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. 2024 Sep;31(9):1386-1393.
doi: 10.1038/s41594-024-01298-3. Epub 2024 May 7.

Target-based discovery of a broad-spectrum flukicide

Affiliations

Target-based discovery of a broad-spectrum flukicide

Daniel J Sprague et al. Nat Struct Mol Biol. 2024 Sep.

Abstract

Diseases caused by parasitic flatworms impart a considerable healthcare burden worldwide. Many of these diseases-for example, the parasitic blood fluke infection schistosomiasis-are treated with the drug praziquantel (PZQ). However, PZQ is ineffective against disease caused by liver flukes from the genus Fasciola because of a single amino acid change within the target of PZQ, a transient receptor potential ion channel in the melastatin family (TRPMPZQ), in Fasciola species. Here, we identify benzamidoquinazolinone analogs that are active against Fasciola TRPMPZQ. Structure-activity studies define an optimized ligand (BZQ) that caused protracted paralysis and tegumental damage to these liver flukes. BZQ also retained activity against Schistosoma mansoni comparable to PZQ and was active against TRPMPZQ orthologs in all profiled species of parasitic fluke. This broad-spectrum activity manifests as BZQ adopts a pose within the binding pocket of TRPMPZQ that is dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPMPZQ and a first-in-class, broad-spectrum flukicide.

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Conflict of interest statement

Competing Interests. JSM, DJS, LS, and TPS have pending patent applications for the compounds described in this study. The remaining authors declare no competing interests.

