3-oxoacyl-ACP reductase from Schistosoma japonicum: integrated in silico-in vitro strategy for discovering antischistosomal lead compounds

Abstract

Background: Schistosomiasis is a disease caused by parasitic worms and more than 200 million people are infected worldwide. The emergence of resistance to the most commonly used drug, praziquantel (PZQ), makes the development of novel drugs an urgent task. 3-oxoacyl-ACP reductase (OAR), a key enzyme involved in the fatty acid synthesis pathway, has been identified as a potential drug target against many pathogenic organisms. However, no research on Schistosoma japonicum OAR (SjOAR) has been reported. The characterization of the SjOAR protein will provide new strategies for screening antischistosomal drugs that target SjOAR.

Methodology/principal findings: After cloning the SjOAR gene, recombinant SjOAR protein was purified and assayed for enzymatic activity. The tertiary structure of SjOAR was obtained by homology modeling and 27 inhibitor candidates were identified from 14,400 compounds through molecular docking based on the structure. All of these compounds were confirmed to be able to bind to the SjOAR protein by BIAcore analysis. Two compounds exhibited strong antischistosomal activity and inhibitory effects on the enzymatic activity of SjOAR. In contrast, these two compounds showed relatively low toxicity towards host cells.

Conclusions/significance: The work presented here shows the feasibility of isolation of new antischistosomal compounds using a combination of virtual screening and experimental validation. Based on this strategy, we successfully identified 2 compounds that target SjOAR with strong antischistosomal activity but relatively low cytotoxicity to host cells.

Figure 1. Expression, purification and identification of rSjOAR protein.

A. SDS-PAGE analysis of SjOAR expression from E. coli BL21 transformed with the recombinant plasmid SjOAR-pET28a. M, molecular weight marker; lane 1, whole lysate of bacterial without induction; lane 2 and lane 3, whole lysate and supernatant of bacteria induced with 1 mM IPTG at 37°C for 6 h. B. Elution profile of SjOAR on a gel chromatography column. BSA with a molecular weight of 67 KDa (monomer) was used as an internal control. The insert figure shows a single band on an SDS-PAGE gel. C. Identification of rSjOAR by western blotting. M, prestained protein marker; lane 1, the purified His-tag fusion SjOAR protein was probed with rabbit anti-His antibody (1:2,000 diluted in PBST buffer). The secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1: 8,000 diluted in PBST buffer). Chromogenic detection was performed using 3, 3′-diaminobenzidine (Sigma, USA). The arrow indicates the target band, assumed to be SjOAR protein.

Figure 2. Enzymatic activity of rSjOAR in vitro.

The concentrations of SjOAR for each reaction were 0.15 µM (circle), 0.30 µM (square), 0.60 µM (triangle), 1.20 µM (diamond) and 2.40 µM (asterisk). The analysis was done in triplicate and repeated three times.

Figure 3. Kinetic analysis of rSjOAR.

A. AcAcCoA concentration was varied with fixed concentrations of NADPH (triangle, 100 µM; diamond, 150 µM; square, 200 µM). B. NADPH concentration was varied with fixed concentrations of AcAcCoA (triangle, 500 µM; diamond 750 µM; square, 1, 000 µM). The rates for each reaction were fitted individually to the Michaelis-Menten equation by non-liner regression and displayed as double-reciprocal plots. The molar absorptivity (ε) of NADPH at 340 nm is 6.22 mM⁻¹cm⁻¹ .

Figure 4. Time-dependent survival rates of adult worms treated with compounds 19, 22, 27 and PZQ.

A, B and C, Adult worms were treated with compounds 19, 22 and 27. Compound concentrations were 10 µg/ml (asterisk), 20 µg/ml (triangle), 40 µg/ml (square) and 80 µg/ml (diamond). 1.6 µg/ml PZQ (dash line with circle mark) was used as a positive control.

Figure 5. Optical microscopy of worms exposed to compounds 19, 22 and 27.

A. The morphology of worms treated with 2% DMSO for 72 h. B. The morphology of worms treated with 10 µg/ml of OAR19 for 48 h. C. The morphology of worms treated with 10 µg/ml of OAR22 for 48 h. D. The morphology of worms treated with 40 µg/ml of OAR27 for 24 h. E. The morphology of worms treated with 10 µg/ml of PZQ for 24 h.

Figure 6. SEM images of the mid-body of adult worms.

A. The morphology of worms treated with 2% DMSO for 72 h. B. The morphology of worms treated with 10 µg/ml of OAR19 for 48 h; C. The morphology of worms treated with 10 µg/ml of OAR22 for 48 h. D. The morphology of worms treated with 40 µg/ml of OAR27 for 24 h; E. The morphology of worms treated with 10 µg/ml of PZQ for 24 h.

Figure 7. The effect of active compounds on rSjOAR enzymatic activity.

OAR19 (triangle), OAR22 (square), OAR27 (diamond) and PZQ (dash line with circle mark). The rSjOAR concentration in each assay was 0.06 µM. Enzymatic activity in the no-inhibitor group was defined as 100%. The inhibition of rSjOAR enzymatic activity in the experimental group was calculated using the following equation: inhibition % = (1−V_i/V₀)×100%, where V₀ is the initial velocity of the no-inhibitor group and V_i is the initial velocity in each experimental group.

Figure 8. Cytotoxicity assay for human liver cells cultured with different concentrations of active compounds.

Columns filled with black, gray and white represent OAR19, OAR22 and OAR27, respectively. The cell activity of the compound-carrier-only group was defined as 100%. The relative cell activity values of other experimental groups were calculated by comparing them with the control group. Data shown in the figure represent three independent experiments.