Comparison of a High-Throughput High-Content IntracellularLeishmania donovani Assay with an Axenic Amastigote Assay
Manu De Rycker,a Irene Hallyburton,a John Thomas,a Lorna Campbell,a Susan Wyllie,b Dhananjay Joshi,a* Scott Cameron,b
Ian H. Gilbert,a Paul G. Wyatt,a Julie A. Frearson,*a Alan H. Fairlamb,a,b David W. Graya
Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, United Kingdoma; Fairlamb Laboratory, Division of BiologicalChemistry and Drug Discovery, University of Dundee, Dundee, United Kingdomb
Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there isan urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-livingforms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiologi-cal situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intra-cellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. Apanel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings showthat for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 di-verse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial com-pounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited asa primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assaymay render it more suitable for hit-discovery.
The leishmaniases are a group of parasitic diseases caused bymembers of the protozoan trypanosomatid genus Leishmania
that have a significant health impact, primarily in the developingworld (1). Current treatments for visceral leishmaniasis includeantimonials, amphotericin B, miltefosine, and paromomycin. Allof these drugs have substantial issues associated with them, in-cluding toxicity, emerging resistance, parenteral administration,high cost and relatively long treatment regimens. Thus, there is anurgent need for the development of new, better medicines (2).Leishmanial parasites have two major life cycle stages: the promas-tigote stage (inside the insect vector) and the amastigote stage(inside the mammalian host). The amastigotes reside in parasito-phorous vacuoles, phagolysosome-like compartments with acidicand hydrolytic conditions (3). Leishmania amastigotes are exqui-sitely adapted to this environment and subvert many of the innatedefense mechanisms of the host cell (4).
For the purpose of drug discovery, many different assays, usingeither promastigotes or amastigotes, have been developed toscreen compound collections to identify new molecules that areactive against Leishmania. The different assays all have advantagesand drawbacks. The most straightforward assays use free-livingparasites to assess the effect of compounds on cell viability. Themain advantage of such assays is that they allow fast and easyscreening of large compound collections, a feature that is oftenrequired for the successful identification of developable hits. Pro-mastigotes have been used routinely for this purpose (5–10), butsince they represent the insect life-stage, there is the risk of iden-tifying compounds that do not affect the relevant disease-causinglife-stage. An alternative is to use axenic amastigotes, i.e., amasti-gotes that have been adapted to grow outside their host cell in agrowth medium that mimics the intracellular conditions (11–14).Such amastigotes can be used for straightforward high-through-put screening in a standard growth assay and have the advantageof being more similar to the disease-relevant parasite stage than
promastigotes (15–17). However, several reports indicate that ax-enic amastigotes are different from intracellular amastigotes bothin terms of protein expression and in terms of drug susceptibility(18–21). Thus, hits from screens with free-living parasites alwayshave to be confirmed using an intramacrophage assay. Further-more, compounds that interfere with the parasite-host interactioncannot be identified by such screens (22). With the advent of newtechnologies, it has now become possible to increase the through-put of the traditionally very labor-intensive intracellular amasti-gote assays. Two main methods are in use for the detection ofintracellular parasites: plate-reader-based methods that rely onreporter constructs (23–25) and microscopy-based methods thatcount parasites directly (8, 26, 27). With the indirect reporter-based assays, there is a risk of artifacts; compounds may interferewith the reporter protein or with the substrate, and no informa-tion is obtained regarding the number of host cells or the distri-bution of amastigotes in macrophages since these are whole-wellreadout assays. Microscopy-based high-content technology en-ables direct counting and circumvent many of these problems.However, this method is complex and has only recently been de-
Received 4 December 2012 Returned for modification 19 February 2013Accepted 1 April 2013
scribed for primary screening of compound libraries (8, 26, 27).These publications show that screening with promastigotes resultsin a large set of hits that show no activity against the intracellularparasites (i.e., false positives) and that it is possible to identifycompounds that show activity against intracellular amastigotesbut not against free-living promastigotes. We present here an al-ternative 384-well microscopy-based assay, which we used tocarry out a large-scale comparison to the axenic amastigote assayto determine the differences in hits identified by both platforms.
