We report herein an electrochemical analysis of the interact

We report herein an electrochemical analysis of the interaction between L1 and sGC, where conventional solution phase voltammetry is combined with a novel technique, the voltammetry of microparticles (VMP), in order to obtain mechanistic information on the deactivation of sGC by L1. The VMP is a solid state electrochemical technique developed by Scholz et al. [14], which provides analytical information for insoluble solids in contact with suitable electrolytes. Apart from its inherently high sensitivity, immobilization of solids onto inert electrode surfaces offers the possibility of blocking secondary reactions interfering the target processes and provides structural constraints which can be often used for mimicking biological barriers and/or conformational constraints in biological systems. This technique is applied here to the formation of films of sGC and L1 on glassy carbon electrodes, following a strategy recently described previously used for monitoring phenethylamine-derived psychotropic drugs [15] and screening potential hemozoin-based antimalarials [16].
Results and discussion We assessed whether canthin-6-one (L1) altered sGC activity in the absence or the presence of the NO donor sodium nitroprusside (SNP). The results shown in Fig. 2, suggested that Sephin 1 canthin-6-one inhibited basal sGC activity (Wilcoxon signed rank test, p < 0.05) in a dose dependent manner (90 ± 11; 70 ± 12, 60 ± 8, and 47 ± 3% of the activity in the absence of L1 at 1, 10, 100 and 300 μmol/L of L1, respectively). At concentrations of 100 and 300 μmol/L of L1 basal sGC activity is significantly reduced (p-value = 0.014 and p-value = 0.031, respectively) (Fig. 2b). In the absence of L1, 100 μM SNP increased the activity of sGC by eight-fold. However, SNP-induced sGC activity was also reduced in the presence of L1 (Wilcoxon signed rank test, p < 0.05) in a dose dependent manner (79 ± 19; 72 ± 18, 37 ± 12, and 32 ± 22% of the activity in the absence of L1 at 1, 10, 100 and 300 μmol/L of L1, respectively) (Fig. 2c). L1 decreased the activation of sGC induced by SNP up to two-three-fold. This effect was statistically significant at 100 μmol/L of L1 (p-value = 0.038). Fig. 3 compares the square wave voltammetric responses at glassy carbon electrode for a solution of sGC in aqueous phosphate buffer in the absence (a) and in the presence (b) of an equimolar amount of L1. The pristine solution of sGC yields an oxidation peak (I) at −0.15 V vs. Ag/AgCl attributable, as inferred upon comparison with heme blanks [16], to the Fe2+ to Fe3+ oxidation of the heme unit of sGC. A second ill-defined anodic wave appears at ca. +0.87 V (II), attributable to the oxidation of cysteine units [20], [21]. In the presence of L1, significant changes in the voltammogram occur: the oxidation signal shows peak splitting with additional peaks at +0.04 (I′) and +0.16 V (I″), whereas the peak II becomes clearly marked. Experiments in sGC solutions at L1-modified graphite electrodes provide several interesting features. Remarkably, upon scanning the potential in the negative direction from the potentials where the process II occurs, this signal becomes considerably enhanced, relative to the I signal, at unmodified electrodes, whereas the reduction signal for L1 at −1.0 V (III) becomes significantly decreased, almost entirely vanishing (Fig. 3). This voltammetric response clearly differs from those expected from the summation of the responses of L1 and sGC, thus denoting that both species interact significantly. In particular, L1 becomes electrochemically silent in the region of potentials where sGC oxidation occurs. A typical negative potential-going voltammogram for a sGC solution in the presence of L1 is shown in Fig. 4a. The observed response is indistinguishable from that recorded for an equivalent solution of L1 in the absence of sGC. This suggests that there are no electrocatalytic effects in solution phase involving a possible reaction between L1 and Fe3+. In contrast, voltammograms obtained on sGC solutions at L1 film-modified electrodes display (Fig. 4b) an additional cathodic peak (IV) at ca. −0.65 V. This peak can be attributed to the canthin-localized reduction of a sGC-canthin adduct. Consistently, peak IV is absent in the voltammetry of sGC films in phosphate buffer (Fig. 5a and b) and is considerably enhanced in voltammograms recorded at sGC films in contact with L1 solutions (see Fig. 5c and d), while the anodic signals become lowered.