direct inhibition of choline kinase by a near-infrared ... ·...

Small Molecule Therapeutics

Direct Inhibition of Choline Kinase by a Near-InfraredFluorescent Carbocyanine

Sean P. Arlauckas, Anatoliy V. Popov, and Edward J. Delikatny

AbstractCholine kinase alpha (ChoK) expression is increasingly being recognized as an important indicator of breast

cancer prognosis; however, previous efforts to noninvasively measure ChoK status have been complicated by

the spectral limitations of in vivomagnetic resonance spectroscopy (MRS) and the complex network of enzymes

involved in choline metabolism. The most effective ChoK inhibitors are symmetric and contain quaternary

ammoniumgroupswithinheterocyclic headgroups connectedbyanaliphatic spacer.Characterization of these

bis-pyridinium and bis-quinolinium compounds has led to phase I clinical trials to assess small-molecule

inhibitors of ChoK for solid tumor treatment.We report the development of a novel carbocyanine dye, JAS239,

whose bis-indolium structure conforms to the parameters established for ChoK specificity and whose spacer

length confers fluorescence in the near-infrared (NIR) window. Fluorimetry and confocal microscopy were

used todemonstrate that JAS239 rapidly enters breast cancer cells independent of the choline transporters,with

accumulation in the cytosolic space where ChoK is active. Radio-tracing and 1HMRS techniques were used to

determine that JAS239 binds and competitively inhibits ChoK intracellularly, preventing choline phosphor-

ylation while inducing cell death in breast cancer cell lines with similar efficacy to known ChoK inhibitors.

Fluorescentmolecules that report onChoK status have potential use as companion diagnostics for noninvasive

breast tumor staging, because NIR fluorescence allows for detection of real-time probe accumulation in vivo.

Furthermore, their ability as novel ChoK inhibitors may prove effective against aggressive, therapy-resistant

tumors. Mol Cancer Ther; 13(9); 2149–58. �2014 AACR.

IntroductionUpregulation of choline kinase alpha (ChoK) has been

correlated with histologic tumor grade and resistance toantiestrogen therapies in breast cancer, ultimately indi-cating a poorer prognosis (1). ChoK catalyzes the conver-sion of choline to phosphocholine (PC), an importantsecond messenger and the first step in the biosynthesisof the predominant membrane phospholipid, phosphati-dylcholine (PtdCho). ChoK activity can be enhanced by anumber of clinically relevant oncogenes (2–4), growthfactors (5–7), transcription factors (8, 9), and carcinogens(10) andChoKoverexpression alone is sufficient to inducemalignant transformation (4). The 9 chemically equivalentprotons in choline yield a strong singlet peakdetectable byproton magnetic resonance spectroscopy (1H MRS). MRSstudies have found increasing PC and total choline (tCho)levels as breast cell lines progress from normal to immor-

talized, to oncogene transformed, to malignant (11). Invivo 1HMRS studies have identified choline accumulationin 83% of breast lesions (12). Furthermore, alterations incholine and lipid metabolism detected by MRS are pre-dictive of tumor response to certain therapies (13, 14).

In vivo 1HMRS is limitedby spectral resolution,with thetCho peak representing a composite resonance consistingof free choline, PC, and glycerophosphocholine (GPC).Phosphorus (31P) MRS is capable of distinguishing thephosphomonoester PC from the phosphodiesterGPC, but31P-MRS is relatively insensitive requiring large voxels invivo. In addition, direct PC measurements are not alwaysaccurate descriptors of ChoK activity because of the cat-abolic formation of PC by the phospholipase and sphin-gomyelinase enzymes (Fig. 1). There have been efforts tohyperpolarize choline with the intention of monitoringChoK activity (15, 16); however, this approach is limitedby the polarization lifetime of 15N and has yet to bedemonstrated feasible in intact cells or in vivo tumormodels. Similar strategies utilizing 11C-choline (17) and18F-choline (18) for positron emission tomography (PET)imaging have been explored but isotope-labeled cholineanalogs run into the problem of being dependent on thecomplex families of proteins responsible for choline trans-port (ChoT: high-affinity choline transporters, cholinetransporter-like proteins, organic cation transporters, andorganic cation/carnitine transporters). Upregulation of

Department of Radiology, University of Pennsylvania, Philadelphia,Pennsylvania.

Corresponding Author: Edward J. Delikatny, Department of Radiology,University of Pennsylvania, 317 Anatomy-Chemistry Building, 3620 Hamil-ton Walk, Philadelphia, PA 19104, USA. Phone: 215-898-3105; Fax: 215-573-2113; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0085

�2014 American Association for Cancer Research.

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these proteins has been demonstrated in some cancers buttheir involvement remains, for the most part, poorlyunderstood (19, 20). An alternative method to noninva-sively detect ChoK statuswould be useful to aid in clinicaltumor assessment.

