Extended data This project contains the following extended data: - Fig S1. Dose response titrations of compounds 15, 16, 33, 35 and 36 against schistosomula. - Fig S2. Compound 33 treatment induces the release of oocytes, spermatozoa and vitelline cells from adult worms. - Fig S3. Fast Blue BB stain (orange-red labelling) showing loss of mature vitellocytes in compound 33-treated female worms compared to the control ones. - Fig S4. Compound 33 treatment reduces stem cell proliferation in adult male worms. - Fig S5. SmLSD1 inhibition causes changes in chromatin structure in S. mansoni male adult worms. - Fig S6. Visualisation of ATAC-seq samples. - Fig S7. Computational preparation of the covalent adduct derived from the interaction of compound 33 with the FAD cofactor. - Fig S8. Chemical space covered by the library of 39 HsLSD1 inhibitors. - Fig S9. Compound 33 treatment inhibits H3K4me2 demethylation in adult male worms. Fig S1. Dose response titrations of compounds 15, 16, 33, 35 and 36 against schistosomula. The five hit compounds were screened against mechanically-transformed schistosomula at 10 µM and lower concentrations (5, 2.50, 1.25 and 0.625 µM). Three independent dose response titrations were performed and each compound concentration was evaluated in duplicate. Each concentration point defines the average mean of the three biological replicates (each of them with two technical replicates). Dose response curves for S. mansoni schistosomula phenotype (P) are presented here using GraphPad Prism (mean +/- SE of mean is indicated for each compound concentration). Estimated EC50s (and corresponding 95% confidence interval) calculated from these dose response curves are summarised in the table underneath. Z´ scores for motility and phenotype for the three screens are reported in Table S3. Fig S2. Compound 33 treatment induces the release of oocytes, spermatozoa and vitelline cells from adult worms. After 72 h incubation, representative images of eggs, oocytes (oc), spermatozoa (sp), mature spermatozoa (ms) and vitelline cells (vc) in tissue culture medium of worm pairs treated with DMSO (A and C) and a sublethal dose of compound 33 (3.13 μM, panels B, D, E and F) were taken. Images were acquired with Olympus microscope (4x for panels A and B), 10x for panels C and D and 20x for panels E and F)). Fig S3. Fast Blue BB stain (orange-red labelling) showing loss of mature vitellocytes in compound 33-treated female worms compared to the control ones. Two representative full-body images of a compound 33-treated female worm vs an untreated female. Fig S4. Compound 33 treatment reduces stem cell proliferation in adult male worms. Male schistosomes were treated with 3.13 μM of compound 33 (n = 6) or DMSO (n = 6) for 48 h and then were labelled with EdU for an additional 24 h. (A) – Schematic of a S. mansoni adult male with representative anterior region (yellow box), gonadal (magenta) and posterior region (brown) of untreated (top row, grey) compared to the compound-treated (bottom row, light brown) worms. Fluorescent microscopic images (6 males per treatment) were acquired on a Leica TCS SP8 super resolution laser confocal microscope fitted with a 40X objective (water immersion, 1,00 zoom factor, Z stack of 60 steps) using the Leica Application Suite X. DAPI stain = blue; EdU+ cells = green. Scale bar represents either 1 mm or 40 μm. Scatter plots illustrate the percentage of proliferative stem cells present in control (DMSO treated worms, n = 6) versus compound 33-treated males (3.13 µM of compound 33, n = 6) in the head region (B), the testes (C) and the tail region (D). Standard errors are shown and a Mann-Whitney test (with ** corresponding to p < 0.0021) was subsequently performed to quantify statistical significance between treatments. Fig S5. SmLSD1 inhibition causes changes in chromatin structure in S. mansoni male adult worms. (A) - Combined metagene ATAC profiles derived from compound 33 – (green) and mock (DMSO, blue) treated S. mansoni adult male worm libraries. The bold lines represent average values (from the 12 replicates), with the standard