Zinc(II)-Dipicolylamine Coordination Complexes as Targeting andChemotherapeutic Agents for Leishmania major

Douglas R Ricea Paola Vacchinab Brianna Norris-Mullinsb Miguel A Moralesb Bradley D Smitha

Department of Chemistry and Biochemistry University of Notre Dame Notre Dame Indiana USAa Eck Institute for Global Health Department of Biological SciencesUniversity of Notre Dame Notre Dame Indiana USAb

Cutaneous leishmaniasis is a neglected tropical disease that causes painful lesions and severe disfigurement Modern treatmentrelies on a few chemotherapeutics with serious limitations and there is a need for more effective alternatives This study de-scribes the selective targeting of zinc(II)-dipicolylamine (ZnDPA) coordination complexes toward Leishmania major one of thespecies responsible for cutaneous leishmaniasis Fluorescence microscopy of L major promastigotes treated with a fluorescentlylabeled ZnDPA probe indicated rapid accumulation of the probe within the axenic promastigote cytosol The antileishmanialactivities of eight ZnDPA complexes were measured using an in vitro assay All tested complexes exhibited selective toxicityagainst L major axenic promastigotes with 50 effective concentration values in the range of 127 to 03 M Similar toxicitywas observed against intracellular amastigotes but there was almost no effect on the viability of mammalian cells includingmouse peritoneal macrophages In vivo treatment efficacy studies used fluorescence imaging to noninvasively monitor changesin the red fluorescence produced by an infection of mCherry-L major in a mouse model A ZnDPA treatment regimen reducedthe parasite burden nearly as well as the reference care agent potassium antimony(III) tartrate and with less necrosis in the lo-cal host tissue The results demonstrate that ZnDPA coordination complexes are a promising new class of antileishmanial agentswith potential for clinical translation

Leishmaniasis is a parasite infection transmitted to mammals bythe bites of infected female phlebotomine sandflies It is en-

demic in more than 70 countries worldwide with approximately12 million people infected and 310 million at risk of infection (1)The disease is associated with impoverished areas of the MiddleEast Southeast Asia and South America Different Leishmaniaspecies are responsible for the two main disease manifestationsvisceral and cutaneous The visceral form mainly targets the liverand spleen resulting in high-grade fever and organ failure It is themost lethal form and the second greatest parasitic killer in theworld (2) The cutaneous form is an infection of the dermis whichproduces inflamed lesions that heal slowly leaving scars or thatcan evolve into mucosal infections which cause oral nasal andpharyngeal tissue damage (3) Consequently patients can sufferdevastating facial disfigurement and aesthetic trauma

Modern treatment regimens for cutaneous infections involvelocal application of antimonial drugs such as meglumine antimo-niate and sodium stibogluconate (4) Antimonial based therapieshave been the primary treatment option since their developmentin the early 1930s (5) These drugs are notable as pioneering ther-apeutics but they exhibit numerous drawbacks Antimonials aregenerally expensive nonspecific and toxic to internal organsRoutine doses cause painful side effects such as vomiting swellingand myalgia (5) Additionally antimonial therapy is prohibited inmany patient populations such as women during pregnancyyoung children and patients suffering from chronic conditionssuch as kidney disease or heart failure (6) Antifungal- and antibi-otic-based drugs such as amphotericin B paromomycin and pen-tamidine isethionate are second-line treatments but severe sideeffects and high cost limit their use (4 7) Anticancer agents suchas miltefosine are promising but relatively expensive potentiallyteratogenic and prone to result in infection relapse after treat-ment (8) Taken together the weaknesses of existing remedies

highlight the need for new classes of effective nontoxic and inex-pensive antileishmanial chemotherapeutics

Cationic molecules are attractive starting points for Leishma-nia drug discovery because they can distinguish the anionic sur-face charge of the parasites from the near-neutral membrane sur-face charge of healthy mammalian host cells Leishmania parasitesare negatively charged due to (i) a thick coating of the anionicpolysaccharide lipophosphoglycan which covers 60 of theparasite surface and (ii) a high fraction of anionic polar lipidswithin the plasma membrane (9ndash13) Certain classes of cationicpeptides and cationic liposomes bearing sterylamine are toxic toLeishmania (14ndash16) In addition aromatic dicationic compoundsstructurally related to pentamidine are active against L infantum(17) and the positively charged small molecule sitamaquine hasundergone phase II clinical trials for treatment of visceral infec-tions (18) The toxic mechanisms of these treatments are not wellunderstood but they appear to occur through a combination offactors including membrane depolarization mitochondrial dis-ruption or DNA damage

The present study evaluates a series of cationic zinc(II)-dipico-lylamine (ZnDPA) coordination complexes for ability to selec-

Received 19 February 2016 Accepted 25 February 2016

Accepted manuscript posted online 29 February 2016

Citation Rice DR Vacchina P Norris-Mullins B Morales MA Smith BD 2016Zinc(II)-dipicolylamine coordination complexes as targeting andchemotherapeutic agents for Leishmania major Antimicrob Agents Chemother602932ndash2940 doi101128AAC00410-16

Address correspondence to Bradley D Smith smith115ndedu or Miguel AMorales miguelmoralesndedu

Supplemental material for this article may be found at httpdxdoiorg101128AAC00410-16

Copyright copy 2016 American Society for Microbiology All Rights Reserved

crossmark

2932 aacasmorg May 2016 Volume 60 Number 5Antimicrobial Agents and Chemotherapy

tively target and kill Leishmania parasites ZnDPA complexes areknown to selectively recognize anionic cell surfaces due to a com-bination of general electrostatic attraction and specific coordina-tion of the zinc cations with the phosphate and carboxylate groupson the polar lipids in the cell membrane (Fig 1) FluorescentZnDPA probes are effective imaging agents for dead and dyingmammalian cells (which expose anionic phosphatidylserine) in arange of cell culture systems and small animal models of disease(19ndash26) ZnDPA probes also target the anionic surfaces of bacte-ria and they have been used for imaging infection in animal mod-els (27ndash33) Some ZnDPA complexes are known to have antibioticactivity due to their capacity to disrupt bacterial membranes (31)

