Guanylate cyclases (GTP pyrophosphate-lyase [cyclizing]; EC 4.6.1.2) catalyze the biosynthesis of guanosine 3',5'-cyclic monophosphate (cGMP) from GTP. While the membrane bound forms are monomers and stimulated by the natriuretic peptides, the soluble guanylate cyclases (sGC) exist as heterodimers consisting of an α₁- and a β₁-subunit and containing heme as a prosthetic group 1. Besides this major species of sGC there exist also reports of homodimeric forms of this enzyme 23. By formation of cGMP as a second messenger, sGC plays an important role in smooth muscle cell relaxation 4, inhibition of platelet aggregation, retinal signal transduction 5 and synaptic transmission 6. The enzyme is strongly activated by NO 7, by the new direct and NO-independent stimulator YC-1 891011, and to a lesser extend by CO 12131411. Thus in several studies YC-1 was shown to be an antithrombotic agent by elevation of cGMP, VASP phosphorylation and inhibiting platelet aggregation 81315161718. Furthermore YC-1 was shown to relax precontracted aortic rings 9, even if they were made tolerant with glyceryl trinitrite 19 and to induce a dose-dependent decrease in systolic blood pressure 19. Interestingly, in addition to the direct activation of the purified sGC by YC-1, an overadditive effect was observed by the combinations of YC-1 and NO or CO 910111820. It was shown that YC-1 is a heme-dependent but NO-independent stimulator of sGC 1113. In contrast to NO and CO which directly interact with the heme group of the enzyme, YC-1 did not change the spectral characteristics of the heme moiety of sGC 11. Therefore it is believed that YC-1 did not interact directly with the heme region of the sGC. Because YC-1 sensitises the enzyme towards their endogenous ligands NO and CO, it is proposed that YC-1 binds to an allosteric site on sGC and thereby reduces the ligand dissociation rates from the heme group 10112021.

Recently, we described the pharmacological profile of a new sGC stimulator – BAY 41-2272 with similar characteristics like YC-1, however, with a distinctly higher potency and no PDE inhibitory activity 22. Using BAY 41-2272 as lead structure of this new pharmacological principle, we synthesized the first photoaffinity label derived from the BAY 41-2272 chemical core structure, characterized it on the purified enzyme, labeled the highly purified sGC, and identified the binding amino acids of this new type of sGC stimulators.

We developed the first photoaffinity label for direct and NO-independent sGC stimulators. Coming from YC-1 and BAY 41-2272 as lead structures of direct and NO-independent sGC stimulators we synthesized the ortho- (BAY 50-6038), meta- (BAY 51-9491) and para-PAL (BAY 50-8364) compounds (Fig. 1) and tested their influence on sGC activity. The meta- and para-PAL showed on the one hand a direct sGC stimulation comparable to YC-1 and on the other hand in combination with NO distinct synergistic effects on sGC activity. The ortho-PAL showed the lowest sGC stimulation and in combination with NO no synergistic effect on sGC activity (Tab. 1). Both under reducing conditions and in the absence of DTT, in the sGC assay a similar sGC activating profile of the three PALs is given (Tab. 1). Because of the more dominant stimulation of sGC by the single compound both in the presence and absence of DTT the meta-PAL compound was chosen as photoaffinitylabel for the following studies (Tab. 1).

Similar to YC-1 and BAY 41-2272, meta-PAL (BAY 51-9491) concentration-dependently stimulates purified sGC and shows in combination with NO a potentiation of sGC activity, while in combination with YC-1, only additive effects could be detected. sGC stimulation by meta-PAL could be nearly completely blocked by ODQ and is dependent of the presence of the prosthetic heme-moiety of sGC (Fig. 3a). As YC-1, meta-PAL does not shift the position of the Soret band of sGC both under basal and NO stimulated conditions (Fig. 3b).

