Fig. 1: CH004 inhibits the activity of hCBS in the in vitro purified enzyme assays under different assay conditions.
a A scheme for the cascade enzymatic reactions catalyzed by CBS or CSE. Cth, cystathionine. b, c Inhibitory effects of CH004 on the activity of hCBS-413 (b) or hCSE (c). The enzyme activities were monitored for various concentrations of CH004 under the standard conditions (50 mM Tris-HCl, pH 8.6; “Materials and Methods” section). The result is shown as percentages of the control (DMSO, 100%). The data are shown as means ± SDs (n = 3). d The dose-dependent inhibition of CH004 on the hCBS-413 activity was measured by the tandem-well-based assay under the standard conditions except using 50 mM Hepes buffer (pH 7.4) instead of 50 mM Tris-HCl (pH 8.6). Means ± SDs (n = 3). e The dose-dependent inhibition of CH004 on the CBS activity measured by methylene blue method. The CBS enzyme assay was performed with a 50 μl assay buffer in an Eppendorf tube under the standard assay conditions for the 192-tandem-well based assay, i.e., 50 μl 50 mM Tris-HCl buffer (pH 8.6) containing 150 nM hCBS-FL, 200 μM S-adenosyl-L-methionine, 4 mM L-Cys and 4 mM d,l-Hcys, in the presence of 0 (DMSO control), 0.01, 0.5, 1, 5, 10 or 100 μM CH004. After 80 min, the assay was stopped by added 10 μL 10% ZnAC and the generated H2S was quantified by N,N-dimethyl-p-phenylenediamine to give an absorbance at 670 nm (for details, see Materials and Methods section; ref. 53). The data are presented as percentages of control (DMSO, 100%) and shown as means ± SDs (n = 3. The dose-dependent curves were fitted to the data points with GraphPad Prism 5. f The interference of CH004 with H2S at micomolar concentrations. CH004 at the indicated concentrations was mixed with 100 μM NaHS in the absence of CBS under the assay conditions (“Materials and Methods” section). The released H2S was quantified with DTNB by using the 192-tandem plate. The data are presented as percentages of control (DMSO, 100%) and shown as means ± SDs (n = 3)
