International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

1. Overall and Crystal Structures of Nucleotidyl Cyclases.

ACs 1–9 differ with respect to sequence and length (between 1080 and 1353 amino acids). ACs 1–9 are single peptide chains consisting of an N-terminal cytosolic domain of varying length, two membrane-spanning domains (TM1, TM2), each with six transmembrane α-helices, and two cytosolic domains C1 and C2 subdivided into catalytic (C1a, C2a) and regulatory (C1b, C2b) subdomains (Fig. 1). ACs 1–9 are activated by Gα_s, and ACs 1–8 are also activated by FSK. The interface of C1a and C2a contains the ATP binding site and a pseudosymmetric FSK site. C1a and C2a, each with ∼190 amino acids, are structurally homologous to each other, among mAC isoforms and with corresponding catalytic domains of sAC, sAC-C1 and sAC-C2, and of sGC, sGCα_cat and sGCβ_cat.

Our present knowledge about structure, function, and regulation of the catalytic domains of mammalian mACs is mainly based on X-ray structure analysis of recombinant C1a and C2a originating from dog AC5 and rat AC2, respectively. Until now, 17 crystal structures have been released in the Brookhaven Protein Databank (PDB). Table 1 presents an overview of the structures, varying in cocrystallized metal ions and ligands of the ATP binding site.

C1a and C2a were expressed and purified as 25-kDa peptide chains forming heterodimers with similar AC activity and regulatory properties as the holoenzymes, whereas homodimers of C1a or C2a are catalytically inactive. The heterodimeric construct was termed 5C1:2C2 and in all cases cocrystallized with FSK or its methylpiperazino derivative as well as with recombinant Gα_s and GTPγS as Gα_s ligand. ATP-site ligands comprise ATP analogs, P-site inhibitors, and 2',3′-substituted purine nucleotide inhibitors.

sAC exhibits an amino acid sequence and domain organization related more closely to cyanobacteria and myxobacteria nucleotidyl cyclases than to mammalian mACs (Buck et al., 1999). In human sAC, the first 470 amino acids comprise a short N-terminal tail as well as C1 and C2 connected by a linker of about 70 residues (Kleinboelting et al., 2014a). The C-terminal domain of variable length (human sAC ∼1140 amino acids) contains a putative autoinhibitory domain, a P-site, and a protein/protein interaction domain. A potential heme-binding domain (sAC-HD) shows some sequence similarities to Per-ARNT-Sim (PAS) domains known as interaction modules in signaling proteins, and more extensive parts of the C-terminal region slightly resemble STAND domains (Signal Transduction ATPases with Numerous Domains) (Middelhaufe et al., 2012; Steegborn, 2014). No clearly defined transmembrane domain is present in the sAC amino acid sequence. A full-length and a short form of sAC were identified by Western blot of testis extracts with the short form encompassing the C1/C2 domain. Initially, the short form was thought to be generated by proteolysis (Buck et al., 1999), this processing being required for expression of catalytic activity. However, it is now established that alternate splicing generates the testis short form (Jaiswal and Conti, 2001), usually termed truncated sAC (tsAC), as well as isoforms reported in other tissues. Some of these variants lack the C1 domain, whereas others have shortened C termini (Geng et al., 2005; Schmid et al., 2007; Chen et al., 2014a).

The catalytic domain structure of human sAC was resolved by X-ray crystallography. At present, the PDB provides 28 sAC crystal structures (Kleinboelting et al., 2014a,b, 2016; Saalau-Bethell et al., 2014; Ramos-Espiritu et al., 2016), including complexes with ATP, α,β-methylene adenosine-5′-triphosphate, cAMP, pyrophosphate, bicarbonate, biselenite, bisulfite, bithionol, and various inhibitors. Comparing structures of 5C1:2C2, of sAC-C1-C2 and of an inactive human sGCα_cat:sGCβ_cat heterodimer (Allerston et al., 2013; Seeger et al., 2014) indicates high structural homology not only of the individual catalytic domains, but also of the whole complexes, and points to a common, well conserved mechanism of cNMP generation facilitated by two metal ions.

2. General Folds and Alignments of Catalytic Domains.

All crystal structures of 5C1:2C2, sGCα_cat:sGCβ_cat, and sAC C1-C2 show a heterodimeric architecture with a pseudo-twofold axis. In Fig. 2, 5C1 and 2C2 are shown in side-by-side comparison denoting secondary structure elements (Zhang et al., 1997), primed in the case of 2C2. A root mean square deviation (rmsd) of only 1.3 Å for 153 structurally equivalent Cα atom pairs indicates the close similarity of 5C1 and 2C2 (Tesmer et al., 1997). More or less analogous fits result from pairwise superposition of 5C1, sGCα_cat, and sAC-C1 as well as 2C2, sGCβ_cat, and sAC-C2, respectively, and of sGCα_cat with sGCβ_cat as well as sAC-C1 with sAC-C2, in particular if only the sections between β1 (β1′) and α5 (α5′) are considered. In contrast, sequence alignments are less similar even if based on structure, but reflect the close evolutionary relatedness of mAC and sGC catalytic domains (amino acid identities: 5C1 versus sGCα_cat 29%, 2C2 versus sGCβ_cat 27%), whereas sAC is quite distant from the other two enzymes (sAC-C1 versus 5C1 14%, versus sGCα_cat 17%, sAC-C2 versus 2C2 14%, versus sGCβ_cat 15%). The amino acid identity between sGCα and sGCβ is high (42%) in contrast to 5C1 and 2C2 (25%) and to the dissimilar sAC-C1 and sAC-C2 domains (12%).

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Fig. 2.

5C1 and 2C2 in side-by-side comparison of superimposed monomers. α-Helices, red; β-sheets, blue; loops and turns, yellow. Models are based on the crystal structure PDB 1CJU (Tesmer et al., 1997, 1999). Secondary structure elements as well as N and C termini are labeled.

Figure 3 presents 5C1:2C2 in complex with Gα_s:GTPγS, FSK, ATP, and Mg²⁺ (Tesmer et al., 1997). The catalytic domains form a “head-to-tail, wreath-like dimer” (Tesmer et al., 1997) with a pseudo-twofold axis. Perpendicular to this axis and in front, the so-called “ventral” surface (Zhang et al., 1997) is visible (see also Fig. 1). Common of each domain are five α-helices surrounding an eight-stranded β-sheet core. Two strands (β1, β4 and β1′, β4′, respectively) are discontinuous. The 5C1-2C2 interface contains a spacious cavity with two pseudosymmetric binding sites for FSK and ATP + 2 Mg²⁺ ions, respectively. The ligands probably approach from the ventral surface because of extensive 5C1-2C2 contacts at the dorsal side. Gα_s interacts nearly exclusively with 2C2 (α1′-α2′ loop, α2′, α3′, α3′-β4′ loop) and projects widely beyond the ventral surface (Fig. 1), suggesting that the dorsal side faces the membrane (Tesmer et al., 1997).

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Fig. 3.

5C1:2C2 in complex with FSK, ATP, Gα_s, and GTPγS. View along the pseudo-twofold axis. 5C1, green; 2C2, cyan; Gα_s helical domain, yellow; ras-like domain, mauve; atom colors: C, gray; N, blue; O, red; P, orange; Mg²⁺, violet. Models are based on the crystal structure PDB 1CJU (Tesmer et al., 1997, 1999) with ATP docked instead of 2′,3′-dideoxy-ATP. Secondary structure elements (Gα_s only contact sites with 5C1:2C2) as well as N and C termini of 5C1:2C2 are labeled.

The structurally most conserved regions between β1 and β4 as well as β1′ and β4′, including the two metal-ion binding carboxylate residues in 5C1, constitute βαββαβ catalytic cores present also in the Pol 1 family of nucleotide polymerases, which catalyze an attack of a 3′-hydroxyl upon a 5′ phosphate, too (Artymiuk et al., 1997). It may be concluded that mammalian nucleotidyl cyclases have evolved from a very remote ancient nucleotide polymerase ancestor although evolutionary convergence cannot be ruled out.

The architecture of 5C1:2C2 (Fig. 3) is very similar to that of sGCα_cat:sGCβ_cat (Allerston et al., 2013) and, to a lesser extent, sAC-C1-C2 (Kleinboelting et al., 2014a). If only the sequences between β1 and α4 (β1′ and α4′, respectively) are considered, more or less close superpositions are possible (Fig. 4). From these superpositions, a structure-based alignment of the catalytic domains of AC1, AC2, AC5, sAC, and sGC was derived (Fig. 5), being essentially in line with a previous comparison of mACs and sGC, published when only crystal structures of 5C1:2C2 were known (Tesmer et al., 1997), and with a recent alignment of 5C1:2C2, sGCα_cat:sGCβ_cat, and different prokaryotic and eukaryotic sAC-C1-C2 domains (Kleinboelting et al., 2014b). Exact mapping of secondary structure elements was based on 5C1:2C2, but α-helical and β-strand segments between α2 and α4 (α2′ and α4′) are nearly identical in size and position in all cases.

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Fig. 4.

Catalytic domains of sGC and sAC aligned with 5C1:2C2. Domains are drawn from β1 to α4 and β1′ to α4′, respectively. Views along the pseudo-twofold axes. sdGCα_cat, sAC-C1; 5C1, green; sGCβ_cat, sAC-C2; 2C2, cyan; atom colors: C, gray; N, blue; O, red; P, orange; Mn²⁺ and Ca²⁺, magenta; secondary structure elements as well as N and C termini are labeled. (A) sGCα_cat:sGCβ_cat in complex with 2′-MANT-3′-dATP and Mn²⁺, model of the intermediate state based on the open state crystal structure PDB 3UVJ (Allerston et al., 2013) and on 5C1:2C2 PDB 1TL7 (Mou et al., 2005) as template (transparent model). (B) sAC in complex with α,β-methylene adenosine-5′-triphosphate, bicarbonate and Ca²⁺, model based on the crystal structure PDB 4CLK (Kleinboelting et al., 2014a), 5C1:2C2 (transparent model) corresponding to Fig. 3.

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Fig. 5.