ChoK has garnered clinical attention as a biomarker oftumor malignancy, leading to its study as a treatmenttarget. Silencing ChoK via RNAi has been shown toreduce cell proliferation (21), enhance the sensitivity ofaggressive cell lines to 5-fluorouracil (22), and reducetumor growth rates (23) in breast cancer models. Thecholine mimetic, hemicholinium-3 (HC-3), has long beenknown to inhibit ChoK, but also interrupts neuronalcholine transport and acetylcholinesterase activity (24).Its neurotoxicity at doses relevant for ChoK inhibition (25)led to the development of molecules capable of blockingcholine phosphorylation without causing respiratoryparalysis (26). The most potent and specific ChoK inhi-bitors to date have been established by the Lacal lab basedon quantitative structure–activity relationship studies ofbis-pyridinium (27) and bis-quinolinium (28) HC-3mimetics (Fig. 2). These studies found that lipophilicityenhances antiproliferative activity but must be optimizedbecause of trade-offs in solubility. The top candidatesidentified were symmetric and featured 2 aromatic het-erocyclic head groups, containing quaternary ammoniumelements, attached by a linker of optimized length (29).Many of the bis-pyridinium and bis-quinolinium struc-

tures feature electron-donating functional groups, whichstabilize the positive charge and increase activity (27).Effective inhibitors have been identified from compoundswith aromatic or aliphatic linkers, which can be used tooptimize the antiproliferative properties.

1,10-((Butane-1,4-diylbis(4,1-phenylene))bis(methylene))bis(4-(dimethylamino)pyridin-1-ium) bromide (MN58b) isthe best-characterized bis-pyridiniumChoK inhibitor, hav-ing been demonstrated to be effective against breast, colon(23), bladder (30), and cervical cancer, as well as squamouscell carcinoma, histiocytic lymphoma, and chronicmyeloidleukemia models (31). As part of their transformation,cancer cells are thought to build an addiction to PC, whichsensitizes them to ChoK-targeted inhibition. MN58b treat-ment causes a temporary cell-cycle arrest in normal cellsbecause of dephosphorylation of the checkpoint proteinpRb. This cue for growth arrest is not seen in tumor cells,which attempt to bypass theKennedy pathway by excisingthe PC group from sphingomyelin for further PtdChoproduction, releasing ceramides as a by-product (32). Theaccumulationof ceramides, in addition toattenuationof theproliferative PC signal, leads to tumor-specific apoptosis(33). TCD-717, developed by TCD Pharma, is the bestcandidate among the bis-quinolinium compounds and hasenteredphase I clinical trials for solid tumor treatment (34).

We sought to test the hypothesis that carbocyanine-derived HC-3 mimetics could be adapted to the estab-lished confines for effective ChoK inhibition (Fig. 2). Thecarbocyanine dyes contain a number of structural simi-larities to existing ChoK inhibitors, including symmetricheterocyclic head groups containing quaternary ammo-niummoieties connectedbyanaliphatic spacer.We report

Figure 1. Mechanisms affecting the intracellular pool of PC: (i) choline(Cho) uptake via Cho transporters (ChoT) and entry into the Kennedypathway of PtdCho biosynthesis (black) by which Cho is phosphorylatedby ChoK (EC 2.7.1.32), then converted to CDP-Cho and PtdCho bycholine phosphate cytidylyltransferase (CTT; EC 2.7.7.15) andphosphocholine diacylglycerol transferase (PCT; EC 2.7.8.2),respectively. The four catabolic routes: (ii)membrane sphingomyelin (SM)hydrolyzed by sphingomyelinase (SMase; EC 3.1.4.12; green) to PC andceramide; (iii) PtdCho deacylation to lyso-PtdCho (LPC), GPC, and Cho(yellow) by phospholipase A2 (PLA2; EC 3.1.1.4), and lysophospholipase(EC 3.1.1.5) or phospholipase A1 (PLA1; EC 3.1.1.32), and GPC:phosphodiesterase (GPC:PDE; EC 3.1.4.2), respectively; (iv) PtdChohydrolysis directly to Cho and phosphatidic acid by phospholipase D(PLD; EC 3.1.4.4; red); (v) PtdCho hydrolysis directly to PC anddiacylglycerol via PtdCho-specific phospholipase C (PLC; EC 3.1.4.3;blue). 1H MR-visible Cho metabolites that contribute to the compositetCho peak are in boxes.

Figure 2. Templates for ChoK-specific inhibitors: the bis-pyridinium (top)and bis-quinolinium (middle) structureswith electron-donating functionalgroups (R) and aromatic or aliphatic linkers. A novel template for ChoKinhibitors is proposed based upon the carbocyanines (bottom), whosefluorescence properties are determined by linker length and aresubstitutable at the A and R positions.

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here a new prototype ChoK inhibitor with both potentantiproliferative activity as well as fluorescence in thenear-infrared (NIR) range. The optical properties of NIRfluorophores are ideal for in vivo optical imaging, ashuman tissue (water, hemoglobin, fat) hasminimal absor-bance in this wavelength range (�650–900 nm). Althoughfluorescent kinase inhibitors in the visible rangehavebeenreported (35–37), our approach using NIR fluorophorespotentially allows for the imaging of kinase expressionand function in vivo.

Materials and MethodsChemistry methods

1H NMR spectra were recorded on a Bruker DMX360 orUNI500 spectrometer in CDCl3 or CD3OD using tetra-methylsilane (TMS) as an internal standard. Chemicalshifts are reported in ppm. Mass spectra were recordedon a Bruker Microflex MALDI-TOF spectrometer. HPLCanalysis was performed as described elsewhere (38). Sol-vents were purchased from Fisher Scientific. 1,4-Diphenyl-butane was purchased from Pfaltz & Bauer, Inc. All otherchemicalswerepurchased fromSigma-Aldrichandusedasreceived. The compounds were named with ChemBio-Draw Ultra (v. 13, CambridgeSoft).

