To further investigate the possible interaction of L with sG

To further investigate the possible interaction of L1 with β1-sGC, a protein-ligand docking experiment was performed. Calculations were carried out with the program GOLD (version 5.2) [18], [34] and performed with the chemPLP scoring function [19], [34]. The ten lowest energy solutions presented a reasonable score of approximatively 58 units. In all solutions, the ligand makes important hydrophobic interactions with the hydrophobic patch located on the roof of the binding site and composed by residues ALA8, LEU9, PHE70, PHE74, LEU142, ILE145 and ILE149, on the one side and the porphyrin ring on the other (Fig. 9). In all poses, several atoms of L1 lay next to the metal ion. Two major binding orientations are observed. In one, both the carbonyl group and several carbon atoms of the phenyl group of L1 stand below 4 A from the iron moiety. On the other, the nitrogen L-161,982 of the pyridine subsystem of L1 is about 3.5 A of the iron, an orientation reminiscent to type I inhibition of cytochromes P450 by nitrogenated compounds in which a direct coordination of the iron takes place with nitrogen atom. In none of these orientations, Cys78 comes close to the ligand although interactions throughout a network of water molecules and/or motion of the Phe74 could nuance this observation. The coexistence of two deactivating pathways could explain the apparent contradiction between the observation9 of maximal NO stimulation with S nitrosation under aerobic conditions and the inhibition of sGC activity by S-nitrosation of specific sGC cysteines (β1C122 and α1C243) [10], because, depending on nitrosating reagents and reaction conditions, different pathways can predominate. The effect of L1 on sGC activity was observed at range of μM. However, further modulations in the canthin-6-one scaffold could increase its effect at lower doses. Inhibition of the NO-cGMP pathway, and particularly sGC activity, alone or combined with other therapies may be useful for the treatment of diseases including refractory hypotension associated with endothelial dysfunction in vasoplegic syndrome, malaria, and Parkinson's disease [35].
Conclusions In summary, the present publication present three different points of view about the inhibition of sGC by canthin-6-one (L1). First of all, the biochemical results show that canthin-6-one inhibited basal sGC activity in a dose dependent manner at μMolar range. The electrochemical analysis of sGC-L1 interactions was achieved by the solution-phase and solid-state voltammetry. The electrochemical data suggest that deactivation of sGC by L1 operates via a coordinating pathway rather than a direct oxidation of the metal center, the L1 coordination possibly does not affect the Fe-His bond, but involves binding to the sGC active site in different orientations. Such mechanisms appear to be strongly influenced by structural/conformational constraints allowing for the occurrence of that oxidative pathway under determined circumstances. Finally, in order to confirm our experimental a molecular modeling study was performed. As the crystal structure of human sGC has not been resolved, the study started by constructing a 3D model using homology modeling. Once the model was established we proceeded to the docking study and the coexistence of two deactivating pathways was confirmed. Apparently, one of these pathways will be predominant depending on the biochemical conditions.
Acknowledgments Spanish Ministry of Economy and Competitiveness (MEC) I+D+I project CTQ2011-28079-CO3-02, supported by the European Regional Development Fund, the European Community for financial support (Marie Curie Early Stage Training Fellowship of the European Community's Sixth Framework Programme: contract BioMedChem), and Institute of Health Carlos III from the MEC. R Rodrigo has a “Miguel Servet” contract funded by the Institute of Health Carlos III (CP09/118). J-D Maréchal wishes to thank the support given by Spanish grant CTQ2011-23336 and the Generalitat de Catalunya grant 2009SGR68 and the European COST Action CM1306. Special thanks to Dr. Rafael Vazquez-Manrique (Sensorineural Disorders, Health Research Institute-La Fe, Valencia, Spain) for many fruitful discussions.