Structure-based sequence alignment of the catalytic domains of sAC, sGC, AC1, AC2, and AC5. Secondary structure elements are primarily derived from 5C1 (green) and 2C2 (cyan), after α5 of sAC1, 5C1 and sGCα, only β7 and β8 are common. The sequence numbers above and below the alignment correspond to 5C1 and 2C2, respectively. Amino acids in contact with Gα_s are highlighted in yellow, underscored residues form the C1-C2 interface of 5C1:2C2 and sAC. Bold-colored amino acids belong to the FSK (5C1:2C2) or bicarbonate (sAC) and nucleotide binding sites and interact with FSK or bicarbonate (magenta), metal ions (red), triphosphate (brown), the ribosyl moiety (green), and the nucleobase (blue), respectively. Amino acids in italics contribute via backbone, hydrophobic, or van der Waals interactions to nucleotide binding. Other interactions are electrostatic (including ionic, hydrogen bonds) and via side chains.

Differences are obvious between lengths of various loops and between secondary structure elements in the N- and C-terminal regions. The exceptionally long insert in 2C2 between β3′ and α3′ is not fully resolved in 5C1:2C2 structures and probably involved in interactions with Gβγ (Chen et al., 1995), a characteristic modulator of AC2. The extended β2′-β3′ loop of sAC-C2 contributes to the conversion of the mAC-FSK site into a binding site for the sAC activator bicarbonate (Kleinboelting et al., 2014a). The α1-α2 and α1′-α2′ regions vary within and between the three structures. In 5C1:2C2, α1 is one helical turn longer than α1′ and connected by a hairpin turn with α2. The long, protruding α1′-α2′ loop is in keeping with its role as part of the Gα_s binding site. By contrast, α1 and α1′ are of similar length (two turns) in sGCα_cat and sGCβ_cat, respectively. Helix α1′ is not present in sAC-C2.

The most striking differences between the sequences and structures appear in the C-terminal regions (after α5 and α5′, respectively). In 5C1, a short β7-strand is connected with β8 by a long Ω loop including two short α-helices which are not present in sGCα_cat and sAC-C1. Nevertheless, the course of sGCα_cat in this region roughly resembles that of 5C1. The β7-strand of sAC-C1 is longer, and the course between β7 and β8 differs compared with 5C1. The β7′-β8′ segments of the three structures are more similar. Two extended β-strands are connected by a reverse turn directed to the triphosphate group of the nucleotide.

3. Interaction of C1 Catalytic Domain of AC5:C2 Catalytic Domain of AC2 with α-Subunit of the Stimulatory G-protein of Adenylyl Cyclase Gs.

The interface between 5C1:2C2 and Gα_s is only about half as large as that between 5C1 and 2C2 (Tesmer et al., 1997), and only a few structural elements are involved in interactions (Figs. 3, 5, and 6). The vast majority of contacts are formed by 2C2. The switch II helix of Gα_s is embedded into a groove enclosed by the α1′-α2′ loop, α2′, and the α3′-β4′ loop, which, together with α3′, also contacts α3 and the α3-β5 loop of Gα_s. 5C1 contributes to mAC-Gα_s interactions only at the N terminus (before β1) approaching Gα_s at the switch II helix and the α3-β5 loop. It has been suggested that Gα_s binding (insertion of the switch II helix between the α1′-α2′ loop and the α3′ helix) is necessary for the transition between an inactive, “open” into an active, “closed” 5C1:2C2 state. As a result of this activating conformational change, the β2-β3 loop is slightly shifted toward nucleotide binding elements in β5′ and α4’, leading to proper alignment of the catalytic residues with the ATP substrate (Tesmer et al., 1997).

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Fig. 6.

Detailed 5C1:2C2-Gα_s interactions. 5C1, green; 2C2, cyan; Gα_s, mauve; heteroatom colors: N, blue; O, red; S, yellow. Hydrogen bonds are drawn as red dashed lines. Model based on the crystal structure PDB 1CJU (Tesmer et al., 1997, 1999). Contacting amino acids (distance <3 Å) and secondary structure elements are labeled.

Seven polar interactions predominate among AC-Gα_s contacts, namely hydrogen bonds of side chains of N905 (α1′-α2′ loop) and C911 (α2′) with Q236 (Gα_s switch II) and of E917 (α2′) with W281 (Gα_s α3-β5 loop), a hydrogen bond of the backbone oxygen of H989 (α3′-β4′ loop) with the terminal NH₂ group of N279 (Gα_s α3-β5 loop), a salt bridge between D921 (α2′) and R280 (Gα_s α3-β5 loop), and contacts of the side chains of R913 (α2′) and N992 (α3′-β4′ loop) with N239 and R232 (Gα_s switch II), respectively. The bottom of the binding groove for switch II is crossed by a hydrophobic patch consisting of M378, F379 (before β1), F898 (α1′-α2′ loop) and L914 (α2′). In particular, I235 and F238 of the switch II helix interact with this patch. At the α3-β5 loop of Gα_s, W281 is stacked with 5C1 F379, and L282 contacts 5C1 M378. A second center of hydrophobic interactions emanates from F991 (α3′-β4′ loop) approaching W234 (switch II), L272, and I276 (α3) of Gα_s. This pattern of AC-Gα_s interactions is common for mAC isoforms, because all respective mAC amino acids are conserved (Fig. 5).

Gα_i inhibits AC5 and AC6 noncompetitively with respect to Gα_s (Taussig et al., 1994a), binding at a site that is opposite to that for Gα_s, i.e., between the α1-α2 loop and the α3 helix in the 5C1 domain (Tesmer et al., 1997; Dessauer et al., 1998).

4. C1 Catalytic Domain of AC5:C2 Catalytic Domain of AC2 Interface.

The interface reflects the pseudosymmetry of 5C1 and 2C2. Figure 7 shows the secondary structure segments and amino acids involved in polar and hydrophobic 5C1-2C2 interactions. Contacts (interatomic distances < 4Å) are formed by 28 amino acids of 5C1 and 33 of 2C2 (Tesmer et al., 1997), from which 17 are located at equivalent domain positions and 7 are identical (Fig. 5).

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Fig. 7.

5C1-2C2 interface. Cα-atoms of all amino acids involved in 5C1-2C2 interactions (distance <4 Å, see Fig. 5) are drawn as balls, hydrogen bonds, and salt bridges of labeled residues as sticks. Colors: H-acceptor/acidic side chain, red; backbone O as H-acceptor, orange; H-donor/basic side chain, dark blue; backbone NH as H-donor, light blue; other contacting amino acids and framework of 5C1, green, of 2C2, cyan.

The rough symmetry of interacting secondary structure elements becomes obvious: contacts of α1-α2 (α1′-α2′) with β1′, β4′ and the β5′-α4′ turn (β1, β4, β5-α4 turn) as well as of the C-terminal α2 (α2′) section and β2 (β2′) with the β4′-β5′ (β4-β5) loop. However, explicit interactions of amino acids are rather asymmetric. Only two charge-assisted hydrogen bonds between D424 (α2) and the backbone NH moieties of A1012 and Q1013 (β4′-β5′ loop) are reflected by positionally identical hydrogen bonds of D923 (α2′) with L499 and R500 (β4-β5 loop). The asymmetry of the contacts correlates with the different shape of the FSK and the ATP binding site. The “upper righthand” interface region is characterized by extensive contacts and additionally stabilized by FSK binding. A hydrogen bond between K436 (β2) and the backbone oxygen of T939 (β2′) may be regarded as borderline between this region and the “lower lefthand” interface region with fewer contacts and a larger cavity involving the ATP and metal binding site. Characteristic of the lower region is a hydrogen bond between T401 and K1065, which keeps the β7′-β8′ loop in an “open” position with respect to α1. This contact is broken on nucleotide binding. Gα_s and FSK are suggested to activate mACs by changing the relative orientation of the catalytic domains while preserving nearly all interfacial contacts (Tesmer et al., 1997). Conformational switches are mainly based on a few alternate interactions of flexible segments (loops) of one domain with core components (helices, strands) of the other domain.

The interface in the structure of apo sAC-C1-C2 (Kleinboelting et al., 2014a) corresponds largely to that in 5C1:2C2 (Fig. 5). Thirty-two amino acids of sAC-C1 and thirty amino acids of sAC-C2 participate in contacts. Nineteen residues, five of which are identical, are located at equivalent domain positions. Interactions between secondary structure elements show the same rough symmetry as in case of 5C1:2C2 (see above and Figs. 4B and 7), and also the asymmetry of contacts is similar. The C1-C2 interface of sAC is stabilized by ten hydrogen bonds. Among them, the following five hydrogen bonds overlap with hydrogen or ionic bonds between 5C1 and 2C2 (Fig. 7): Y38 (β1)-E308 (α2′) with K386-E907, E84 (α2)-H398 (β4′-β5′ loop) with D424-A1012, F96 (β2)-K334 (β2′) with K436-T939, G158 (β4-β5 loop)-Q315 (α2′) with G498 - N916 and D159 (β4-β5 loop)-Y318 (α2′) with L499-D923. The structure of apo sAC-C1-C2 resembles the “closed” state of 5C1:2C2 with no contact between α1 and the β7′-β8′ loop.

5. Forskolin Binding Site.

FSK binds in the “upper” pseudosymmetric region of the ventral cleft close to Gα_s (Fig. 3). The binding site is located between β1, β3, and the β5-α4 loop of 5C1 on one hand and α1′ and the β2′-β3′ loop of 2C2 on the other hand. Figure 8 presents the binding mode of FSK in more detail. The backbone oxygen of V506 (β5), the side chain of T512 (α4), and the backbone NH moieties of S508 (β5-α4 loop) and S942 (β2′-β3′ loop) form four direct hydrogen bonds with hydroxy and carboxy groups, respectively, of FSK. The side chain of S942 is further involved in a water-mediated H-bond network bridging the O1 and O4 atoms in FSK. Hydrophobic contacts with V511 (α4), K896 (α1′), I940, G941 (β2′-β3′ loop) and a couple of aromatic amino acids, namely F394 (β1), Y443 (β3), W507 (β5), F895 (α1′), and Y899 (α1′-α2′ loop) complete the binding mode. Whereas FSK stabilizes 5C1-2C2 interactions by one order of magnitude (Sunahara et al., 1997), it does not affect the K_m value of ATP (Dessauer et al., 1997). However, V_max increases 60-fold (Sunahara et al., 1997), indicating that FSK induces a conformational change of the ATP site leading to more efficient cAMP synthesis. Probably contacts between the β2-β3 and the β2′-β3′ loop link amino acids of the FSK site with residues contributing to catalysis (Tesmer et al., 1997).