Chemical synthesis1,4-Bis(4-bromomethylphenyl)butane. 1,4-Diphenyl-

butane (2 g, 9.51 mmol) was added to a mixture of 48%aqueous HBr (4.15 mL, 39.9 mmol) and glacial AcOH (50mL), followed by 1,3,5-trioxane (0.57 g, 6.34 mmol) andhexadecyltrimethylammonium bromide (52 mg, 0.143mmol). The mixture was stirred such that only a singlelayer could be seen then heated to a gentle reflux for8 hours. The volatiles were removed under reduced pres-sure. The product was isolated by column chromatogra-phy (silica gel, hexane/benzene 1/1, v/v). The isolated1,4-bis(4-bromomethylphenyl)-butane (1.959 g, yield52%) was a white solid, mp 119 to 121�C (from acetone).1H NMR (500 MHz, CDCl3, d ppm) 7.04 ppm (AB, A ¼7.18, B ¼ 6.89, JAB ¼ 9 Hz, 8H), 4.53 (s, 4H), 2.66 (m, 4H),1.62 (m, 4H).MN58b The mixture of 4-dimethylaminopyridine

(238.0 mg, 1.948 mmol) and 1,4-bis(4-bromomethylphe-nyl)butane (385.9 mg, 0.974 mmol) in dry ethanol (30 mL)was heated and stirred at 160�C in a 45 mL Parr autoclaveequipped with a magnetic stirring bar for 3 hours. Thewhite solid product was precipitated twice from EtOHinto Et2O, dissolved in deionized water and lyophilized.Yield: 96%, melting point (mp) 103 to 106�C (fromwater).1H NMR (360 MHz, CD3OD-, d ppm): 8.21 (dt, J¼ 7.6 Hz,J¼ 2.9 Hz, 4H, H-2pyr), 7.29 (dm, J¼ 7.9 Hz, 4H, Ph), 7.23(dm, J¼ 7.9Hz, 4H, Ph), 7.00 (dm, J¼ 7.6Hz, 4H,H-3pyr),5.33 (s, 4H, CH2N

þ), 3.25 (s, 12H, N(CH3)2), 2.64 (m, 4H,CH2C6H4), and 1.62 (m, 4H, C–CH2–C).1-(2-Hydroxyethyl)-2,3,3-trimethyl-3H-indol-1-ium

chloride. The mixture of 2,3,3-trimethyl-3H-indole(1,592.3mg, 10mmol) and 2-chloroethan-1-ol (1,610.2mg,

20mmol) in dry ethanol (15mL)was heated and stirred at160�C in a 45mLParr autoclave equippedwith amagneticstirring bar for 4 hours. The purple solid product wasprecipitated twice from EtOH with Et2O. Yield 86%. 1HNMR (360 MHz, CD3OD, d ppm): 7.88 ppm (m, 1H), 7.77(m, 1H), 7.65 (m, 2H), 4.68 (m, 2H), 4.06 (m, 2H), 1.63 (s,6H), labile OH and N ¼ C–CH3 protons are in exchangewith CD3OD. MALDI-TOF, m/z: (M-Cl)þ 204.35, calcu-lated for C13H18NO 204.14.

1-(2-Hydroxyethyl)-2-((1E,3E,5E)-7-((Z/E)-1-(2-hydro-xyethyl)-3,3-dimethylindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-3,3-dimethyl-3H-indol-1-ium chloride (JAS239). Asolution of acetic anhydride (112.3 mg, 1.1 mmol) in CH2

Cl2 (2 mL) was added to a cooled (�20�C), stirredsuspension of N-((1E,3E,5Z)-5-(phenylimino)penta-1,3-dien-1-yl) benzenaminium chloride (142.4 mg, 0.5 mmol)and triethylamine (222.6 mg, 2.2 mmol) in CH2Cl2 (10mL). The resulting clear solution was stirred for another3 hours at room temperature (RT) and concentratedunder high vacuum. The residue containing N-phenyl-N-((1E,3E,5Z)-5-(phenylimino)penta-1,3-dien-1-yl)acet-amide was dissolved in ethanol (5.0 mL) and addeddrop-wise to a refluxing solution of 1-(2-hydro-xyethyl)-2,3,3-trimethyl-3H-indol-1-ium chloride (360.0mg, 1.5 mmol) and anhydrous sodium acetate (200 mg,2.5 mmol) in ethanol (100 mL). The mixture was refluxedfor 5 hours and concentrated. The product JAS239 wasisolated by column chromatography [silica gel, ethylacetate–methanol (0%–100%)]. Yield 19.9%. 1H NMR(360 MHz, CD3OD, d ppm): 7.93 [t (dd), J ¼ 13.1 Hz,2H]; 7.58 [t (dd), J¼ 12.8 Hz, 1H]; 7.46 (d, J¼ 7.2 Hz, 2H);7.38 (td, J ¼ 7.4 Hz, J ¼ 1.1 Hz, 2H); 7.29 (d, J ¼ 7.9 Hz,2H); 7.23 (td, J ¼ 7.2 Hz, J ¼ 0.7 Hz, 2H); 6.53 [t (dd), J ¼12.6 Hz, 2H]; 6.35 (d, J ¼ 13.7 Hz, 2H); 4.21 (t, J ¼ 5.8 Hz,4H); 4.92 (t, J ¼ 5.8 Hz, 4H); 1.70 (s, 12H). MALDI-TOF,m/z: (M–Cl)þ 469.56, calculated for C31H37N2O2 469.28.HPLC: one peak retention time ¼ 15.22 minutes.

