Development of a movement-based <i>in vitro</i> screening assay for the identification of new anti-cestodal compounds

<div><p>Intestinal cestodes are infecting millions of people and livestock worldwide, but treatment is mainly based on one drug: praziquantel. The identification of new anti-cestodal compounds is hampered by the lack of suitable screening assays. It is difficult, or even impossible, to evaluate drugs against adult cestodes <i>in vitro</i> due to the fact that these parasites cannot be cultured in microwell plates, and adult and larval stages in most cases represent different organisms in terms of size, morphology, and metabolic requirements. We here present an <i>in vitro</i>-drug screening assay based on <i>Echinococcus multilocularis</i> protoscoleces, which represent precursors of the scolex (hence the anterior part) of the adult tapeworm. This movement-based assay can serve as a model for an adult cestode screen. Protoscoleces are produced in large numbers in Mongolian gerbils and mice, their movement is measured and quantified by image analysis, and active compounds are directly assessed in terms of morphological effects. The use of the 384-well format minimizes the amount of parasites and compounds needed and allows rapid screening of a large number of chemicals. Standard drugs showed the expected dose-dependent effect on movement and morphology of the protoscoleces. Interestingly, praziquantel inhibited movement only partially within 12 h of treatment (at concentrations as high as 100 ppm) and did thus not act parasiticidal, which was also confirmed by trypan blue staining. Enantiomers of praziquantel showed a clear difference in their minimal inhibitory concentration in the motility assay and (R)-(-)-praziquantel was 185 times more active than (S)-(-)-praziquantel. One compound named MMV665807, which was obtained from the open access MMV (Medicines for Malaria Venture) Malaria box, strongly impaired motility and viability of protoscoleces. Corresponding morphological alterations were visualized by scanning electron microscopy, and demonstrated that this compound exhibits a mode of action clearly distinct from praziquantel. Thus, MMV665807 represents an interesting lead for further evaluation.</p></div

Activity assessment of praziquantel and enantiomers on <i>E</i>. <i>multilocularis</i> protoscoleces.

<p><i>E</i>. <i>multilocularis</i> protoscoleces were exposed to various concentrations (100 ppm till 0.0006 ppm, 1 to 3 dilution series) of the PZQ-enantiomers (R)-(-)-PZQ and (S)-(-)-PZQ or the racemic mixture of both (PZQ). After 12 h of incubation, motility was assessed (A) and corresponding pictures of the morphological changes at 0.02 ppm are shown (B). (A) 1% DMSO was set to 100% relative movement and all other drug-responses were calculated accordingly. Note that all forms of PZQ never led to a complete inhibition of protoscolex movement. (R)-(-)-PZQ and the racemic mixture exhibited higher activities than the (S)-(-)-PZQ, with minimal inhibitory concentrations (MICs) indicated by asterisks (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.g001" target="_blank">Fig 1</a> for compound structures and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.s004" target="_blank">S1 Fig</a> for other measurement time points). Experiments shown here were repeated three times.</p

Isolation and activation of protoscoleces for movement-based assay.

<p><i>E</i>. <i>multilocularis</i> protoscoleces were purified from metacestode material that was grown in gerbils. (A) Subsequent to purification, protoscoleces were evaginated by incubation in various concentrations of DMSO for 3 hours (1, 2.5, 5, 10, and 20%), pepsin (0.5 mg/mL, pH 2) for 3 hours, pepsin (0.05%, pH 2.0, 20 minutes) and Na-taurocholate (0.2%, 3 hours), or in a DMEM control, all performed in triplicates at 37°C. Pictures were taken of each well and the number of in- and evaginated protoscoleces was counted manually. Mean values and standard deviations are given. Highest evagination rates were reached with 20% DMSO. (B) Effects of the induction of evagination on subsequent movement were also analyzed. Mean movement rates (absolute number of changed pixels) and standard deviations are shown. Highest motility rates were measured after activation in 10% DMSO as compared to 20% DMSO, pepsin or pepsin and Na-taurocholate, where motility was reduced. (C) Micrographs of SEM of protoscoleces incubated in DMEM only (control), or activated with 10% DMSO, pepsin only, or pepsin and Na-taurocholate. Based on these pre-trials, evagination was induced by incubation in 10% DMSO in all subsequent experiments. The experiments in A and B were repeated three times and one representative plot is shown each.</p

Activity assessment of various drugs against <i>E</i>. <i>multilocularis</i> protoscoleces.