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Fig. 8.

Detailed 5C1:2C2-FSK interactions. 5C1, green; 2C2, cyan; FSK and water, gray; heteroatom colors: N, blue; O, red. Hydrogen bonds are drawn as red dashed lines. Model based on the crystal structure PDB 1CJU (Tesmer et al., 1997, 1999). Contacting amino acids (distance < 3.5 Å) and secondary structure elements are labeled.

The bicarbonate binding site of sAC overlaps with that of FSK at 5C1:2C2 (see Figs. 4B and 5). According to the structure of human sAC in complex with bicarbonate (Kleinboelting et al., 2014a), the bicarbonate ion forms charge-assisted hydrogen bonds with the backbone NH moieties of V167 (β5) and M337 (β2′-β3′ loop) as well as salt bridges with K95 (β2) and R176 (α4). The bicarbonate binding site of sAC is too small to accommodate FSK (Kleinboelting et al., 2014a).

6. Nucleotide Binding Site.

The nucleotide binding site in the “lower” pseudosymmetric region of the ventral cleft is more spacious than the FSK site, and interfacial contacts are weaker than in the “upper” region. The resulting high conformational flexibility facilitates ATP binding, catalysis, regulation, as well as cAMP and pyrophosphate dissociation. Moreover, not only Gα_s but also ATP binding is necessary to transfer the inactive open into the active closed 5C1:2C2 state. In Fig. 9, an apo-AC 5C1:2C2 structure is aligned with an ATP bound structure (Tesmer et al., 1997). The closed state of the latter results from translocation and 7°-rotation of 5C1 around the center of the heterodimer, closing the catalytic site by moving α1 toward α4′ and the β7′-β8′ loop of 2C2. Nevertheless, all interfacial contacts except two between α1 and the β7′-β8′ loop are conserved in both states. With two inhibitory Ca²⁺ ions instead of Mg²⁺, 5C1:2C2 binds ATP without switching into a closed state (Mou et al., 2009). The substituents of 2′,(3′)-O-(N-methylanthraniloyl) nucleoside 5′-triphosphates and 2′,3′-O-(2,4,6-trinitrophenyl-NTPs bind between α1 and the β7′-β8′ loop, preventing full closure of the catalytic site (Mou et al., 2005, 2006). As a consequence, the respective complexes represent intermediate (between closed and open) states of 5C1:2C2. In case of the sGCα_cat:sGCβ_cat heterodimer, similar differences of an open and a GTP-bound closed conformation have been suggested (Allerston et al., 2013).

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Fig. 9.

Closed and open 5C1:2C2 conformations. Alignment of the complex with ATP, Mg²⁺, and FSK with the open inactive state of apo-5C1:2C2. View along the pseudo-twofold axes. Closed state 5C1, green; 2C2, cyan; open state 5C1, yellow; 2C2, purple; atom colors: C, gray; N, blue; O, red; P, orange; Mg²⁺, violet. Models are based on the crystal structures PDB 1CJU with ATP docked instead of 2′,3′-dideoxy-ATP and 1AZS, respectively (Tesmer et al., 1997, 1999). Unfitting secondary structure elements as well as N and C termini are labeled.

The structural similarity between mammalian mACs, sAC, and sGC in the nucleotide binding regions is exceptionally high. On one hand, common binding modes of ATP, GTP, and metal ions as well as common interactions and structural transformations indicate an evolutionarily well conserved catalytic mechanism of cNMP generation. On the other hand, differences observed in crystal structures result from substrate and inhibitor selectivity and from the flexibility of the ribosyl moiety and the triphosphate chain. The common nucleotide binding region of mACs, sAC and sGC and raw positions of aligned amino acids interacting with ATP and GTP, respectively, are shown in Fig. 10.

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Fig. 10.

Common nucleotide binding site of mAC, sGC and sAC. Essential amino acids are drawn as space fill models of Cα and Cβ atoms; catalytic domain 1, green; catalytic domain 2, cyan; metals A and B, red balls; interactions of metal ions, red; triphosphate, orange; ribosyl moiety, green; nucleobase, blue. The model is based on the crystal structure PDB 1CJU (Tesmer et al., 1997, 1999) with ATP docked instead of 2′,3′-dideoxy-ATP. Common secondary structure elements and amino acids of the binding site are labeled.

Metal-, triphosphate-, ribosyl-, and nucleobase-cyclase interactions are clearly distinguishable (Fig. 5). Two aspartates in β1 and the β2-β3 loop, respectively, coordinate metal ions A and B (Mn²⁺ or Mg²⁺ in native enzymes). The three phosphate moieties interact with conserved basic residues (Arg, Lys) in β4, α4′ and the β7′-β8′ loop. A relatively spacious cavity with the β2-β3 loop as well as β5′ and α4′ as lateral margins and with β2′ and the β2′-β3′ loop as roof accommodates the entire nucleoside moiety. Only two replacements between mACs and sGC, namely Lys by Glu (β2′) and Asp by Cys (β5′), are required to reverse adenine into guanine selectivity (Sunahara et al., 1998; Tucker et al., 1998).

Figure 11A shows detailed interactions between ATP and 5C1:2C2. Both Mg²⁺ ions are coordinated with D396 and D440, MgA additionally with the α-phosphate and the 3′-OH group of ATP, MgB with all phosphate moieties and with the backbone oxygen of I397. The three phosphates are integrated in a compact network of charge-assisted hydrogen bonds and salt bridges: the position and charge of the α-phosphate is stabilized by R1029 (α4′), the β-phosphate interacts with the backbone NH functions of F400 and T401 (α1), and the γ-phosphate with G399 (backbone NH, β1-α1 loop), R484 (β4), and K1065 (β7′-β8′ loop). The ribosyl moiety forms two hydrogen bonds between the ring oxygen and N1025 (α4′) and between the 2′-OH group and S1028 (α4′).

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Fig. 11.

Detailed interactions of mACs and sAC with ATP. C1, green; C2, cyan; ATP, gray; heteroatom colors: N, blue; O, red; P, orange;, hydrogen bonds are drawn as red dashed lines, contacting amino acids (distance <3.5 Å) and secondary structure elements are labeled. (A) 5C1:2C2-ATP interactions, Mg²⁺, violet, model based on the crystal structure PDB 1CJU (Tesmer et al., 1997, 1999) with ATP docked instead of 2′,3′-dideoxy-ATP. (B) sAC-ATP interactions, Na⁺, violet; w, water oxygen; model based on the crystal structure PDB 4USW (Kleinboelting et al., 2014b).

A two-metal-ion catalytic mechanism, previously derived from crystal structures in complex with ATP analogs and various divalent cations (Tesmer et al., 1999), was recently corroborated by density functional theory calculations of 5C1:2C2-ATP complexes (Hahn et al., 2015). Metal A acts as Lewis acid facilitating deprotonation of the 3′-OH group. Water-mediated transfer of the proton to the γ-phosphate is followed by changes in the coordination of both metal ions and in the conformation of ATP. The resulting shorter distance facilitates the nucleophilic attack of the 3′-oxygen on the α-phosphorus atom. The subsequent, concerted phosphoryl transfer step is rate-limiting and includes a transition state tightened by R1029. K1065 contributes to shifting the proton transfer equilibrium.

Binding of the nucleobase to mACs is relatively strong compared with sAC and sGC. The adenine moiety is involved in hydrogen bonds of the N1 nitrogen with K938 (β2′) and of the 6-NH₂ group with D1018 and the backbone oxygen of I1019 (β5′). Van der Waals contacts of the purine nucleus with L438 and G439 (β2-β3 loop) and with F889 (β1′) align the nucleobase between both domains.

The spacious nucleotide binding site of mACs is promiscuous for different nucleobases and accommodates various ligands, e.g., P-site inhibitors, 2′- and 3′-MANT as well as TNP [2′,3′-O-(2,4,6-trinitrophenyl)]-substituted nucleotides, with often high affinity (Seifert et al., 2012). The general binding mode of these inhibitors corresponds to that of ATP, but flexible fit of the phosphate and ribosyl moieties (ring pucker) leads to slightly different and specific interactions. However, NTPs other than ATP do not act as mAC substrates (Sunahara et al., 1998; Hasan et al., 2014), because coherent dynamic transformations rather than tight interactions account for kinetic processes. The high substrate selectivity of mACs with respect to variation of the nucleobase and the triphosphate moiety depends on specific stable and/or transient hydrogen bonds and on distinct flexibility compatible with a concerted catalytic mechanism.

Figure 11B demonstrates similarities and differences of the binding mode of ATP at sAC compared with that at mACs. The structure of the complex (Kleinboelting et al., 2014b) contains only one Na⁺ (or Ca²⁺) ion located somewhat between the reference positions of metals A and B in 5C1:2C2. The triphosphate moiety adopts a specific conformation with partially different interactions than in the case of 5C1:2C2. The α-phosphate interacts with R416 (α4′) and K451 (β7′-β8′ loop); the β-phosphate with S49 (β1), K144 (β4), and Na⁺; and the γ-phosphate forms three hydrogen bonds with G50 (backbone NH, α1), T52 (α1), and N412 (α4′) and contacts also Na⁺. R176 (α4), in fact pointing to the regulatory bicarbonate site, was modeled as in the sAC complex with α,β-methylene-ATP (Kleinboelting et al., 2014a), because this amino acid together with R416 (α4′) probably takes part in catalysis. The adenine base adopts a syn conformation in contrast to the anti conformation in most other mammalian nucleotidyl cyclase structures resolved so far. The 6-NH₂ group is involved in a hydrogen bond with the backbone oxygen of V406 (β5′) and in a water-mediated interaction with K334 (β2′). During catalysis, a transient direct contact between K334 and the 6-NH₂ substituent may be formed (Kleinboelting et al., 2014a). The catalytic constants of ATP and GTP as sAC substrates indicate that base discrimination takes place during turnover rather than by substrate binding (Kleinboelting et al., 2014b). K_m values for ATP for sAC are ∼20 times higher than those for mACs (Kleinboelting et al., 2014a). Figure 11 provides possible reasons: lower affinity of ATP at sAC may be due to fewer explicit hydrogen bonds compared with 5C1:2C2. Specifically, the ribosyl moiety forms no hydrogen bonds corresponding to those with N1025 (sAC N412) and S1028 (sAC A415) in the case of 5C1:2C2.