To optimize the irradiation conditions for the following photoaffinity studies, the NH- and CH-insertion qualities of meta-PAL (BAY 51-9491) were studied in the presence of different solution compounds by irradiation under 254 or 365 nm. As shown in Tab. 2, NH-insertion was more effective than CH-insertion under our conditions. UV 254 nm was chosen as irradiation source. In addition, the time dependency of the photoactivation of the meta-PAL (BAY 51-9491) under UV 254 nm (40 Watt, distance of 3 cm) was studied by thin layer chromatography (Rf of meta-PAL = 0.7 and of photolysed meta-PAL = 0.5). The meta-PAL was completely destroyed after irradiation in PAL buffer within 3 min. In the presence of glucose oxidase as model protein, meta-PAL was completely destroyed after an irradiation time of about 18 min (data not shown). Therefore the irradiation time for the labeling studies was chosen with 15 min under 254 nm (40 Watt) at a distance of 3 cm.

For enzyme labeling, ³H-meta-PAL was synthesized as described (Fig. 2). The binding of ³H-meta-PAL to highly purified sGC was studied both in the presence of different concentrations of sGC as different concentrations of ³H-meta-PAL, and also in the presence of unlabeled meta-PAL (100 μM). As shown in Fig. 4, ³H-meta-PAL binds concentration-dependently almost exclusively to the α₁-subunit of the sGC after irradiation. The radiolabeling of the β₁-subunit is only weakly visible in the autoradiogramm after reaction of sGC with 200 μCi ³H-meta-PAL. In the presence of unlabeled meta-PAL (100 μM) the covalent binding of the ³H-meta-PAL to the α₁-subunit is obviously diminished in the presence of 20 or 2 μCi ³H-meta-PAL.

In addition, we examined the effects of different sGC modulators on binding of ³H-meta-PAL on purified sGC (Fig. 5). Both unlabeled meta-PAL (100 μM and 10 μM), YC-1 (100 μM), BAY 41-2272 22 but also ODQ (100 μM) diminished concentration-dependently the binding of ³H-meta-PAL to the α₁-subunit. ODQ inhibit the binding of the ³H-meta-PAL to the α₁-subunit, but in this case weak unspecific labeling of the β₁-subunit and the α₁-subunit was detected. ³H-meta-PAL processed with sGC without irradiation showed no insertion of radioactivity (Fig. 5).

To identify the binding residues of the ³H-meta-PAL at the α₁-subunit, sGC was labeled with ³H-meta-PAL and the labeled protein was fragmented by CNBr cleavage. The resulting protein fragments were separated by electrophoresis (10–20% SDS-PAGE), transferred to a PVDF membrane, stained with Coomassie-blue and exposed to Imaging-Plates (Fig. 6). The resulted autoradiogramm shows five highly labeled protein fragments (CNBr I-V) with molecular weights of 51.8 / 35.9 / 30.2 / 11.7 and 6.2 kDa (Fig. 6) which were identified and excised from the Coomassie-blue stained Western blots. Sequencing of this bands showed, that they contain different fragments of both the α₁- as the β₁-subunit (Tab. 4). For better separation of peptides in the lower molecular weight range, the same fragments were separated on a TRIS-Tricine-gel. In this autoradiogramm we detected three highly labeled protein fragments (CNBr VI-VIII) with molecular weights of 14.7 / 11.7 and 6.2 kDa (Fig. 7). Before sequencing of the different labeled sGC fragments, both intact subunits of sGC were sequenced. We observed, that the first 20 coded amino acids of the α₁-subunit probably belong to the presequence. The β₁-subunit corresponded to the published sequence. Because of the selective labeling of the α₁-subunit by the ³H-meta-PAL (Fig. 4 and 5), in Tab. 3 only the theoretically CNBr fragments of the α₁-subunit (AS 20-690) are aligned and signed after their position. It is known that CNBr fragments show different migration properties in SDS-PAGE related to the used molecular weightmarkers. Therefore in the figures we used the calculated molecular weights from sequencing to mark the labeled CNBr fragments. The sequenced bands are marked in Fig. 6 and 7. The results of the sequencing of the different fragments are shown in Tab. 4. The overview of the determined α₁-sequences shows, that the ³H-meta-PAL specifically binds to the CNBr VIII = CNBr-2 fragment of the α₁-subunit (Tab. 3 and 4) with the amino acids 236–290.

