Inhibitors of membranous adenylyl cyclases

Abstract

Membranous adenylyl cyclases (mACs) constitute a family of nine isoforms with different expression patterns. Studies with mAC gene knockout mice provide evidence for the notion that AC isoforms play distinct (patho)physiological roles. Consequently, there is substantial interest in the development of isoform-selective mAC inhibitors. Here, we review the current literature on mAC inhibitors. Structurally diverse inhibitors targeting the catalytic site and allosteric sites (e.g. the diterpene site) have been identified. The catalytic site of mACs accommodates both purine and pyrimidine nucleotides, with a hydrophobic pocket constituting a major affinity-conferring domain for substituents at the 2'- and 3'-O-ribosyl position of nucleotides. BODIPY-forskolin stimulates ACs 1 and 5 but inhibits AC2. However, so far, no inhibitor has been examined at all mAC isoforms, and data obtained with mAC inhibitors in intact cells have not always been interpreted cautiously enough. Future strategies for the development of the mAC inhibitor field are discussed critically.

Fig. 1. Multiple sequence alignment of the C1 and C2 subunits of mAC isoforms and of sGC α₁- and β₁ subunits

Sequences are identified with human (hAC), murine (muAC) or human soluble GCS (hsGC) and a unique GenBank identification number. The full sequence of AC1 is shown as reference sequence. A, C1 domain of ACs and α₁-subunit of hsGC; B, C2 domain of ACs and β₁-subunit of hsGC. The conserved residues correspond to the C1 and C2 subunits of other AC isoforms and hsGC subunits and are indicated as “.”; amino acid differences are indicated in the one-letter code, and sequence gaps are indicated as “-”. The secondary structures correspond to the C1 from AC5 (VC1) and C2 from AC2 (IIC2) constructs that were determined by X-ray crystallography and are shown above the alignment; arrows represent β strands, and the cylinders represent α helices. Other structural elements, such as random coils and turns, are represented by a solid line. The functional residues in C1 and C2 subunits are indicated: bold, substrate binding; bold/italic, FS binding. The underlined residues indicate the hydrophobic region important for binding to (M)ANT- und TNP groups of 2′,3′-O-ribosyl-substituted nucleotides.

Fig. 1. Multiple sequence alignment of the C1 and C2 subunits of mAC isoforms and of sGC α₁- and β₁ subunits

Sequences are identified with human (hAC), murine (muAC) or human soluble GCS (hsGC) and a unique GenBank identification number. The full sequence of AC1 is shown as reference sequence. A, C1 domain of ACs and α₁-subunit of hsGC; B, C2 domain of ACs and β₁-subunit of hsGC. The conserved residues correspond to the C1 and C2 subunits of other AC isoforms and hsGC subunits and are indicated as “.”; amino acid differences are indicated in the one-letter code, and sequence gaps are indicated as “-”. The secondary structures correspond to the C1 from AC5 (VC1) and C2 from AC2 (IIC2) constructs that were determined by X-ray crystallography and are shown above the alignment; arrows represent β strands, and the cylinders represent α helices. Other structural elements, such as random coils and turns, are represented by a solid line. The functional residues in C1 and C2 subunits are indicated: bold, substrate binding; bold/italic, FS binding. The underlined residues indicate the hydrophobic region important for binding to (M)ANT- und TNP groups of 2′,3′-O-ribosyl-substituted nucleotides.

Fig. 1. Multiple sequence alignment of the C1 and C2 subunits of mAC isoforms and of sGC α₁- and β₁ subunits

Sequences are identified with human (hAC), murine (muAC) or human soluble GCS (hsGC) and a unique GenBank identification number. The full sequence of AC1 is shown as reference sequence. A, C1 domain of ACs and α₁-subunit of hsGC; B, C2 domain of ACs and β₁-subunit of hsGC. The conserved residues correspond to the C1 and C2 subunits of other AC isoforms and hsGC subunits and are indicated as “.”; amino acid differences are indicated in the one-letter code, and sequence gaps are indicated as “-”. The secondary structures correspond to the C1 from AC5 (VC1) and C2 from AC2 (IIC2) constructs that were determined by X-ray crystallography and are shown above the alignment; arrows represent β strands, and the cylinders represent α helices. Other structural elements, such as random coils and turns, are represented by a solid line. The functional residues in C1 and C2 subunits are indicated: bold, substrate binding; bold/italic, FS binding. The underlined residues indicate the hydrophobic region important for binding to (M)ANT- und TNP groups of 2′,3′-O-ribosyl-substituted nucleotides.