Cell culturesTriple-negative human-derived breast cancer MDA-

MB-231 cells (ATCC) were maintained in DMEM (Med-iatech) supplemented with 10% FBS (HyClone Laborato-ries), 1% penicillin/streptomycin (Mediatech), and 1% L-glutamine (Mediatech) at 37�C in a humidified atmo-sphere (5% CO2). Genetically modified MCF7 breast can-cer cells overexpressing ChoKa were provided by ZaverBhujwalla and Tariq Shah of Johns Hopkins University;further information on the design and characterizationof the empty vector (MCF7 EV) and Chk-4 clone (MCF7CKþ) transfected cell lines is available in their originalpublication (39). These cells were cultured inMEM (Med-iatech) supplemented with 10% FBS, 1% penicillin/strep-tomycin, 1% L-glutamine, and 400 mg/mL G418 sulfateselection agent (Mediatech). G418was not included in theculture medium during experiments. All cell lines usedwere tested regularly for mycoplasma. Cells receivedfrom ATCC in November of 2011 were used within 6months of resuscitation, and authorized by the cell bank

NIR Fluorescent Choline Kinase Inhibitor

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via the COI assay, STR analysis, and BacT/ALERT 3D.Cells received from the Bhujwalla lab were tested formycoplasma upon receipt inMarch of 2012.Western blotswere used to establish the overexpression of ChoKa. Cellswere frozen in liquid nitrogen and only used at lowpassage numbers.

14C-choline ChoK activity assayCellswere plated at 1.5� 106 cells/well in 6-well dishes

and allowed to grow for 48 hours. Media was aspiratedand replaced with fresh media containing varying con-centrations of MN58b, JAS239, or ethanol control, fol-lowed 1 hour later by treatment with 0.5 mCi/mL of[methyl-14C]-choline chloride (Perkin Elmer). At the indi-cated timepoints, cells were rinsed with ice-cold PBS(Mediatech) andfixed in16% trichloroacetic acid. Sampleswere scraped, collected in centrifuge tubes, washed 3� indiethyl ether, lyophilized, and resuspended in water forTLC separation using a solvent system of 0.9% NaCl:methanol:ammonium hydroxide (50:70:5, v/v/v). Quan-tification of the water-soluble choline metabolites wasperformed using autoradiography with a FujiFilm FLA-7000 in accordance with previously established protocols(31). The Rf values for the choline metabolites were 14C-choline (0.07), 14C-PC (0.14), and 14C-GPC (0.39). In highlymetastatic MDA-MB-231 cells that have high constitutiveChoK expression (21), it takes approximately 17 hours for14C-choline to pass through the Kennedy pathway andbe recycled by PLA enzymes to become detectable as14C-GPC.

Cell viability studiesCells were plated at 1.5� 106 cells/well in 6-well plates

and incubated for 2 days. Media was removed andreplaced with fresh media containing MN58b or JAS239at varying concentrations. At the indicated time, cellswere rinsed, trypsinized, resuspended in fresh mediacontaining Trypan Blue, and counted using a Neubauerhemacytometer.

1H MRS studies of cellular extractsMCF7 cells (�30 � 106) were harvested using trypsin

and washed 2� with PBS. Dual-phase methanol/chlo-roform/water extraction was performed in a glass cen-trifuge tube as described elsewhere (40) and the aque-ous phase roto-evaporated, lyophilized, and resus-pended in D2O containing 0.7 mmol of trimethylsilylpropionate (Aldrich) as standard. Fully relaxed 1HNMR spectra were acquired using an 11.7 T VarianINOVA high-resolution NMR spectrometer and a 90�

pulse width (relaxation delay: 6 seconds; repetitiontime: 10.73 seconds; number of scans: 64; data size: 32K; spectral width: 6,000 Hz; temp: 30�C; total acquisitiontime: 11 minutes 28 seconds). Spectra were analyzedusing Mnova Lite 5.2.5 software with 0.5 Hz apodiza-tion. The choline (3.20 ppm), PC (3.22 ppm), and GPC(3.23 ppm) peaks were integrated and compared withTSP (0.00 ppm) values for quantification.