The objective of this study was to determine whether ZnDPAcomplexes can selectively target L major a common species re-sponsible for most cutaneous leishmaniasis infections in the Mid-dle East and North Africa (34) A fluorescently labeled ZnDPAprobe was used for promastigote staining experiments and a li-brary of eight ZnDPA complexes was evaluated for in vitro toxicityagainst L major promastigotes amastigotes and mammaliancells A chemotherapeutic candidate was chosen for further activ-ity testing against intracellular amastigotes murine macrophagesand a mouse model of cutaneous leishmaniasis To facilitate mea-surements of in vivo efficacy a fluorescence imaging method wasdeveloped using a genetically modified L major strain that ex-pressed the red fluorescent protein mCherry A mouse footpadinfection model enabled direct comparison of treatment efficacyfor a ZnDPA complex and a standard antimonial chemotherapeu-tic agent

MATERIALS AND METHODSMaterials The targeted fluorescent ZnDPA probe mSeek nontargetedfluorescent Control Dye and all ZnDPA complexes tested were synthe-sized as previously reported (22 33) Microscopy slides and coverslipswere purchased from Thermo Scientific Unless otherwise stated all re-agents and solvents were purchased from Sigma-Aldrich

Leishmania culture conditions Leishmania major axenic promasti-gotes (MHOMJL80Friedlin) a fluorescent transgenic L major strainconstitutively expressing mCherry (mCherry-L major) and a fluorescenttransgenic strain of mCherry-L donovani axenic amastigotes were main-tained at 27degC in M199 medium supplemented with 10 fetal bovineserum (FBS Atlanta Biolabs) (35)

In vitro toxicity assay and EC50 calculation Toxicity against L majorpromastigotes and intracellular amastigotes was assessed using the fluo-rometric resazurin-based method CellTiter-Blue (Promega) as previouslydescribed (35) For promastigotes 106 cellsml were seeded in a 96-wellplate and incubated in the presence of increasing concentrations ofZnDPA complex for 72 h at 27degC along with the appropriate solventcontrols Afterward 20 l of CellTiter-Blue reagent was added to 100 l of

the cell culture followed by a 4-h incubation period at 37degC and thefluorescence was measured (excitation 555 nm emission 580 nm) usinga Typhoon FLA 9500 laser scanner (GE Healthcare) and analyzed withImageQuant TL software (GE Healthcare) The output fluorescence val-ues were background subtracted from wells containing medium alone andnormalized to wells containing untreated L major Normalized fluores-cence values were plotted against the ZnDPA complex concentration us-ing GraphPad Prism 5 software and fitted to the following curve to deter-mine the 50 effective concentration (EC50)

y a b a

1 x frasl cd (1)

where a is the estimated bottom of the curve b is the estimated top of thecurve c is the 50 effective concentration (EC50) and d is the Hill coef-ficient

L major intracellular amastigote viability was evaluated using a back-differentiation method with RAW2647 murine macrophages and infec-tive-stage mCherry parasites (36) Briefly 106 metacyclic promasti-gotesml were added to wells in a 96-well plate seeded with 105

macrophagesml The infection was performed over 8 h in RPMI 1640medium at a multiplicity of infection of 10 metacyclic parasites per mac-rophage (101) Free parasites were removed by one wash with RPMI 1640medium At 24 h postinfection ZnDPA compound 7 was added in in-creasing concentrations and incubated for 48 h at 37degC followed by a 12 hof incubation at 26degC to facilitate back differentiation from viable amas-tigotes to promastigotes To detect the remaining viable parasites 30 l ofCellTiter-Blue reagent were incubated for 4 h with 200 l of culture at37degC followed by a fluorescence measurement and sample analysis asdescribed above Each assay was performed in triplicate

Mammalian cell culture and toxicity CHO-K1 (Chinese hamsterovary) cells purchased from the American Type Culture Collection(CCL-61) were spread into 96-microwell plates and grown to confluenceof 85 in RPMI or F-12K media supplemented with 10 FBS and 1streptavidin L-glutamate at 37degC and 5 CO2 CHO cell viability wasmeasured by using a 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazo-lium bromide (MTT) cell viability assay The number of living cells isdirectly correlated to the amount of reduced MTT which is monitored byabsorbance at 570 nm Only active reductase enzymes in viable cells canperform the reduction reaction A Vybrant MTT cell proliferation assay(Invitrogen Eugene OR) was performed according to the manufacturerrsquosprotocol The CHO cells were treated with incremental concentrations ofa ZnDPA complex (0 to 50 M) and incubated for 24 h at 37degC Themedium was replaced with 100 l of F-12K medium containing MTT (12mM) and incubated at 37degC and 5 CO2 for an additional 4 h An SDS-HCl detergent solution was added and the absorbance of each well wasread at 570 nm and normalized to wells containing cells but no addedZnDPA complex (n 3)

Host cell viability was tested using macrophages isolated from theintraperitoneal cavities of three BALBc mice Using an insulin syringewith a 24-gauge needle 5-ml aliquots of Dulbecco modified Eagle me-dium (DMEM Life Technologies) supplemented with 10 FBS were in-jected and then extracted from the peritoneal cavities of euthanized miceThe fluid was centrifuged and the supernatant was discarded The pel-leted macrophage cells were used to seed 105 macrophageml in a 96-wellplate and incubated in the presence of increasing concentrations ofZnDPA compound 7 for 48 h at 37degC in the presence of 5 CO2 Viabilitywas measured with the Cell-Titer Blue assay as described above

Promastigote and amastigote fluorescence microscopy Samples ofmCherry-L major axenic promastigotes and mCherry-L donovani axenicamastigotes (107ml) were fixed with 1 formalin in 15-ml microcentri-fuge tubes followed by centrifugation (3000 rpm 5 min) The fixed par-asites were resuspended in 1 ml of sterile phosphate-buffered saline (145mM NaCl [pH 74]) and treated for 15 min with green-emitting fluores-cent ZnDPA probe mSeek (5 to 10 M) and two drops of NucBlue FixedCell ReadyProbes reagent (Thermo Fisher) The samples were washed