Fig. 1. Multiple sequence alignment of the C1 and C2 subunits of mAC isoforms and of sGC α₁- and β₁ subunits

Sequences are identified with human (hAC), murine (muAC) or human soluble GCS (hsGC) and a unique GenBank identification number. The full sequence of AC1 is shown as reference sequence. A, C1 domain of ACs and α₁-subunit of hsGC; B, C2 domain of ACs and β₁-subunit of hsGC. The conserved residues correspond to the C1 and C2 subunits of other AC isoforms and hsGC subunits and are indicated as “.”; amino acid differences are indicated in the one-letter code, and sequence gaps are indicated as “-”. The secondary structures correspond to the C1 from AC5 (VC1) and C2 from AC2 (IIC2) constructs that were determined by X-ray crystallography and are shown above the alignment; arrows represent β strands, and the cylinders represent α helices. Other structural elements, such as random coils and turns, are represented by a solid line. The functional residues in C1 and C2 subunits are indicated: bold, substrate binding; bold/italic, FS binding. The underlined residues indicate the hydrophobic region important for binding to (M)ANT- und TNP groups of 2′,3′-O-ribosyl-substituted nucleotides.

Fig. 2. Crystal structures and general pharmacophore model of VC1·IIC2 in complex with MANT- or TNP-nucleotides or P-site inhibitors

All data presented in this Figure refer to VC1:IIC2. A, Detailed views of the substrate binding site in the complex of Gαs–activated mAC with the competitive inhibitors MANT-GTP (PDB:1TL7) [23], MANT-ATP (PDB:2GVZ) [24], MANT-ITP (PDB:3G82) [24] and TNP-ATP (PDB:2GVD) [25], and two Mn²⁺ ions. Structures of inhibitors as bound to their respective complexes with Gαs·VC1:IIC2 are superimposed. The molecular surface was calculated using PYMOL (DeLano Scientific, San Carlos, CA, USA), based on the atomic coordinates of the Gαs·VC1:IIC2·TNP-ATP complex. Ligands are shown as stick models. Carbon atoms are colored magenta for MANT-GTP, cyan for MANT-ATP, yellow for MANT-ITP, and gray for TNP-ATP, nitrogens blue, oxygens red, sulfur yellow, and phosphorous green; the two Mn²⁺ ions are shown as metallic orange spheres. The secondary structures of VC1 and IIC2 domains are shown in tan and mauve, respectively. Ligands and two metal ions occupy the interdomain cleft between the C1 and C2 domains. Inhibitors prevent transition of the enzyme from the catalytically inactive open conformation to the catalytically active closed conformation because the MANT- and TNP-groups act like rigid body movement-impairing wedges. The MANT- and TNP groups insert into a hydrophobic pocket close to the catalytic site, providing substantial binding energy and giving rise to hydrophobicity-dependent fluorescence increases. Substitution of the 3′-hydroxyl group in MANT- and TNP-nucleotides prevent the 3′:5′-ATP cyclization reaction. B, Comparative views of substrate binding site of the Gαs-activated mAC complex with the prototypical non-competitive/uncompetitive P-site inhibitor, 2′,5′-dideoxy-3′-ATP (PDB:1CUL) [38]. Atoms are colored according to panel A. The Mg²⁺ ions are shown as metallic limeyellow spheres. Note that 2′,5′-dideoxy-ATP, while occupying the catalytic site, in contrast to MANT- and TNP-nucleotides, does not exploit the hydrophobic pocket. Right-most panels show views of the binding pocket for ribose substitutes of inhibitors and are rotated ∼80° relative to the view shown on the left-most panels. The ribose substituents of inhibitor molecules are positioned between the α4 helix of IIC2 and α1- α2 helices of VC1. C, Structures of MANT-ATP, MANT-GTP, MANT-ITP, and TNP-ATP, as bound to their respective complexes with Gαs·VC1:IIC2 are superimposed and colored as above. Average K_i values are indicated, corresponding to contributions from each type of functional group, derived from singular value decomposition analysis (SVD) D, SVD analysis of the κ_i values for independent components from a set of AC inhibitors. SVD analysis was performed as described [16] using published K_i values as basis [22,24,28]. P, monophosphate; PP diphosphate; PPP for triphosphate; PPSP, [γ-thio]triphosphate; PPNP, [β,γ-imido]triphosphate.