ChoK activity 1H MRS assayMCF7 cells (�20� 106) were trypsinized, washed 2� in

ice-cold PBS, and homogenized on ice in 4 volumes of 100mmol/L Tris-HCl (pH 8.0) containing 10 mmol/L DTTand 1mmol/L EDTA inD2O as described previously (41).Samples were ultrasonicated 2� for 30 seconds at 4�C,centrifuged for 30 minutes, and the supernatant trans-ferred to an NMR tube. ATP (Sigma-Aldrich), cholinechloride (Sigma-Aldrich), and MgCl2 (Sigma-Aldrich) inTris-HCl/D2O buffer were added to the sample with finalconcentrations of 10, 2.5, and 10mmol/L, respectively. 1HMR spectra were recorded immediately at 10-minuteintervals for 500 minutes (90o pulse length, relaxationdelay: 6.6 seconds; repetition time 10 seconds; number ofscans: 60; data size: 16 K; spectral width: 6,000 Hz; temp:30�C; total acquisition time 8 hours, 20 min). Choline andPC peaks were fit from Fourier transformed spectra usingMnova Lite 5.2.5. Data were fit to the linear equation, y ¼mxþ b, where x is time, y is the area of the PC or Cho peak,and m, the slope, gives the rate of conversion. To reducethe amount of JAS239 required to treat 20 � 106 cells(grown in 4 � 150 cm2 flasks containing 25 mL mediaeach), treatment occurred after cells were pooled andcentrifuged, thus 53 nmol of JAS239 were present in the530 mL NMR sample, equivalent to a concentration of530 nmol/L if treatment were applied to the cells beforetrypsinization.

Immunoblot of cell extractsMCF7 EV and CK cells were plated in 10-cm dishes

(1.0 � 106 cells) and grown for 24 hours. Proteins wereextracted on ice using radioimmunoprecipitation lysisbuffer (Abcam) fortifiedwith a cOmpleteMini, EDTA-freeprotease inhibitor cocktail (Roche) and quantified usingthe BCA Protein Assay Kit (Pierce). Approximately 30 mgtotal protein was resolved on a 10% SDS-PAGE gel, trans-ferred to a nitrocellulose membrane, blocked using nonfatmilk, and blotted for ChoKa (Abcam) or GAPDH andimaged using Luminata Western Chemiluminescent HRPsubstrates (Millipore). Bands from each of three separateexperiments were quantified using ImageJ software.

Fluorimetry analysisMDA-MB-231 cells were grown for 48 hours in 6-well

plates (1.5 � 105 cells/well). Each well was aspirated andreplaced with fresh media containing JAS239 with orwithout choline and incubated for 2 hours. Samples werewashed in PBS, lysed in DMSO, and the fluorescencemeasured using a Molecular Devices Spectra Max M5plate reader (excitation 640 nm, emission 770 nm).

Confocal microscopyMDA-MB-231 cellswereplated at 1.5� 105 cells/dish in

35-mm glass-bottom dishes coated with poly-D-lysine(MatTek Corp.). After 2 days of incubation, cells weretreated with fresh DMEM containing no phenol red and 2mmol/L JAS239 and/or 5 mmol/L of the nuclear targetingdye SYTO9 dye (Life Technologies). The cells were

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subsequently imaged using a Zeiss LSM 510 META con-focal microscope with excitation 633 nm and emissionfilter 650 to 790 nm.

Statistical analysisData are reported as the mean � standard deviation

(SD) of three separate experiments (n ¼ 3). Probability(P) values were measured using a 2-tailed t test; P-values < 0.05 were considered statistically significant.

ResultsThe structural similarities between the bis-pyridi-

nium/quinolinium ChoK inhibitors and the carbocya-nine dyes led us to hypothesize that similar pharmaco-logic activity could exist between the 2 classes of com-pounds. To test our hypothesis, we designed a carbo-cyanine dye substituted with choline-like Nþ

CH2CH2OH moieties at the nitrogen atom of the indo-linium ring. The synthesis of the carbocyanine precur-sor, 1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indol-1-iumchloride, was performed in ethanol within an autoclaveat high temperature to shorten the reaction time con-siderably from previous reports in the literature (42).Stable 2-chloroethan-1-ol was used as the alkylatingagent, rather than the unstable and more expensivebromo or iodo derivatives, to increase the yield andstability of the intermediates. In the final step, wemodified a previously reported procedure for the syn-

thesis of C7-carbocyanine (42) to run at a higher tem-perature by replacing methanol with ethanol as thesolvent. This allowed a reduction in the condensationtime from 16 to 5 hours (Fig. 3A). The reaction can besupervised by colorimetric monitoring as depictedin Fig. 3A. The final compound, JAS239, demonstratedNIR fluorescence, with excitation maximum at 740 nmand emission at 770 nm.

To compare the 2 classes of compounds,we synthesizedMN58b as a positive control. The synthesis of MN58b hasbeen reported in 2 papers where it was referred to asCompound 41 (43, 44). In this article,we include adetaileddescription of the synthesis of the intermediate 1,4-bis(4-bromomethylphenyl)butane (Fig. 3B), whichwas not pro-vided in the original papers (45, 46) or in either of thepapers describing the synthesis of MN58b (43, 44). Inaddition,wehave improvedupon the synthesis ofMN58b(Fig. 3B) by using an autoclave method following thephase transfer catalyzed bromomethylation (47). Thisdecreases the reaction time from 192 hours (44) to 3 hourswith 50% yield. The melting point of our MN58b productdiffers from that in ref. 44, where it is reported as 161�C to163�C, but is consistent the values of 98�C to 100�C whenthe compound was crystallized from water (43).

The novel NIR fluorophore JAS239 was able to inhibitthe phosphorylation of 14C-labeled choline in MDA-MB-231 triple-negative breast cancer cells with anIC50 of 4.6 mmol/L at 17 hours of treatment, comparableto the IC50 of 2.3 mmol/L observed for MN58b (Fig. 4A).