MATERIALS AND METHODSChemicals. The following chemicals were obtained from Sigma: ampho-tericin B, ketoconazole, atovaquone, pentamidine, paromomycin, milte-fosine, phorbol-12-myristate-13-acetate (PMA), 4=,6-diamidino-2-phe-nylindole (DAPI), formaldehyde, thimerosal, and resazurin. Nifurtimoxwas a gift from Bayer, Argentina. Nourseothricin (Lexsy NTC) was ob-tained from Jena Bioscience. Sodium stibogluconate was obtained fromMerck. HCS Cellmask Deep Red was obtained from Invitrogen. Thesmall-molecule library used in the present study contained 15,659 diversecompounds, dissolved at 10 mM in 100% dimethyl sulfoxide (DMSO).This library was assembled using the criteria for a diverse compoundcollection outlined elsewhere (28) and stored under low-oxygen and low-humidity conditions.
Cells and cell lines. THP-1 (human monocytic leukemia) cells wereobtained from the Health Protection Agency Culture Collections (catalogno. 88081201). The cells were screened for mycoplasma and maintainedin RPMI plus 10% (vol/vol) heat-inactivated fetal bovine serum (FBS).MRC-5 pd30 (human fetal lung) cells were obtained from the HealthProtection Agency Culture Collections (catalog no. 84101801) and testedfor mycoplasma. The cells were maintained in minimal essential mediumplus 10% (vol/vol) FBS. L. donovani (MHOM/SD/62/1S-CL2D, LdBOB)(11, 29) axenic amastigotes and promastigotes were maintained as de-scribed previously (29). Every 5 weeks, the parasites were cycled betweendevelopmental stages. For amastigote differentiation, 20 �l of metacyclicpromastigote culture was inoculated in amastigote media. For the reverseprocess, 20 �l of dense amastigote culture was inoculated in promastigotemedia.
Cloning and expression of eGFP in LdBOB. The enhanced green flu-orescent protein (eGFP) gene from Aequorea victoria (Swiss-Prot acces-sion no. AAB02576) was submitted to GenScript (Piscataway, NJ) forOptimumGene codon optimization to facilitate expression in Leishmaniaspp. The resulting synthetic gene (see Data File S1 in the supplementalmaterial), cloned into pUC57, was flanked by SmaI sites at the 5= and 3=ends of the gene. The pUC57-eGFP construct was then digested withSmaI, and the fragment was cloned into the pIR1SAT expression vector(30), resulting in a pIR1SAT-eGFP construct. Mid-log-phase L. donovanipromastigotes (WT, LdBOB) were transfected with pIR1SAT-eGFP usingthe human T cell Nucleofector kit and Nucleofector (Amaxa, programV-033). After transfection, the cells were allowed to grow 16 to 24 h inmodified M199 medium (29) with 10% FBS prior to drug selection withnourseothricin (100 �g ml�1). Cloned cell lines were generated by limit-ing dilution and maintained in selective medium. Clones expressing highlevels of eGFP were then selected using fluorescence microscopy and con-verted to axenic amastigotes for use in the screening assays.
Assays. (i) Axenic L. donovani assay. LdBOB axenic amastigotes wereincubated for 72 h with compounds, followed by a resazurin-based read-out. A more detailed description of the assay can be found in the supple-mental material.
(ii) Intramacrophage L. donovani assay. An intracellular Leishmaniaassay using eGFP expressing LdBOB amastigotes was performed. PMAdifferentiated THP-1 cells were infected overnight with axenic amasti-gotes at a multiplicity of infection of 5. After infection, compounds wereadded to the plates, followed by a 3-day incubation and microscopy-basedreadout. Further details can be found in the supplemental material.
(iii) MRC-5 assay. MRC-5 cells were incubated for 72 h with com-pounds, followed by a resazurin-based readout. A more detailed descrip-tion of the assay can be found in the supplemental material.
Data analysis. All data were processed using IDBS ActivityBase. Rawdata were converted into percent inhibitions through linear regression bysetting the high-inhibition control as 100% and the no-inhibition controlas 0%. Quality control criteria for passing plates were as follows: z= � 0.5,signal-to-background ratio (S:B) � 3, and percent coefficient of variation(%CV) of the no-inhibition control � 15. The formula used to calculate z= isas follows: z= � 1 � {[3 � (SDhigh � SDlow)]/[ABS(Meanhigh � Meanlow)]}(31), where SDhigh is the standard deviation of the maximum inhibition con-trols, SDlow is the standard deviation of the vehicle-treated controls, ABSstands for absolute value, Meanhigh is the mean of the maximum inhibitioncontrols, and Meanlow is the mean of the vehicle-treated controls. Curve fit-ting was carried out using the following four-parameter equation: y � A �{[B � A]/[1 � (C/x)D]}, where A is the percent inhibition at the bottom, B isthe percent inhibition at the top, C is the 50% effective concentration (EC50),D is the slope, x is the inhibitor concentration, and y is the percent inhibition.If curve definition was poor, B was fixed to 100. For the determination of thereference compound panel potency, all experiments were carried out with aminimum of three independent repeats.