Figures

Extended Data Figure 1.
Extended Data Figure 1.. Dose-response curves of Different Northern Hemisphere Analogs.
Responses of Sm.TRPMPZQ to the molecules are shown in blue circles, Fh.TRPMPZQ in red circles, and control (untransfected cells) red diamonds. All data are presented as mean ± SEM. N = 3 biological replicates comprised of technical duplicates except for the following: untransfected cells, technical duplicates; compounds 4 and 7, Sm.TRPMPZQ and Fh.TRPMPZQ, n = 9 biological replicates comprised of technical duplicates and compound 9, Sm.TRPMPZQ and Fh.TRPMPZQ, n = 4 biological replicates comprised of technical duplicates.
Extended Data Figure 2.
Extended Data Figure 2.. Dose-response curves of Different Southern Hemisphere Analogs.
Responses of Sm.TRPMPZQ to the molecules are shown in blue circles, Fh.TRPMPZQ in red circles, and control (untransfected cells) red diamonds. All data are presented as mean ± SEM. N = 3 biological replicates comprised of technical duplicates except for the following: untransfected cells, technical duplicates; compound 27, Fh.TRPMPZQ, n = 4 biological replicates comprised of technical duplicates; compound 30, Sm.TRPMPZQ, n = 9 biological replicates comprised of technical duplicates.
Extended Data Figure 3.
Extended Data Figure 3.. Dose-response curves of Different Aromatic Core Analogs.
Responses of Sm.TRPMPZQ to the drugs are shown in blue circles, Fh.TRPMPZQ in red circles, and control (untransfected cells) red diamonds. All data are presented as mean ± SEM of n = 3 biological replicates comprised of technical duplicates except for untransfected cells which are presented as technical duplicates.
Extended Data Figure 4.
Extended Data Figure 4.. Effects of BZQ on F. hepatica after prolonged culture.
Tegumental damage in F. hepatica after extended treatment with BZQ. Immature liver flukes were treated for 72 h with BZQ (12.5 μM) or DMSO (0.5%) as control and ventral surfaces were imaged by scanning electron microscopy. Large collapsed blisters (arrows) were found after exposure (two identical replicates are shown). Scale bars = 500 μm (top row) and 10 μm (bottom row).
Extended Data Figure 5.
Extended Data Figure 5.. Effects of various benzamidoquinazolinones on F. hepatica.
(A-C) Motility of (A) adult, (B) triclabendazole (TCBZ)-sensitive immature, and (C) TCBZ-resistant immature F. hepatica after treatment with compound 1 (gold triangles) and compound 6 (purple triangles) compared with application of TCBZ (open circles) or DMSO (1.25%, open grey squares). Panel A: DMSO and both TCBZ, n = 3 flukes each; compounds 1 and 6, n = 4 flukes each. Panel B: DMSO and TCBZ, n = 4 flukes each; compound 1, n = 10 flukes; compound 6, n = 9 flukes. Panel C: DMSO, n = 6 flukes; TCBZ, n = 4 flukes; compound 1, n = 10 flukes; compound 6, n = 9 flukes; all data presented as mean ± SEM. (D-I) Tegumental damage in F. hepatica. Immature liver flukes were treated for 24 h with DMSO (control, D&E), Cmpd 1 (6.25 μM, F&G) or compound 6 (6.25 μM, H&I). 1 and 6 caused bleb formation (arrows) after exposure. Images are representative of three replicates. Scale bars = 500 μm (top row) and 10 μm (bottom row).
Figure 1.
Figure 1.. Functional profiling of TRPMPZQ orthologs.
Representative Ca2+ flux traces depicting the effect of (A, B) (±)-PZQ or (D, E) compound 1 in HEK293 cells stably expressing (A,D) Sm.TRPMPZQ or (B,E) Fh.TRPMPZQ. Cells were treated with increasing concentrations (0–30 μM) of each drug added after ~20 s of sampling the baseline fluorescence emission. (C&F) Concentration-response curves resulting from activation of Sm.TRPMPZQ (blue circles, n=6 biological replicates composed of technical duplicates) or Fh.TRPMPZQ (red circles, n = 6 biological replicates composed of technical duplicates) by (C) (±)-PZQ or (F) compound 1. Control responses in HEK293 cells lacking TRPMPZQ are shown (grey diamonds, n = 3 biological replicates composed of technical duplicates). (G) Schematic of modifiable regions on compound 1. Three regions on the N-benzamidoquinazolinone core were targeted for modification. The Northern Hemisphere (green), Southern Hemisphere (orange), and the aromatic core (pink). (H) Chemical structure of the optimized benzamidoquinazolinone, BZQ, after SAR studies. (I) Concentration-response curves for BZQ in HEK293 cells stably expressing Sm.TRPMPZQ (blue circles, n = 13 biological replicates comprised of technical duplicates) or Fh.TRPMPZQ (red circles, n = 23 biological replicates comprised of technical duplicates), compared to control responses (grey diamonds, n = 2 biological replicates comprised of technical duplicates). (J) Concentration-response curves for BZQ in cells transiently expressing various TRPMPZQ orthologs (all samples, n=3 biological replicates comprised of technical duplicates). These were: Schistosoma mansoni (Sm.TRPMPZQ, closed blue circles), Fasciola hepatica (Fh.TRPMPZQ, closed red circles), Schistosoma haematobium (Sh.TRPMPZQ, open purple squares), Schistosoma japonicum (Sj.TRPMPZQ, open green triangles), Fasciola gigantica (Fg.TRPMPZQ, open gold hexagons), Echinostoma caproni (Ec.TRPMPZQ, open grey diamonds), Clonorchis sinensis (Cs.TRPMPZQ), and Opisthorchis viverrini (Ov.TRPMPZQ, open black diamonds). All concentration-response curves were normalized to the maximum response observed at each channel and represent the mean ± SEM.