1. Cellular Localization.

Although many signaling processes can differently alter activity of each AC isoform, there is a single, primary signal output by ACs 1–10, i.e., production of the second messenger cAMP. This simple fact belies both the intuitive notion and the experimental observation that not all AC isoforms regulate identical cellular functions. To explain this complication, one must evoke the concept of cAMP signaling compartmentation (Ostrom et al., 2012; Cooper and Tabbasum, 2014). Indeed, specific AC isoforms localize in different cellular compartments where they associate with a subset of GPCRs and form distinct complexes with downstream effectors. ACs 3, 5, 6, and 8 associate with caveolar and lipid raft microdomains, whereas the other mACs are localized in nonraft plasma membrane domains (Schwencke et al., 1999; Fagan et al., 2000; Ostrom et al., 2000, 2002; Smith et al., 2002). This isoform-specific localization is remarkably stable across different cell types, species, and activation states (Ostrom and Insel, 2004). ACs partner with GPCRs and downstream effectors that are similarly localized, creating discrete signaling complexes. It is attractive to imagine that these complexes allow the common second messenger, cAMP, to generate a wide array of specific responses, even in a single cell. This is especially true when one considers the large number of different GPCR expressed in a single cell that use cAMP as their primary intracellular signal.

GPCRs display some AC-specific coupling that reflects the colocalization of receptors and AC isoforms in lipid raft or nonraft plasma membranes. By using overexpression or knockdown of an AC isoform, specific GPCR-AC coupling was unmasked. AC6 overexpression in a number of cells, including airway smooth muscle, lung fibroblasts, and neonatal myocytes, enhances signaling by β-adrenoceptors (βARs) without affecting signaling by a number of other Gα_s-coupled receptors (Ostrom et al., 2000, 2002; Liu et al., 2008; Bogard et al., 2011). In each case, receptor coupling could be explained by the colocalization or exclusion of the receptor in lipid rafts (where AC6 resides). This concept of colocalization can be even more complex in cardiac myocytes. Because of specialized T-tubule and sarcolemmal membrane environments in these specialized cells, AC and GPCR localization is even more specific. AC5 is localized primarily in T-tubules, whereas AC6 resides in sarcolemmal membranes (Timofeyev et al., 2013). The β₁-adrenoceptor (β₁AR), but not the β₂-adrenoceptor (β₂AR), couples effectively to AC5 (Tsunematsu et al., 2015). This mirrors the differential distribution of the β₁AR and β₂AR in T-tubules (Cros and Brette, 2013).

In airway smooth muscle cells, β₂AR signaling is selectively increased when AC6 is overexpressed and these components colocalize in lipid rafts (Bogard et al., 2011). In contrast, prostanoid EP receptors couple only to AC2 in nonraft domains. Unique cellular responses can be mapped to individual AC isoforms in these cells. By examining gene expression after FSK stimulation in cells overexpressing either AC2 or AC6, interleukin 6 expression is increased by AC2-produced cAMP, whereas somatostatin expression was regulated by cAMP emanating from AC6 (Bogard et al., 2014). Studies using Förster resonance energy transfer-based biosensors to detect cAMP in different subcellular domains have provided direct evidence of these signaling compartments (Agarwal et al., 2011, 2014). These findings show that cAMP is generated in discrete places by individual ACs and that the response machinery in these locations differs enough to yield contrasting regulation in the nucleus.

Specific examples of receptor regulation of AC activity depending on colocalization in membrane domains continue to emerge. Dopamine D1 receptors preferentially regulate AC3, AC5, and AC6 activity and this depends on interaction with lipid rafts, whereas dopamine D5 receptors are more nonraft localized and couple to just AC5 (Yu et al., 2014). Family B GPCRs also seem to depend upon colocalization of receptor and AC, as evidenced by the observation that the pituitary AC-activating polypeptide type 1 receptor was unable to signal to downstream effectors when AC6 (but not AC7) expression was knocked down (Emery et al., 2015). Prostacyclin receptor inhibition of platelet activation depends upon intact lipid rafts (Raslan and Naseem, 2015), which is a known location of both prostacyclin receptor and AC5/6 expression (Liu et al., 2008).

Coupling of GPCRs to specific AC isoforms is highly regulated. In early pregnancy, α₂-adrenoceptors inhibit uterine smooth muscle AC activity but then switch to being stimulatory during midterm pregnancy (Zhou et al., 2000). An upregulation of uterine AC2 expression at about the same time renders it likely that the switch in α₂ adrenoceptor signaling is due to the altered AC isoform expression (Zhou et al., 2007). Signaling by a coexpressed receptor can even dictate coupling between a specific GPCR and AC. Activation of prostaglandin EP1 receptors facilitates dopamine D1 receptor activation of cAMP production (Ehrlich et al., 2013). This effect depends on Gβγ signaling and expression of AC7, a Gβγ-sensitive isoform, and may explain how prostaglandin E2 can regulate neuronal dopamine signaling.

Lipid rafts exert a more complex function than just providing an assembly point for GPCR and ACs. AC regulation by capacitive calcium entry depends on intact lipid rafts and is specific to raft-localized AC isoforms (Cooper et al., 1995; Fagan et al., 1998, 2000; Smith et al., 2002). Disruption of lipid rafts causes a loss of coupling between capacitive calcium entry and calcium-regulated AC isoforms, AC6 and AC8. Thus, lipid rafts facilitate the juxtaposition of calcium-sensitive AC isoforms and capacitive calcium entry channels.

Given that AC isoforms are either lipid raft localized or not, a structural component must exist in all ACs to confer an affinity for one domain or the other. Intuition leads one to consider sequence-specific protein-protein or protein-lipid interactions and posttranslational modifications as likely mechanisms. Evidence of sequence-specific interactions come from studies making chimeras between raft and nonraft isoforms (Crossthwaite et al., 2005) as well as reductionist approaches examining fragments of ACs (Thangavel et al., 2009). Both of these approaches found evidence for roles of the intracellular C1 and C2 domains in targeting ACs to lipid rafts. These studies imply that AC isoforms have key protein-protein interactions that confer their lipid raft localization. However, protein-lipid interactions may be just as important. Mutation of three N-linked glycosylation sites on an extracellular loop of AC8 causes this lipid raft-localized isoform to appear in nonraft membranes (Pagano et al., 2009).

With intracellular protein-protein interactions facilitating lipid raft localization of AC, it is tempting to implicate a known scaffolding protein and resident of these domains, caveolin. However, caveolins are not responsible for anchoring ACs in lipid rafts. Specifically, AC isoforms have the same expression and localization in cardiac fibroblasts isolated from both caveolin-1 knockout and wild-type mice (Thangavel et al., 2009). Additionally, cell lines that naturally lack caveolin expression still display lipid raft-localized AC isoforms (Thangavel et al., 2009).

sAC has been detected in a variety of organelles including nuclei, microtubules, cilia, and mitochondria (Zippin et al., 2003). Functional data supporting the expression of an active enzyme are available particularly for mitochondria and cilia of the airway epithelium. Intramitochondrial sAC activation is associated with an increase in cAMP measured by an immunodetection assay (Acin-Perez et al., 2009) or by monitoring Förster resonance energy transfer of mitochondria-targeted cAMP sensors (Di Benedetto et al., 2013). The increase in cAMP leads to PKA-mediated phosphorylation of respiratory chain enzymes (Acin-Perez et al., 2009; Di Benedetto et al., 2013) and an increase in ATP.

2. Organization of Adenylyl Cyclases with Downstream Effectors.

A long-standing idea is that cAMP signaling is “tailored” in various cell types via the combinatorial expression of AC and PDE isoforms (Houslay and Milligan, 1997). However, a greater level of organization of signaling is achieved by A kinase anchoring proteins (AKAPs). Via their defining PKA binding motif, AKAPs organize cAMP-dependent protein kinase (PKA) signaling to a wide variety of other proteins (Colledge and Scott, 1999). They scaffold the kinase and its substrates and bring together a diverse array of other proteins involved in various signaling pathways. Several diverse members of the AKAP family interact with ACs in an isoform-specific manner and regulate cAMP signaling (Bauman et al., 2006). To date, the specific AKAP-AC interactions that have been defined are AKAP9/Yotiao with ACs 1, 2, 3, and 9 (Piggott et al., 2008), mAKAP with ACs 2 and 5 (Kapiloff et al., 2009) and AKAP79/150 with ACs 2, 3, 5, 6, 8, and 9 (Efendiev et al., 2010; Delint-Ramirez et al., 2011; Shen and Cooper, 2013).

The role of specific AC-AKAP associations in signal transduction was reviewed by Dessauer (2009). AKAP9/Yotiao has complex effects on AC regulation that depend on the AC isoform. AKAP9 has a pure scaffolding function, with no alteration of enzyme activity, when combined with AC1 and AC9 (Piggott et al., 2008). By contrast, this same AKAP inhibits the activity of AC2 and AC3 when these components interact. AC8 forms a complex with AKAP5 (AKAP79/150) and the effect appears to be a decrease in AC8 sensitivity to Ca²⁺ (Willoughby et al., 2010). But many more elements of the cAMP signaling cascade can be localized to a given site of signal generation. AKAP5 also interacts with the β₂AR and AC5 and AC6 (in addition, of course, to binding PKA) (Bauman et al., 2006). With this arrangement, PKA rapidly phosphorylates the β₂AR to initiate desensitization, receptor translocation and even G-protein switching (Daaka et al., 1997), but the G-protein switch is discussed controversially (Friedman et al., 2002). PKA also inhibits AC5 and AC6 via phosphorylation (Beazely and Watts, 2006) and alters AC8 sensitivity to store-operated calcium entry; regulation by PKA is facilitated by AKAP5 (Delint-Ramirez et al., 2011). In each case, the AKAP-AC interaction establishes critical feedback regulation in the signaling cascade.