FIG 1 Association of ZnDPA coordination complexes with phosphorylatedamphiphiles on the cell surface

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May 2016 Volume 60 Number 5 aacasmorg 2933Antimicrobial Agents and Chemotherapy

twice with fresh buffer to reduce background fluorescence dispersed intosolution and then placed on slides coated with L-lysine Preliminary mi-crographs of promastigotes were acquired using a Nikon EclipseTE2000-U epifluorescence microscope with a 60 objective and a Pho-tometrics Cascade 512B CCD Fluorescence images were captured usingUV (excitation 34080 nm emission 43585 nm) GFP (45090 50050)and Cy3 (53550 61075) filter sets Micrographs of amastigotes wereacquired using an Applied Precision DeltaVision OMX epifluorescencemicroscope with a 63 objective and similar filter sets

Confocal scanning laser microscopy of mCherry-L major promasti-gotes was performed using a Nikon A1R confocal microscope to examinethe cellular localization of mSeek Cell staining was carried out using theprocedure above Twenty sequential planar images were taken over a2-m Z-scan range (01 m apart) using a 60 microscope objective andblue red and green wavelength filters The internalization of mSeekwithin macrophages was evaluated using RAW2647 murine macro-phages Briefly mSeek (10 M) was added to a single well in a six-wellplate seeded with 105 macrophagesml followed by incubation for 24 h at37degC Fluorescence microscopy was performed using a Nikon EclipseTE2000-U epifluorescence microscope with a 40 objective and a Pho-tometrics Cascade 512B CCD Fluorescence images were captured usingthe GFP (45090 50050) and Cy3 (53550 61075) filter sets

Promastigote flow cytometry Fixed L major axenic promastigoteswere stained with Control Dye or mSeek (5 M final concentration) sus-pended in sterile phosphate-buffered saline for 10 min at 25degC followedby three additional wash steps The samples were injected into a BeckmanFC-500 flow cytometer equipped with a Biosense flow cell and a 6-Wargon ion laser The excitation laser was tuned for an 480-nm emission(500 mW) and the emission light was measured using a 530-nm passabsorbance filter Histograms were generated using FlowJo IX softwareand represent a total of 50000 to 100000 events each Fluorescence andcount data were normalized to ldquopolychromaticrdquo calibration beads

Fluorescence imaging of cutaneous leishmaniasis in mouse footpadAll animal experiments used protocols that were approved by the NotreDame Institutional Animal Care and Use Committee (IACUC no 15-10-2708) Stationary mCherry-L major promastigotes were resuspended infresh M199 media Cohorts of BALBc mice (female 4 weeks old CharlesRiver Laboratories) were transferred to a sterile hood Each mouse wasanesthetized with isoflurane followed by skin sterilization with 70 eth-anol and a subcutaneous injection of 108 parasites into the left hind foot-pad Two weeks later the mice were anesthetized and subjected to planarfluorescence imaging using an IVIS Lumina (Xenogen) equipped with a150-W quartz tungsten halogen 21-V bulb for excitation with the follow-ing fluorescence acquisition parameters DsRed fluorescence (excitation500 to 550 nm emission 575 to 650 nm) acquisition time 3 s binning22 F-stop 2 and field-of-view 10 by 10 cm After imaging footpadthickness (lesion size) was measured using a Vernier caliper and the in-fected footpad was photographed using a Canon PowerShot 121 MPdigital camera The 16-bit TIFF images of each living mouse at the differ-ent time points were sequentially opened using the ImageJ 140g softwareThe images were cropped to focus only on the infected footpad and thenconverted to an image stack using the ldquoconvert images to stackrdquo softwarecommand The stack of images was background subtracted using the roll-ing ball algorithm (radius 250 pixels) Next the image stack was set to theldquoFirerdquo fluorescence intensity scale (under the ldquoLookup Tablesrdquo menu)which color-codes the fluorescence counts contained in each pixel Thestack of images was converted into a montage using the ldquoConvert Stack toMontagerdquo command A calibration bar was added to the montage usingthe ldquoCalibration Barrdquo command and the resulting image was saved as aTIFF file Infection burden was quantified by the measuring the meanpixel intensity within a region of interest drawn around each infectedfootpad After each time point a cohort of mice (n 4) were sacrificedand the infected footpads were harvested to measure parasite burden us-ing the limiting dilution method

Quantification of parasite burden in mouse footpad The followingprocedure was adapted a previously published method (37) Infectedfootpads were removed by severing the mouse ankle just above the bridgeof the foot with surgical scissors Severed footpads were sterilized with70 ethanol and placed in a Falcon tube submerged in ice water contain-ing 5 ml of chilled DMEM The footpads were taken to a sterile culturehood sliced into small pieces using a disposable scalpel within a sterilepetri dish and homogenized in the original DMEM buffer using a glassTeflon homogenizer (Thomas Scientific) A 500-l aliquot of the homog-enized footpad solution was serially diluted in a 12-well plate with eightwells containing 45 ml of M199 medium per well Finally 100 l fromeach 12-well plate was dispensed into a separate marked row of a 96-wellplate and placed into a 27degC incubator for 5 to 10 days (n 3) Aftercloudiness developed within the wells indicating parasite growth a mi-croscopic evaluation using an Amscope B10 binocular biological micro-scope was performed to determine the numbers of Leishmania-positiveand Leishmania-negative wells The parasite load in the mouse footpadwas determined from serial dilution calculations

Treatment of cutaneous leishmaniasis in mouse footpad SixteenBALBc mice were inoculated with mCherry-L major as described aboveand allowed to form an infection over a 14-day period After infection themice were separated into cohorts of four and administered one of thefollowing treatment regimens saline (150 mM NaCl five dosesweek)antimonial [55 mM potassium antimony(III) tartrate five dosesweek]or ZnDPA compound 7 (see Fig 3 80 M) at a frequency of two or fivedoses per week All treatments were 30-l intralesional injections of agentdissolved in sterile saline (pH 74) and administered after the skin wassterilized with a 70 ethanol wipe The injections were administered atdifferent sites around the lesion to reduce injury from the needle punctureand to spread treatment throughout the infected footpad Planar fluores-cence imaging of each cohort was performed periodically throughout the12-day treatment period using the following acquisition parametersDsRed fluorescence (excitation 500 to 550 nm emission 575 to 650 nm)acquisition time 4 s binning 22 F-stop 2 and field-of-view 10 by 10cm After treatment the mice were sacrificed and the infected footpadswere harvested to measure parasite burden using the limiting dilutionmethod described above (n 3 one mouse was euthanized before thetreatment ended)