Figure 2.
Figure 2.. Electrophysiological analysis of BZQ action.
(A) Representative whole-cell current (pA) versus time (s) plot of Fh.TRPMPZQ expressing HEK293 cell perfused with different concentrations of compound 1 (1 nM to 100 μM) prior to addition of LaCl3 (1 nM to 10 mM). Extracellular solution: 140 mM NaCl, 5 mM glucose, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4 with NaOH. Intracellular solution: 130 mM CsF, 10 mM CsCl, 10 mM NaCl, 10 mM EGTA, 10 mM HEPES, pH 7.4 with NaOH. (B) Concentration response curves for BZQ and 1 from experiments such as shown in (A) recorded from Fh.TRPMPZQ (red) or Sm.TRPMPZQ (blue) expressing HEK293 cells. Data are shown as mean ± SEM, n = 6 individual whole-cell current recordings per curve, except for compound 1 on Sm.TRPMPZQ (n = 7 individual whole-cell current recordings). (C) Representative cell-attached recordings from Fh.TRPMPZQ or Sm.TRPMPZQ expressing HEK293 cells in the presence of (±)-PZQ (10 μM) or BZQ (10 μM) in the bath solution. Bath solution: 145 mM NaCl, 10 mM HEPES, 1 mM EGTA, pH 7.4. Pipette solution: 145 mM NaCl, 10 mM HEPES, 1 mM EGTA, pH 7.4. c, closed state. Holding voltage, 60mV. (D) Current-voltage plot for Sm.TRPMPZQ (blue) activated by BZQ (closed circle, 10 μM, n = 3 independent recordings from different cells) or (±)-PZQ (open circle, 10 μM, n = 3 independent recordings from different cells)) and Fh.TRPMPZQ activated by BZQ (red, 10 μM, n = 4 independent recordings from different cells). Data are shown as mean ± SEM. (E) Single channel open probability (Popen) of Fh.TRPMPZQ (red) or Sm.TRPMPZQ (blue) activated by BZQ or (±)-PZQ (each at 10 μM) in the bath solution. Data are shown as mean ± SEM, n = 6 indepent single-channel recordings for each group.
Figure 3.
Figure 3.. Effects of BZQ and (±)-PZQ on parasitic flukes.
(A) Exposure of S. mansoni and (B) F. hepatica to (±)-PZQ or BZQ compared with DMSO (1–1.25%, control). A rapid contraction of schistosomes to (±)-PZQ (0.5 μM) or BZQ (0.5 μM) was apparent. BZQ (6.25 μM), but not (±)-PZQ (50 μM), caused spastic paralysis of adult liver flukes. Panel A: representative images of n = 5 individual experiments per condition; Panel B: representative images of DMSO (n = 4 flukes), PZQ (n = 3 flukes), and BZQ (n = 4 flukes). (C-H) Studies of the ultrastructure of BZQ-treated flukes. Transmission electron microscopy of drug-induced damage to S. mansoni tegument (C) without treatment or (D) after treatment with BZQ (1 μM). Scanning electron microscopy of drug-induced damage to immature F. hepatica tegument after treatment with: (E&F) DMSO (1.25%, control) or (G&H) BZQ (6.25 μM, 24 h exposure). BZQ caused blebs to occur on the fluke surface (arrows). S. mansoni images are representative of 6 worms in each condition, with 6 images generated per worm. F. hepatica images are representative of: DMSO, n = 3 worms, 7 total images and BZQ, n= 3 worms, 17 total images. (I-K) Motility of (I) adult (n = 4 flukes per condition), (J) triclabendazole (TCBZ)-sensitive immature (DMSO and BZQ, n = 8 flukes; TCBZ, n = 4 flukes), and (K) TCBZ-resistant immature F. hepatica (DMSO, n = 5 flukes; BZQ, n = 6 flukes; TCBZ, n = 4 flukes) after treatment with BZQ (blue triangles) or triclabendazole (black/green circles) compared with application of DMSO (1.25%, control, grey squares). Motility scores are reported as the mean ± SEM. (L) Dose-response curve for motility of adult (triangles, n = 4 flukes) and immature (circles, n = 8 flukes) F. hepatica treated with BZQ. Data are reported as mean ± SEM. (M) BZQ activity in a murine model of schistosomiasis. Mice, infected with schistosomes, were treated at 7 weeks post-infection with either BZQ or (±)-PZQ. Mice were dosed once daily with each drug for three sequential days (50 mg/kg, intraperitoneally), and worm burden was evaluated on the fourth day after initiation of treatment. Worm burden was reduced by treatment with BZQ or (±)-PZQ compared with either untreated or vehicle-treated mice as described in the methods. N = 13 mice per group; data are shown as mean ± SD and analyzed using the two-tailed Mann-Whitney test. Individual significance is depicted in the figure. Scale bars for (A) = 250 μm, (B) = 1 mm; (C&D) = 1 μM, (E&G) = 500 μm, (F&H) = 10 μm.
Figure 4.
Figure 4.. TRPMPZQ engagement by BZQ.
In silico binding pose for (A&B) (R)-PZQ and (C&D) BZQ in Sm.TRPMPZQ. Concentration-response curves for (E) (±)-PZQ and (F) BZQ in specified Sm.TRPMPZQ mutants. WT = blue circles, Sm.TRPM[N1388]APZQ = orange circles, Sm.TRPM[T1389A]PZQ = green circles, Sm.TRPM[R1514A]PZQ = open purple circles, Sm.TRPM[Y1678A]PZQ = open pink circles. Curves are presented as mean ± SEM of (E) biological duplicates comprised of technical duplicates and (F) biological triplicates comprised of technical duplicates.

Update of

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