<p>Various standard compounds with described activity (niclosamide (B) and nitazoxanide (C)) or no activity (albendazole (D), monepantel (E)) against adult cestodes were assessed concerning their effects on <i>E</i>. <i>multilocularis</i> protoscolex movement (100 to 0.4 ppm, 1 to 3 dilution series) and morphology (shown for 33.3 ppm). Protoscoleces treated with 1% DMSO (A, solvent control) were set to 100% relative movement and all other drug-incubations were calculated accordingly. Each drug was assessed in 6 replicas after 12 h of drug-incubation and mean percentages and standard deviations are given. Further time points are given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.s005" target="_blank">S2 Fig</a>, compound structures and MICs (as indicated here by asterisks) are given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.g001" target="_blank">Fig 1</a>. In addition, also effects of the novel compound, MMV665807, were assessed (F) by means of impact on motility and morphology. Mean percentages of movement and standard deviations are given. The MIC is indicated by an asterisk. Further time points are given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.s005" target="_blank">S2 Fig</a>. All these experiments were repeated at least 3 times.</p

Scanning electron microscopical assessment of protoscoleces treated with praziquantel and MMV665807.

<p>Protoscoleces were incubated in (A) 1% DMSO (control), (B) praziquantel (PZQ), or (C) MMV665807 (the latter two both with 1% DMSO) for 12 hours at 100, 10, 1 and 0.1 ppm and subsequently subjected to scanning electron microscopy (SEM). (A) depicts intact protoscoleces with rostellum (r), including hooks covered by a filamentous network, four muscular suckers (ms) and the protoscolex body (b). The whole protoscolex is covered in microtriches (mt) as shown in the enlarged view of a muscular sucker on the right. (B) Noticeable effects of PZQ on protoscoleces were seen at all concentrations tested (contraction of body and neck region (indicated by arrows), bleb formation (bf), as magnified in the picture most to the right), but the muscular suckers, hooks (h) and microtriches were largely unaffected. (C) MMV665807 led overall to less contraction compared to PZQ, but it induced a striking loss of microtriches and hooks from the rostellum (hl, see especially second picture at 100 ppm) at high concentrations (100, 10 and partially 1 ppm). At low concentrations (0.1 ppm), microtriches and rostellum with hooks were fully intact.</p

Assay conditions for motility-based assessment of protoscolex movement.

<p><i>E</i>. <i>multilocularis</i> protoscoleces were purified from metacestodes and activated for 3 h in 10% DMSO. (A) Protoscoleces were distributed into 384-well plates and incubated in various concentrations of DMSO (0, 0.1, 0.3, 1, 3, and 10%) and in 6 replicas each for 12 h, in order to determine the optimal percentage of DMSO to be used for drugs in subsequent tests. The movement per protoscolex was plotted against the respective DMSO concentrations and it shows that protoscolex movement is not significantly reduced up to 3% of DMSO. (B) In order to assess the correlation of protoscolex number per test well and movement, these two parameters were plotted against the total number of protoscoleces per well. A clear linear correlation between number and movement was observed (r = 0.86, R² = 0.75). (C) For determining the optimal assay temperature, protoscoleces were incubated in 1% DMSO at various temperatures (25, 30, 37, and 41°C) with 10 replicas per temperature. As shown, highest movement levels were reached at 37°C, and this was statistically significant. (A-C) The movement of protoscoleces was measured by assessing the changed pixels between two pictures within a 10 seconds interval. (A) and (C) experiments were repeated three times and one exemplary plot is shown.</p

Structures and minimal inhibitory concentrations of drugs tested in the study.

<p>Representative minimal inhibitory concentrations (MICs) are given after 12 h of drug incubation. They were determined by calculating the concentration where the slope of the dose-response curve changed significantly and are given in ppm and μM for all drugs (see also Figs <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.g004" target="_blank">4</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.g005" target="_blank">5</a>). For inactive drugs, no MIC was calculated, (stated by a minus). Asterisks indicate the MICs of PZQ and derivatives that never totally inhibited motility (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.g004" target="_blank">Fig 4</a>).</p

Assessment of viability of <i>E</i>. <i>multilocularis</i> protoscoleces treated with various drugs.

