Structure of cinaciguat (BAY 58-2667) bound to Nostoc H-NOX domain reveals insights into heme-mimetic activation of the soluble guanylyl cyclase

Abstract

Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58-2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58-2667. The 2.3-A resolution structure of BAY 58-2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58-2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58-2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58-2667 binding causes a rotation of the alphaF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His(105)-heme covalent bond. The structure provides insights into how BAY 58-2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.

FIGURE 1.

sGC activators and their mechanism of activation. A, chemical structures of three sGC activators: nitric oxide (NO), BAY 58–2667, and protoporphyrin IX. For comparison, the molecular structure of heme, which is not an activator because it can form a covalent inhibitory bond with His¹⁰⁵ via its iron atom, is also shown. B, NO activation of sGC via binding of NO to the distal face of the 5-coordinated (5c) heme leads to breakage of the proximal Fe–His¹⁰⁵ bond, a critical step in NO activation. C, activation of sGC by BAY 58–2667 requires displacement of the heme such that BAY 58–2667 can now occupy the heme pocket. This heme removal is facilitated by weakening of the Fe–His¹⁰⁵ bond by heme oxidation. 6c, 6-coordinated.

FIGURE 2.

Structure of BAY 58–2667 bound to H-NOX. A, Ns H-NOX (gold ribbon diagram) bound to BAY 58–2667 (blue stick structure). Also shown is the side chain of His¹⁰⁵ (stick) that is part of the αF helix. B, electron density of BAY 58–2667. Unbiased 2.3-Å resolution | F_o| − |F_c| omit density (green, contoured at 3σ) and the final refined 2|F_o| − |F_c| density (dark gray, contoured at 1.25σ) are shown. C, stereo figure showing interactions of BAY 58–2667 with nearby H-NOX residues. Hydrogen bonds are shown as dashed lines.

FIGURE 3.

BAY 58–2667 binds to H-NOX heme cavity and mimics heme/protein interactions. A, binding of BAY 58–2667 (cyan sticks and transparent spheres) to heme cavity. B, stereo figure showing superposition of BAY 58–2667 (cyan) and heme (green) as bound to H-NOX structures. (Only the protein for the BAY 58–2667 complex structure is depicted for clarity.) Hydrogen bond interactions by the carboxylates of BAY 58–2667 with the YxSxR motif residues are shown as dashed lines. C, structural comparison of BAY 58–2667 and heme (in their conformation when bound to H-NOX).

FIGURE 4.

H-NOX conformational changes upon BAY 58–2667 activation. A, superposition of BAY 58–2667 H-NOX structure (cyan) onto the heme H-NOX structure (gray). (Heme is depicted as transparent spheres.) Proteins are shown as Cα traces revealing the rotation of the αF helix as illustrated by the broken lines along the helix axes and the black arrows pointing in the direction of the rotation. B, close-up view of the H-NOX shifts upon BAY 58–2667 activation. Most prominent are the ∼0.7-Å downward shift of His¹⁰⁵ in the middle of the αF helix and the ∼1.0-Å shift of Phe¹¹² at the C-terminal end of αF.

FIGURE 5.

Mutagenesis of the postulated communicating activation region of the H-NOX domain in sGC. A, close-up surface view of sGC H-NOX domain homology model showing relative orientation of residues 40–45 and 111–116 (pink). The observed activation shift of helix αF (blue), including residues Ile¹¹¹ (red), would cause subsequent shifts in this region hypothesized to be sensed by the rest of sGC. Surface residues targeted for mutagenesis are shown in red, and the heme is shown in magenta sticks. B, basal (gray) and NO-stimulated (white) WT and sGC mutants expressed in COS-7 cells. Basal activities were similar between WT and mutants but not for R40A (for which basal activity was close to background values). NO-stimulated activity of cytosols showed that mutants have a reduced response to NO stimulation compared with WT. The NO donor SNAP was used at 100 μm. Experiments were repeated three times, from two independent transfections, with each measurement done in duplicate and expressed in pmol/min·mg ± S.E.

FIGURE 6.

Structure-function analysis of BAY 58–2667 derivatives. Close-up view of BAY 58–2667 bound to heme pocket of Ns H-NOX. Hydrogen bonds are depicted as black dashed lines. Heme pocket residues important for explaining the SAR relationships of BAY 58–2667 analogs are also depicted. Individual chemical moieties of BAY 58–2667 are labeled 1–5 and are described under “Discussion.”