Non-covalent inhibitors of thioredoxin glutathione reductase with schistosomicidal activity in vivo

Abstract

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms.

Fig. 1. From small molecules fragments bound to the doorstop pocket to the inhibitors designed in this study.

a A simplified picture of the enzymatic mechanism of TGR is shown (for a comprehensive TGR mechanism see refs. ^, ). In the reductive half-reaction 2 eq of NADPH are consumed to produce the EH₄ species, the 4-electron state, the one competent for substrate reduction in the oxidized half-reaction. After the initial reduction of the oxidized enzyme (E_ox) to EH₂, the 2-electron reduced state, TGR oscillates between EH₂ and EH₄ during turn-over. b TGR homodimer is shown in cartoons and each subunit is differently colored. The FAD cofactor is in green sticks. The doorstop pocket adjacent to the NADPH binding site is shown as a pink surface in one subunit. c A magnification of the doorstop pocket with representative bound fragments identified by X-ray crystallography (PDB ID 6FTC – magenta and PDB ID 6FP4 – cyan and yellow models); the molecular surface of the doorstop pocket is colored according to its hydrophobic features (green = hydrophobic; magenta = hydrophilic). Subpockets A-C are outlined in different colors and for each fragment the PDB ID is reported. d The TGR inhibitors designed in this study. e Gameplan non-polar hypotheses (magenta sticks) generated for chimera molecule (green) built using the X-ray fragments found in subpockets A-C in PDB ID 6FP4 and PBD ID 6FTC and connected by a short CH₂CH₂ linker to facilitate calculations and analysis. The binding site surface is colored with VIDA hydrophobicity palette, brown is hydrophobic, and blue is hydrophilic. f SZMAP grid results for TGR-chimera (gray sticks) complex processed with WaterOrientation VIDA extension show the most probable probe positions (polar substituents - yellow bubbles, non-polar substituents - purple bubbles) and the corresponding free energy values and the location of the water molecules (red bubbles) found in the X-ray structures of TGR.

Fig. 2. Inhibition of TGR in newly transformed schistosomula (NTS) visualized with TRFS-Green.

Representative images from two independent experiments in bright field (BF), green fluorescent protein filter (GFP, λ_EX = 438 nm, λ_EM = 538 nm), and overlay (OVL) of newly transformed schistosomula (NTS) after 2 hr culture with inhibitor (AF @ 3 µM, other compounds @ 30 µM) followed by addition of TRFS-Green (10 µM) for 4 hr. Source data are provided as a Source Data file. Scale bar = 200 µm.

Fig. 3. Functional characterization of the inhibitors.

a Reversibility of TGR inhibitors. Activity (5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) reduction) of the enzyme (3.7 nM) incubated with 250 µM inhibitor for 15 min was determined (red bars). TGR (370 nM) was incubated with 250 µM compound and 100 µM NADPH for 15 min. The sample was diluted 100-fold and the activity was determined immediately (blue bars) and after 60 min (light grey bars). AF = auranofin. Average ± standard deviation (n = 3) shown. b NADPH dependence of inhibitors. Activity of TGR after exposure to 6 (green circles), 7 (blue squares), or 8 (black triangles) with or without initial incubation with NADPH compared to control TGR incubated with NADPH and without inhibitor. Average ± standard deviation (n = 3) shown. c Time dependence of inhibition. Inhibition of DTNB reduction by TGR in the presence of 50 µM fast inhibitors 6 (orange), 7 (blue), 8 (black) compared to TGR incubated without compound. Average ± standard deviation (n = 3) shown. d Time dependence of inhibition. Time-dependent activity (DTNB reduction) of TGR in the presence of 50 µM slow inhibitors 1 (purple), 3 (orange), 4 (black), 5 (blue) compared to TGR activity incubated in the absence of compounds. Average ± standard deviation (n = 3) shown. e Compound effect on thermal stability of TGR. Melting temperature without (black) or with addition of 500 µM NADPH (red) of TGR alone or with 100 µM inhibitor 1, 3, 5, 7, 8, 9 or control compounds 11 or 12. Average ± standard deviation (n = 3) shown. f Oxidase activity after incubation with inhibitors. Consumption of NADPH (ΔA₃₄₀/min) by TGR after exposure to inhibitors for 15 min in presence of NADPH. Average ± standard deviation (n = 3) shown except control n = 12. g Production of superoxide by TGR after incubation with inhibitors. Superoxide production was determined by measuring consumption of pyrogallol red (ΔA₅₄₀/min) without added superoxide dismutase (blue circles) and with added superoxide dismutase (black squares) Average ± standard deviation, n = 3 except Auranofin n = 2, TRi-1 with SOD, n = 2, compound 4 without SOD, n = 2, and compound 6 with SOD, n = 2. Source data are provided as a Source Data file.

Fig. 4. Cryo-EM structure of the TGR-9 complex.

a Refined cryo-EM map of the TGR-9 complex at 3.6 Å and its superposition with the resulting PDB model. The additional density ascribed to compound 9 and to the FAD are shown as pink and green continued surfaces, respectively. b Magnification of the doorstop pocket with 9 in two orientations (conformation 1 in magenta sticks; conformation 2 in pink sticks); the subpockets A-C are indicated as well as FAD shown as green sticks. c Interactions of the two conformers of compound 9 with surrounding amino acids.

Fig. 5. Proposed mechanism of inhibition for the compounds described in this study.

a structural superposition between TGR in complex with 9 (in grey, PDB ID 8A1R) and TGR in complex with NADPH (in cyan, PDB ID 2 × 99). b structural superposition between TGR in complex with 9 and human GR in complex with NADP⁺ (in yellow, PDB ID 3DK4). In both panels the FAD cofactors belonging to the different enzymes are in green sticks, while the two conformers of 9 are in pink and in magenta sticks. c hypothetical mechanism of inhibition for the noncompetitive (9) and uncompetitive (3, 7, and 8) inhibitors, considering the possibility that the destabilized NADP⁺-TGR(H) reduced forms of the enzyme may specifically accommodate uncompetitive inhibitors due to the presence of conformational states more suitable for compound binding. EH₂ and EH₄ represent the reduced species with 2 and 4 electrons, respectively, populated during the enzymatic reduced half-reaction; EH₄ is the species competent for substrate reduction in the oxidative half-reaction. During catalysis, TGR oscillates between the 2-electron reduced state and the 4-electron state. The mechanism has been depicted considering the catalytic mechanism of TGR^,.

Fig. 6. Schistosomicidal efficacy in mice.

a Adult worm and liver egg burdens after compound treatments targeting adult worms 42 days after infection. b Adult worm and liver egg burdens after compound treatments targeting juvenile worms 21 days after infection. c Images of livers from a mouse treated with compound 2 at 100 mg/kg 42 days after infection and from an untreated mouse, showing reduction in the number of granulomas. d Images of livers from a mouse treated with compound 2 at 100 mg/kg 21 days after infection and from an untreated mouse, showing reduction in the number of granulomas. The number of mice in each treatment, n = 5. A two-tailed student t-test was used to determine significance, with the cutoff set to p < 0.05 in each comparison between mice treated with inhibitors and the control. Source data are provided as a Source Data file.