Figure 3. Synthetic pathway for the preparation of ChoK inhibitors. A, JAS239 synthesis: (a) 2-chloroethan-1-ol, autoclave 160�C, 4 hours; (b) Ac2O, Et3N,DCM,�20�C-RT, 3 hours; (c) AcONa, EtOH, reflux, 5 hours. B, MN58b synthesis: (d) HBr, cetyltrimethylammonium bromide [C16H33N(CH3)3Br], acetic acid,reflux, 8 hours; (e) N,N-dimethylpyridin-4-amine, EtOH, autoclave 160�C, 3 hours.






Treatment with 10 or 20 mmol/L JAS239 for 17 hours orlonger resulted in a statistically significant reduction inMDA-MB-231 viability as measured by Trypan Blueexclusion (Fig. 4B). The antiproliferative EC50 of JAS239at 24 hours is 13.3 mmol/L. Figure 4C shows that thereduction in choline phosphorylation occurred as earlyas 2 hours after treatment, before any significant loss incell viability was observed at 10 and 20 mmol/L. By 17hours, significant losses in both ChoK activity and cellviability were found (Fig. 4D). Addition of 2 mmol/Lexogenous nonlabeled choline to MDA-MB-231 cellswas able to rescue the inhibition of ChoK by JAS239(10 mmol/L), whereas 0.2 mmol/L had no effect(Fig. 4E). By 17 hours, exogenous choline at 0.2 and 2mmol/L was unable to outcompete JAS239 (Fig. 4F).

Poorly metastatic MCF7 breast cancer cells overexpres-sing ChoKa variant-1 (CKþ) and their empty vector con-trols (EV) were analyzed by NMR to assess baselinecholine metabolite levels. 1H NMR spectra of the water-soluble fraction of chloroform:methanol:water extracts ofMCF7 cells (Fig. 5A) confirms that ChoK overexpressionresulted in increased PC levels (Fig. 5B) compared withWTMCF7s (P ¼ 0.006), whereas the empty vector had noeffect (P ¼ 0.286). The NMR spectra of cytosolic prepara-tions of MCF7 cells in Fig. 5C demonstrate the rise in thePCpeak (3.22ppm)at the expense of the cholinepeak (3.20ppm) after choline, ATP, andMg2þ ions were added. ThePC and choline peak integrals were plotted over time(Fig. 5D) and regressions fit to the linear portion of thecurve to estimate ChoK activity. In the presence of sub-

micromolar concentrations of JAS239, a 26.2% reductionin ChoK activity (P ¼ 0.02) was observed in MCF7 cells(Fig. 5E). ChoK overexpressing MCF7 CKþ cells demon-strated no significant change in ChoK activity (P ¼ 0.20)when exposed to the same concentration of JAS239 (Fig.5E). Western blots were used to confirm the increase inChoKa expression in MCF7 CKþ cells. A representativeblot from MCF7 EV and CK cells are shown in Fig. 5F.Quantified protein expression levels demonstrate a 2.3-fold increase in ChoKa levels in MCF7 CKþ cells.

The uptake of JAS239 was analyzed by fluorimetryusing MDA-MB-231 cells treated for 2 hours at varyingconcentrations. Fluorescence accumulation was linearwithin the entire therapeutically relevant dose range(Fig. 6A). Addition of exogenous choline did not obstructthe uptake of JAS239 into these cells (Fig. 6B). MDA-MB-231 cells treatedwith JAS239 for 2 hours and stained for 30minutes with the nuclear stain SYTO9 were analyzedusing fluorescence confocal microscopy. Figures 6C–Eshow a distinct separation of SYTO9 (Fig. 6C) and JAS239(Fig. 6D) fluorescence, indicating that the localization ofthe novel probe was confined to the cytosolic space,appearing diffusely in the cytoplasm, as well as in punc-tate perinuclear spots (Fig. 6E), indicating possible organ-elle accumulation.

DiscussionThe goal of this study was to develop an NIR fluo-

rescent ChoK inhibitor by exploiting the structural

Figure 4. JAS239 is a competitive inhibitor of ChoK in MDA-MB-231 cells. A, cell treatment for 17 hours with MN58b or JAS239 inhibits 14C-cholinephosphorylationwith an IC50 of 2.3 and 4.6mmol/L, respectively. B, cell viability determined by Trypanblue exclusion in response to JAS239, where z indicatescell reduction (P < 0.05) versus untreated control at 2 and 17 hours and # indicates cell reduction (P < 0.05) versus untreated control at 17 and 24 hours. C, 2-hour exposure to JAS239 caused inhibition of 14C-choline phosphorylationwith no effect on cell viability. D, after 17 hours exposure to JAS239, both inhibitionof 14C-choline phosphorylation and reduction in viability is observed. E, exogenous choline addition at high concentrations rescues 14C-cholinephosphorylation in cells treated with 10 mmol/L of JAS239 for 2 hours. F, by 17 hours, exogenous choline addition does not significantly change 14C-cholinephosphorylation in cells treated with 10 mmol/L of JAS239. Data plotted as % control � SD for three separate experiments. �, P < 0.05.