To more closely specify the binding site of the ³H-meta-PAL, the single PTH-cycles from the sequencing of the CNBr VIII fragment (Fig. 7 and Tab. 4) were collected, lyophilised and the radioactivity of each fraction was counted. Beside an unspecific "wash out", we determined a distinct increase of radioactivity in the cycles 3 and 8 (> 10-fold over basal measured activity), which correspond to the cysteines 238 and 243 (Fig. 8). Thereby ³H-meta-PAL is bound to sGC after irradiation by the cysteines 238 and 243 of the α₁-subunit of the sGC (Fig. 9).

The putative target sequence of bovine sGC differs from rat and human by the substitution of an arginine at position 238 instead of cysteine. Therefore we investigated if BAY 41-2272 is active against the bovine enzyme or in isolated bovine vascular preparations. We prepared crude sGC containing fraction from bovine lung by performing the homogenization and ion-exchange steps of the method described earlier 11 with a specific basal activity of 0.15 nmol/mg/min. Using this preparation we examined the characteristic of sGC stimulation by BAY 41-2272 alone and in the presence of high concentrations of DEA/NO (Fig. 10). This study shows that BAY 41-2272 stimulates directly the crude sGC-containing fraction from bovine lung in a concentration dependent manner. In combination, BAY 41-2272 and the NO donor DEA/NO potentiates over a wide range of concentrations.

To identify the binding site of the new class of direct and NO-independent sGC stimulators like YC-1 and the recently described more potent compound BAY 41-2272 22 we focus in this paper in addition to results, already shown in Stasch et al. 22, on the detailed description of the development of the first photoaffinity label by introducing a azidobenzoic acid at different positions (ortho-, meta-, and para-PAL) into the BAY 41-2272 core structure. As shown, all tested photoaffinitylabels stimulate the purified enzyme with similar characteristics as YC-1. sGC activity increases from the ortho- to the para- to the meta-PAL compound. In contrast to the ortho-PAL compound, the meta- and para-PAL compounds show synergistic effects on sGC activity in combination with NO as described for YC-1 and BAY 41-2272 1122 (Tab. 1). Therefore we conclude from these structure-activity-relationship, that the meta- and para-PALs in contrast to the ortho-PAL match structurally with the new binding site of the sGC, essentially regarding to the synergistic activation of sGC in combination with its physiological stimulator NO and thereby are useful tools in the identification of the new binding site in sGC. In addition to Stasch et al.22, we demonstrate here different potential PALs, which differ in their chemical structure and sGC stimulation characteristics.

In forward to avoid an unproductive destruction of the PAL during the labeling of the enzyme 34, stimulation of sGC was not only tested under standardized conditions 11 but also verified under modified conditions in the absence of DTT. Even under this adapted conditions used for the labeling of the enzyme, the PAL compounds stimulate the purified enzyme with similar properties as YC-1 and BAY 41-2272. Under these conditions meta-PAL reveals to be the most potent compound with the same characteristics on stimulation of sGC as YC-1 and BAY 41-2272. As expected, meta-PAL shows no influence on the position of the Soret band of sGC both under basal and NO-stimulated conditions. Thereby meta-PAL belongs to the new class of direct, NO-independent sGC stimulators, which stimulate sGC by a heme-dependent mechanism without a direct interaction with the heme-moiety 1122.

