Furthermore we observed a striking difference between BR

Furthermore, we observed a striking difference between BR 11257 and activators from the dicarboxylic MAPK Inhibitor Library type in thermofluor measurements on thermostability. The dicarboxylic acids cinaciguat [30] and BAY 60-2770 (see Fig. 3) induced a significant melting temperature increase of sGC. In combination with the non-hydrolysable nucleotide 2′-MANT-3′-dGTP, they transferred the enzyme into a state of high thermodynamic stability, likely reflecting the formation of a dead-end-complex [30]. In contrast, the monocarboxylic acid BR 11257 did not show any stabilisation of sGC in thermofluor measurements. Neither BR 11257 alone nor BR 11257 in combination with the non-hydrolysable nucleotide 2′-MANT-3′-dGTP increased the melting temperature of sGC. Accordingly, there are clear thermodynamic differences between the BAY 60-2770-bound enzyme and the BR11257-bound. The formation of the high stability state requires ligand binding at both HNOX domain and the catalytic domains. Accordingly, the stabilised conformation is likely based on an interaction between the HNOX and catalytic domains, which were described to be in close proximity [36], [47], [48], [49]. sGC’s HNOX domain is divided into two subdomains, between which the heme/activator binding site is located [50]. A likely candidate for mediating the stabilising interaction between HNOX and catalytic domain is the small N-terminal HNOX subdomain, as it is located next to the catalytic domain [47] and seems to be quite flexible [36], [50], [51]. Furthermore, the first α helix of this subdomain contains tyrosine 2, which binds to the benzoic acid carboxylate moiety of dicarboxylic acid activators [24]. This carboxyl group is absent in the monocarboxylic acid derivative BR11257, which could explain the absence of stabilisation with BR 11257. In line with this concept of the number of carboxylic acids, the anthranilic acid HMR 1766 without any carboxylic acid was also not able to increase the thermostability of sGC (data not shown). Activity measurements with purified sGC indicated that thermostabilisation goes along with increased catalytic activity under thermal stress [30]. A stabilisation of sGC in a physiological environment was described by Stasch et al., who observed increased sGC protein levels in endothelial cells after treatment with cinaciguat [19]. The intracellular sGC level is regulated by a continuous steady state between subunit degradation and new formation [52], [53]. By stabilisation of sGC, dicarboxylic activators might disturb this physiological balance. Activity measurements demonstrated no differences in binding affinities between BAY 60-2770 and BR11257. However, the intrinsic activity of BAY 60-2770 was twice as high as the intrinsic activity of BR11257. When a combination of both drugs was added to native enzyme, the measured activity was between the sGC activity levels of each drug alone (Fig. 7). The lack of an additive effect in the activity measurements indicates that both drugs bind to the same site of the enzyme. Consequently, BR 11257 acts as partial agonist in comparison to BAY 60-2770. Partial agonists are known to bind and activate their target with lower efficacy relative to a full agonist [54]. Clinically, partial agonists can be used to activate receptors to give a desired submaximal response when inadequate amounts of the endogenous ligand are present, or they can reduce the overstimulation of receptors when excess amounts of the endogenous ligand are present [55]. However, no statement can be made about a partial agonistic behaviour of BR11257 in comparison to the physiological ligand NO: The physiological ligand NO and the sGC activators target different types of the enzyme. Whereas an NO activation of sGC requires the heme in the HNOX domain of the enzyme, activators preferentially stimulate oxidised or heme-free sGC. In consequence, a direct comparison in in vitro studies is difficult. In contrast to sGC activators, the physiological ligand NO causes no long-term activation of the enzyme. Furthermore, excess amounts of NO lead to posttranslational modifications and alterations in the redox state which are shown to affect sGC’s sensitivity towards NO [41], [56], [57], [58], [59]. Several groups gave insights into S–nitrosylation-dependent sGC desensitization [59], [60], [61]. Moreover, referring to partial agonism, one should remember the concept of the receptor reserve which was shown to be relevant in sGC [62]: There are more receptors on a cell or tissue than needed for a maximal response of a full agonist. If the dose of a partial agonist will be increased, the drug can activate the receptor reserve thus causing a maximal response [63]. This can lead to a preferential effect of partial agonists on tissues or organs with a high receptor reserve [63]. Further in vivo experiments are needed to evaluate this possibility for BR 11257. Nevertheless, the partial agonistic behaviour and the non-stabilising effect of BR11257 are interesting new differences compared to the old class of dicarboxylic acids and may also be relevant for other monocarboxylic acid derivatives.