Fig 1.
Identification of a diaminothienopyrimidine series from an ex vivo T. muris motility screen.
(a) Structure of the hit compound, which was given the identifier OX02926. (b) Hit expansion by testing of structurally-related compounds using library material, assay concentration 100μM. Significance was determined by a two-sided Mann-Whitney test compared to DMSO-only controls, adjusted for multiple comparisons using the Bonferroni method (for test compounds n = 5, each replicate on different assay plates, each point indicates one assay well). Blue bar indicates mean movement score. (c) Structures and identifiers of additional active compounds from this class. (d) Structures and PubChem CID accession numbers for the two compounds that were not significantly active in this assay.
Fig 2.
Synthetic route to putative hit compounds.
(a) Substituted 2-phenoxyethan-1-amine (1.0 equiv.), DIPEA (2.0 equiv.), 1,4-dioxane, 80°C, 3 hours. (b) Alkyl amine (10.0 equiv.), iPrOH, 100°C, 16–24 hours.
Fig 3.
Concentration-response curves for resynthesized DATPs in the T. muris ex vivo adult motility assay.
n = 4 or 5 wells per concentration per compound, each replicate on a different 96-well plate using worms from different mice. Blue line indicates concentration-response curve fitted with the 3-factor log-logistic model using drc [16]. Figure in parenthesis indicates EC50 estimate ± standard error from this model. OX03143 did not clearly form a sigmoidal curve in the range of concentrations used in this assay so we report the EC50 estimate as > 80μM.
Fig 4.
Properties and activities of resynthesized diaminothienopyrimidines, and other anthelmintics.
RMM: relative molecule mass. HBA: number of hydrogen bond acceptors. HBD: number of hydrogen bond donors. tPSA: topological polar surface area, calculated using DataWarrior [20]. ROTB: number of rotatable bonds. a Data from [15]. b Data from [21]. c Data from [12].
Fig 5.
Concentration-response curves for resynthesized DATPs in the C. elegans growth assay.
n = 5 wells per concentration per compound, each replicate on a different 96-well plate. Blue line indicates concentration-response curve fitted with the 4-factor log-logistic model using drc [16]. Figure in parenthesis indicates EC50 estimate ± standard error from this model.
Table 1.
Summary of the cytotoxicity in a mouse epithelial cell line of the DATP series.
Mouse CMT-93 rectal epithelial cells were used for this assay. Maximum tested concentration was 100 μM. n = 8, error range (in parentheses) shows 95% confidence interval. EC50 values in the adult Trichuris paralysis assay are shown for comparison.
Fig 6.
Reduced worm burden in mice given T. muris eggs that had been treated with diaminothienopyrimidines.
(a) Embryonated eggs were soaked in compound for 14 days, washed in water and then used in either in vitro or in vivo hatching assays. (b) Treatment with DATPs reduced the ability of embryonated eggs to hatch in E. coli bacterial suspension after 24 hours. A one-way ANOVA showed a significant difference between treatment groups (F(5,26) = 25.95 p<0.0001) with a post-hoc Dunnett’s compared to DMSO control (**** = p<0.0001) n = 7 (DMSO), n = 5 (DATP compounds) (c) SCID mice were infected with 40 eggs and worm burden assessed at day 15 post infection. The experiment was carried out in two batches, with n = 5 and n = 9 mice respectively in each of the control and treatment groups. Data were normalised for each batch relative to the mean of the DMSO-only control group for that batch. Blue line indicates mean for each treatment group. A two-way ANOVA showed a significant effect of treatment [F(1,24) = 9.569, P = 0.00497] but no effect of batch [F(1,24) = 0.083, P = 0.77618] or interaction [F(1,24) = 0.083 0.77618]. A post-hoc Tukey HSD test showed that the OX02926-treated group was significantly different from the DMSO control group (P = 0.0050).
Fig 7.
Unembryonated T. muris eggs treated with diaminothienopyrimidines have altered embryonation.
Unembyronated eggs were soaked in 100 μM compound (unless specified otherwise) at 26°C (unless specified otherwise) for the duration of the embryonation process (56–60 days) and then embryonation determined and eggs imaged using an Olympus BX63 microscope. Scale bar indicates 10 μm. (a) Typical embryonated egg and (b) unembryonated egg. (c) treatment with DATPs increased the incidence of unembryonated eggs. Representative pictures of (d) DMSO, (e) OX02925, (f) OX02926, (g) OX03143 and (h) OX03147 100 μM and (i) OX03147 1 μM soaked T. muris eggs. (j) Unembyronated eggs soaked in OX03147 at room temperature for 56 days and embryonation determined. (k) Unembyronated eggs soaked in OX03147 at 26°C for 56 days and larval length calculated using ImageJ.