Effects of detergents on the West Nile virus protease activity

Abstract

Detergents such as Triton X-100 are often used in drug discovery research to weed out small molecule promiscuous and non-specific inhibitors which act by aggregation in solution and undesirable precipitation in aqueous assay buffers. We evaluated the effects of commonly used detergents, Triton X-100, Tween-20, Nonidet-40 (NP-40), Brij-35, and CHAPS, on the enzymatic activity of West Nile virus (WNV) protease. Unexpectedly, Triton X-100, Tween-20, and NP-40 showed an enhancement of in vitro WNV protease activity from 2 to 2.5-fold depending on the detergent and its concentration. On the other hand, Brij-35, at 0.001% enhanced the protease activity by 1.5-fold and CHAPS had the least enhancing effect. The kinetic analysis showed that the increase in protease activity by Triton X-100 was dose-dependent. Furthermore, at Triton X-100 and Tween-20 concentrations higher than 0.001%, the inhibition of compound B, one of the lead compounds against WNV protease identified in a high throughput screen (IC(50) value of 5.7+/-2.5 microM), was reversed. However, in the presence of CHAPS, compound B still showed good inhibition of WNV protease. Our results, taken together, indicate that nonionic detergents, Triton X-100, Tween, and NP-40 are unsuitable for the purpose of discrimination of true versus promiscuous inhibitors of WNV protease in high throughput assays.

Figure 1. WNV protease activity in presence of different detergents

Panel A. Standard protease assays were carried out using the tripeptide substrate, t-butyl-oxycarbonyl (Boc)-Gly-Lys-Arg-7-amino-4-methylcoumarin (AMC) as described under Materials and Methods in the absence of any detergent or in the presence of Triton-X100, Tween-20, or CHAPS at the indicated concentrations of 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², 10⁻¹, or 1 % in the assay mixture. Panel B. The experimental conditions for the assay were the same as in Panel A except that NP-40, Brij-35, or CHAPS was used at the indicated concentrations. Panel C. The conditions of the assay were the same except that the effects of Triton X-100 and CHAPS at indicated concentrations on the WNV protease activity using a tetrapeptide substrate, N-Carbobenzyloxy-Val-Lys-Lys-Arg-4-Methoxy-b-naphthylamide as described under Materials and Methods. The percent protease activity was plotted compared to that of the no-detergent control set at 100%.

Figure 1. WNV protease activity in presence of different detergents

Panel A. Standard protease assays were carried out using the tripeptide substrate, t-butyl-oxycarbonyl (Boc)-Gly-Lys-Arg-7-amino-4-methylcoumarin (AMC) as described under Materials and Methods in the absence of any detergent or in the presence of Triton-X100, Tween-20, or CHAPS at the indicated concentrations of 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², 10⁻¹, or 1 % in the assay mixture. Panel B. The experimental conditions for the assay were the same as in Panel A except that NP-40, Brij-35, or CHAPS was used at the indicated concentrations. Panel C. The conditions of the assay were the same except that the effects of Triton X-100 and CHAPS at indicated concentrations on the WNV protease activity using a tetrapeptide substrate, N-Carbobenzyloxy-Val-Lys-Lys-Arg-4-Methoxy-b-naphthylamide as described under Materials and Methods. The percent protease activity was plotted compared to that of the no-detergent control set at 100%.

Figure 1. WNV protease activity in presence of different detergents

Panel A. Standard protease assays were carried out using the tripeptide substrate, t-butyl-oxycarbonyl (Boc)-Gly-Lys-Arg-7-amino-4-methylcoumarin (AMC) as described under Materials and Methods in the absence of any detergent or in the presence of Triton-X100, Tween-20, or CHAPS at the indicated concentrations of 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², 10⁻¹, or 1 % in the assay mixture. Panel B. The experimental conditions for the assay were the same as in Panel A except that NP-40, Brij-35, or CHAPS was used at the indicated concentrations. Panel C. The conditions of the assay were the same except that the effects of Triton X-100 and CHAPS at indicated concentrations on the WNV protease activity using a tetrapeptide substrate, N-Carbobenzyloxy-Val-Lys-Lys-Arg-4-Methoxy-b-naphthylamide as described under Materials and Methods. The percent protease activity was plotted compared to that of the no-detergent control set at 100%.

Figure 2. Effect of Triton X-100 on K_m and V_max values of WNV protease

Different WNV substrate concentrations (0 – 2000 μM) were used to determine WNV protease activity in the presence and absence of Triton X-100 in standard protease assays as described under Materials and Methods. The experiments were repeated three times and the error bars represent the standard deviation of the mean.

Figure 3. Effect of Triton X-100 on the inhibition of WNV protease by compound B

A. The assays were performed as described under Materials and Methods in the presence and absence of Triton X-100 and 2 μM aprotinin (light grey bars) or 20 μM compound B (dark grey bars). At Triton X-100 concentrations at 0.5 CMC (CMC=0.02%), the inhibition of compound B is reduced ~85%. B. The concentrations of compound B were varied as indicated. The protease assays were performed at two different Triton X-100 concentrations (0.001 %-grey bars and 0.01%-white bars) along with no-detergent control (black bars). The experiments in panels A and B were repeated at least three times with similar results. The error bars represent standard deviation of the mean.