Histology Separate cohorts of uninfected BALBc (n 3) were givenintradermal footpad injections of saline or ZnDPA compound 7 at a fre-quency of five doses per week for 2 weeks as described above After thetreatment period the mice were euthanized and the treated footpadswere excised fixed and then embedded and flash frozen in OCT (Tissue-Tek) Footpad tissue was sliced (8-m thickness) at 17degC and the slicesadhered to Unifrost microscope slides (Azer Scientific USA) they werethen fixed with chilled acetone for 10 min and air dried for an additional20 min Tissue sections were stained with hematoxylin-eosin and imagedusing a Nikon 90i uprightwidefield equipped with a 40 objective lensand color camera

RESULTSFluorescence microscopy studies Selective targeting of a ZnDPAcomplex to axenic promastigotes and amastigotes was assessedusing fluorescence microscopy and flow cytometry The studiesused mCherry-L major promastigotes and mCherry-L donovaniaxenic amastigotes which stably expressed the red fluorescentmCherry protein (excitation 587 nm emission 610 nm) in thecytoplasm (see Fig S1 in the supplemental material) (38) L don-ovani axenic amastigotes were studied because there are no cur-rent methods for culturing L major axenic amastigotes The par-asites were fixed with 1 formalin prior to imaging to ensuremembrane integrity and arrest cellular mobility for high-resolu-tion fluorescence micrographs Aliquots of parasites were incu-bated with mSeek a green-emitting fluorescent ZnDPA probe

Rice et al

2934 aacasmorg May 2016 Volume 60 Number 5Antimicrobial Agents and Chemotherapy

(Fig 2A) for 10 min prior to three wash steps The treated parasiteswere first examined using widefield fluorescence microscopywhich revealed strong and uniform staining by the mSeekthroughout the parasites including the promastigote flagellum(see Fig S2 and S3 in the supplemental material) Additional sam-ples of probe stained promastigotes were imaged with a confocalmicroscope which permitted three-dimensional fluorescence im-aging Confocal micrographs of the parasite interior showed

clearly that the mSeek was dispersed throughout the cytoplasmcolocalizing with cytoplasmic mCherry (Fig 2B) The microscopyshowed negligible promastigote staining by the nontargeted Con-trol Dye that lacked a ZnDPA complex In addition flow cytom-etry histograms of parasites treated with mSeek indicated 3-fold-higher fluorescence than parasites treated with Control Dye(Fig 2C) To determine macrophage uptake murine macro-phages were incubated for 24 h with mSeek followed by fluores-cence microscopy The probe fluorescence was dispersedthroughout the macrophage cytoplasm with noticeably higherintensity than the background macrophage autofluorescence (seeFig S4 in the supplemental material)

FIG 3 Structures of ZnDPA complexes tested for L major toxicity and EC50

values

TABLE 1 In vitro activity of ZnDPA (compound 7) against mammaliancells and L major promastigotes and amastigotes

Evaluation model and cell type Mean EC50 (M) SDa

Cellular evaluationCHO-K1 50Peritoneal macrophages 10

Parasite evaluationAxenic promastigotes 23 02Intracellular amastigotes 47 01

a For at least three replicates

FIG 2 (A) Structures of green-emitting fluorescent ZnDPA probe mSeek andcontrol dye (B) Confocal micrographs of fixed mCherry-L major promasti-gotes stained with DAPI (4=6=-diamidino-2-phenylindole) and mSeek (5 M)for 10 min prior to red blue and green fluorescence imaging The montage isslice 10 (60 magnification) from a series of 20 slices acquired through a 2-mZ-scan range (01 m between each slice) (C) Representative flow cytometryhistograms of L major cells with green fluorescence (n 3)

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May 2016 Volume 60 Number 5 aacasmorg 2935Antimicrobial Agents and Chemotherapy

Parasite activity screening and host cell toxicity The antil-eishmanial activities of eight ZnDPA complexes were evaluatedagainst L major promastigotes (22) The inhibitory activity wasdetermined using a CellTiter-Blue assay which measures the abil-ity of living cells to convert a redox dye (resazurin) into a fluores-cent end product (resorufin) (see Fig S5A in the supplementalmaterial) The results in Fig 3 show that all complexes were activeagainst L major with EC50s between 127 and 03 M In contrastthere was hardly any toxic effect against mammalian cells Stan-dard MTT assays (see Fig S5B in the supplemental material) usingChinese hamster ovary (CHO) cells indicated that compounds 14 7 and 8 caused negligible toxicity up to 50 M whereas theother four complexes reduced cell viability 30 at 50 M Al-though ZnDPA compound 7 was not the most active complex itwas chosen as the chemotherapeutic candidate for further evalu-ation against amastigotes and murine macrophages due to itsstructural simplicity and ease of production The intracellularamastigote EC50 measured using the back-transformationmethod was 5 M with a murine macrophage toxicity of 10M (Table 1) Histological analyses of uninfected mouse footpadstreated with compound 7 were also performed to complement thein vitro toxicity results After a 2-week treatment regimen of cuta-neous footpad injections (see below) the mice were sacrificed andthe footpads were harvested for hematoxylin-eosin staining and avitality assessment Compared to the control group injected withsaline no cellular or nuclear morphological changes in the cuta-neous footpads were observed (see Fig S6 in the supplementalmaterial) Inflammatory foci that could indicate signs of cutane-ous toxicity were not observed

