Fig 1.
Schematic showing possible treatment strategies: post-infection treatment and breaking the Trichuris life cycle in the environment.
Trichuris 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 in vivo while worms are at larval and adult stages.
Fig 2.
Hit compounds identified in a screen for molecules that paralyse whole T. muris parasites.
(A) Movie frames showing complete worm paralysis. A montage of frames for control (top row wells) and hit compound treated (bottom row wells) is displayed; each frame is 4 seconds apart (B) Volcano plot of primary screen (n = 4, Mann-Whitney-Wilcoxon test, compounds tested at 100μM) showing significant reduction in worm movement for 20 candidate compounds. (C) Of the 20 hit compounds from the primary screen that were selected for further study, 14 were active in the secondary screen (P < 0.05, Mann-Whitney-Wilcoxon test, corrected for multiple comparisons by Holm’s method. *** = P < 0.005, ** = P < 0.01, * = P < 0.05.) (D) 13 of the active compounds contain the same core dihydrobenzoxazepinone group. R1 = Aryl or Cycloalkyl. R2 = Aryl or Heteraryl.
Fig 3.
Elaboration of initial active compounds by characterising a diverse set of 192 dihydrobenzoxazepinones with the same core structure.
(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 100μM. (C) Relationship between activity and predicted hydrophobicity (cLogP) of each dihydrobenzoxazepinone.
Fig 4.
Synthetic route to putative hit compounds.
(A) i. R1CHO (1.5 equiv.), AcOH (0.5 equiv.), NaBH(OAc)3, CH2Cl2, 0°C to RT, 2 days; ii. LiAlH4 (1M in THF, 3.5 equiv.); iii. chloroacetyl chloride (2.0–4.0 equiv.), NEt3 (2.0–4.4 equiv.), THF, 0°C to RT, 16 h; iv. 10N NaOH (aq.), RT, 2 h; v. R2B(OH)2 (1.3 equiv.), Pd(PPh3)4 (5 mol%), 1.5M NaHCO3 (aq.) (3 equiv.), DMF, 150 oC (m.w.), 15 min. (B) Structures of resynthesized compounds.
Table 1.
Structures of active dihydrobenzoxazepinones that were resynthesized, with selected calculated properties.
RMM: relative molecular mass. #HBA: number of hydrogen bond acceptors. #HBD: number of hydrogen bond donors. tPSA: topological polar surface area. (Calculated using DataWarrior [28]).
Fig 5.
Resynthesized dihydrobenzoxazepinones show dose-dependent attenuation of ex vivo T. muris adult motility.
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.
Table 2.
Dihydrobenzoxazepinones cause limited cytotoxicity against mouse epithelial cell lines demonstrating selectivity for the pathogen target.
Mouse CMT-93 rectal epithelial cells were used for this assay. Maximum tested concentration was 100μM. n = 8, error range (in parentheses) shows 95% confidence interval. EC50 values in the adult Trichuris paralysis assay (Fig 5) are shown for comparison.
Fig 6.
T. muris eggs treated with dihydrobenzoxazepinone OX02983 are less infective in vivo.
(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 in vivo. 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.