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

Abstract

1. 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 (alpha/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. 2. 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. 3. Binding studies with radiolabelled BAY 58-2667 show a high affinity site on the enzyme. 4. Using photoaffinity labelling studies we identified the amino acids 371 (alpha-subunit) and 231 - 310 (ss-subunit) as target regions for BAY 58-2667. 5. 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. 6. 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. 7. This novel type of sGC activator is a valuable research tool and may offer a new approach for treating cardiovascular diseases.

Figure 1

Effects of BAY 58-2667 on sGC activity. (A) Structure of BAY 58-2667. (B) Stimulation of purified sGC by BAY 58-2667 in the absence and presence of DEA/NO (10 and 100 nM) and ODQ (10 μM). The specificity is expressed as fold stimulation versus basal activity in the presence of Mg²⁺: 111 nmol min⁻¹ mg⁻¹ for the purified sGC. (C) Stimulation of haem free sGC by BAY 58-2667 in the absence and presence of ODQ. The specificity of sGC is expressed as fold stimulation versus basal activity in the presence of Mg²⁺: 169 nmol min⁻¹ mg⁻¹ for the haem-free sGC. Each value represents the mean±s.e.mean from three (B) and five (C) independent experiments performed in duplicate. (D) Haem spectra of sGC in the presence of BAY 58-2667 and DEA/NO. These data are representative of three independent determinations.

Figure 2

Receptor binding studies. (A) Structure of ³H-BAY 58-2667. (B) Saturation binding of ³H-BAY 58-2667 on sGC in the absence and presence of ODQ (10 μM). (C) Competition binding of ³H-BAY 58-2667 on sGC in the absence and presence of ODQ (10 μM) by BAY 58-2667. Unspecific binding was maximum 20% of total binding. Each value represents the mean±s.e.mean from four independent experiments performed in duplicate.

Figure 3

Photoaffinity labelling. (A) Structure of the ³H-photoaffinity label (³H-PAL). (B) Autoradiogram of photoaffinity-labelled sGC after separation by SDS – PAGE (10%). lane 1: 15 μg (100 pmol) sGC with 5 μCi ³H-PAL after irradiation; lane 2: as lane 1 with 15 μCi ³H-PAL; lane 3: as lane 1 with 50 μCi ³H-PAL; lanes 4 – 6: as lanes 1 – 3, but in the presence of a 50 fold excess of PAL. (C) control, as lane 3 but without irradiation. (C) Autoradiogram of photoaffinity-labelled sGC after separation by SDS – PAGE (10%). Control: 10 μg sGC with 15 μCi ³H-PAL after irradiation: lane 1: as control with a 50 fold surplus PAL; lane 2: as control with BAY 41-2272 (250 μM), lane 3 as control with DEA/NO (2.5 μM); lane 4: as control with ODQ (250 μM). (D) Autoradiogram of photoaffinity-labelled sGC after separation by SDS – PAGE (10%). Control: 10 μg sGC with 15 μCi ³H-PAL after irradiation; lane 1: as control with a 50 fold surplus ODQ; lane 2: as control with haem-free sGC (final concentration 0.5% Tween 20). (E) 2D-PAGE of CNBr fragments. The first dimension was performed in a tube gel and the second dimension was run on a 16% Tris/glycine gel. The gel was blotted to a PVDF membrane, Coomassie stained, and autoradiography was performed. Labelled spots were highlighted in red for α-subunit fragments and blue for β-subunit fragments. On the right side the theoretical cleavage fragments of both subunits with the first amino acids are shown. The labelled positions are highlighted in yellow.

Figure 4

Pharmacological effects of BAY 58-2667 in vitro. (A) Inhibition of phenylephrine (30 μg ml⁻¹) induced contractions of isolated rabbit saphenous arteries by BAY 58-2667, BAY 41-2272, SNP and SIN-1. Values are means±s.e.mean of six, 16, eight and seven experiments. (B) Effects of BAY 58-2667 and GTN on phenylephrine-induced contractions of isolated rabbit saphenous arteries from normal and tolerant rabbits. Values are means±s.e.mean of eight and 11 vessels. ^*P<0.05 and ^** P<0.01 vs normal saphenous artery rings. (C) Effect of BAY 58-2667 and GTN on coronary perfusion pressure at the rat heart Langendorff preparation. (D) The inhibitory effects of BAY 58-2667 on U 46619, collagen, ADP, TRAP-6-induced platelet aggregation in human platelet rich plasma and on thrombin-induced platelet aggregation in washed human platelets. The final concentrations of U 46619, collagen, ADP, TRAP-6 and thrombin were 1, 1 – 2, 30 – 50, and 5 μg ml⁻¹, respectively. The effects were expressed as percentage inhibition of platelet aggregation compared to vehicle control. Each value represents the mean±s.e.mean from eight (U46619), 14 (collagen), five (ADP), five (TRAP-6), four (thrombin) experiments.

Figure 5

Pharmacological effects of BAY 58-2667 in vivo. (A) Effect of BAY 58-2667 (0.3, 1.0, 3.0 and 10 mg kg⁻¹ p.o.) and clopidrogel (3.0 mg kg⁻¹ p.o.) on the thrombus formation in the FeCl₃ arterial thrombosis rat model (n=10 per group). ^*** P<0.001 compared with the values in the untreated controls. (B) Rat tail bleeding time after oral administration of BAY 58-2667 (0.3, 1.0, 3.0, and 10 mg kg⁻¹), acetylsalicylic acid (30 mg kg⁻¹) or vehicle (n=10 per group). ^*** P<0.001 compared with the values in the untreated controls. Haemodynamic effects of intravenous bolus injections of (C) GTN (3 μg kg⁻¹) and (D) BAY 58-2667 (30 μg kg⁻¹). The relative effects of each compound on mean arterial blood pressure (MAP), heart rate (HR), diastolic pulmonary artery pressure (PAP), and on mean right atrial pressure (RAP) are shown. Mean arterial blood pressure before administration (at time 0): GTN 90±1.7; BAY 58-2667 89.3±3.6 mmHg; heart rate: GTN 103±5.9; BAY 58-2667 97.3±6.8 beats min⁻¹, diastolic pulmonary artery pressure: GTN 7.7±1.1; BAY 58-2667 7.5±1.1 mmHg; mean right atrial pressure: GTN 2.8±0.4; BAY 58-2667 2.6±0.5 mmHg (n=4 per group). (E) Effect of orally administered BAY 58-2667 (1.0, 3.0 and 10 mg kg⁻¹) on mean arterial blood pressure and (F) on heart rate of spontaneously hypertensive rats. Values depicted represent changes in heart rate and mean arterial blood pressure (n=6 per group). The heart rate at baseline was 318±16, 288±9, 297±7 and 302±6 b.p.m., respectively, and the mean arterial blood pressure at baseline was 122±11, 130±10, 130±5 and 139±7 mmHg, respectively. Values are mean±s.e.mean.