AKAPs can also regulate other ACs in an isoform-specific manner via targeting protein kinase C (PKC) phosphorylation. PKC increases activity of certain ACs (ACs 1, 2, 3, 5, 7) but inhibits the activity of others (AC6) (Sunahara et al., 1996; Lai et al., 1997). AKAP5 orchestrates muscarinic receptor activation of cAMP production by recruitment of PKC to a complex containing the PKC-stimulated AC2 (Shen and Cooper, 2013). PDE4 and PKA are also key elements in the complex. βAR regulation of postsynaptic AMPA-type glutamate receptors in mouse forebrain slices depends upon AKAP5 anchoring both PKA and AC (Zhang et al., 2013). Although the AC isoform in this model was not defined, these studies demonstrate that long-term potentiation is lost without this AKAP5-mediated organization.

AKAP-AC interactions have other pathophysiological implications (Efendiev and Dessauer, 2011). Mutations of the potassium channel KCNQ1 subunit interrupt its binding to Yotiao, causing a loss of PKA regulation of the channel, alteration in the action potential, and arrhythmias (Marx et al., 2002). Moreover, AKAP-AC complexes serve to sensitize bound PKA effectors to the effects of cAMP, shifting the concentration/response curves by two orders of magnitude (Efendiev et al., 2010). Furthermore, PKA/PDE4D feedback contributes to local regulation of ACs (Mika and Conti, 2016).

1. Group I (Adenylyl Cyclases 1, 8, and 3).

1. Group II (Adenylyl Cyclases 2, 4, and 7).

3. Group III (Adenylyl Cyclases 5 and 6).

4. Group IV (Adenylyl Cyclase 9).

Reports for AC9 knockout phenotypes are lacking. However, microRNAs targeting AC9 (miR-142-3p, miR-181a, and miR-181b) point to a decrease in AC9 expression in sepsis (Risoe et al., 2011) and potential roles in regulating cell proliferation in cervical cancer cells and differentiation of promyelocytic leukemia (Yang et al., 2014; Zhuang et al., 2014). Several reports suggest roles for AC9 in immune function. miR-142-3p restricts cAMP production in regulatory T cells (Huang et al., 2009), whereas knockdown or overexpression of AC9 in human neutrophils alters chemotaxis (Liu et al., 2010). In newly diagnosed patients with acute myelogenous leukemia and in those with a disease relapse, AC9 expression is lower than in cases of remission (Zhuang et al., 2014), suggesting that AC9 could be used as biomarker for diagnosis and clinical course of certain leukemia forms. In cervical carcinoma cells, miR-181b downregulates AC9 and promotes proliferation (Yang et al., 2014), pointing to a more general role of AC9 in cancer biology. AC9 is the functionally predominant AC isoform in neutrophils and monocytes (Mahadeo et al., 2007; Brunskole Hummel et al., 2013; Werner et al., 2014, 2016). Finally, association of AC9 with Yotiao and certain potassium channels in heart may point to a role in cardiac repolarization and certain forms of long-QT syndrome (Li et al., 2012). ADCY9 gene polymorphisms are linked to different diseases including asthma (Small et al., 2003; Kim et al., 2011b), pancreatic cancer (Lee and Gyu Song, 2015), mood disorders (Toyota et al., 2002), body weight (Berndt et al., 2013), and susceptibility to malaria (Manjurano et al., 2012; Maiga et al., 2013). ADCY9 gene polymorphisms determine the efficacy of dalcetrapib, increasing high-density lipoprotein, on atherosclerosis (Tardif et al., 2015).

5. Soluble Adenylyl Cyclase (Adenylyl Cyclase 10).

Two mouse knockout lines for this gene have been generated [deletion of the first exon of the sAC gene, which prevents the expression of the C1 domain (C1KO)] (Esposito et al., 2004) and deletion of exons 12–14 that code for the C2 domain (C2KO) (Chen et al., 2013a). Both lines lead to similar phenotypes in the testis, including a complete loss of bicarbonate-sensitive AC activity in the testis and spermatozoa and undetectable cAMP levels in isolated spermatozoa, giving rise to infertility because of defective sperm motility (Esposito et al., 2004; Xie et al., 2006; Chen et al., 2013a). Additional phenotypes of these mouse lines have been reviewed elsewhere (Schmid et al., 2014; Levin and Buck, 2015). Reed et al. (2002) identified 18 base substitutions in the human sAC gene in a population of patients with absorptive hypercalciuria and increased propensity to kidney stones. Additional ADCY10 gene polymorphisms suggest association with oligoazoospermia, asthenozoospermia, osteporosis, and pancreatic cancer (Herrinton et al., 2011).

1. General Aspects.

The FSK binding site in mACs constitutes the classic target for stimulatory drugs. Initially, FSK was characterized in membrane preparations containing a mixture of various mAC isoforms (Daly, 1984). ACs 1–8, but not AC9, are activated by FSK (Premont et al., 1996; Yan et al., 1998; Onda et al., 2001; Cumbay and Watts, 2004) (Table 3). There is marked synergism between Gα_s and FSK with respect to increasing V_max of catalysis, characterized in detail with the 5C1:2C2 system (Sunahara et al., 1997; Mou et al., 2005). For identification of small molecule AC activators, low basal catalytic activity is acceptable.

The lack of FSK-activation of AC9 has been used to identify this isoform as functionally dominant AC in human myeloid cells (Mahadeo et al., 2007). Specifically, human monocytes and neutrophils express AC activity that is effectively stimulated by the β₂AR and histamine H₂ receptor, whereas FSK is almost ineffective at increasing cAMP levels in these cell types (Brunskole Hummel et al., 2013; Werner et al., 2014, 2016). At the mRNA level, several AC isoforms including AC9 are expressed in myeloid cells (Mahadeo et al., 2007; Brunskole Hummel et al., 2013; Werner et al., 2014, 2016). The molecular basis for the FSK resistance of AC9 is attributable to the presence of a tyrosine at position 1082, corresponding to a leucine in other AC isoforms (Yan et al., 1998). FSK can also be coupled to agarose and used as an affinity ligand for purification of mACs (Pfeuffer et al., 1985, 1989; Pfeuffer, 1991).

Despite intense efforts in the AC research community, no endogenous ligand for the FSK binding site has been identified so far (Laurenza et al., 1989; Seifert et al., 2012). Reports on the occurrence of an endogenous FSK-like molecule have not yet been confirmed (Putnam et al., 2007). Thus, the physiologic relevance of the FSK binding site is still elusive.

2. Structure-activity Relationships of Diterpenes.

A limited set of systematic structure-activity relationship studies for FSK was performed at recombinant ACs 1, 2, and 5, representatives of the three major AC families, and the purified catalytic mAC subunits 5C1:2C2 (Table 5). Figure 12 shows the structures of the compounds examined. In the presence of Mn²⁺, which is very often used in AC assays because it confers higher catalytic activity to these systems (Gille et al., 2004; Mou et al., 2005), FSK is the most potent and most efficacious diterpene at all ACs studied (Pinto et al., 2008). AC1 is the most sensitive isoform, but the difference in potency to the least sensitive AC isoform, AC2, is just 10-fold. In general, modifications such as the introduction of a hydrophilic N-methylpiperaziono-γ-butyryloxy group, the removal of the 7-acetyl group or 7,6-isomerization of the acetyl group result in losses in potency and efficacy. Loss of the 9-hydroxyl group in FSK results in decreases in potency and efficacy, particularly for AC2. The removal of the 1-hydroxyl group in FSK is detrimental for AC activation, pointing to the crucial importance of a hydrogen bond between the 1-hydroxyl group and Val506 for activation (Pinto et al., 2008, 2009). Some degree of AC isoform selectivity of FSK and FSK derivatives was also reported in other studies (Sutkowski et al., 1994; Onda et al., 2001), but all available studies are incomplete to some extent, i.e., not all AC isoforms were studied, and complete concentration-response curves were not routinely generated.

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Fig. 12.

Structures of diterpenes. Pharmacological data for selected diterpenes are listed in Table 5. FSK, forskolin; DMB, 7-deacetyl-7-(N-methylpiperazino-butyryloxy); 6A7DA, 6-acetyl-7-deacetyl; 7DA, 7-deacetyl; 9d, 9-deoxy; 1d, 1-deoxy; BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. Diterpenes are highly lipophilic. To increase water solubility, hydrophilc substituents were introduced at the 6′-position (colforsin daropate) and the 7′-position (DMB-FSK) of the diterpene ring. In Japan, colforsin daropate is used clinically for treatment of heart failure. BODIPY-FSK is a fluorescent FSK derivative that shows inverse agonist activity at AC2 and partial agonist activity at ACs 1 and 5.

The poor water solubility constitutes a serious problem in the pharmacological analysis of diterpenes, requiring dimethyl sulfoxide concentrations of up to 3% (w/v) to fully solubilize compounds (Pinto et al., 2008, 2009). Although such high solvent concentrations may be compatible with cell-free AC activity assays, experiments with intact cells generally do not tolerate dimethylsulfoxide concentrations higher than 1% (Brunskole Hummel et al., 2013). The high hydrophobicity of FSK also delays its dissociation from ACs and potentially prolongs drug action (Pinto et al., 2009). One strategy to circumvent these problems is the introduction of water-soluble FSK derivatives (Laurenza et al., 1987; Onda et al., 2001) (Fig. 12).

