisk denotes a statistically significant difference from the respective control (* P < 0.05, *** P < 0.001, one-way ANOVA, followed by Tukey’s test).


44

Page 45 of 51

B

A

30

Concentration in prostate (nmol/g)

Concentration in plasma (nmol/ml)

30

25

20

15

10

5

0

25

20

15

10

5

0

0

30

60

90

120

150

180

210

240

270

300

0

30

60

90

Time (min)

120

150

180

210

240

270

300

210

240

270

300

210

240

270

300

Time (min)

D

C 30

Concentration in kidney (nmol/g)

Concentration in liver (nmol/g )

50 45 40 35 30 25 20 15 10 5 0

25

20

15

10

5

0

0

30

60

90

120

150

180

210

240

270

300

0

30

60

90

Time (min)

120

150

180

Time (min)

E

F 0.30 5

0.25

Concentration in heart (nmol/g)

Concentration in brain (nmol/g)

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


0.20

0.15

0.10

0.05

0.00

4

3

2

1

0

0

30

60

90

120

150

180

210

240

270

300

0

30

Time (min)

60

90

120

150

180

Time (min)


45


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

Page 46 of 51

Figure 7. Plasma (A), prostate (B), liver (C), kidney (D), brain (E) and heart (F) concentrationtime curves (up to 300 min) of compound 1 after a single dose of 23 µmol/kg i.p. in mice. The concentrations are presented as mean ± SEM (n=3).


46

* 0.014

***

0.012

*

Kin (mL/s/g)

0.010 0.008 0.006 0.004 0.002

10

er f

us io n C m wit tr h i 10 l 18 n a f 0 0 µM m ter in in je af c te r i tion nj ec t io n

0.000

op

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


C

Page 47 of 51

Figure 8. Inhibition of 0.157 µM [14C]-L-leucine uptake across the blood-brain barrier by 100 µM compound 1. The reversibility of the uptake inhibition by compound 1, was determined by injecting 23 µmol/kg (i.p.) of the compound and performing the brain perfusion with [14C]-Lleucine 10 min and 180 min after the injection. The data is presented as mean ± SD, (n=3). An asterisk denotes a statistically significant difference from the respective control (* P < 0.05, *** P < 0.001, one-way ANOVA, followed by Tukey’s test).


47


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Page 48 of 51

SCHEMES Scheme 1. Conversion of compound 3 to LAT1 inhibitor 1. a) EDC, DMAP, CH2Cl2/DMF, reflux, 20 h, 77%; b) ethylenediamine, THF/DMF, reflux, 10 min, 70%.


48

Page 49 of 51

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


TABLES Table 1. Reduction of MCF-7 Cell Viability after 24, 48 and 72 h incubation (mean ± SD, n=3). Reduction of cell viability (%) 24 h

48 h

72 h

Compound 1 25 µM

13.9 ± 0.6

-a

34.8 ± 1.5

Compound 1 100 µM

22.9 ± 4.7

14.8 ± 3.5

47.7 ± 9.0

BCH 100 µM

13.9 ± 6.3

-a

24.1 ± 11.0

100 µM Bestatin

3.4 ± 0.8

7.5 ± 8.5

-a

Bestatin 100 µM + compound 1 25 µM

24.7 ± 4.0

40.5 ± 13.2

52.6 ± 2.0

Bestatin 100 µM + compound 1 100 µM

-b

42.7 ± 13.3

68.6 ± 1.2

Bestatin 100 µM + BCH 100 µM

30.8 ± 7.5

-a

3.13 ± 6.1

Cisplatin 100 µM

-a

-a

-a

Cisplatin 100 µM + compound 1 25 µM

-b

-b

49.7 ± 9.5

Cisplatin 100 µM + compound 1 100 µM

65.0 ± 3.6

60.4 ± 4.6

81.9 ± 1.6

Cisplatin 100 µM + BCH 100 µM

35.9 ± 3.2

-a

42.7 ± 7.4

a

Viability above control. b High variance among samples.


49


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

Page 50 of 51

Table 2. Pharmacokinetic Parameters of Compound 1 in Plasma and Tissues Calculated from in Vivo Data after a Single Dose of 23 µmol/kg i.p. in Mice (n=3). AUC0-480 min

Cmax

(nmol/g×min) Plasma

AUCu, 0-480 min

Cu, max

(nmol/g) a (%)

(nmol/g×min)

(nmol/g) (min)

(min)

795

19.5 ± 8.6

0.98

7.8

0.19

10

44.9

Prostate

1275

24.1 ± 3.1

1.44

18.4

0.34

30

12.0

Liver

1930

38.7 ± 8.9

0.98

18.9

0.38

10

52.8

Kidney

1426

22.5 ± 3.9

1.66

23.7

0.37

10

41.8

Brain

11

0.2 ± 0.1

1.03

0.1

0.02

30

71.6

Heart

249

4.9 ± 0.1

1.62

4.0

0.08

30

19.8

a

fu, tissue

tmax

t½β

Cmax values are presented as mean ± SEM (n = 3)


50

Page 51 of 51

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Table of Contents Graphic and Synopsis


51

Type Amino Acid Transporter 1 (LAT1) - ACS Publications - American

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