errors in shades (light green for compound 33, light blue for DMSO, light grey for the overlapping area of the first two). X-axis in kilobase (kb). TSS = Transcription start site, TES = transcription end site. −2.0 represents 2 kb upstream of the TSS, and 2.0 represents 2 kb downstream of the TES. Y-axis represents the averaged intensity value of the ATAC-Seq enrichment, which is based on the number of aligned reads but also the average distance between reverse and forward reads that were converted into ‘peak scores’ by MACS and a background correction with Poisson Pvalue (-log10(pvalue) using control as lambda and treatment as observation. (B) - Pie chart illustration of genomic distribution for the ATAC-seq peaks differentially found between mock and compound treated male worms. The percentage of ATAC-seq peaks is provided for each genomic feature: exonic (1st exon or other exons), intronic (1st intron or other introns), distal intergenic (regions located between genes - more than 300 bp downstream the end gene and more than 3 kb upstream, promoter region, downstream region (up to 300 bp downstream the end gene). (C) - Heatmap of differentially accessible ATAC-seq peaks (p-value < 0.05) in adult male worms derived from DMSO- (n = 12) versus compound 33-treated male worms (n = 12). Clustering method: average linkage; Distance Measurement Method: Manhattan; clustering applied to rows and columns. Heatmap represents row-based z-scores of DESeq2 normalized Tn5 insertion counts for each differentially accessible ATAC-seq peak. Every line represents a peak, 2,107 peaks in total (peaks were not labelled for readability reasons). High (ATAC-seq up) and low (ATAC-seq down) chromatin accessibility is indicated in yellow and blue, respectively. Fig S6. Visualisation of ATAC-seq samples. Representative IGV screenshot of the DMSO (depicted in purple) and compound 33 (depicted in pink) ATAC-seq peaks derived from the female libraries. Differential ATAC-seq peaks tracks are represented with solid dark red boxes. (A) - Snapshot of the IGV browser showing smp_312640 (Locus: SM_V7_4:10754393-10755036), which showed an ATAC-seq positive signal (accessible chromatin) found in the compound 33-treated female samples. This gene was found differentially expressed in the late female germ cell cluster. (B) - Snapshot of the IGV browser showing smp_200410 (Locus: SM_V7_1:31057921-31059724), which showed an ATAC-seq positive signal (accessible chromatin) found in the DMSO female samples. This gene was found differentially expressed in the GSC cluster. Fig S7. Computational preparation of the covalent adduct derived from the interaction of compound 33 with the FAD cofactor. (A) - Chemical structure of compound 33. (B) - Stick representation of the chemical structure of the cofactor FAD. (C) - Stick representation of the covalent adduct of compound 33 with the flavin ring of the cofactor. Fig S8. Chemical space covered by the library of 39 HsLSD1 inhibitors. The scatter plot shows the distribution of the calculated logP (cLogP) vs the molecular weight (MW, g/mol) of the 39 compounds under study in this investigation. Each compound is shown as orange dot where the five most active compounds are shown in green. The reference compound (Tranylcypromine, compound 1) of this family of LSD1 inhibitors and the two more closely related derivatives (GSK-LSD1 and ORY-1001 - compounds 2 and 3, respectively) are labelled for comparison to the five most active anti-schistosomal compounds presented in this study (in green). The anti-schistosomal controls used in the ex vivo assays (praziquantel and auranofin) are also indicated in purple. Fig S9. Compound 33 treatment inhibits H3K4me2 demethylation in adult male worms. Visualisation of H3/H3K4me2 marks in adult male worm histone extracts (derived from 20 individuals, three biological replicates) after 72 h incubation with 3.13 µM of compound 33. Western blots of each biological replicate are reported here showing the H3 (loading controls) and H3K4me2 abundances of each experimental replicate.
|Dyddiad y'i gwnaethpwyd ar gael||05 Maw 2023|