Fig. 2. Crystal structures and general pharmacophore model of VC1·IIC2 in complex with MANT- or TNP-nucleotides or P-site inhibitors

All data presented in this Figure refer to VC1:IIC2. A, Detailed views of the substrate binding site in the complex of Gαs–activated mAC with the competitive inhibitors MANT-GTP (PDB:1TL7) [23], MANT-ATP (PDB:2GVZ) [24], MANT-ITP (PDB:3G82) [24] and TNP-ATP (PDB:2GVD) [25], and two Mn²⁺ ions. Structures of inhibitors as bound to their respective complexes with Gαs·VC1:IIC2 are superimposed. The molecular surface was calculated using PYMOL (DeLano Scientific, San Carlos, CA, USA), based on the atomic coordinates of the Gαs·VC1:IIC2·TNP-ATP complex. Ligands are shown as stick models. Carbon atoms are colored magenta for MANT-GTP, cyan for MANT-ATP, yellow for MANT-ITP, and gray for TNP-ATP, nitrogens blue, oxygens red, sulfur yellow, and phosphorous green; the two Mn²⁺ ions are shown as metallic orange spheres. The secondary structures of VC1 and IIC2 domains are shown in tan and mauve, respectively. Ligands and two metal ions occupy the interdomain cleft between the C1 and C2 domains. Inhibitors prevent transition of the enzyme from the catalytically inactive open conformation to the catalytically active closed conformation because the MANT- and TNP-groups act like rigid body movement-impairing wedges. The MANT- and TNP groups insert into a hydrophobic pocket close to the catalytic site, providing substantial binding energy and giving rise to hydrophobicity-dependent fluorescence increases. Substitution of the 3′-hydroxyl group in MANT- and TNP-nucleotides prevent the 3′:5′-ATP cyclization reaction. B, Comparative views of substrate binding site of the Gαs-activated mAC complex with the prototypical non-competitive/uncompetitive P-site inhibitor, 2′,5′-dideoxy-3′-ATP (PDB:1CUL) [38]. Atoms are colored according to panel A. The Mg²⁺ ions are shown as metallic limeyellow spheres. Note that 2′,5′-dideoxy-ATP, while occupying the catalytic site, in contrast to MANT- and TNP-nucleotides, does not exploit the hydrophobic pocket. Right-most panels show views of the binding pocket for ribose substitutes of inhibitors and are rotated ∼80° relative to the view shown on the left-most panels. The ribose substituents of inhibitor molecules are positioned between the α4 helix of IIC2 and α1- α2 helices of VC1. C, Structures of MANT-ATP, MANT-GTP, MANT-ITP, and TNP-ATP, as bound to their respective complexes with Gαs·VC1:IIC2 are superimposed and colored as above. Average K_i values are indicated, corresponding to contributions from each type of functional group, derived from singular value decomposition analysis (SVD) D, SVD analysis of the κ_i values for independent components from a set of AC inhibitors. SVD analysis was performed as described [16] using published K_i values as basis [22,24,28]. P, monophosphate; PP diphosphate; PPP for triphosphate; PPSP, [γ-thio]triphosphate; PPNP, [β,γ-imido]triphosphate.

Fig. 2. Crystal structures and general pharmacophore model of VC1·IIC2 in complex with MANT- or TNP-nucleotides or P-site inhibitors

All data presented in this Figure refer to VC1:IIC2. A, Detailed views of the substrate binding site in the complex of Gαs–activated mAC with the competitive inhibitors MANT-GTP (PDB:1TL7) [23], MANT-ATP (PDB:2GVZ) [24], MANT-ITP (PDB:3G82) [24] and TNP-ATP (PDB:2GVD) [25], and two Mn²⁺ ions. Structures of inhibitors as bound to their respective complexes with Gαs·VC1:IIC2 are superimposed. The molecular surface was calculated using PYMOL (DeLano Scientific, San Carlos, CA, USA), based on the atomic coordinates of the Gαs·VC1:IIC2·TNP-ATP complex. Ligands are shown as stick models. Carbon atoms are colored magenta for MANT-GTP, cyan for MANT-ATP, yellow for MANT-ITP, and gray for TNP-ATP, nitrogens blue, oxygens red, sulfur yellow, and phosphorous green; the two Mn²⁺ ions are shown as metallic orange spheres. The secondary structures of VC1 and IIC2 domains are shown in tan and mauve, respectively. Ligands and two metal ions occupy the interdomain cleft between the C1 and C2 domains. Inhibitors prevent transition of the enzyme from the catalytically inactive open conformation to the catalytically active closed conformation because the MANT- and TNP-groups act like rigid body movement-impairing wedges. The MANT- and TNP groups insert into a hydrophobic pocket close to the catalytic site, providing substantial binding energy and giving rise to hydrophobicity-dependent fluorescence increases. Substitution of the 3′-hydroxyl group in MANT- and TNP-nucleotides prevent the 3′:5′-ATP cyclization reaction. B, Comparative views of substrate binding site of the Gαs-activated mAC complex with the prototypical non-competitive/uncompetitive P-site inhibitor, 2′,5′-dideoxy-3′-ATP (PDB:1CUL) [38]. Atoms are colored according to panel A. The Mg²⁺ ions are shown as metallic limeyellow spheres. Note that 2′,5′-dideoxy-ATP, while occupying the catalytic site, in contrast to MANT- and TNP-nucleotides, does not exploit the hydrophobic pocket. Right-most panels show views of the binding pocket for ribose substitutes of inhibitors and are rotated ∼80° relative to the view shown on the left-most panels. The ribose substituents of inhibitor molecules are positioned between the α4 helix of IIC2 and α1- α2 helices of VC1. C, Structures of MANT-ATP, MANT-GTP, MANT-ITP, and TNP-ATP, as bound to their respective complexes with Gαs·VC1:IIC2 are superimposed and colored as above. Average K_i values are indicated, corresponding to contributions from each type of functional group, derived from singular value decomposition analysis (SVD) D, SVD analysis of the κ_i values for independent components from a set of AC inhibitors. SVD analysis was performed as described [16] using published K_i values as basis [22,24,28]. P, monophosphate; PP diphosphate; PPP for triphosphate; PPSP, [γ-thio]triphosphate; PPNP, [β,γ-imido]triphosphate.