Not surprisingly, potencies and efficacies of ligands differ to some extent between 5C1:2C2 on one hand and ACs 1, 2, and 5 on the other, but the critical importance of the 1-hydroxyl group for activation was retained (Pinto et al., 2009). Interestingly, fluorescence spectroscopy studies with MANT-nucleotides as sensors revealed that FSK derivatives without the 1-hydroxyl group can still bind to AC (Pinto et al., 2009), suggesting that formation of the hydrogen bond between the 1-hydroxyl group of FSK and Val506 is important for the actual activation step of the enzyme. 1-Deoxy-FSK prevents activation of 5C1:2C2 by FSK, supporting the view that the former compound still binds to the FSK binding site.

The most notable selectivity among FSK derivatives for AC isoforms was observed for BODIPY-FSK, characterized by a large bulky fluorescent substituent at the 7′-position of the diterpene ring. Although BODIPY-FSK is a partial activator of AC1 and AC5, BODIPY-FSK decreases the basal activity of AC2 (Pinto et al., 2008). AC2 is characterized by a high degree of ligand-independent constitutive activity (Pieroni et al., 1995; Pinto et al., 2008). By analogy to the situation described for GPCRs (Seifert and Wenzel-Seifert, 2002), BODIPY-FSK could be considered as inverse agonist at AC2. When Mn²⁺ is substituted by Mg²⁺, potencies and efficacies change, the most remarkable difference being a profound increase in inverse agonistic efficacy of BODIPY-FSK at AC2 (Erdorf et al., 2011). These data also raise the question about the possible physiologic relevance of the high constitutive activity of AC2. The selectivity of BODIPY-FSK for AC2 inhibition, in conjunction with RT-PCR studies and analysis of MANT-nucleotides, was used to define AC2 as the functionally predominant AC isoform in rat B103 neuroblastoma cells (Kinast et al., 2012). Intriguingly, upon loss of hypoxanthine phosphoribosyl transferase, the causative enzyme defect of Lesch-Nyhan disease (Fu et al., 2014), AC2 function is substantially impaired (Kinast et al., 2012).

3. Off-target Effects of Diterpenes.

Thus, although the selectivity of currently available FSK derivatives for defined AC isoforms is far from satisfying, proof of principle has been provided that ligand selectivity could be obtained, particularly by the introduction of large substituents. However, it should be kept in mind that FSK does not only bind to mACs but also to other important pharmacological targets such as glucose transporters, P-glycoprotein multidrug resistance transporters, ion channels, and nuclear receptors such as pregnane X receptor (Joost et al., 1988; Morris et al., 1991; Ding and Staudinger, 2005; Pavlikova et al., 2010; Kinast et al., 2012; Angel-Chavez et al., 2015). Such interactions of FSK are of concern for clinical application, specifically for long-term systemic treatment in conjunction with other drugs, increasing the risk of drug/drug interactions. Large dissociations between K_d values for [³H]FSK binding and EC₅₀ values of FSK for AC activation support the notion that multiple FSK targets exist (Seamon et al., 1984). Limited structure-activity relationship studies have been undertaken to dissociate interaction of FSK analogs with ACs and off-target sites (Joost et al., 1988; Pfeuffer and Pfeuffer, 1989; Appel et al., 1992). When using BODIPY-FSK as a tool, nonspecific effects of the compound should be kept in mind as well (Kinast et al., 2012).

4. Clinical Applications of Diterpenes.

A water-soluble FSK derivative, colforsin daropate hydrochloride, also referred to as NKH477 (Onda et al., 2001), is used for treatment of acute heart failure in Japan (Hosono, 1999) (Fig. 12). Colforsin exhibits moderate selectivity for AC5 relative to ACs 2 and 3, but its complete pharmacological profile at all mAC isoforms is not known (Toya et al., 1998; Onda et al., 2001). However, the primary goal of colforsin development was not as an AC5-selective inhibitor but rather to obtain a compound with high water solubility to prevent unwanted effects in the CNS. Colforsin daropate hydrochloride may also be useful for the treatment of septic syndrome and subarachnoid hemorrhage (Oishi et al., 2012; Suzuki et al., 2012). The local use of FSK for treatment of glaucoma is being explored, too (Majeed et al., 2015). Moreover, the dermatological application of FSK to protect the skin against ultraviolet light and induce persistent tanning is under investigation (Spry et al., 2009; Scott et al., 2012; Sikora et al., 2015). Furthermore, FSK derivatives are explored as anticancer compounds (Ponnam et al., 2014). Lastly, Coleus forskohlii extracts with varying and undefined FSK content are widely and uncritically used as dietary supplement for weight reduction (Rios-Hoyo and Gutierrez-Salmean, 2016). The available clinical evidence (Godard et al., 2005) is insufficient to recommend the use of FSK for treatment of obesity.

1. General Aspects.

Most inhibitory compounds bind to the catalytic site. Such compounds may act either as noncompetitive, activity-dependent inhibitors, so-called P-site inhibitors, or as competitive inhibitors that are actually also activity-dependent (Seifert et al., 2012). Figure 13 shows representative competitive mAC inhibitors [(M)ANT- and TNP-nucleotides] and noncompetitive P-site inhibitors. For more structures and detailed descriptions of individual inhibitor profiles, the reader is referred to Seifert et al. (2012). So far, there is no known selective inhibitor for a given mAC isoform with clinical relevance. In contrast to the development of AC activators (see section B.1), the development of AC inhibitors requires a reasonably high catalytic activity to begin with, because otherwise inhibition cannot be detected analytically. Therefore, all typical AC inhibitor studies are conducted under optimal stimulatory AC assay conditions. Should an AC isoform-selective inhibitor be identified, it would have to be verified that the inhibitor and the corresponding ADCY gene knockout show very similar effects. Conceptually, this is an important control to exclude off-target effects of small molecule inhibitors.

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Fig. 13.

Structures of representative inhibitors targeting the catalytic site of mACs. Pharmacological data for competitive inhibitors are listed in Table 6, and pharmacological data for noncompetitive P-site inhibitors are listed in Table 7. The (M)ANT group can spontaneously isomerize between the 2′- and 3′-ribosyl position, provided that there is only substituent and a free hydroxyl group. The different bases (A, adenine; G, guanine; I, hypoxanthine; X, xanthine; C, cytosine; U, uracil) constitute substituent Y at the ribosyl moiety. The different phosphate chains (mono-, di-, and triphosphate and hydrolysis-resistant phosphorothioates or β,γ-imidophosphates) constitute substituent X at the ribosyl moiety. (M)ANT- and TNP-nucleotides do not cross the plasma membrane and can only be used in cell-free systems or in electrophysiological studies in which compounds are injected into the cell via the patch pipette. Experimental problems with the membrane-permeable P-site inhibitors are the relatively low potency, insufficient selectivity and high lipophilicity, requiring the use of high concentrations of organic solvents.

The catalytic site possesses three pharmacophoric sites, i.e., the polyphosphate-binding site, the base-binding domain, and a hydrophobic pocket between the latter two sites (Mou et al., 2005, 2006; Hübner et al., 2011). In terms of inhibitor affinity, the hydrophobic pocket exhibits the largest impact on inhibitor affinity, followed by the phosphate-binding domain and the base-binding domain (Seifert et al., 2012). Accordingly, the catalytic site of ACs exhibits a broad base specificity and accepts numerous purine and pyrimidine bases, xanthine being an exception for extremely poor binding (Mou et al., 2006).

2. P-site Inhibitors.

The first identified mAC inhibitors were P-site inhibitors; adenosine analogs that are typically noncompetitive or uncompetitive with respect to substrate ATP (Londos and Wolff, 1977). These compounds were characterized in the classic cell-free AC assay with [α-³²]ATP as substrate. Representative P-site inhibitors, ordered by potency, include: 2′,5′-dideoxyadenosine-3′-tetraphosphate > 2′,5′-dideoxy-3′-ATP > 2′,5′-dideoxy-3′-ADP > 2′,5′-dideoxy-3′-AMP > 2′-deoxy-3′-AMP > 3′-AMP > 2′-deoxyadenosine > adenosine (Johnson et al., 1989; Desaubry et al., 1996; Dessauer et al., 1999; Seifert et al., 2012). P-site inhibitors containing a phosphate residue are too hydrophilic to cross the plasma membrane and cannot be used in intact-cell studies. The requirement for an intact adenine ring reflects the fact that P-site inhibitors bind within the same enzyme pocket as substrate ATP (Tesmer et al., 1997; Tesmer et al., 2000). These inhibitors have revealed important insights into the mechanism of AC catalytic activity. Kinetic analysis of the forward and reverse (i.e., formation of ATP from cAMP and PP_i) reactions of mAC show that the cAMP product is preferentially released from the enzyme before PP_i (Dessauer and Gilman, 1997). In the basal state or under low levels of activity, the slow step of the reaction is dictated primarily by the cyclization of ATP to cAMP. But upon activation of the enzyme, the catalytic rate speeds up and the release of PP_i becomes the slow step in the reaction. P-site inhibitors bind to the catalytic pocket of AC and mimic a product-bound transition state of the enzyme that often includes bound PP_i. In the basal state, the catalytic site is generally open. Activators of the enzyme (e.g., Gα_s and FSK) increase the affinity between the C1 and C2 domains and result in a more closed conformation (Whisnant et al., 1996; Yan et al., 1996; Tesmer and Sprang, 1998). Structures of AC with bound P-site inhibitors exhibit an even further closed conformation. This explains the uncompetitive nature of their inhibition; they require substrate binding and catalysis to form an enzyme-PP_i-inhibitor dead-end complex. Moreover, P-site inhibitors are far more potent on stimulated forms of mAC, where release of PP_i is the slow step and the concentration of steady-state enzyme-PP_i complex increases (Dessauer and Gilman, 1997).