RESULTSHigh-content assay design. Figure 1A shows an overview of theassay, a more detailed description of which can be found in Ma-terials and Methods. Plates were imaged on an automated micro-scope, and the images were analyzed with an image analysis algo-rithm that we designed using GE Incell Investigator software (Fig.1B and see also the supplemental material). Examples of curvesobtained for the standard compounds amphotericin B and milte-fosine are given in Fig. 1C. Note that the increase in THP-1 countsafter treatment with 0.1 to 1 �M amphotericin B is a reproducible,compound-specific effect. The DMSO tolerance of the assay wasdetermined, and there was no detectable effect of DMSO on theassay at 0.5% DMSO, the concentration routinely used in ourscreens (see Fig. S1 in the supplemental material).
Assay characterization. To further characterize the assay, thelevel of Leishmania infection was assessed by looking at the num-ber of amastigotes per cell and the number of infected cells. Figure2A shows a typical distribution for the number of amastigotes permacrophage at 72 h postinfection (mean � 7.2 and mode � 2amastigotes/infected macrophage). The assay also allows determi-nation of amastigote replication inside the host cell during thetime frame of the assay. Replication was assessed over 7 days bycounting the average number of amastigotes per macrophage atmultiple time points. Since there is effectively no division of thedifferentiated THP-1 cells, this gives a good indication of intracel-lular parasite replication. Over the 7 days we only observed limitedLeishmania growth, as shown in Fig. 2B (doubling time � 12days). In contrast, cell division is rapid and exponential in theaxenic system (doubling time, 5.8 h, Fig. 2C).
To assess the suitability of the assay for high-throughputscreening, we determined standard screening statistics across alarge number of plates (Table 1). The data show that the assay isrobust and has low variability and a high S:B. We also monitoredthe potency of amphotericin B, which was always included as ref-erence on potency plates, and found it to be highly reproducible.Using 384-well plates, we achieved a throughput of 7,680 wells perbatch while maintaining high-quality performance statistics. Thereproducibility of the assay was further assessed by carrying out aset of potency determinations on two separate occasions, and a
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high level of correlation was observed, as shown in Fig. 2D (R2 �0.88, slope � 0.9).
Reference compound panel. A panel of reference compounds(amphotericin B, atovaquone, miltefosine, paromomycin, so-dium stibogluconate, ketoconazole, pentamidine, and nifurti-mox) was tested against intracellular amastigotes, as well as axenicamastigotes, and MRC-5 cells, a human cell-line used for initialtoxicity determinations. The results are shown in Table 2 and arebroadly in line with previously reported values (15, 20, 32, 33, 34).The clinically used compounds amphotericin B, miltefosine, andparomomycin show very similar activities against axenic and in-tracellular amastigotes, whereas most other compounds show asignificant dropoff from the axenic to the intramacrophage assay(Fig. 3).
Axenic versus intramacrophage assay. In order to comparethe axenic L. donovani assay with the intramacrophage assay, wescreened a diverse compound set (�16,000 compounds) in bothassays. For the axenic screen, a top concentration of 3 �M drugwas used. Preliminary experiments with the intramacrophage as-say showed that the hit rate at this concentration was very low, andinstead we screened the set at 50 �M in this assay. Figure 4A showsthe hit frequency distributions for both assays. Hits were definedas showing �70% inhibition of amastigote growth, with the ad-ditional criterion of �50% inhibition of THP-1 cell counts for theintramacrophage assay. In the axenic screen 381 hits were identi-fied (a hit rate of 2.4% at 3 �M). In the intracellular assay, a totalof 213 compounds showed �70% inhibition of the amastigotecount (1.4% of the compounds at 50 �M). Of these, 128 showed�50% inhibition of THP-1 cell counts, thus leaving 85 hits and ahit rate of 0.5% (Fig. 4B). Only 17 compounds were hits in boththe axenic and the intramacrophage screens, whereas 85 com-pounds showed activity against the THP-1 cells and the axenicamastigotes, indicating that these may be generally toxic com-pounds (Fig. 4C).