Fig. 3. Model of the interactions of BODIPY-FS and various P-site inhibitors with mAC

Molecular modelling studies were performed using the Surflex module in SYBYL 8.1 (Tripos Associates, St. Louis MO, 2010). To dock BODIPY-FS (in A, B), the Surflex protomol file was defined by residues within 5.0 Å of the known diterpene site plus any cationic residues within 15.0 Å of the diterpene site. To dock non-nucleoside P-site inhibitors (in C), the protomol file was defined by residues within 5.0 Å of the catalytic site. Atoms of each ligand are represented as sticks according to standard CPK coloring except for boron (black; present only in A and B) and carbon atoms (specified below). A, Interaction of BODIPY-FS (white C atoms) with AC5 showing the relative position of co-crystallized ATPαS (green C atoms). B, Interaction of BODIPY-FS (white C atoms) with AC2 showing the relative position of co-crystallized ATPαS (green C atoms). C, Interactions of ATPαS (white carbons), PMC6 (green carbons), AraAde (black carbons), NKY80 (pink carbons) and NB001 (brown carbons) with VC1:IIC2. In all cases above, the receptor surface is represented as a solvent-accessible Connolly surface, colored as follows: lipophilic regions are yellow, polar oxygens are red, polar nitrogens are blue, donatable protons are cyan, and polarized alkyl or aryl moieties are white. Approximate locations of the α-carbon atoms of key residues are labeled for reference. Note that interactions of the P-site inhibitors shown in C with sites in mACs other than the catalytic site cannot be excluded, particularly in light of the fact that our modeling does not explain the reported mAC isoform-specificity of the compounds (see Table 1). Moreover, crystal structures of VC1:IIC2 with the above-mentioned P-site inhibitors, in contrast to MANT-nucleotides, TNP-nucleotides and 2′,5′-dd-3′-ATP (see Fig. 2 and Table 1) are not yet available.

Fig. 3. Model of the interactions of BODIPY-FS and various P-site inhibitors with mAC

Molecular modelling studies were performed using the Surflex module in SYBYL 8.1 (Tripos Associates, St. Louis MO, 2010). To dock BODIPY-FS (in A, B), the Surflex protomol file was defined by residues within 5.0 Å of the known diterpene site plus any cationic residues within 15.0 Å of the diterpene site. To dock non-nucleoside P-site inhibitors (in C), the protomol file was defined by residues within 5.0 Å of the catalytic site. Atoms of each ligand are represented as sticks according to standard CPK coloring except for boron (black; present only in A and B) and carbon atoms (specified below). A, Interaction of BODIPY-FS (white C atoms) with AC5 showing the relative position of co-crystallized ATPαS (green C atoms). B, Interaction of BODIPY-FS (white C atoms) with AC2 showing the relative position of co-crystallized ATPαS (green C atoms). C, Interactions of ATPαS (white carbons), PMC6 (green carbons), AraAde (black carbons), NKY80 (pink carbons) and NB001 (brown carbons) with VC1:IIC2. In all cases above, the receptor surface is represented as a solvent-accessible Connolly surface, colored as follows: lipophilic regions are yellow, polar oxygens are red, polar nitrogens are blue, donatable protons are cyan, and polarized alkyl or aryl moieties are white. Approximate locations of the α-carbon atoms of key residues are labeled for reference. Note that interactions of the P-site inhibitors shown in C with sites in mACs other than the catalytic site cannot be excluded, particularly in light of the fact that our modeling does not explain the reported mAC isoform-specificity of the compounds (see Table 1). Moreover, crystal structures of VC1:IIC2 with the above-mentioned P-site inhibitors, in contrast to MANT-nucleotides, TNP-nucleotides and 2′,5′-dd-3′-ATP (see Fig. 2 and Table 1) are not yet available.