Cutaneous leishmaniasis BALBc mouse model A cutaneousleishmaniasis animal model was developed by inoculating cohortsof female BALBc mice with stationary mCherry-L major (108) inthe left hind footpad (39) Lesion progression was monitored byimaging the red fluorescence emission of the mCherry-L majoramastigotes and also by measuring the thickness of inflamed foot-pads with a Vernier caliper The cohorts were examined weekly for5 weeks with a single cohort (n 4) sacrificed every week forfootpad harvesting and parasite counting Figure 4 shows repre-sentative fluorescence images recorded weekly from infectedmice

A linear correlation was observed between fluorescence inten-sity and lesion size (Fig 5A) and also fluorescence intensity andparasite burden in the footpad at each weekly time point (Fig 5B)To prove that the red fluorescence at the site of infection was dueto mCherry synthesis by the viable transgenic parasites a control

FIG 4 (A) Representative whole-body red fluorescence image of a BALBc mouse harboring a footpad infection of mCherry-L major (B) Red fluorescenceintensity (top) and color photographs (bottom) of untreated mouse footpad infection after inoculation with 108 mCherry-L major promastigotes Thefluorescence intensity scale bar applies to all footpad images and is given in arbitrary units

FIG 5 (A) Plot comparing red fluorescence mean pixel intensity (MPI) forinfected mouse footpads in living mice (black) and footpad thickness (lesionsize) (orange) over time Each data point represents the means the standarderrors (n 4) (B) Plot of red fluorescence MPI for infected footpads in livingmice and parasite counts in harvested footpads (n 4) Each point representsmeans the standard errors

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2936 aacasmorg May 2016 Volume 60 Number 5Antimicrobial Agents and Chemotherapy

experiment inoculated a separate cohort of mice with a L majorstrain lacking the mCherry transcript and allowed infections todevelop over 35 days As expected fluorescence imaging of theinfection sites showed no measurable red fluorescence (see Fig S7in the supplemental material)

Treatment of cutaneous leishmaniasis model The mouseleishmaniasis model described above was used with footpad infec-tions of mCherry-L major Treatment efficacy experiments com-pared the antileishmanial activity of compound 7 to the standardagent potassium antimony(III) tartrate (antimonial) or no che-motherapeutic agent (saline) (5) Separate cohorts with 14-dayfootpad infections were given a daily intralesional injection ofsaline compound 7 (01 mgkg) or antimonial (5 mgkg) for 5days followed by 2 days of recovery An additional cohort wasgiven two doses per week of compound 7 (01 mgkg) followed byfour recovery days The red fluorescence emission from the cuta-neous lesions was imaged four times over a 12-day period for alltreatment regimens (Fig 6)

Region-of-interest analysis of the fluorescence pixel intensitymaps revealed a progressive loss in fluorescence in mice givencompound 7 (five doses per week) compared to mice given saline(Fig 7A) No significant decrease in Leishmania burden was ob-served in mice receiving only two weekly doses of compound 7The diminished parasite burden was confirmed after dissection ofthe infected footpads and counting viable promastigotes differen-tiated from amastigotes after limiting dilution assay (Fig 7B) Theparasite burden was 70 less in the cohort treated with fivedoses of compound 7 compared to saline-treated animals In ad-dition the physical appearances of the treated footpads were sig-nificantly different at the conclusion of treatment (Fig 7C) Anti-

monial treated footpads displayed cutaneous necrosis andscabbing as expected (40) Conversely footpads treated withcompound 7 displayed some minor local inflammation but noobvious cutaneous reaction to the treatment

DISCUSSION

Fluorescence microscopy with mSeek a green-emitting fluores-cent ZnDPA probe found that ZnDPA has high affinity for Lmajor parasites Confocal micrographs show diffuse internaliza-tion of the probe within the parasites colocalizing with the cyto-solic mCherry reporter protein (Fig 2 see also Fig S2 and S3 inthe supplemental material) The cytosolic distribution contrastswith that seen in planktonic bacteria where mSeek localizes pri-marily in the bacterial envelope with no internalization (33) Invitro toxicity assays with eight different ZnDPA complexes re-vealed strong to moderate antileishmanial activity with minimalmammalian cell cytotoxicity (Fig 3 see Fig S5 in the supplemen-tal material) Although ZnDPA compound 7 was not the mostactive complex its structural simplicity and ease of productionmade it the most attractive choice for studies in the mouse footpadinfection model The ability to noninvasively monitor changes inthe red fluorescence produced by the mCherry-L major infectionsgreatly facilitated the treatment efficacy experiments Five weeklyintralesional doses of compound 7 produced 70 reduction inparasite burden compared to an untreated cohort (Fig 5 to 7) Thedose amount of compound 7 was 50 times lower than the com-parative dose of antimonial agent and yet the reduction in infec-tion burden was very similar over a 12-day period Furthermoretreatment with compound 7 produced significantly less host tissuedamage at the treatment site compared to antimonial treatment

FIG 6 Representative red fluorescence intensity images of BALBc mouse footpads after inoculation with mCherry-L major promastigotes (108) and treatedwith saline at five dosesweek (A) antimony(III) tartrate at five dosesweek (B) and compound 7 at either two dosesweek (C) or 5 dosesweek (D) over a 12-dayperiod The mice were inoculated 14 days prior to the treatment start point (day 0) All doses were 30-l intralesion injections of aqueous solutions containingsaline (150 mM NaCl) antimony(III) tartrate (50 mgkg) or compound 7 (01 mgkg) The fluorescence intensity scale bar applies to all images and is given inarbitrary units

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May 2016 Volume 60 Number 5 aacasmorg 2937Antimicrobial Agents and Chemotherapy

In vitro activity and toxicity measurements revealed that com-pound 7 is quite active against intracellular amastigotes and con-siderably less toxic against murine macrophages (Table 1) Thehigh tolerance of the ZnDPA complex matches previous observa-tions of no obvious acute murine toxicity (22 27) However morein vivo studies are needed to fully evaluate host toxicity and tomore accurately measure the therapeutic window

The mechanism of ZnDPA action against L major is not pres-ently clear and may be multifactorial One possibility is that theZnDPA complexes disrupt the parasite membrane Alternativelythe ZnDPA complexes may alter metal cation concentrationswithin the cytosol Zinc homeostasis is needed to maintain criticalphysiological processes (41 42) and several studies report thatLeishmania parasites are sensitive to doses of metal cations in-cluding zinc copper and rhenium (43ndash47) Various zinc-sulfon-amide complexes have low micromolar EC50s against L major andL amazonensis (48-50) and a large screening study reported thatvarious heterocyclic metal-binding compounds are highly activeagainst L donovani (51)