The results of screening cascades performed after the primaryscreens are shown in Fig. 4D and E. Hits from the axenic screenwere first tested for potency in both the axenic model and theMRC-5 counterscreen. Compounds showing selectivity over theMRC-5 cells (319 compounds in total) were then progressed topotency testing in the macrophage assay. To quickly remove inac-tive compounds at this stage, we triaged compounds by assessingtheir potency using a single-replicate, 5-point, one-in-three dilu-tion curve. Compounds showing activity were then further pro-gressed to full 10-point duplicate potency testing. The majority ofthe axenic hits dropped out at this stage, showing no activity in themacrophage assay (278 compounds, 87% of the axenic hits). Afterapplication of selectivity rules comparing intramacrophage po-tency with THP-1 and MRC-5 cell activity, six hits remained.
The 85 hits from the primary intramacrophage screen wereprogressed into 10-point potency testing and MRC-5 counter-screening, after which 7 hits were retained. Of these, 6 were thesame as the ones identified using the axenic cascade. The addi-tional hit (compound 2) was not very potent against the intracel-lular amastigotes, with a pEC50 of 5 (EC50 � 10 �M). Since axenicpotency testing was initially carried out with a top concentrationof 16.6 �M, we may not have picked up such a low level of activityin the axenic primary screen. As a result, this compound was re-tested in the axenic assay with a top concentration of 50 �M andreturned a potency value in the same range as seen in the intracel-lular assay (pEC50 � 4.7). The structures for all 7 hits from both
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FIG 1 Intracellular Leishmania assay. (A) Graphical representation of assayprotocol. The x axis represents the time in hours. Details regarding each stepcan be found in Materials and Methods. (B) Image analysis. Pseudocoloredraw images are shown and marked with their respective dyes/fluorophores(CMdr, cytoplasm marker [HCS Cellmask Deep Red]). The result of imageanalysis is shown in the bottom right panel (cytoplasm, green outline; amas-tigotes, yellow outline). (C) Representative dose-response curves for miltefos-ine and amphotericin B. Ten-point potency curves for the indicated com-pounds were created and tested in the intracellular Leishmania assay. Potencyvalues for the compounds are given in Table 2. The data are from at least fourreplicates, shown as purple diamonds, and the curve is fitted to the averagevalues (blue circles). The data points for Leishmania inhibition that are omit-ted from the curve fit for miltefosine because of toxicity to THP-1 cells aremarked as boxed crosses.
screening campaigns with their potencies in the various assays aregiven in Table 3.
For a total of 110 compounds from this screen, we have carriedout full 10-point potency determinations in both the axenic andthe intramacrophage systems. Comparison of these shows that forthe majority of compounds there was a significant reduction inpotency from the axenic to the intramacrophage assay, with 80compounds showing a �10-fold drop in activity from the axenicto the intramacrophage assay (includes compounds with no activ-ity intramacrophage assay) (Fig. 5A). No compound was mark-edly more potent in the intramacrophage assay.
A total of 206 compounds were tested for potency in the intra-
macrophage Leishmania assay and the human counterscreen lineMRC-5. Of these, 145 showed no activity against both THP-1 andMRC-5 cell lines. Comparison of the remaining 61 compoundsrevealed that, in general, the MRC-5 cells are more sensitive thanthe THP-1 cells, with 47 compounds showing no activity againstTHP-1 cells while being active against MRC-5 cells, and with 28compounds being �3-fold more active against MRC-5 cells thanTHP-1 cells (Fig. 5B).
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FIG 2 Assay characterization. (A) Frequency distribution of intracellular amastigote count. At the end of the intracellular Leishmania assay, the number ofamastigotes in 19,738 infected cells was counted using the automated image analysis algorithm, and a frequency distribution was plotted. (B) Growth curve forintracellular amastigotes. Cells were plated and infected in 384-well plates, and one plate was fixed at each time point. The values represent the number ofamastigotes per infected cell (i.e., the averages and standard deviations from the entire plate). A linear regression was calculated (R2 � 0.93). (C) Growth curvefor axenic amastigotes in 384-well plates. Axenic amastigotes were counted at the indicated time points. The y axis shows the number of amastigotes per ml(�1,000). An exponential curve was fitted to time points 0 to 55 h (R2 � 0.999). The results from triplicate experiments are shown. (D) Replicate potencies. Thepotency of 85 compounds was tested on two separate occasions in the intracellular Leishmania assay, and the potencies of the replicates were plotted against eachother. A linear regression was calculated (R2 � 0.88).
