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. 2012 May 1;52(9):1897-902.
doi: 10.1016/j.freeradbiomed.2012.02.046. Epub 2012 Mar 8.

Off-target thiol alkylation by the NADPH oxidase inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870)

Qi-An Sun  1 , Douglas T Hess, Benlian Wang, Masaru Miyagi, Jonathan S Stamler
Affiliations

Off-target thiol alkylation by the NADPH oxidase inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870)

Qi-An Sun et al. Free Radic Biol Med. .

Abstract

Specific inhibitors of the production of reactive oxygen species (ROS) by the NADPH oxidases (Nox's) are potentially important therapeutic agents in the wide range of human diseases that are characterized by excessive ROS production. It has been proposed that VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3- triazolo[4,5-d]pyrimidine), identified as an inhibitor of Nox2 by small-molecule screening, may serve as an example of such an agent. Here we show that VAS2870 inhibits ROS production in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle, previously identified with Nox4, and thereby abrogates O(2)-coupled redox regulation of the ryanodine receptor-Ca(2+) channel (RyR1). However, we also find that VAS2870 modifies directly identified cysteine thiols within RyR1. Mass spectrometric analysis of RyR1 exposed in situ to VAS2870 and of VAS2870-treated glutathione indicated that thiol modification is through alkylation by the benzyltriazolopyrimidine moiety of VAS2870. Thus, VAS2870 exerts significant off-target effects, and thiol alkylation by VAS2870 (and closely related Nox inhibitors) may in fact replicate some of the effects of ROS on cellular thiol redox status. In addition, we show that SR-localized Nox4 is inhibited by other thiol-alkylating agents, consistent with a causal role for cysteine modification in the inhibition of ROS production by VAS2870.

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Figures

Fig. 1
Fig. 1
Both DPI (A) and VAS2870 (B) inhibit ROS production (as assessed by DHE or DCF fluorescence), and (A,C) RyR1 activity (as assessed by [3H]ryanodine binding), in isolated SR vesicles. Means ± SEM, n = 3 for A–C. In A left panel, *p < 0.03 re 0 μM DPI at 20% O2. In A right panel, *p < 0.01 re 0 μM DPI at 20% O2. In B left panel, *p < 0.025 re 0 μM VAS at 20% O2; § p < 0.017 re 0 μM VAS at 1% O2. In B right panel, *p < 0.04 re 0 μM VAS. In C, *p < 0.02 re 0 μM VAS at 20% O2; § p < 0.01 re 0 μM VAS at 1% O2.
Fig. 2
Fig. 2
(A) In isolated SR vesicles, the number of free thiols within RyR1 (as assessed by monbromobimane labeling) is reduced at 21% O2 versus 1% O2. The pO2 –dependent loss of thiols is prevented by DPI (100 μM). In contrast, exposure to VAS2870 (20 μM) reduces the number of free thiols regardless of pO2. Means ± SEM, n = 5; *p < 0.18 re control. (B) Exposure of purified RyR1 to VAS2870 (10 μM) results in a loss of free thiols (as assessed by monobromobimane labeling). Note that SR vesicles were prepared at ambient pO2 (21% O2) prior to purification of RyR1. Means ± SEM, n = 4; *p = 0.031.
Fig. 3
Fig. 3
(A) Following treatment of SR vesicles with VAS2870, mass spectrometric analysis (LC-MS/MS) of trypsin-digested RyR1 identified six peptides containing a Cys residue bearing a modification of 209.2 amu, consistent with adduction by the benzyl-triazolopyrimidine moiety of VAS2870 (see Fig. 5 for further analysis). (B) By example, the spectrum shown identifies Cys2326 as a site of modification by VAS2870.
Fig. 4
Fig. 4
Exposure of GSH to VAS2870 results in the rapid loss of free thiols. Means ± SEM, n = 3; *p < 0.01.
Fig. 5
Fig. 5
Mass spectrometric analysis (LC-ESI-MS) of the modification of GSH by VAS2870. (A and B) Extracted ion current (EIC) chromatograms and corresponding mass spectra are shown for, respectively, VAS2870 and a mixture of VAS2870 and GSH (20 min incubation). Note different scales for ordinates. (C) The mass spectrometric analysis indicates that the thiol of GSH is alkylated by the benzyl-triazolopyrimidine moiety of VAS2870, with the thio-benzoxazoyl moiety of VAS2870 serving as leaving group.
Fig. 6
Fig. 6
Production of ROS by isolated SR vesicles (as assessed by DHE fluorescence) is inhibited by the thiol alkylating agents monobromobimane (MBB), at left, and iodoacetamide (IA), at right. Means ± SEM, n = 4. In left panel, *p < 0.01 re 0 μM MBB at 20% O2; ◆ p = 0.23 re 0 μM DPI at 1% O2 (i.e., MBB eliminates pO2-dependent DHE fluorescence); § p < 0.05 re 0 μM MBB at 1% O2. In right panel, *p < 0.05 re 0 μM IA at 20% O2; ◆ p = 0.2 re 0 μM IA at 1% O2 (i.e., IA eliminates pO2-dependent DHE fluorescence).
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