P-site inhibitors that have 3′-polyphosphate moieties, such as 2′,5′-dideoxy-3′-ATP, bind in the absence of PP_i but still display noncompetitive kinetics (Desaubry et al., 1996; Desaubry and Johnson, 1998; Tesmer et al., 2000). The 3′-polyphosphates bind in the pyrophosphate pocket of the catalytic site and serve to increase the potency of these molecules by 100–1,000-fold. Because these inhibitors do not require bound PP_i, one might expect a competitive pattern of inhibition, as is observed for ATP analogs (Gille and Seifert, 2003). However, P-site inhibitors still require for binding a conformation that resembles a product-like transition state and thus are more potent on the active forms of the enzyme. The affinity of mAC for all P-site inhibitors increases in the presence of Mn²⁺ versus Mg²⁺, similar to the reduction of the K_m for ATP with Mn²⁺. The tight binding of these inhibitors at the active site renders these important tools for understanding the function of these enzymes and the development of future cell-permeable inhibitors (Seifert et al., 2012).

3. Structure-activity Relationships and Binding Modes of 2′,(3′)-O-(N-Methylanthraniloyl)- and 2′,3′-O-(2,4,6-Trinitrophenyl)-nucleotides.

MANT- and TNP-nucleotides explore all three pharmacophoric domains in ACs and are competitive inhibitors (Gille and Seifert, 2003). Some of the compounds studied exhibit nanomolar affinities that enabled their use for crystallographic studies (Mou et al., 2005, 2006; Hübner et al., 2011) and fluorescence spectroscopy studies monitoring ligand-specific conformational changes (Pinto et al., 2009, 2011; Suryanarayana et al., 2009a) with purified 5C1:2C2. Unfortunately, because of the fact that MANT-nucleotides bind to many proteins with a nucleotide binding site (see, e.g., Remmers et al., 1994; Franks-Skiba and Cooke, 1995; Scheidig et al., 1995; Dayan et al., 1997; Hiratsuka, 2003; Zielke et al., 2015), their potential as starting point for the development of AC isoform-selective drugs is very limited.

The binding modes of 2′- and 3′-MANT as well as TNP-substituted nucleotides at mACs are roughly similar to the binding mode of ATP (Fig. 11). In the complex of 5C1:2C2 with 3′-MANT-GTP (Mou et al., 2005), the ribosyl moiety adopts a 2′-endo conformation with an axial orientation of the MANT group (Fig. 14A). Its wedge-like fit between α1, α2, and the β5′-α4′ loop prevents the formation of the fully closed conformation. The phenyl ring is involved in hydrophobic interactions with L412 (α2) and van der Waals contacts with the backbone of W1020, G1021, and N1022 (β5′-α4′ loop). With a 3′-endo conformation of the ribosyl moiety corresponding to an equatorial 3′- and an axial 2′-orientation, 2′-MANT, and even 2′,3′-bis-MANT nucleotides may bind in a similar mode.

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Fig. 14.

Interactions of 5C1:2C2 with selected MANT- and TNP-nucleotides. 5C1, green; 2C2, cyan; heteroatom colors: N, blue; O, red; P, orange; Mn²⁺, violet. Nucleobase-2C2 hydrogen bonds are drawn as red dashed lines, contacting amino acids (distance < 3.5 Å) and secondary structure elements are labeled. (A) Binding mode of 3′-MANT-GTP, model based on the crystal structure PDB 1TL7 (Mou et al., 2005) C and selected H atoms of 3′-MANT-GTP, gray. (B) Binding mode of TNP-ATP, model based on the crystal structure PDB 2GVD (Mou et al., 2006), C and selected H atoms of TNP-ATP, mauve. The gray shadow image shows 3′-MANT-GTP from a superposition of both binding sites (root-mean-square deviation of Cα atoms, 0.44 Å).

The triphosphate conformation in 3′-MANT-GTP differs from that in ATP. In particular, α- and β-phosphates interact only with metal ions. The γ-phosphate forms three hydrogen bonds with the backbone NH functions of F400 and T401 (α1) and with K1065 (β7′-β8′ loop), but does not contact R484 in contrast to ATP. The ribosyl moieties of 3′−MANT-GTP and ATP also interact differently with 5C1:2C2. In case of 3′-MANT-GTP, a weak hydrogen bond of the 2′-OH group with N1025 (α4′) and distant van der Waals contacts with the side chains of F400 (α1) and V1024 (α4′) are formed. The hydrogen bonds of the adenylyl moiety in the 5C1:2C2-ATP complex with K938 (β2′) and D1018 (β5′) are reproduced by the oxygen, the N1 atom and the 2-NH₂ group, respectively, of the guanine base. In both cases, the base adopts an anti conformation. In contrast, a syn position of the adenylyl moiety and no specific enzyme-base interactions are characteristic for the binding mode of 3′-MANT-ATP in complex with 5C1:2C2 (Mou et al., 2006).

TNP-ATP binds roughly similar to 3′-MANT-GTP (Mou et al., 2006), i.e., the pockets occupied by the three components nucleobase, triphosphate, and substituent are common (Fig. 14B). The fit of the trinitrophenyl ring between L412 (α2) and the backbone of W1020, G1021, and N1022 (β5′-α4′ loop) corresponds to that of the 3′-MANT substituent. The 2-nitro group is involved in a hydrogen bond with the side chain amide of N1022. N1025 (α4′) also interacts with the 2-nitro group and not with the ribosyl fragment, which is almost planar with two axial oxygens in 2′- and 3′-position. Interactions and conformation of the triphosphate moiety of TNP-ATP differ from those of 3′-MANT-GTP with respect to the β- and γ-phosphate. In case of TNP-ATP, the β-phosphate forms two hydrogen bonds with the backbone NH of F400 (α1), and the γ-phosphate interacts with R484 (β4) and K1065 (β7′-β8′ loop). In contrast to 3′-MANT-ATP, the adenine base in TNP-ATP adopts an anti position. A single hydrogen bond is formed between the 6-NH₂ group and the side chain of S1028 (α4′). Taken together, Fig. 14B is suggestive of flexible fits of MANT- and TNP-nucleotides to partially different components of the binding pocket as major cause of mAC promiscuity for competitive inhibitors.

Table 6 lists the structure-activity relationships for selected (M)ANT- and TNP-nucleotides at ACs 1, 2, and 5 as representatives of the three major mAC families, purified catalytic subunits 5C1:2C2, sAC and purified sGCα₁β₁, also referred to as GC1. In general, AC2 is the least sensitive mAC isoform toward TNP- and (M)ANT-nucleotides, but selectivity for ACs 1 and 5 is at best 10-fold (Gille et al., 2004; Suryanarayana et al., 2009a; Pinto et al., 2011). In addition, TNP- and (M)ANT-nucleotides are very potent sGC inhibitors (Gille et al., 2004; Suryanarayana et al., 2009a; Dove et al., 2014). The potent sGC inhibition by (M)ANT-nucleotides is not surprising in light of the high conservation of the catalytic domains of these enzymes (Sunahara et al., 1998; Dove et al., 2014). Given the beneficial clinical effects of allosteric sGC activators and sGC stimulators (Stasch et al., 2011, 2015), sGC inhibition would be clearly an off-target effect of mAC inhibitors (Dove et al., 2014). However, there is evidence for pharmacological distinction between mACs and sGC with (M)ANT- and TNP-nucleotides (Dove et al., 2014) (Table 6).

In addition to nucleotidyl cyclases, (M)ANT- and TNP-nucleotides bind to many other nucleotide-binding enzymes and signal-transducing guanine nucleotide-binding proteins (see e.g., Remmers et al., 1994; Franks-Skiba and Cooke, 1995; Scheidig et al., 1995; Dayan et al., 1997; Hiratsuka, 2003; Zielke et al., 2015), rendering AC specificity difficult. Application of (M)ANT- and TNP-nucleotides in intact cells is also impossible because the compounds are too hydrophilic to penetrate the plasma membrane. Electrophysiological experiments with intracellularly injected MANT-GTP pointed to a role of AC5 in the regulation of voltage-dependent calcium channels in cardiomyocytes (Rottlaender et al., 2007), but studies with the structurally closely related nucleotide MANT-ITP revealed unexpected off-target effects (Hübner et al., 2011b), although MANT-ITP is the most potent mAC inhibitor known so far (Hübner et al., 2011a). Intriguingly, bulky bis-MANT-nucleotides exhibit strong preference for the Bordetella pertussis AC toxin CyaA relative to mACs, constituting the basis for the development of bacterial toxin inhibitors (Geduhn et al., 2011) (Table 6).

4. Membrane-permeable Membrane Adenylyl Cyclase Inhibitors.

The issues with impermeability of the plasma membrane for nucleotides can be avoided by applying membrane-permeable nucleotide prodrugs (Laux et al., 2004), but this strategy has not yet found broad application in the field, leaving many of the most potent AC inhibitors with little cellular utility. As an alternative strategy, membrane-permeable and hence lipophilic nucleoside analogs can be used in intact cell studies. In general, these compounds are much less potent than MANT/TNP-nucleotides because they only explore the base-binding domain of ACs (Seifert et al., 2012; Brand et al., 2013). These compounds act as noncompetitive P-site inhibitors. The lipophilicity of compounds may require high concentrations of organic solvents in cell experiments so that nonspecific solvent effects become a problem (Brunskole Hummel et al., 2013), but this aspect has not been generally considered in cell studies.

Three compounds, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22,536), and vidarabine (also referred to as adenine 9-β-d-arabinofuranoside or 9-β-d-arabinofuranosyl-adenine), have been systematically studied at all nine mAC isoforms, several native membrane systems, and sAC (Brand et al., 2013; Bitterman et al., 2013; Braeunig et al., 2013; Braeunig et al., 2014). Table 7 lists the pIC₅₀ values of these compounds for inhibition of ACs and pK_i values for interaction with adenosine receptors. Some compounds have been studied with defined ACs by several independent research groups. Overall, there is reasonably good agreement in the obtained pIC₅₀ values (Table 7). SQ22,536, NKY80, and vidarabine exhibit some degree of selectivity among AC isoforms, AC2 being relatively insensitive compared with ACs 5 and 6 (Bitterman et al., 2013). ACs 8 and 9 and sAC are very resistant to inhibition by SQ22,536, NKY80, and vidarabine. pIC₅₀ values for sGC of the compounds listed in Table 7 are not available. A recent study reported on the identification of noncompetitive inhibitors with some selectivity for AC2 relative to ACs 1 and 5, compound 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide (SKF-83566) being a representative (Conley et al., 2013). However, SKF-83566 is also a potent dopamine D1 receptor antagonist (O’Boyle and Waddington, 1984).

cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride (MDL 12,330A) was one of the first AC inhibitors to be introduced into experimental pharmacology (Hunt and Evans, 1980) and is still widely used. The compound is membrane-permeable and has been reviewed critically (Seifert et al., 2012). MDL 12,330A cannot be recommended for intact-cell studies because of its low potency and pleiotropic effects. The compound has only been incompletely characterized so far at defined AC isoforms. MDL 12,330A inhibits a lysate of mAC with a pIC₅₀ of 3.4 and purified sAC with a pIC₅₀ of 3.6 (Bitterman et al., 2013). Thus, MDL 12,330A does not discriminate between mACs and sAC, and the potency of the compound is lower than the potency of many membrane-permeable P-site inhibitors (Table 7).