TABLE 1 Performance statistics of the intramacrophage assay
Performance statistic Avg SD (n)a
Z-factor 0.68 0.09 (518)%CV 9.8 2.8 (518)S:B 90 51 (518)Amphotericin B potency (pEC50) 6.74 0.18 (41)% Infected cells 81 (19,738)Throughput 20 � 384 wells per batcha Values are averages the standard deviation except as noted otherwise in column 1. nis the number of plates for the Z-factor, the percent coefficient of variation (%CV), andthe signal-to-background ratio (S:B), the number of curves for amphotericin B potency,and the number of cells for the percent infected cells.
TABLE 2 Potencies for reference compound panel
Compound
pEC50 {�log(EC50[M])}a
Axenic INMAC THP1 MRC5
Amphotericin B 7.06 (0.17) 7.13 (0.05) 5.68 (0.1) 5.08 (0.12)Miltefosine 5.61 (0.2) 6.03 (0.17) 4.33 (0.08) 4.3 (0.09)Atovaquone 5.37 (0.11) 5.36 (0.12) �3.8 4.56 (0.13)Paromomycin 5.29 (0.37) 5.18 (0.14) �2.5 �2.5Ketoconazole 7.57 (0.22) 4.89 (0.1) 4.33 (0.07) 4.29 (0.07)Pentamidine 6.3 (0.25) 4.88 (0.21) 4.09 (0.07) 4.8 (0.1)Nifurtimox 5.39 (0.34) 4.62 (0.09) �3.8 3.98 (0.06)Sodium stibogluconate �2.9 3.82 (0.17) 2.98 (0.05) 3.09 (0.13)a Results are expressed as the pEC50 with standard deviations given in parentheses,except for sodium stibogluconate, where the result is given as the �log(EC50[�g ofSb/ml]).
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A 384-well intramacrophage Leishmania donovani assay. Thereis an urgent need for new medicines for leishmaniasis. In terms ofhit discovery, this parasitic disease poses a significant challenge,since the disease-causing stage resides in an acidic vacuole (theparasitophorous vacuole) inside the host’s macrophages. High-content screening using automated microscopy provides a suit-able platform for assessing such intracellular parasites, and re-cently both a 96-well and a 384-well high-content assay have beenreported (26, 27). We have also developed a 384-well microscopy-based intracellular Leishmania assay with sufficient capacity toallow primary screening and potency follow-up of medium-sizedsmall-molecule libraries. To achieve sufficient throughput, ro-bustness, and reproducibility, a number of alterations were madeto the traditional direct counting assay (reviewed in reference 35).Typically, ex vivo amastigotes are used to infect primary macro-
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FIG 3 Comparison of reference compound activity between the axenic andintramacrophage assays. The fold differences between the potencies, as re-ported in Table 2, are displayed.
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FIG 4 Screening cascades. (A) Frequency distributions for 15,659 compound diverse library screen against axenic (left panel) and intracellular (center and rightpanels) Leishmania. The abscissa shows the percent inhibition (PI). Three populations are shown on each frequency diagram: negative controls (red), positivecontrols (green), and test compounds (blue). Positive controls on the left panel cannot be seen since they are fully covered by the blue line for the test compoundsat 100%. (B) Venn diagram for intramacrophage primary screen hits. INMAC, compounds that showed �70% inhibition of intracellular Leishmania; THP-1,compounds that showed �50% inhibition of THP-1 cell count. (C) Comparison of hits from intramacrophage and axenic primary screen. THP-1, compoundsthat showed �50% inhibition of THP-1 cell count; axenic, compounds that showed �70% inhibition in the axenic primary screen; INMAC*, compounds thatshowed �70% inhibition of intracellular Leishmania and �50% inhibition of the THP-1 cell count. (D and E) Hit selection in the axenic (D) and intracellular(E) screening cascades. Numbers in green are compounds that progressed to the next step; numbers in red are compounds that were removed from the cascade(no activity, low activity, or toxicity). SP, single point primary screen; Pot, ten-point dose-response curve in duplicate; EC50, concentration at which a 50% effectis seen; tox, �10-fold window between antileishmanial EC50 and THP-1 EC50.
