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Schistosoma mansoni lysine specific demethylase 1 (SmLSD1) is a druggable target involved in parasite survival, oviposition and stem cell proliferation

Schistosomiasis is a chronically-debilitating neglected tropical disease (NTD) that predominantly affects people living in resource-poor communities of tropical and subtropical countries. Schistosoma mansoni, one of three species responsible for most human infections, undergoes strict developmental regulation of gene expression that is carefully controlled by both genetic- and epigenetic- processes. As inhibition of S. mansoni epigenetic machinery components has been shown to impair key transitions throughout the parasite’s digenetic lifecycle, this knowledge is currently fuelling the search for new epi-drug - based anthelmintics. In this study, the anti-schistosomal activity of 39 re-purposed Homo sapiens Lysine Specific Demethylase 1 (HsLSD1) inhibitors was investigated on key life cycle stages associated with both definitive (schistosomula, juvenile worms, sexually-mature adults) and intermediate host (miracidia) infection. The most active compound (compound 33; e.g. schistosomula phenotype EC50 = 4.370 µM; adult worm motility EC50 = 2.137 µM) was subsequently used to provide further insight into the critical role of S. mansoni lysine specific demethylase 1 (SmLSD1) in adult worm oviposition and stem cell proliferation. Here, compound 33 treatment of adult schistosomes led to significant defects in egg production, intra-egg vitellocyte/ovum packaging and gonadal/neoblast stem cell proliferation. A greater abundance of H3K4me2 marks accompanied these phenotypes and supported specific inhibition of SmLSD1 in adult schistosomes by compound 33. In silico screening indicated that compound 33 likely inhibits SmLSD1 activity by covalently reacting with the FAD cofactor. This work suggests that evaluation of HsLSD1 - targeting epi-drugs could have utility in the search for next-generation anti-schistosomals. The ability of compound 33 to inhibit parasite survival, oviposition, H3K4me2 demethylation and stem cell proliferation warrants further investigations of this compound and its epigenetic target. This data further highlights the importance of histone methylation in S. mansoni lifecycle transitions. Author summary Affecting over 200 million people, schistosomiasis is a chronic disease caused by the parasitic worm Schistosoma mansoni. The frontline drug for schistosomiasis treatment is praziquantel. Owing to the concern surrounding praziquantel insensitivity or resistance developing, current research is directed towards the identification of novel drugs. We have focused our search for compounds that affect essential aspects of schistosome biology including parasite movement, fertility, cell proliferation and survival. Since all of these functions are potentially influenced by epigenetic regulation of gene expression, we investigated the activity of compounds that alter histone methylation status. In this report, we show that S. mansoni Lysine Specific Demethylase 1 (SmLSD1), a histone demethylase, is critical to miracidia-to-sporocyst transitioning, adult worm motility, egg production and parasite survival. Inhibition of SmLSD1 with compounds developed to inhibit the human paralog show promising potential as novel anti-schistosomal agents.


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In this regard, two anti-schistosomal drug discovery strategies stand out. One involves 87 the 're-purposing' of approved drugs for new indications [11,12]. A second strategy involves 88 the de novo design of drugs using either a ligand-or target-based molecular modelling 89 approach [13,14]. Considering that epigenetic pathways play an important role in regulating 90 schistosome phenotype [15], controlling development [16] and responding to environmental 91 stresses [17,18], pharmacologic inhibition of the key protein components of these epigenetic 92 regulators by re-purposed or de novo designed compounds clearly defines a promising control 93 strategy [11,15,19,20].

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Using both combinatorial chemistry and drug re-purposing, we and others have 95 pursued the investigation of protein methylation components as next generation drug targets 96 for schistosomiasis [19][20][21][22] due to growing evidence of their impact on schistosome 97 development and reproduction [23]. In the current investigation, we further explore the S.

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Discovered in 2004, HsLSD1 is a histone H3K4 mono-and di-methyl demethylase that 102 employs flavin adenine dinucleotide (FAD) as a cofactor [29]. LSD1 has been extensively 103 explored as a drug target due to an enzymatic activity associated with protein complexes 104 involved in diverse biological processes [28,30]. Indeed, dysregulation of LSD1 function has

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HsLSD1 inhibitors differentially affect schistosomula phenotype and motility 127 A small library of LSD1 inhibitors was acquired through commercial and collaborative 128 sources or from information derived from a recent large-scale RNAi investigation of S. mansoni 129 gene function [38] (Fig 1 and S1 Table). This collection included the only FDA-approved LSD1

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The selected compounds (10 µM final concentration) were initially co-cultured with 143 schistosomula for 72 h. At this concentration, each compound was screened at least three 144 times (biological replicates) and, in each screen, the effect of the compounds on 145 schistosomula phenotype and motility was assessed twice (technical replicates). These whole 146 organism assays were quantified using the in-house facility Roboworm [20,50,51]. For each 147 screen, the calculated Z´ scores for both phenotype and motility metrics were within 148 acceptable ranges (S2 Table) as previously described [52]. Upon screening (Fig 2), five 149 compounds (15, 16, 33, 35 and 36, in red) were always identified as hits on both metrics 150 (phenotype Fig 2A and motility Fig 2B) when compared to negative (0.625% DMSO) and 151 positive (10 µM Auranofin -AUR in 0.625% DMSO) controls [53].