Moreover we characterize the insertion characteristics of the meta-PAL. Concomitant with the literature, our meta-PAL showed more potent NH- than CH-insertion qualities and thereby is qualified for protein conjugation reactions 35. During irradiation with UV light (254 nm), under our adapted conditions the meta-PAL completely disintegrated within a time of maximal 18 min under release of nitrogen, detected by thin-layer-chromatography. Beyond the fast reaction of azido compounds after irradiation with UV, the quality of a photoaffinitylabel depends on the stability of the compound and the radioactive label and of the covalent binding properties of the compound at or at least in the environment of the binding site 35. Our compound is stable for several weeks stored at 70°C (data not shown). Moreover, under reducing conditions, where the sGC is distinctly stimulated by the meta-PAL, and thereby the active conformation of the sGC should be provided, the ³H-meta-PAL binds not only concentration-dependently but also specifically to the α₁-subunit of the sGC. Ideally, a classical receptor binding study would be the best aproach to characterize the specific as well as unspecific binding to sGC. However, due to the physikochemical properties of the radioligand (e.g. low solubility, lipophilic character, low specific activity) and the difficulty that the receptor sGC is a soluble cytosolic enzyme, it was not possible to separate free and bound radioligand. For this reasons we were not able to establish this study. Binding to the α₁-subunit of the enzyme was inert versus all used analytical steps (e.g. TCA precipitation, acid CNBr proteolyse, SDS-PAGE) and thereby underlines the quality of this new compound.

In a further study, the inhibition of this specifically binding of the ³H-meta-PAL to the α₁-subunit of the sGC by several sGC modulators was examined. Thereby we could show that the ³H-meta-PAL binds only after irradiation to the α₁-subunit of the sGC, implicating that the ³H-meta-PAL binds as a consequence of irradiation and not by an unspecific interaction to the enzyme. After preincubation with the purified sGC, the unlabeled meta-PAL(10 and 100 μM) as well as YC-1 (100 μM) or BAY 41-2272 22 inhibited the insertion of the ³H-meta-PAL in the α₁-subunit, underlining the specificity of the binding. As shown in Fig. 4, incubation with 200 μCi (125 μM) ³H-meta-PAL in the presence of 100 μM unlabeled PAL showed only slight competition due to the small access of unlabeled compound. For this reason further competition studies were performed with 12.5 μM ³H-meta-PAL (Fig. 5). In addition, the specific inhibitor of sGC, ODQ 36, strongly diminished the binding of the ³H-meta-PAL to the α₁-subunit. In this case there could be detected an equivalent binding of the ³H-meta-PAL both to the α₁- and the β₁-subunit. It is described that ODQ inhibits sGC activity by oxidation of the heme moiety from Fe²⁺ to Fe³⁺ or by competition with the NO binding site 3738. Therefore we propose that after interaction with ODQ the native heme environment of the sGC is changed (e.g. by oxidation), destroying the binding site for direct and NO-independent sGC stimulators and therefore ³H-meta-PAL unspecifically binds to both subunits of the sGC. The slightly stronger labeling of the beta subunit would result in this context from the possible higher available concentration of free meta-PAL. This explanation is underlined by the observation, that the heme-depleted enzyme is insensitive towards stimulation by meta-PAL and other direct and NO-independent sGC stimulators like BAY 41-2272 or YC-1 1322. Recently, Martin et al. 46 showed that the stimulation of sGC by the new type of sGC stimulators like YC-1 has both heme-dependent and heme-independent components. The stimulation of sGC by YC-1 is not completely blocked by ODQ, in particular in higher concentrations of sGC stimulators. They conclude, that the binding of ODQ and YC-1 are not mutually exclusive processes and that their binding sites do not overlap. These data corroborate our findings.