phages. However, this is difficult to achieve for a large-scalescreening assay. Instead, we opted to use axenic amastigotes. Theadvantage of axenic amastigotes over metacyclic promastigotes isthat they do not elicit an oxidative burst when entering the mac-rophage (4). This oxidative burst may result in the killing or sen-sitizing of the phagocytosed promastigotes, which may potentiatedrug action. In the host, most spreading of the disease occursthrough amastigotes and, as such, they may provide a better dis-ease model (36). We used L. donovani strain 1S-CL2D, which wasshown to retain in vivo infectivity and exhibits characteristics of exvivo amastigotes (11). In terms of the mammalian host cell, it
would be preferable to use primary macrophages, ideally of hu-man origin. However, it is difficult to obtain a reliable and homo-geneous supply at a scale suitable for routine screening. Instead,PMA-differentiated THP-1 cells were chosen, which are consid-ered a suitable model for human macrophages (37–39). The use ofnonprimary cells for our assay necessitates follow-up experimentsusing ex vivo amastigotes and primary macrophages during thehit-to-lead stage of a compound series since cell-line-specific ef-fects have been described both for Leishmania strains and host celllines (32, 40).
A key challenge in developing the intramacrophage assay
TABLE 3 Hits identified in screening campaign
Structure ID Assay pEC50
DDD00023040 INMAC (Leishmania) 5.94
INMAC (THP-1) 4.38
Axenic 6.3
MRC-5 �4.3
DDD00055223 INMAC (Leishmania) 4.99
INMAC (THP-1) �4.3
Axenic 4.73
MRC-5 �4.3
DDD00055671 INMAC (Leishmania) 4.82
INMAC (THP-1) 4.47
Axenic 5.88
MRC-5 �4.3
DDD00077687 INMAC (Leishmania) 4.91
INMAC (THP-1) 4.54
Axenic 5.83
MRC-5 �4.3
DDD00080409 INMAC (Leishmania) 5.23
INMAC (THP-1) 4.38
Axenic 5.93
MRC-5 4.31
DDD00084972 INMAC (Leishmania) 4.7
INMAC (THP-1) �4.3
Axenic 4.63
MRC-5 �4.3
DDD00085002 INMAC (Leishmania) 4.63
INMAC (THP-1) 4.3
Axenic 4.55
MRC-5 4.3
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proved to be the optimization of the washing step after infection.Since the axenic amastigotes were able to proliferate outside themacrophages, it was crucial to remove the majority of amastigotesthat had not been phagocytosed without damaging the THP-1cells. They would otherwise provide a reservoir for continuousinfection, which would make interpretation of the results difficult.In addition, their presence would complicate the imaging of theintracellular parasites. As detailed above, there are two significantdifferences with the previously published 384-well high-contentLeishmania assay (27): the use of axenic amastigotes for infectioninstead of metacyclic promastigotes and the removal of the infec-tion inoculum by a washing step. Whether either of these changeshas a major impact on the relevance of the assay is difficult to saywithout careful side-by-side comparison, but it is worth pointingout that the clinically used compounds paromomycin and sodiumstibogluconate show activity in the assay presented here, whilethey do not in the assay described by Siqueira-Neto et al. (27). Thismay indicate a higher physiological relevance for the assay pre-sented here.
Advantages of a high-content approach. A microscopy-basedassay, as presented here, has many advantages over plate-reader-based assays. A microscopy-based assay mimics the traditionaldirect-counting assay and provides cell-by-cell analysis. This al-lows the determination of parameters such as the number of in-fected cells and the number of amastigotes per individual cell. Thisinformation provides a more direct understanding of the assayresults and reduces the occurrence of artifacts. The assay also re-ports the number of THP-1 cells that may give a first indication oftoxicity of the compounds tested. However, since differentiatedTHP-1 cells are less sensitive to compound toxicity than activelygrowing human cells, a secondary mammalian counterscreen isstill advisable (see Fig. 5B). For correct interpretation of the Leish-mania inhibition data, it is essential to take into account theTHP-1 cell count, since the Leishmania percent inhibition valuebecomes unreliable at drug concentrations that kill the majority ofthe macrophages. This is demonstrated at the top concentration ofthe miltefosine dose-response curve shown in Fig. 1C. This figurealso reveals that amphotericin B has a beneficial effect on the num-ber of THP-1 cells in the 0.1 to 1 �M range. This could potentially
be explained by the known immunomodulatory effects of ampho-tericin B on THP-1 cells (41) or its ability to activate the survival-and growth-promoting kinase Akt/PKB (42, 43).