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Reassuringly, and in line with previous studies [20], GSK-LSD1 (compound 2) failed to 153 affect either schistosomula motility or phenotype. Amongst the five hits, compounds 33, 35 154 and 36 appeared to be more potent than compounds 15 and 16 (i.e., schistosomula motility 155 and phenotype scores for the first three compounds were lower than the latter two, Fig 2). A 156 subsequent dose-response titration of these five compounds confirmed this observation (Z´ 157 scores for both phenotype and motility metrics of each screen summarised in S3 Table)

Five HsLSD1 inhibitors affect adult worm motility and IVLE production 163
The effect of anti-schistosomula compounds (15, 16, 33, 35 and 36) on adult male and 164 female schistosome pairs (7 weeks old) was next explored to expand their anti-schistosomal 165 applicability (Fig 3). Here, all compounds had a lethal effect (i.e., absence of parasite motility 166 and gut peristalsis for 30 seconds associated with parasite detachment) on the parasite at the 167 highest concentrations tested (50 and 25 µM, Fig 3A). A similar observation was recorded for 168 all compounds at 12.50 µM; the only exception being compound 16, which severely inhibited 169 parasite motility, but did not lead to lethality. Upon further adult worm titrations, and consistent 170 with the schistosomula screens, compound 33 displayed the greatest activity and inhibited 171 schistosome motility at concentrations as low as 6.25 µM. At 3.13 μM, the effect of all 172 compounds was minimal except for compound 33 (S1 Movie); at lower concentrations, the 173 treated worms started recovering when compared to the control (Fig 3A).

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In vitro laid egg (IVLE) production was also affected by co-cultivation with all five 175 compounds (Fig. 3B). Unsurprisingly, based on motility readouts (Fig 3A), no eggs were  191 inhibitors of this study (Fig 4 and S2 Movie).

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Particularly, eggs derived from schistosome cultures co-incubated with sub-lethal 193 concentrations of this chemical (3.13 µM, which did not significantly reduce worm motility - Fig   194  3A) were analysed using confocal microscopy and compared to IVLE derived from DMSO-195 treated worms. Even though there were no evident phenotypic abnormalities (lateral spine and 196 oval shape were both present) in the IVLEs analysed (Fig 4A), a significant difference in 197 overall egg volume was observed in compound 33 treated worms (Fig 4B). Moreover,

Compound 33 inhibits adult worm stem cell proliferation and SmLSD1 activity 202
Due to a previous role ascribed for LSD1 in maintaining mammalian stem cell function

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EdU labelling was performed on both male (Fig 5 and S3 Movie) and female (S3 Fig and S4 206 Movie) worms treated with a sublethal concentration (3.13 µM) of compound 33 for three days.

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For each worm (n = 6), three different anatomical regions (anterior region, gonadal showed an average 56% reduction in the relative ratio of H3/H3K4me2 marks (compared to 220 the DMSO treated control) indicating specific inhibition of LSD1 activity (i.e., accumulation of 221 H3K4me2 in the drug-treated worms (Fig 5E)).

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Compound 33 reduces juvenile worm viability and miracidia transformation 224 The activity of compound 33 was further explored on two other important S. mansoni 225 life cycle stages, the immature juvenile worms and the snail-infective miracidia. Firstly, juvenile

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(3 weeks old) schistosomes were subjected to dose response titrations of this compound for 227 72 h, after which both parasite motility and viability was assessed (Fig 6). 228 At the highest concentration tested (20 µM), compound 33 significantly reduced 229 parasite movement when compared to the negative (DMSO) control (Fig 6A). A similar 230 observation was recorded for AUR-treated parasites (at 15 µM). Furthermore, when visualised 231 for propidium iodide (PI) uptake at 536 nm (Figs 6B and 6C), both treatments were associated 232 with increased fluorescence, providing confirmation of juvenile worm death. In line with other 233 reports [6, 60, 61], PZQ only showed partial activity on juvenile worms (Fig 6A), and as 234 confirmed by a lower uptake of PI (Fig 6B), these parasites where not all dead. At lower 235 concentrations of compound 33 (10, 5 and 2.50 µM), reductions in both motility ( Fig 6A) and 236 viability (Fig 6C) were still noted when compared to the DMSO control. However, at lower 237 concentrations (1.25 µM and 0.63 µM), juveniles started recovering normal movement and 238 mortality was reduced.