In addition, the ³H-meta-PAL labeled sGC was fragmented by CNBr digest to identify the binding site of the ³H-PAL at the α₁-subunit more closely. In this study, the chemical digest of the labeled sGC shows five highly labeled protein fragments on the Photo-Imaging-Plate of the gradient gel in the molecular range between 6.2 and 51.8 kDa and three highly labeled bands in the Photo-Imaging-Plate of the TRIS-Tricine-gel in the molecular range between 6.2 and 14.7 kDa. These bands were identified and excised from the Coomassie-blue stained blot and sequencing of these bands showed, that they contain different fragments of both the α₁- as the β₁-subunit. Because the ³H-meta-PAL binds specifically to the α₁-subunit of the sGC and thereby all identified β₁-sequences could be discarded, the overview of the determined α₁-sequences shows, that the ³H-meta-PAL binds specifically to the CNBr VIII fragment, consisting of the amino acids 236–290 of the α₁-subunit. To more closely identify the binding site of the ³H-meta-PAL, the single PTH-cycles from the sequencing of the CNBr VIII fragment were collected and the cysteines 238 and 243 of the α₁-subunit were detected as binding sites of ³H-meta-PAL.

As shown in Fig. 9, these cysteines are conserved between the amino acid sequences of rat and human sGC 3940 and this region could play an important role in regulation of sGC activity by this type of sGC. However, the putative target of bovine sGC differs from rat and human by the substitution of an arginine at position 238 instead of cysteine. As shown in previous studies with YC-1 11 and verified in this study using crude bovine lung derived sGC extracts, we know that cysteine 238 is not essential for the stimulation of sGC by neither YC-1, nor BAY 41-2272, nor the PAL compound. However, is must be considered that the potencies of BAY 41-2272 on human, rat and bovine sGC could be different. For such comparison, the highly purified guanylate cyclases from these three species are needed. The mechanism of sGC stimulation is rather more complicated than simple interaction with one or two amino acids since the heme moiety bound to the His-105 of the β₁-subunit is essential for activating the enzyme by YC-1, BAY 41-2272 and the PAL compound. There is a distance of 9 Å between the photolabile azido group and the pharmacophore of the compound. For this reason a covalent binding of the ³H-PAL to reactive cysteines slightly outside the binding pocket would not be surprising. Nevertheless, we assumed that Cys-238 and Cys-243 are in the direct region of access of ³H-PAL because no other labeled amino acids have been detected and a diffusion over a wide distance can be excluded.

Recent studies with YC-1 implify that YC-1 binds to an allosteric site on sGC and thereby increases the maximal catalytic rate and sensitises the enzyme towards its gaseous activators NO and CO 1113. Studies about the kinetics and equilibra of sGC in the presence of YC-1 led to a mechanistic model, which attributes a crucial role to the proximal bond that connects the heme iron to the histidine 105 of the β₁-subunit of sGC 4142, but also requires protein control of the distal environment 43. Firstly a small shift in the soret absorption feature of the sGC by YC-1 is observed. It is discussed, that this small shift could be evoked by the replacement of the His-105 as the proximal heme ligand by another base, either another amino acid side chain or even YC-1 itself 43.

In contrast to 21 who postulated the YC-1 binding site in analogy to the forskolin binding site in the adenylate cyclase within the catalytic region of the α₁- subunit of the sGC in the region of cysteine 596, we showed that the new generated photoaffinitylabel for direct and NO-independent sGC stimulators binds to the cysteines 238 and 243 in the N-terminus of the α₁-subunit of the sGC and thereby in the near of the heme binding region. Hobbs 44 postulated an intramolecular sixth ligand of the heme, which oscillates on and off the sixth coordinate, thereby conferring some sort of ligand specificity (i.e. NO and CO). The binding of direct sGC stimulators to this site could block this intramolecular binding site and thereby simplify the binding of NO and CO to the enzyme. This model is concomitant with the sensitising of the enzyme towards NO by this class of stimulators. Very recently our findings have been supported 45 showing the important role of an additional heme binding domain for sGC activation by the NO-independent sGC stimulator YC-1. This domain is in the α-subunit in vicinity of the PAL labeled region.

In summary, using photoaffinity labelling, we identified the region of the cysteines 238 and 243 in the α₁ subunit of sGC as the target for NO-dependent sGC stimulators. However, the relevance of the identified region as a regulatory unit remains to be confirmed by mutational analysis and co-crystallization studies.