Although the high-content assays discussed here provide alarge amount of information, they do not take into account thehost microenvironment, which could influence parasite behaviorand drug action. Recently, a study demonstrated that, by usingsplenic explants and a reporter gene, it is possible to further im-prove the physiological relevance of the intracellular assay, albeitat a lower throughput (44).
Comparison of intracellular assay with axenic amastigote as-say. Other groups have shown that assaying free-living promasti-gotes results in high false-positive rates (i.e., the promastigote ac-tivity does not translate into activity against intracellularamastigotes) and may not identify intracellular stage-specificcompounds (8, 26, 27, 44). Here, we compared the intracellularassay to the axenic assay, which has been proposed as an alterna-tive model to promastigotes for primary screening (15, 17, 45).The rationale behind this comparison was to see whether the ax-enic assay has a place as a primary screening assay to allow for ahigher throughput, followed by confirmation in the macrophageassay. In the first instance, we tested a panel of reference com-pounds in both assays. We found that all clinically used com-pounds show similar activity in the axenic and intracellular assays,except for sodium stibogluconate, which shows no activity in ouraxenic assay (Table 2). The literature contains a lot of discussion,and conflicting results, regarding the effect of pentavalent anti-mony (SbV) on axenic amastigotes, with some groups showingactivity against all stages (46) and others showing activity againstaxenic and intracellular amastigotes but not promastigotes (15,47, 48), while others showed activity only against intracellularamastigotes (20, 36, 49). The evidence indicates that the reductionof SbV to trivalent antimony (SbIII) is critical for toxicity and thatthis only happens in fully differentiated amastigotes, so it is pos-sible that our axenic amastigotes retain some promastigote traits,including an inability to reduce SbV.
This small panel of reference compounds does not allow us todraw any conclusions regarding the general relationship betweenthe axenic and intracellular assays. To do this, we carried out a
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FIG 5 (A) Comparison of compound potency in axenic and intracellular assays. Compounds on the blue line show equal activity in both assays, and compoundson the right-hand side of the line are more potent in the axenic assay. Red diamonds, no activity in intramacrophage assay (pEC50�4.78, 82 compounds); greensquares, activity in both assays (28 compounds). (B) Comparison of compound potency against THP-1 cells and MRC-5 cells. Red diamonds, active againstMRC-5 and no activity against THP-1 (pEC50�4.78) (47 compounds); green triangles, activity against both cell lines (10 compounds); blue squares, activeagainst THP-1 and no activity against MRC-5 (pEC50 � 4.78) (4 compounds).
parallel primary screen of a library of nearly 16,000 diverse com-pounds in both systems. We observed high hit rates in the axenicassay compared to the intracellular assay, even though wescreened at much lower compound concentrations. As expected,many of the axenic hits did not show activity in the intracellularassay, and the compounds that did show activity across both plat-forms showed a significant dropoff of activity from the axenicassay to the intracellular assay, without any clear correlation. Afterprogressing the compounds through the two screening cascades(Fig. 4D and E), we obtained the same hit compounds. Our ob-servations allowed us to draw several important conclusions. (i)The axenic assay is much more sensitive than the intracellularassay, and the lack of correlation between the two assays limits theuse of the axenic assay as a predictor of intracellular activity. (ii)The use of the axenic assay as a primary platform, followed byintracellular follow-up, is highly inefficient. The cascade takes a lotlonger (7 weeks versus 4 weeks), and the majority of the com-pounds that progressed to the intracellular assay showed no activ-ity, so that valuable throughput is wasted. (iii) When stringentpotency criteria are applied to axenic hits before progression to theintracellular assay, there is a significant risk of losing interestingcompounds, since there is no correlation between axenic and in-tracellular potency. Thus, it seems clear that running the intracel-lular assay as a primary platform is the preferred route for findingnew hits.