The World Health Organization has recommended combina-tion therapy as a strategy to increase the therapeutic life span ofdrugs and delay the emergence of resistance (1) Thus a questionfor future studies is whether ZnDPA coordination complexes canact in synergy with other therapeutic agents against cutaneousleishmaniasis It is likely that ZnDPA complexes will have activityagainst other Leishmania species genetically similar to L majorand L donovani as well as against other trypanosomatids includ-ing Trypanosoma cruzi (52ndash54) Another goal for future studies isto determine whether ZnDPA coordination complexes have ac-tivity against models of visceral Leishmaniasis FluorescentZnDPA probes such as mSeek should be very helpful in determin-ing systemic biodistribution and we are encouraged by our pre-vious work indicating that the biodistribution of ZnDPA probes iseasily altered by rational structural modification (22ndash28)

ACKNOWLEDGMENTS

We thank M Leevy and S Chapman of the Notre Dame Integrated Im-aging Facility for technical assistance with the imaging systems

FUNDING INFORMATIONThis work was funded in part by the Defense Threat Reduction Agency(grant HDTRA1-13-1-0016 to BDS) the National Institutes of Health(NIH grants R01GM059078 to BDS and T32GM075762 to DRR) theIndiana Clinical and Translational Institute funded by the NIH NationalCenter for Advancing Translational Science Clinical and TranslationalSciences Award and the Eck Institute for Global Health (to MAM)

REFERENCES1 World Health Organization 2012 Leishmaniasis World Health Orga-

nization Geneva Switzerland httpwwwwhointleishmaniasisen2 Desjeux P 2001 The increase in risk factors for leishmaniasis worldwide

Trans R Soc Trop Med Hyg 95239 ndash243 httpdxdoiorg101016s0035-9203(01)90223-8

3 Hartley MA Drexler S Ronet C Beverley SM Fasel N 2014 Theimmunological environmental and phylogenetic perpetrators of meta-static leishmaniasis Trends Parasitol 30412ndash 422 httpdxdoiorg101016jpt201405006

4 Singh N Kumar M Singh RK 2012 Leishmaniasis current status ofavailable drugs and new potential drug targets Asian Pac J Trop Med5485ndash 497 httpdxdoiorg101016S1995-7645(12)60084-4

5 Haldar AK Sen P Roy S 2011 Use of antimony in the treatment ofleishmaniasis current status and future directions Mol Biol Int 2011571242 httpdxdoiorg1040612011571242

FIG 7 (A) Red fluorescence mean pixel intensities for separate treatment cohortsof living BALBc mouse footpads infected with mCherry-L major (n 4) Eachbar represents the means the standard errors (B) Parasite counts in footpadsharvested after 12-day treatments with saline treatment (gray) or compound 7(blue) Bars represent the means the standard errors (n 3) (C) Representativephotographs of mouse footpads after 12 days of different treatments

Rice et al

2938 aacasmorg May 2016 Volume 60 Number 5Antimicrobial Agents and Chemotherapy

6 Machado-Pinto J Pinto J da Costa CA Genaro O Marques MJModabber F Mayrink W 2002 Immunochemotherapy for cutaneousleishmaniasis a controlled trial using killed Leishmania (Leishmania)amazonensis vaccine plus antimonial Int J Dermatol 4173ndash78 httpdxdoiorg101046j1365-4362200201336x

7 Mishra M Biswas UK Jha DN Khan AB 1992 Amphotericin versuspentamidine in antimony-unresponsive kala-azar Lancet 3401256 ndash1257 httpdxdoiorg1010160140-6736(92)92952-C

8 Dorlo TP Balasegaram M Beijnen JH de Vries PJ 2012 Miltefosine areview of its pharmacology and therapeutic efficacy in the treatment ofleishmaniasis J Antimicrob Chemother 672576 ndash2597 httpdxdoiorg101093jacdks275

9 Wassef MK Fioretti TB Dwyer DM 1985 Lipid analyses of isolatedsurface membranes of Leishmania donovani promastigotes Lipids 20108 ndash115 httpdxdoiorg101007bf02534216

10 Wanderley JL Thorpe PE Barcinski MA Soong L 2013 Phosphatidyl-serine exposure on the surface of Leishmania amazonensis amastigotesmodulates in vivo infection and dendritic cell function Parasite Immunol35109 ndash119 httpdxdoiorg101111pim12019

11 Glew RH Saha AK Das S Remaley AT 1988 Biochemistry of theLeishmania species Microbiol Rev 52412ndash 432

12 Turco SJ Descoteaux A 1992 The lipophosphoglycan of Leishmaniaparasites Annu Rev Microbiol 4665ndash94 httpdxdoiorg101146annurevmi46100192000433

13 Weingartner A Kemmer G Muller FD Zampieri RA Gonzaga dosSantos M Schiller J Pomorski TG 2012 Leishmania promastigotes lackphosphatidylserine but bind annexin V upon permeabilization or milte-fosine treatment PLoS One 7e42070 httpdxdoiorg101371journalpone0042070

14 Mendez-Samperio P de la Rosa-Arana JL 2013 Antimicrobial peptidesas parasiticidal against human trypanosomatids mechanisms of actionand current status in development J Egypt Soc Parasitol 43195ndash208 httpdxdoiorg10128160006377

15 Dey T Anam K Afrin F Ali N 2000 Antileishmanial activities ofstearylamine-bearing liposomes Antimicrob Agents Chemother 441739 ndash1742 httpdxdoiorg101128aac4461739-17422000

16 Afrin F Dey T Anam K Ali N 2001 Leishmanicidal activity of stearyl-amine-bearing liposomes in vitro J Parasitol 87188 ndash193 httpdxdoiorg1016450022-3395(2001)087[0188LAOSBL]20CO2