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similarities between the existing bis-symmetric inhibi-tors and the carbocyanine dyes. Taking into consider-ation the structural properties established by the Lacal

group for ChoK-specific inhibition (29), we developedan autoclave method for carbocyanine synthesis, whichsuccessfully reduced the time required to alkylate the

Figure 5. ChoK inhibition and rescue in 1H NMR assays. A, expanded 1H NMR spectra of the aqueous portion of chloroform:methanol:water extractsprepared from ChoKa overexpressing MCF7s (CKþ) or empty vector (EV) control. B, concentration per cell of NMR-detectable water-solublecholine metabolites in wild-type (WT), empty-vector (EV), and ChoKa overexpressing (CKþ) MCF7s. C, superimposed 1H spectra obtained over500 minutes following addition of choline, Mg2þ, and ATP to cytosolic extracts of MCF7 WT cells show increases in PC at the expense of choline. D,corresponding PC and Cho peak integrals plotted over time. E, regression of the linear portion of D yields an estimate of ChoK activity, which isreduced by 530 nmol/L of JAS239 in empty vector (MCF7 EV) cells, but unaffected in overexpressing (MCF7 CKþ) cells. F, representative immunoblotfor ChoKa and GAPDH (loading control) of MCF7 EV versus CK cells and the quantified ChoKa/GAPDH expression levels confirming ChoKaoverexpression. Error bars represent � SD for three separate experiments.�, P < 0.05.

Figure 6. Uptake and subcellularlocalization of JAS239. A, MDA-MB-231 cells treated with varyingconcentrations of JAS239 for 2hours demonstrate linear increasesin relative fluorescence intensity(RFI; excitation 640 nm, emission770 nm). B, fluorimetry studydemonstrating that addition ofexogenous choline to MDA-MB-231 cells could not outcompeteuptake of JAS239. Fluorescenceconfocal micrographs show MDA-MB-231 cells stained for 30minutes with the nuclear stainSyto9 (C), 2 mmol/L of JAS239 (D),and merged images (E). Error barsrepresent � SD for three separateexperiments.






ammonium group of the indolium moiety by 11 hours.Using this new method, libraries of promising fluores-cent agents can now be synthesized and screened fordiagnostic and therapeutic applications in cancer mod-els. The recently reported anticancer activity of substi-tuted benzo[d]thiazoles (48), which are common termi-nal moieties (together with 3,3-dimethyl-3H-indole andbenzo[d]oxazole) in carbocyanine dyes represent onesuch potential application. In this study, choline-mimet-ic hydroxyethyl alkyl groups were attached at theammonium sites within the heterocyclic head groupson either side of a 7-carbon spacer. The 7-carbon spacingis important because it imparts fluorescence in the NIRrange, outside the range of autofluorescence. The result-ing JAS239 built using the carbocyanine template wasanalyzed as an NIR-fluorescent ChoK inhibitor. We thenadapted the autoclave method to establish a rapid andhigh-yield synthesis of MN58b, the most-effective ChoKinhibitor in the literature.

The number and variety of previously reported ChoKinhibitors suggests that there exists a certain degree offlexibility in the functional groups and spacers, whichcan yield effective candidate therapeutics. The majorconcerns for analogs of HC-3 in vivo are that they (i) willnonspecifically target normal tissue; (ii) will disruptcholine uptake into normal cells, which can lead toneuronal toxicity; or (iii) will be prone to DNA inter-calation, resulting in ChoK-independent cell death. Theinherent fluorescence of a carbocyanine-based inhibitorgreatly simplifies the process of excluding candidatesthat inhibit 14C-choline phosphorylation, but are notproperly delivered to the intended tissue or subcellularcompartment. The ability to track the cellular uptake ofthese fluorophores using fluorimetry and confocalmicroscopy also permits the determination of potentialinteractions with choline transporters, which couldaffect the interpretation of the efficacy of ChoK inhibi-tion in vitro. This is also important because compoundsthat inhibit choline transport have been shown to lead torespiratory paralysis in animals. The broad excitationrange of JAS239 and its fluorescence in the NIR windowwherein breast tissue is relatively transparent makes itpossible to study (i) plasma half-life, biodistributionand tumor localization; (ii) cellular uptake pathwaysand kinetics; and (iii) subcellular localization by in vitroand ex vivo microscopy.

Using 14C-choline as a radiotracer, JAS239was found toinhibit ChoK activity at concentrations comparable toMN58b, as indicated by reduced production of 14C-PC.ChoK inhibition occurred at 2 hours, before the onset ofcell death, and was reversible by addition of unlabeledcholine, demonstrating that JAS239 acts as a competitiveinhibitor. After 17 hours, choline addition was unable toreverse the effects of JAS239 on choline metabolism,suggesting that irreversible alterations to the Kennedypathway had already occurred.

A 1H NMR activity assay was used to explore ChoKkinetics in response to a pulse of choline in the presence of

excess ATP and Mg2þ. Choline depletion in MCF7 cellswas precisely mirrored by increased PC, confirming theinactivation of competing enzymes by the reducing envi-ronment of the cell lysis buffer. The enzymatic rate ofChoK determined by this assay (6.25 nmol PC produced/106 cells/h) was consistent with the observation that 14C-choline phosphorylation upon cell entry is nearly instan-taneous. Choline phosphorylation was inhibited byJAS239 but could be rescued by ChoK overexpression,thus confirming that ChoK is the primary target of JAS239that leads to the observed reduction in choline phosphor-ylation. This assay measures choline phosphorylation inthe absence of choline transporters, suggesting that thedecrease in PC levels by JAS239 is independent of theiraction. Moreover, only nanomolar concentrations of thisprobe were needed to inhibit ChoK in cell lysates, indi-cating that functional modifications to the probe that leadto increased uptake into intact cells may further improveefficacy.