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Although drug screens were mainly focused on intra-mammalian parasitic stages, we 240 were also interested in whether the most potent LSD1 inhibitor affected schistosome 241 developmental forms that interact with the intermediate molluscan host [62]. Hence, free-242 swimming miracidia were exposed to compound 33 (in a dose response titration) for 48 h and 243 in vitro transformation into sporocysts was assessed (Fig 7).

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Upon titration, a significant inhibition in miracidia to sporocyst transformation was 245 found for parasites treated with 50 -5 µM of compound 33. In fact, no movement or flame cell 246 activity (related to parasite death) was observed at 50, 25 and 10 µM concentrations. At 5 µM, compound docking were performed (Fig 8).

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As previously published [19], the homology model of the parasite enzyme was 285 generated using HsLSD1 (PDB entry: 2V1D, Fig 8A) [67]. In order to predict the binding mode was embedded between two residues (Tyr1053 and Thr1121, Fig 8C). These two residues 296 together define the aromatic cage, which has been observed in other amine oxidase enzymes 297 (including LSD1) and contributes to the active site hydrophobic shielding from the influx of 298 external solvents [68,69]. In addition to this, we also observed the orientation of the compound 299 towards another conserved residue, the invariant lysine (Lys889), which has been investigated 300 for its role in catalysis as well as in proton transfer (acting as an active-site base of LSD1) [70,  (Fig 3), the most active anti-schistosomula compound (in this case compound 319 33) also demonstrated the strongest effects (decreased worm motility and IVLE production; between 400 and 600 g/mol, but variable lipophilicities. These two properties do not 327 adequately explain differences in activity among the five most potent compounds or between 328 them and the remaining less active ones.

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The presence a para-fluorophenyl substitution on the cyclopropyl ring of the 330 tranylcypromine derivatives was identified as a common feature of the five most active 331 compounds (S4 Table). Considering the mechanism of action of these compounds, they are 332 all capable of forming a covalent adduct with FAD that contains the fluorophenyl ring. Though 333 the para-fluro substitution may be important for potency, this structural feature cannot explain 334 the differences in activity among the five compounds. However, for a more detailed structure-  Table).

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in adult worms and the viability assays of other synthetic LSD1 inhibitors [19,21]. In addition 346 to an egg laying deficit, compound 33 significantly reduced the overall volume of IVLEs as well 347 as the packaging of fertilised ova/vitellocytes into them (Fig 4 and S2 Fig) compound 33 treated males (Fig 5E). In support of this contention, it was previously 356 demonstrated that global accumulation of H3K4me2 in spr 5 (lsd1 homologue) C. elegans null 357 mutants led to progressive sterility in progeny due to dis-regulation of spermatogonia-358 associated genes [75]. When taken together, our data supports previous observations [16] 359 and highlights an essential role for post-translational histone modifications in the control of 360 oviposition.

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In addition to affecting schistosomula and adult worm phenotypes, we also 362 demonstrated that compound 33 markedly reduced the transformation of miracidia into 363 germinal cell-enriched sporocysts (Fig 7)

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Similar to its effect on germinal stem cells, compound 33 also inhibited adult neoblast 381 proliferation in both sexes (Fig 5 and S3 Fig)

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Ligand preparation, protein preparation and molecular docking 417 From the chemical structure of compound 33 (an N-alkylated tranylcypromine 418 derivative) and the cofactor FAD (S5A Fig and S5B Fig, respectively), the covalent adduct

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The obtained N5 adduct was saved in a sdf format prior to processing by the Lig Prep

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For sample normalisation, the membranes were stripped by incubation at 50°C for 1 h 603 in 62 mM TRIS-HCl pH 6.8, 2% SDS, 0.8% Beta-Mercaptoethanol, then washed with distilled 604 water several times until complete disappearance of the Beta-Mercaptoethanol smell. The

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Lot GR103803-1), 1:1000 diluted in 5% non-fat dried milk in TBST. Detection of H3 signal by 607 secondary antibody, image acquisition and analysis were all performed as described above.

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Statistics 610 All statistical analyses were performed using a nonparametric Student's t-test (Mann-

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Whitney test, two samples) or a two-way ANOVA followed by Least Significant Difference 612 post-hoc correction (more than two samples).   Table S2.     Table. 760 761 S2 Figure

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Confocal microscopy, quantification and data visualisation was performed similarly to Fig 5. 777 Standard errors are shown and Mann-Whitney t-test (with **** corresponding to p < 0.0001)

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was subsequently performed to explore a statistical difference between the treatments.

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In these series of optical sections, three different anatomical regions (anterior region, gonadal

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Comparison between the negative control (DMSO, first row) and the drug treated (compound 832 33, 3.13 µM, second row) parasites is provided.