17 Rosypal AC Hall JE Bakunova S Patrick DA Bakunov S Stephens CEKumar A Boykin DW Tidwell RR 2007 In vitro activity of dicationiccompounds against a North American foxhound isolate of Leishmaniainfantum Vet Parasitol 145207ndash216 httpdxdoiorg101016jvetpar200701005

18 Loiseau PM Cojean S Schrevel J 2011 Sitamaquine as a putativeantileishmanial drug candidate from the mechanism of action to the riskof drug resistance Parasite 18115ndash119 httpdxdoiorg101051parasite2011182115

19 Hanshaw RG Lakshmi C Lambert TN Smith BD 2005 Fluorescentdetection of apoptotic cells using a zinc coordination complex with aselective affinity for membrane surfaces that are enriched in phosphati-dylserine Biophys J 88341andash341a httpdxdoiorg101002cbic200500149

20 Koulov AV Stucker KA Lakshmi C Robinson JP Smith BD 2003Detection of apoptotic cells using a synthetic fluorescent sensor for mem-brane surfaces that contain phosphatidylserine Cell Death Differ 101357ndash1359 httpdxdoiorg101038sjcdd4401315

21 Lakshmi C Hanshaw RG Smith BD 2004 Fluorophore-linked zinc(II)dipicolylamine coordination complexes as sensors for phosphatidyl-serine-containing membranes Tetrahedron 6011307ndash11315 httpdxdoiorg101016jtet200408052

22 Plaunt AJ Harmatys KM Wolter WR Suckow MA Smith BD 2014Library synthesis screening and discovery of modified zinc(II)-bis(dipicolylamine) probe for enhanced molecular imaging of cell deathBioconjug Chem 25724 ndash737 httpdxdoiorg101021bc500003x

23 Smith BA Akers WJ Leevy WM Lampkins AJ Xiao S Wolter WSuckow MA Achilefu S Smith BD 2010 Optical imaging of mammaryand prostate tumors in living animals using a synthetic near infrared zinc(II)-dipicolylamine probe for anionic cell surfaces J Am Chem Soc 13267ndash 69 httpdxdoiorg101021ja908467y

24 Smith BA Gammon ST Xiao S Wang W Chapman S McDermott RSuckow MA Johnson JR Piwnica-Worms D Gokel GW Smith BDLeevy WM 2011 In vivo optical imaging of acute cell death using a

near-infrared fluorescent zinc-dipicolylamine probe Mol Pharm 8583ndash590 httpdxdoiorg101021mp100395u

25 Smith BA Harmatys KM Xiao S Cole EL Plaunt AJ Wolter WSuckow MA Smith BD 2013 Enhanced cell death imaging using multi-valent zinc(II)-bis(dipicolylamine) fluorescent probes Mol Pharm 103296 ndash3303 httpdxdoiorg101021mp300720k

26 Smith BA Xie BW van Beek ER Que I Blankevoort V Xiao S ColeEL Hoehn M Kaijzel EL Lowik CW Smith BD 2012 Multicolorfluorescence imaging of traumatic brain injury in a cryolesion mousemodel ACS Chem Neurosci 3530 ndash537 httpdxdoiorg101021cn3000197

27 Leevy WM Gammon ST Jiang H Johnson JR Maxwell DJ JacksonEN Marquez M Piwnica-Worms D Smith BD 2006 Optical imagingof bacterial infection in living mice using a fluorescent near-infrared mo-lecular probe J Am Chem Soc 12816476 ndash16477 httpdxdoiorg101021ja0665592

28 Leevy WM Gammon ST Johnson JR Lampkins AJ Jiang H MarquezM Piwnica-Worms D Suckow MA Smith BD 2008 Noninvasiveoptical imaging of staphylococcus aureus bacterial infection in living miceusing a Bis-dipicolylamine-Zinc(II) affinity group conjugated to a near-infrared fluorophore Bioconjug Chem 19686 ndash 692 httpdxdoiorg101021bc700376v

29 Leevy WM Johnson JR Lakshmi C Morris J Marquez M Smith BD2006 Selective recognition of bacterial membranes by zinc(II)-coordination complexes Chem Commun httpdxdoiorg101039b517519d1595-1597

30 Leevy WM Serazin N Smith BD 2007 Optical imaging of bacterialinfection models Drug Discov Today Dis Models 491ndash97 httpdxdoiorg101016jddmod200707001

31 OrsquoNeil EJ Jiang H Smith BD 2013 Effect of bridging anions on thestructure and stability of phenoxide bridged zinc dipicolylamine coordi-nation complexes Supramol Chem 25315ndash322 httpdxdoiorg101080106102782013776170

32 Xiao S Abu-Esba L Turkyilmaz S White AG Smith BD 2013 Mul-tivalent dendritic molecules as broad spectrum bacteria agglutinationagents Theranostics 3658 ndash 666 httpdxdoiorg107150thno6811

33 Rice DR Gan H Smith BD 2015 Bacterial imaging and photodynamicinactivation using zinc(II)-dipicolylamine BODIPY conjugates Pho-tochem Photobiol Sci 141271ndash1281 httpdxdoiorg101039c5pp00100e

34 Desjeux P 2004 Leishmaniasis current situation and new perspectivesComp Immunol Microbiol Infect Dis 27305ndash318 httpdxdoiorg101016jcimid200403004

35 Vacchina P Morales MA 2014 In vitro screening test using Leishmaniapromastigotes stably expressing mCherry protein Antimicrob AgentsChemother 581825ndash1828 httpdxdoiorg101128AAC02224-13

36 Hendrickx S Boulet G Mondelaers A Dujardin JC Rijal S Lachaud LCos P Delputte P Maes L 2014 Experimental selection of paromomy-cin and miltefosine resistance in intracellular amastigotes of Leishmaniadonovani and L infantum Parasitol Res 1131875ndash1881 httpdxdoiorg101007s00436-014-3835-7

37 Titus RG Marchand M Boon T Louis JA 1985 A limiting dilution assayfor quantifying Leishmania major in tissues of infected mice Parasite Im-munol 7545ndash555 httpdxdoiorg101111j1365-30241985tb00098x