5. Adenylyl Cyclase 1 Inhibitors and Modulation of Calmodulin.

A nucleotidyl cyclase that has attracted considerable attention as drug target is AC1. It was proposed that AC1 inhibition could be used to treat various neuropsychiatric disorders such as neuropathic pain (Wang et al., 2011a). 5-[[2-(6-Amino-9H-purin-9-yl)ethyl]amino]-1-pentanol (NB001) was suggested to inhibit AC1 in intact cells selectively (Wang et al., 2011a), but this proposition was not confirmed with recombinant AC1 in membrane preparations (Brand et al., 2013). Thus, the precise mechanism of action of NB001 remains elusive.

AC1 forms a unique interface with its activator CaM compared with other mammalian CaM targets (Vorherr et al., 1993; Diel et al., 2008; Masada et al., 2012; Lübker and Seifert, 2015; Lübker et al., 2015a,b). Principally, such a unique protein-protein interaction interface could be exploited pharmacologically. In this context, it is interesting to note that calmidazolium chloride, which is very potent at inhibiting numerous CaM-regulated effectors, only very ineffectively inhibits AC1 (Lübker and Seifert, 2015). However, potent and selective inhibitors of the AC1-CaM interaction have not yet been identified in a small-scale screening study (Lübker and Seifert, 2015). Calmidazolium also inhibits AC9, probably by binding to the catalytic site (Haunso et al., 2003). Currently, there is no clinical use of AC1 inhibitors.

Ageing processes are related to the oxidation of methionine residues in CaM (Bigelow and Squier, 2005), and oxidized CaM is less efficient than native CaM at activating AC1 (Lübker et al., 2015a,b). Thus, one could envisage that reduction of oxidized methionine residues in CaM by increased expression of methionine sulfoxide reductase is therapeutically useful in CNS diseases associated with impaired learning and memory (Moskovitz and Oien, 2010; Lübker et al., 2015a,b).

6. Adenylyl Cyclase 5 Inhibitors.

Currently, there is great interest in AC5-selective inhibitors. Based on experiments with AC5 knockout mice (see section II.E.3.a), it has been proposed that selective AC5 inhibition may be beneficial in the treatment of heart failure and other age-related diseases (Vatner et al., 2013; Bravo et al., 2016). However, AC5 is broadly expressed in many organs, including kidney (Erdorf and Seifert, 2011) and brain (Kim and Han, 2009), so that substantial organ toxicity could emerge as a result of global AC5 inhibition (Table 3). Most notably, AC5 inhibition in the brain could result in Parkinson-like symptoms (Iwamoto et al., 2003; Seifert, 2014). In addition, the available competitive and noncompetitive AC inhibitors do not discriminate between AC5 and the closely related AC6 (Gille et al., 2004; Brand et al., 2013) (Table 7). This is supported by the observation that vidarabine shows very similar potency in heart membranes from wild-type mice and AC5 knockout mice (Table 7). The lack of discrimination between ACs 5 and 6 is problematic, because AC6 also plays an important functional role in the heart (Göttle et al., 2009; Tang et al., 2013).

Another complication constitutes the fact that several P-site inhibitors show significant affinity to adenosine receptors (Klotz and Kachler, 2016) (Table 7) and inhibit cellular phosphorylation cascades via an as yet unknown mechanism (Emery et al., 2013), so that interpretation of pharmacological data as AC5 inhibition is very problematic. In some instances, because of solubility issues and low compound potency, it has been impossible to obtain sufficiently high inhibitor concentrations in intact cells to observe AC inhibition (Brunskole Hummel et al., 2013). Similarly, it is questionable whether the required micromolar concentrations of P-site inhibitors can be obtained in the intact organism without off-target effects (Seifert, 2016). Overall, NKY80, SQ22,536, and vidarabine, which are very widely used as “selective mAC inhibitors” (or even more problematic as “selective AC5 inhibitors”) (e.g., Rangel-Barajas et al., 2011; Bravo et al., 2016; Liu and Steketee, 2016; Wada et al., 2016; Wei et al., 2016), should be used with great caution as pharmacological tools, and any observed effects should be carefully checked for off-target actions (Seifert, 2014, 2016). It is particularly important to confirm that the AC inhibitor used actually prevents a GPCR- or FSK-mediated cAMP increase (Brunskole Hummel et al., 2013). Because vidarabine constitutes an FDA-approved antiviral drug, the clinical use of this compound for AC5 inhibition has been proposed (Vatner et al., 2013; Bravo et al., 2016). Unfortunately, vidarabine possesses a high potential for toxic effects so that systemic clinical use would potentially be dangerous (Seifert, 2014, 2016).

A newly emerging potential clinical application of AC5 inhibitors is the treatment of extrapyramidal movement disorders such as FDFM, caused by gain-of-function mutations in the ADCY5 gene (Chen et al., 2012c, 2014b). Thus, reduction of the pathologically increased catalytic activity of the R418W AC5 mutant and A726T AC5 mutant may be beneficial. However, like with wild-type ACs, selectivity of inhibitors is a concern. As a first step toward the development of mutant-selective AC5 inhibitors, a comprehensive pharmacological characterization of AC5 mutants with the available tools (Figs. 12 and 13; Tables 6 and 7) is required.

7. Soluble Adenylyl Cyclase Inhibitors.

In general, MANT- and TNP-nucleotides inhibit purified sAC much less potently than ACs 1, 2, and 5 and purified 5C1:2C2 and sGC (Table 6), but data sets are incomplete. Along the same line, membrane-permeable P-site inhibitors, often used in intact cell studies, inhibit sAC considerably less potently than most mAC isoforms (Table 7). The membrane-impermeable P-site inhibitor 2′,5′-dideoxy-3′-ATP (Fig. 15) is less potent at inhibiting sAC than purified 5C1:2C2 and various mAC isoforms (Table 8), fitting to the data on membrane-permeable P-site inhibitors.

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Fig. 15.

Structures of representative inhibitors targeting sAC. Pharmacological data for inhibitors are listed in Table 8. All compounds are noncompetitive sAC inhibitors. 2CE, KH7, and RU-0204277 are membrane-permeable. KH7 has been broadly used in intact cell studies, but cell toxicity is of concern. RU-0204277 is a recently developed compound with less toxic liability than KH7 and binds to the bicarbonate site of sAC. 2CE exhibits different potencies at purified sAC and in intact cells. 2′,5′-dideoxy-3′-ATP is a classic P-site inhibitor related to SQ 22536, NKY80, and vidarabine (for structures, see Fig. 13 and for pharmacological data see Table 7) that also potently inhibits mACs and cannot penetrate the plasma membrane.

Considerable effort has been directed toward the development of sAC inhibitors to probe the role of sAC in cell physiology. Catechol estrogens are noncompetitive sAC inhibitors, binding to a hydrophobic region in the vicinity of the catalytic site (Steegborn et al., 2005). 2-Hydroxyestradiol (2CE) is a prototypical compound of this class (Fig. 15). 2CE exhibits selectivity toward AC1, 2, and 5, but not AC7 (Table 8). The potency of 2CE in intact cells and cell-free systems differs for as yet unknown reasons (Steegborn et al., 2005; Bitterman et al., 2013).

(±)-2-(1H-Benzimidazol-2-ylthio)propanoic acid 2-[(5-bromo-2-hydroxyphenyl)methylene]hydrazide (KH7) is the most widely used sAC inhibitor in intact-cell studies (Kumar et al., 2009; Schmid et al., 2010; Kolodecik et al., 2012; Bitterman et al., 2013; Dey et al., 2014; Hasan et al., 2014; Nickols et al., 2015; De Rasmo et al., 2015; Sosa et al., 2016). Caution must be exerted when interpreting intact-cell studies with KH7 because of toxic effects of this compound in mitochondria (Di Benedetto et al., 2013) and β cells (Tian et al., 2011). KH7 inhibits purified sAC with similar potency as 2CE and shows selectivity when examined against a lysate containing mACs (Table 8). However, data for individual mAC isoforms are not available. The exact binding site of KH7 on sAC is not known.

6-Chloro-N⁴-cyclopropyl-N⁴-[(thiophen-3-yl)methyl]pyrimidine-2,4-diamine (RU-0204277) has recently emerged as a novel sAC inhibitor from a high-throughput screening strategy (Ramos-Espiritu et al., 2016). RU-0204277 binds to the bicarbonate-binding site of sAC and exhibits potency comparable to the potencies of 2CE and KH7 (Ramos-Espiritu et al., 2016) (Table 8). At a concentration of 50 μM, the substance did not affect ACs 1, 2, 5, 8, and 9. Given the IC₅₀ for sAC of ∼10 μM, it will be necessary to examine inhibitor concentrations of 100–500 μM to assess specificity more comprehensively. Initial studies indicate that RU-0204277 is also useful for intact-cell studies and devoid of toxicity (Ramos-Espiritu et al., 2016).

Inhibition of sGC is a problematic property when aiming at the clinical application of mAC and sAC inhibitors (Stasch et al., 2011, 2015; Dove et al., 2014). Unfortunately, pIC₅₀ values for sGC of the compounds listed in Table 8 are not yet available.