Assay type versus compound mode of action. It is worth con-sidering why so few axenic hits translate into intracellular ac-tivity. There are inherent differences between the transcrip-tomes and proteomes of axenic and intracellular amastigotes(19, 50), and these could affect drug action. Two other obviousdifferences are rate of replication and free-living versus intra-cellular localization. In the assays described here, we see fastexponential growth of the axenic amastigotes and almost nogrowth of the intracellular amastigotes (Fig. 2C and D). This islikely to have a dramatic effect on their respective drug sensi-tivities, with the replicating cells being much more sensitive toinhibitors of cell division, energy metabolism, etc. In addition,a starting density significantly below the detection limit is usedin the axenic assay because of the high growth rate. This resultsin the assay not only identifying cytocidal compounds but alsogrowth-slowing compounds (51), which will also contribute tothe high false-positive rate, since only cytocidal compoundswere identified in our intramacrophage assay. We anticipatethat to achieve the current target product profile for visceralleishmaniasis compounds need to be cytocidal rather thanstatic or growth slowing (52). Comparing in vitro growth to thesituation in vivo is difficult, since many other factors play a role(in particular, the clearance of amastigotes by the immune sys-tem and differences between rodent and human physiology).Bradley and Kirkley (53) reported an �20-fold increase in tis-sue parasite counts over the first 8 days of infection in mice,which means that a minimum of four rounds of cell divisiontake place during this window. Hence, the rate of amastigotedivision in vivo is likely to be somewhere in between what wesee in the intramacrophage and axenic assays. When consider-ing false-positive rates for the axenic assay, it is important tokeep in mind that these rates should be based on the goldstandard comparator. Here, we used the intracellular Leishma-nia assay as the comparator, but it would be better to comparethe findings to those obtained in an in vivo animal model (or to
human clinical data). However, we did not have sufficient com-pounds with appropriate efficacy and absorption, distribution,metabolism, and excretion properties to carry out such a com-parison.
The localization of the parasites inside the parasitophorousvacuole presents a serious hurdle for small molecules; to exerttheir action, they need to cross three membranes and do this atboth neutral and acidic pH. This means that even if a compoundcould kill intracellular amastigotes, it may not be able to reachthem. Although such compounds will not be identified using theintracellular assay as a primary platform, they may nevertheless beof interest since further chemical optimization may allow thesemolecules to reach the vacuole and exert their effect, or alternativedelivery strategies could be used. It is likely that for these reasonsthe intracellular assay may not detect a subset of potentiallyinteresting compounds and, in view of the dearth of new leadsfor leishmaniasis, we may have to consider alternative assaysthat will yield more hits. To understand the effect of the fastaxenic growth on the hit rate in this assay, it would be veryinformative to devise an axenic amastigote assay under muchless stimulating growth conditions. Such an assay may havelower false-positive rates and could be of use for high-through-put screening. It would also allow the use of a higher startingdensity so that it only reports cidal compounds, again poten-tially reducing the false-positive rate.
Conclusion. Combining high throughput with physiologicalrelevance is a challenge for organisms with a complicated life cyclesuch as Leishmania. Ultimately, the only relevant readout is suc-cess in the clinic. The intramacrophage assay presented here re-ports activity for all clinically used compounds and, as such, ap-pears to be a good in vitro model for predicting clinical activity.The acquisition of clinical data for new drugs that derive from thecurrent leishmaniasis drug discovery drive will allow further as-sessment of the suitability of this assay as a primary drug discoveryplatform.
ACKNOWLEDGMENTS
We thank Daniel James for data management, Michael Thomas for helpwith preparing Table 3, and Mascha Brinkkötter for assistance in thegeneration of the L. donovani fluorescent reporter strain. We thank Jean-Robert Ioset and Eric Chatelain at DNDi for their advice and for criticallyreading the manuscript.
This study was funded by the Drugs for Neglected Diseases Initiative,through grants from several donors (Department for International Devel-opment, United Kingdom; Swiss Agency for Development and Coopera-tion, Switzerland; Médecins Sans Frontières/Doctors Without Borders;International and Gesellschaft für Technische Zusammenarbeit, Ger-many). This study was also supported by Wellcome Trust strategic award083481. A.H.F., S.W., and S.C. are supported by the Wellcome Trust(079838 and 083481).
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