Werner Schroeder

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle

Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (α/ß) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. Through a high‐throughput screen we identified BAY 58‐2667, an amino dicarboxylic acid which potently activates sGC in an NO‐independent manner. In contrast to NO, YC‐1 and BAY 41‐2272, the sGC stimulators described recently, BAY 58‐2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem deficient. Binding studies with radiolabelled BAY 58‐2667 show a high affinity site on the enzyme. Using photoaffinity labelling studies we identified the amino acids 371 (α‐subunit) and 231 – 310 (ß‐subunit) as target regions for BAY 58‐2667. sGC activation by BAY 58‐2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not influenced by nitrate tolerance. BAY 58‐2667 shows a potent antihypertensive effect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic effects of BAY 58‐2667 and GTN are very similar on the arterial and venous system. This novel type of sGC activator is a valuable research tool and may offer a new approach for treating cardiovascular diseases.

NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272

BackgroundThe most important receptor for nitic oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase.ResultsWe developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the α1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236–290 of the α1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.ConclusionsOur data demonstrate that the region surrounding the cysteines 238 and 243 in the α1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.

Epitope-specific monoclonal antibodies against human c-terminal procollagen α1(III)-propeptide

We have generated monoclonal antibodies against recombinant C-terminal human procollagen alpha1(III) propeptide (PIIICP), produced in E. coli in high yields. The monoclonal antibodies were screened for epitope specificity using recombinant truncated PIIICP. Several antibodies were identified which recognized different regions of the PIIICP molecule. The ability of the antibodies to detect PIIICP antigens in human cell line lysates and supernatants was demonstrated. As PIIICP antigens are a key marker of extracellular matrix metabolism, the monoclonal antibodies described here should be of value for clinical and basic research.