38 Goyard S Segawa H Gordon J Showalter M Duncan R Turco SJBeverley SM 2003 An in vitro system for developmental and geneticstudies of Leishmania donovani phosphoglycans Mol Biochem Parasitol13031ndash 42

39 Calvo-Alvarez E Guerrero NA Alvarez-Velilla R Prada CF RequenaJM Punzon C Llamas MA Arevalo FJ Rivas L Fresno M Perez-Pertejo Y Balana-Fouce R Reguera RM 2012 Appraisal of a Leishmaniamajor strain stably expressing mCherry fluorescent protein for both invitro and in vivo studies of potential drugs and vaccine against cutaneousleishmaniasis PLoS Negl Trop Dis 6e1927 httpdxdoiorg101371journalpntd0001927

40 Oliveira LF Schubach AO Martins MM Passos SL Oliveira RVMarzochi MC Andrade CA 2011 Systematic review of the adverse ef-fects of cutaneous leishmaniasis treatment in the new world Acta Trop11887ndash96 httpdxdoiorg101016jactatropica201102007

41 Carvalho S Barreira da Silva R Shawki A Castro H Lamy M Eide DCosta V Mackenzie B Tomas AM 2015 LiZIP3 is a cellular zinc trans-porter that mediates the tightly regulated import of zinc in Leishmania

Chemotherapeutic Agents for Leishmania major

May 2016 Volume 60 Number 5 aacasmorg 2939Antimicrobial Agents and Chemotherapy

infantum parasites Mol Microbiol 96581ndash595 httpdxdoiorg101111mmi12957

42 Al-Mulla Hummadi YM Al-Bashir NM Najim RA 2005 The mecha-nism behind the antileishmanial effect of zinc sulfate II Effects on theenzymes of the parasites Ann Trop Med Parasitol 99131ndash139 httpdxdoiorg101179136485905X19937

43 Sanchez-Delgado RA Anzellotti A 2004 Metal complexes as chemo-therapeutic agents against tropical diseases trypanosomiasis malaria andleishmaniasis Mini Rev Med Chem 423ndash30 httpdxdoiorg1021741389557043487493

44 Ramirez-Macias I Maldonado CR Marin C Olmo F Gutierrez-Sanchez R Rosales MJ Quiros M Salas JM Sanchez-Moreno M 2012In vitro anti-leishmania evaluation of nickel complexes with a triazolopy-rimidine derivative against Leishmania infantum and Leishmania brazil-iensis J Inorg Biochem 1121ndash9 httpdxdoiorg101016jjinorgbio201202025

45 Caballero AB Salas JM Sanchez-Moreno M 2014 Metal-based thera-peutics for leishmaniasis In Leishmaniasis trends in epidemiology diag-nosis and treatment InTech Rijeka Croatia httpwwwintechopencombooksleishmaniasis-trends-in-epidemiology-diagnosis-and-treatmentmetal-based-therapeutics-for-leishmaniasis

46 Fattahi Bafghi A Noorbala M Noorbala MT Aghabagheri M 2014Anti-leishmanial effect of zinc sulphate on the viability of Leishmaniatropica and L major promastigotes Jundishapur J Microbiol 7e11192httpdxdoiorg105812jjm11192

47 Najim RA Sharquie KE Farjou IB 1998 Zinc sulfate in the treatment ofcutaneous leishmaniasis an in vitro and animal study Mem Inst OswaldoCruz 93831ndash 837 httpdxdoiorg101590s0074-02761998000600025

48 Hassan Khan NU Zaib S Sultana K Khan I Mougang-Soume BNadeem H Hassan M Iqbal J 2015 Metal complexes of tosyl sulfon-amides design X-ray structure biological activities and molecular dock-ing studies RSC Adv 530125ndash30132 httpdxdoiorg101039c4ra16124f

49 da Silva LE Joussef AC Pacheco LK da Silva DG Steindel MRebelo RA Schmidt B 2007 Synthesis and in vitro evaluation ofleishmanicidal and trypanocidal activities of N-quinolin-8-yl-arylsulfonamides Bioorg Med Chem 157553ndash7560 httpdxdoiorg101016jbmc200709007

50 da Silva LE de Sousa PT Maciel EN Nunes RK Eger I Steindel MRebelo RA 2010 In vitro antiprotozoal evaluation of zinc and coppercomplexes based on sulfonamides containing 8-aminoquinoline ligandsLett Drug Des Discov 7679 ndash 685 httpdxdoiorg102174157018010792929586

51 Pena I Pilar-Manzano M Cantizani J Kessler A Alonso-Padilla JBardera AI Alvarez E Colmenarejo G Cotillo I Roquero I de Dios-Anton F Barroso V Rodriguez A Gray DW Navarro M Kumar VSherstnev A Drewry DH Brown JR Fiandor JM Julio-Martin J 2015New compound sets identified from high-throughput phenotypic screen-ing against three kinetoplastid parasites an open resource Sci Rep 58771httpdxdoiorg101038srep08771

52 Zhang WW Mendez S Ghosh A Myler P Ivens A Clos J Sacks DLMatlashewski G 2003 Comparison of the A2 gene locus in Leishmaniadonovani and Leishmania major and its control over cutaneous infection JBiol Chem 27835508ndash35515 httpdxdoiorg101074jbcM305030200

53 Rogers MB Hilley JD Dickens NJ Wilkes J Bates PA Depledge DPHarris D Her Y Herzyk P Imamura H Otto TD Sanders M SeegerK Dujardin JC Berriman M Smith DF Hertz-Fowler C Mottram JC2011 Chromosome and gene copy number variation allow major struc-tural change between species and strains of Leishmania Genome Res 212129 ndash2142 httpdxdoiorg101101gr122945111

54 Toledo A Martın-Saacutenchez J Pesson B Sanchiz-Marın C Morillas-Maacuterquez F 2002 Genetic variability within the species Leishmania infan-tum by RAPD A lack of correlation with zymodeme structure MolBiochem Parasitol 119257ndash264 httpdxdoiorg101016s0166-6851(01)00424-8

Rice et al

2940 aacasmorg May 2016 Volume 60 Number 5Antimicrobial Agents and Chemotherapy