<p>(A) Trypan blue staining of protoscoleces was performed after 12 h of drug-incubation in triplicates (PZQ and enantiomers, niclosamide, nitazoxanide, MMV665807, albendazole, monepantel, concentrations from 100 to 0.4 ppm, 1 to 3 dilution series) and the mean percentages and standard deviations of protoscolex viabilities are shown. Protoscoleces incubated in the solvent DMSO at 1% served as control (dotted line). PZQ did not reduce the viability of protoscoleces, whereas niclosamide, nitazoxanide and MMV665807 did. The experiment was repeated 3 times and one exemplary plot is shown. (B) shows the correlation between effects on motility and viability (both in % of DMSO) for differently treated protoscoleces after 12 h. The correlation follows an exponential curve for all active drugs except PZQ. Respective functions and R² values are given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.s007" target="_blank">S1 Table</a>. The same legend applies for both subfigures.</p

Effects of monepantel on <i>C. elegans</i> and rescue of mutant <i>acr-23</i>.

<p>A) Adult N2 hermaphrodite with hatched embryos on 60 µM monepantel. L1 and L2 larvae are paralyzed and coiled (carcass of mother is outlined). B) Higher resolution of an arrested L2 larva. The germline is outlined. C) <i>acr-23</i>(<i>cb27</i>) animals carrying an extrachromosomal array containing wild-type <i>acr-23</i> grown on standard NGM. Transgenic animals have a rolling phenotype (red arrowhead; non transgenic worms are denoted by a black arrowhead in C and D). D) In presence of monepantel, only non-rolling siblings are mobile and fertile. Transgenic animals are immobilized and dying or dead. Panels C) and D) show plates with the progeny of one single rolling parent incubated for 6 days at 20°C. E) Phenotypes of homozygotic and heterozygotic <i>acr-23(cb27). </i><i>eDf43</i> is a deficiency that spans <i>acr-23</i> and neighboring genes. Depicted adult worms are one heterozygote, with dominant pharyngeal GFP expression (<i>nT1g</i>), and one non-glowing <i>acr-23(cb27)</i> homozygote, both grown on 20 µM monepantel. White arrows indicate all embryos present in the uterus. Similar results were obtained with <i>acr-23(ok2804)/nT1g</i> heterozygotes (not shown). Asterisks indicate the head. Bar, 50 µm. F) <i>acr-23</i> mutants make fewer reversals. The rate of reversal events, where worms shift from forward to backward locomotion or vice versa, was measured in starved worms crawling on food-free NGM plates at 20°C. <i>n</i> is the number of movies analyzed, each tracking 15 to 60 individuals over at least 1 minute. *, P-value = 6.4E-5 versus N2 by Student's T-test (two-tailed). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003524#s3" target="_blank">Results</a> are expressed as mean with s.e.m as error bars.</p

Effect of <i>ric-3</i> on the response to monepantel.

<p>A) Dose response to monepantel. The average number of progeny reaching adulthood after 3 days is plotted versus the concentration of monepantel. <i>ric-3(md158)</i> mutants are partially resistant to low doses of monepantel. B) <i>ric-3(md158)</i> adult grown on 1 µM monepantel (bottom) compared to a sibling grown in absence of the drug (top). The reduced length is certainly caused by muscle contraction due to monepantel. White arrows and arrowheads point at the heads and vulvae, respectively. C) ACR-23::GFP protein levels in wild-type (lanes 1,2) and <i>ric-3</i> (lanes 3,4) animals after 2 hours of exposure to 10 µM monepantel (lanes 2,4). α-Tubulin was used to verify equal loading. D) Growth of wild-type and mutant strains on monepantel. Light gray (0 µM), dark gray (20 µM) and black (50 µM) bars represent the growth rate after 9 days of culture. In the absence of monepantel, wild-type (N2) worms grow much faster than any of the mutants tested. Genotypes are indicated. Values for growth rates are defined in the methods section (1, poor growth; 5, robust growth). <i>acr-23</i> single mutants grew to level 5 in less than 5 days, whereas <i>ric-3; acr-23</i> double mutants reached level 2 in absence of monepantel, and level 4 in presence of the drug. E) Growth rates after 9 days on the inactive R-enantiomer of monepantel. F) <i>ric-3(md158); acr-23(cb27)</i> adult grown in absence of monepantel. G) Adult <i>ric-3(md158); acr-23(cb27)</i> sibling grown on 20 µM monepantel. In both panels (F and G), one germline arm is outlined. Arrowheads and arrows point at oocytes and early embryos, respectively. The outlined germline arm shows robust production of gametes (panel G). Scale bar, 50 µm.</p