Structures of active dihydrobenzoxazepinones that were resynthesized, with selected calculated properties.

<p>RMM: relative molecular mass. #HBA: number of hydrogen bond acceptors. #HBD: number of hydrogen bond donors. tPSA: topological polar surface area. (Calculated using DataWarrior [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005359#pntd.0005359.ref028" target="_blank">28</a>]).</p

<i>T</i>. <i>muris</i> eggs treated with dihydrobenzoxazepinone OX02983 are less infective <i>in vivo</i>.

<p>(A) Experimental scheme: embryonated eggs were soaked in compound, and then used to infect mice by oral gavage. At day 15 post-infection, mice were culled and worm burden assessed. (B) Treatment with OX02983 reduced the ability of embryonated eggs to establish infection <i>in vivo</i>. Blue bar indicates median worm burden. A one-way ANOVA (worms ~ treatment) showed a significant difference between treatment groups (F(3,48) = 8.3, P< 0.0005). Differences between groups were determined using a post-hoc Tukey HSD test (** = P<0.005, *** = P<0.0001). n = 14 (DMSO), 16 (water), 10 (OX02983), 12 (OX03153). (C) Dihydrobenzoxazepinones do not act by blocking embryonation. Eggs were incubated with 100μM compounds or DMSO-alone control for 56 days. Embryonation was then quantified. No significant differences between groups were detected: one-way ANOVA (embryonation ~ treatment, F(2,12) = 0.60, P = 0.57). Blue bar indicates median percentage embryonation.</p

Resynthesized dihydrobenzoxazepinones show dose-dependent attenuation of <i>ex vivo T</i>. <i>muris</i> adult motility.

<p>Single adult worms were incubated for 24 hours in wells containing media plus compound. Motility was quantified using an automated phenotyping platform. EC₅₀ ± standard error shown in parentheses. n = 5, except OX03144 where n = 10. Curve fitted using the four parameter log-logistic model.</p

Schematic showing possible treatment strategies: post-infection treatment and breaking the <i>Trichuris</i> life cycle in the environment.

<p><i>Trichuris</i> infection could be prevented by using novel drugs to 1) target the prevention of egg embryonation in the external environment, 2) reduce the infectivity of embryonated eggs prior to ingestion, or 3) treat existing infections <i>in vivo</i> while worms are at larval and adult stages.</p

Elaboration of initial active compounds by characterising a diverse set of 192 dihydrobenzoxazepinones with the same core structure.

<p>(A) Screening and elaboration procedure. (B) Relationship between activity and molecular weight of each dihydrobenzoxazepinone. Activity is the minus log₂ movement reduction compared to DMSO-only controls when assayed at 100<b>μ</b>M. (C) Relationship between activity and predicted hydrophobicity (cLogP) of each dihydrobenzoxazepinone.</p

Identification of a diaminothienopyrimidine series from an <i>ex vivo T</i>. <i>muris</i> motility screen.

<p><b>(a)</b> Structure of the hit compound, which was given the identifier <b>OX02926</b>. <b>(b)</b> Hit expansion by testing of structurally-related compounds using library material, assay concentration 100<b>μ</b>M. 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. <b>(c)</b> Structures and identifiers of additional active compounds from this class. <b>(d)</b> Structures and PubChem CID accession numbers for the two compounds that were not significantly active in this assay.</p

Unembryonated <i>T</i>. <i>muris</i> eggs treated with diaminothienopyrimidines have altered embryonation.

<p>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. <b>(a)</b> Typical embryonated egg and <b>(b)</b> unembryonated egg. <b>(c)</b> treatment with DATPs increased the incidence of unembryonated eggs. Representative pictures of <b>(d)</b> DMSO, <b>(e) OX02925</b>, <b>(f) OX02926</b>, <b>(g) OX03143</b> and <b>(h) OX03147</b> 100 μM and <b>(i) OX03147</b> 1 μM soaked <i>T</i>. <i>muris</i> eggs. <b>(j)</b> Unembyronated eggs soaked in <b>OX03147</b> at room temperature for 56 days and embryonation determined. <b>(k)</b> Unembyronated eggs soaked in <b>OX03147</b> at 26°C for 56 days and larval length calculated using ImageJ.</p

Properties and activities of resynthesized diaminothienopyrimidines, and other anthelmintics.

<p>RMM: relative molecule mass. HBA: number of hydrogen bond acceptors. HBD: number of hydrogen bond donors. tPSA: topological polar surface area, calculated using DataWarrior [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006487#pntd.0006487.ref020" target="_blank">20</a>]. ROTB: number of rotatable bonds. ^a Data from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006487#pntd.0006487.ref015" target="_blank">15</a>]. ^b Data from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006487#pntd.0006487.ref021" target="_blank">21</a>]. ^c Data from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006487#pntd.0006487.ref012" target="_blank">12</a>].</p

Summary of the cytotoxicity in a mouse epithelial cell line of the DATP series.

<p>Mouse CMT-93 rectal epithelial cells were used for this assay. Maximum tested concentration was 100 μM. <i>n =</i> 8, error range (in parentheses) shows 95% confidence interval. EC₅₀ values in the adult <i>Trichuris</i> paralysis assay are shown for comparison.</p

Reduced worm burden in mice given <i>T</i>. <i>muris</i> eggs that had been treated with diaminothienopyrimidines.

<p><b>(a)</b> Embryonated eggs were soaked in compound for 14 days, washed in water and then used in either <i>in vitro</i> or <i>in vivo</i> hatching assays. <b>(b)</b> Treatment with DATPs reduced the ability of embryonated eggs to hatch in <i>E</i>. <i>coli</i> 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) <i>n =</i> 7 (DMSO), <i>n =</i> 5 (DATP compounds) <b>(c)</b> 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 <b>OX02926</b>-treated group was significantly different from the DMSO control group (P = 0.0050).</p