Theuptake of JAS239 into intactMDA-MB-231 cellswasanalyzed by fluorimetry and was found to be both rapidand linearly proportional to the concentration of probeadded; no quenching of signal intensity was observedeven when high therapeutic doses were used. JAS239seems to enter cells independent of the choline transpor-ters, as exogenous choline addition had no effect on probeuptake. The ability to separate ChoK expression fromcholine uptake is an advantage to using fluorescent ChoKinhibitors. Reported observations made using MRS andPET measure the effects of both choline transport andphosphorlyation together. Our approach may yield acompanion diagnostic that would aid in identifyingpatients best suited for ChoK inhibitor-based therapies.The inherent optical properties of this small moleculeinhibitor allow for study of subcellular localization byfluorescence confocal microscopy. JAS239 collects in thecytosolic space where ChoK is active (49) and is excludedfrom the nucleus where DNA intercalation, mutagenesis,and nonspecific cell death would otherwise be confound-ing factors.

Against pure yeast ChoK, the average reported IC50s ofthe bis-pyridinium and bis-quinolinium ChoK inhibitorswere 37.1 and 33.9 mmol/L, respectively; the antiproli-ferative activities, EC50s, in HT-29 cells were 19.7 and 3.7mmol/L, respectively (27, 28). Although determined indifferent systems, the measured IC50 of 4.6 mmol/L forJAS239 represents potency comparable or better than 85%of the reported bis-pyridinium–based ChoK inhibitors,and 61% of the bis-quinolinium compounds. The EC50

values are also comparable, although determined usingdifferent assays in cell lines of different tissue origin. Theaction of JAS239 against MDA-MB-231 cells suggestsChoK inhibition may be an effective strategy in triple-negative breast cancers, which are often therapy resistant.Its potency suggests that further modifications basedupon the carbocyanine template, and subsequent struc-ture–activity characterization, may yield compoundshighly specific to ChoK.

Arlauckas et al.





Our results have indicated that JAS239 enters breastcancer cells independent of the choline transportersand competes with choline at the active site of ChoK.The development of a small molecule kinase inhibitorwith NIR-fluorescence confers the ability to noninva-sively track the biodistribution of this probe with NIRoptical imaging (50), greatly reducing the number ofanimals required for detailed pharmacokinetics stud-ies. ChoK expression can be up to 15-fold higher inmalignant compared with nontransformed breast cells(21), thus imaging agents targeted to this biomarkermay benefit from substantial signal-to-noise.We describe here a novel strategy for reporting ChoK

expression, which has been clinically correlated to his-tologic tumor grade and estrogen receptor status inbreast cancer. The simplified chemical synthesis andreaction monitoring technique reported here makespossible the production of carbocyanine-based librariesto be screened for ChoK inhibition and accumulation inbreast tumors. As the interest in ChoK inhibitors forcancer therapy expands, these compounds have theunique potential to serve as companion diagnostics toidentify patients most likely to benefit from this therapyand to make ongoing minimally invasive evaluations oftheir response.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S.P. Arlauckas, A.V. Popov, E.J. DelikatnyDevelopment ofmethodology: S.P. Arlauckas, A.V. Popov, E.J. DelikatnyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.P. Arlauckas, E.J. DelikatnyAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): S.P. Arlauckas, A.V. Popov, E.J. DelikatnyWriting, review, and/or revision of the manuscript: S.P. Arlauckas, A.V.Popov, E.J. DelikatnyAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.P. Arlauckas, A.V. PopovStudy supervision: A.V. Popov, E.J. Delikatny

AcknowledgmentsThe MCF7 cell lines used were generously provided by Dr. Zaver

Bhujwalla and Dr. Tariq Shah at Johns Hopkins University. Theauthors thank Mansi Shinde of the Pharmacology Graduate Groupat the University of Pennsylvania for assistance with molecular biol-ogy techniques. 1H-NMR spectra of cell extracts were acquired usingthe 11.7 T high-resolution MR spectrometer at the Small AnimalImaging Facility (SAIF) of the University of Pennsylvania. Confocalmicroscopy images were taken with assistance from James Hayden ofthe Wistar Cancer Institute. Mass spectrometry was performed inMass Spectrometry Facility of the Department of Chemistry at theUniversity of Pennsylvania.

Grant SupportThis work was supported by NIH R01-CA129176 (to E.J. Delikatny),

T32-GM008076 (to S.P. Arlauckas), F31-CA180328 (to S.P. Arlauckas), andDoD Breast Cancer Concept Award BC076631 (to E.J. Delikatny).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 29, 2014; revisedMay 19, 2014; accepted June 23, 2014;published OnlineFirst July 15, 2014.

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2014;13:2149-2158. Published OnlineFirst July 15, 2014.Mol Cancer Ther Sean P. Arlauckas, Anatoliy V. Popov and Edward J. Delikatny CarbocyanineDirect Inhibition of Choline Kinase by a Near-Infrared Fluorescent

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