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, 24 (3), 911-6

The Benzimidazole Based Drugs Show Good Activity Against T. Gondii but Poor Activity Against Its Proposed Enoyl Reductase Enzyme Target


The Benzimidazole Based Drugs Show Good Activity Against T. Gondii but Poor Activity Against Its Proposed Enoyl Reductase Enzyme Target

Craig Wilkinson et al. Bioorg Med Chem Lett.


The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors targeting the NAD(+) bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD(+) and its availability in the natural environment makes the NADH complex form of ENR an attractive target. Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella ENR, but despite good efficacy against Toxoplasma gondii, the IC50 for T. gondii ENR is poor, with no inhibitory activity at 1 μM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the ENR enzyme and as such we believe that there may be significant off target effects for this family of inhibitors.

Keywords: Benzimidazole; Enoyl reductase; Toxoplasma; Triclosan.


Figure 1
Figure 1
(A) Refined TgENR/NADH crystal structure with the 2Fobs-Fcalc density map (blue) and 1Fobs-1Fcalc density map (green) contoured at 1.6σ and 4.0σ, respectively. (B) The same view as in (A) but with the FtENR/NADH/benzimide complex superimposed in red to show the predicted position of the benzimide inhibitor. (C) Representative density of the refined TgENR/NADH structure with waters and glycerol modeled into the inhibitor binding site with the map contoured at 1.5σ. For AC, nitrogen, oxygen, chlorine, phosphorous and carbon are colored blue, red, green, orange and yellow (TgENR)/red(FtENR), respectively. (D) Overlay of FtENR (blue sidechains) and TgENR (green sidechains) inhibitor binding sites with bound inhibitor (yellow), NADH cofactor (green) shown in stick format. The position of the critical Tyr residue which forms a hydrogen bond to the bound inhibitor is shown along with those residues which are significant different between species.
Figure 2
Figure 2. Efficacy and toxicity of compound 1–3 against T. gondii
(A) Growth of RH-YFP in human Foreskin fibroblasts (HFF) measured as fluorescence intensity. HFF were infected with differing numbers of RH-YFP tachyzoites as indicated, with uninfected control HFF also shown. HFF infected with 3200 RH-YFP tachyzoites treated with pyrimethamine/sulfadiazine (p/s) or 0.1% DMSO are additional positive and negative controls, respectively (left panel). The effect of compound 1–3 against Toxoplasma tachyzoites was determined by measurement of RH-YFP fluorescence as shown in the right hand panel. HFF were infected with 3200 RH-YFP and treated with compounds 1–3 at the concentration indicated. Toxicity was measured using WST stain. (B) The effect of compounds 1–3 on Toxoplasma growth was determined by infecting confluent fibroblasts with YFP expressing Pru strain T. gondii tachyzoites and measuring YFP florescence at 72 hours. Compounds 1 and 3 effectively reduced parasite growth however at 10μM (p<0.005). (C) Compounds 1–3 were incubated with confluent luciferase expressing PC3-Luc cells to determine their toxicity against prostate cancer cells. 1% Triton X was included as a positive control for cytotoxicity. After 96 hours luciferin (150μg/ml) was added and cytotoxicity was determined by a reduction in bioluminescent activity from the cells. Neither compounds 1–3, nor the vehicle control resulted in a reduction of bioluminescent activity from the PC3-Luc cells. (D) The viability of host HFF cells was assessed by WST-1 staining after 72 hours of incubation of indicated compounds at 10μM concentration. Effect of various concentrations of DMSO present in the HFF culture medium served as a standard toxicity curve.
Scheme 1
Scheme 1
Reagents and conditions: (a) NaH, KI, DMF, 0°C warming to RT, 16h, 40–60%.

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