Reference Work Entry

Encyclopedia of Molecular Pharmacology

pp 28-37

Adenylyl Cyclases

  • Roger A. JohnsonAffiliated withDepartment of Physiology and Biophysics, State University of New York

Abbreviations Used Within the Text

Ado

adenosine

cAMP

adenosine‐3′:5′ monophosphate

2′‐d‐Ado

2′‐deoxyadenosine

2′‐d‐2‐F‐Ado

2′‐deoxy‐2‐fluoro‐adenosine

3′‐d‐Ado

3′‐deoxyadenosine (cordycepin)

2′,5′‐dd‐Ado

2′,5′‐dideoxyadenosine

2′,3′‐dd‐Ado

2′,3′‐dideoxyadenosine

2′,5′‐dd‐2‐F‐Ado

2′,5′‐dideoxy‐2‐fluoro‐adenosine

2′,5′‐dd‐2,5′‐di‐F‐Ado

2′,5′‐dideoxy‐5′‐fluoro‐2‐fluoro‐adenosine

9‐CP‐Ade

9‐(cyclopentyl)‐adenine

9‐THF‐Ade

9‐(tetrahydrofuryl)‐adenine (SQ22,536)

9‐Ara‐Ade

9‐(arabinofuranosyl)‐adenine

9‐Xyl‐Ade

9‐(xylofuranosyl)‐adenine

2′‐d‐Xyl‐Ade

9‐(2‐deoxyxylosyl)‐adenine

2′,5′‐dd‐Xyl‐Ade

9‐(2,5‐dideoxyxylosyl)‐adenine

2′‐d‐3′‐AMP

2′‐deoxyadenosine‐3′‐monophosphate

2′‐d‐3′‐ADP

2′‐deoxyadenosine‐3′‐diphosphate

2′‐d‐3′‐ATP

2′‐deoxyadenosine‐3′‐triphosphate

2′‐d‐3′‐AMPS

3′‐(thiophosphoryl)‐2′‐deoxyadenosine

2′,5′‐dd‐3′‐AMP

2′,5′‐dideoxyadenosine‐3′‐monophosphate

2′,5′‐dd‐3′‐ADP

2′,5′‐dideoxyadenosine‐phos‐phate‐3′‐diphosphate

2′,5′‐dd‐3′‐ATP

2′,5′‐dideoxyadenosine‐3′‐triphosphate

2′,5′‐dd‐3′‐A4P

2′,5′‐dideoxyadenosine‐phos‐phate‐3′‐tetraphosphate

2′,5′‐dd‐3′‐AMPS

3′‐(thiophosphoryl)‐2′,5′‐dideoxyadenosine

5′‐APP(CH2)P

adenosine 5′‐(β(‐methylene)‐triphosphate

β‐L‐5′‐ATP

β‐L‐adenosine‐5′‐triphosphate

β‐L‐2′,3′‐dd‐5′‐ATP

β‐L‐2′,3′‐dideoxyadenosine‐5′‐triphosphate

PMEA

9‐[(2‐phosphonylmethoxy(ethyl)]‐adenine

PMEApp

9‐[(2‐diphosphorylphosphonylmethoxy(ethyl)]‐adenine

PMEAp(NH)p

9‐[(2‐iminodiphosphorylphosphonylmethoxy(ethyl)]‐adenine

PMPA

9‐[(2‐phosphonylmethoxy)propyl]‐adenine

PMPApp

9‐[(2‐diphosphorylphosphonylmethoxy(propyl)]‐adenine

2′,5′‐dd‐3′‐AMP‐bis(Me‐SATE)

2′,5′‐dideoxyadenosine‐3′‐(acetyl‐2‐thioethyl)‐phosphate

2′,5′‐dd‐3′‐AMP‐bis(t‐Bu‐SATE)

2′,5′‐dideoxyadenosine‐3′‐(pivaloyl‐2‐thioethyl)‐phosphate

2′,5′‐dd‐3′‐AMP‐bis(Ph‐SATE)

2′,5′‐dideoxyadenosine‐3′‐(phenyl‐2‐thioethyl)‐phosphate

2′,5′‐dd‐2F‐Ado‐3′‐P‐bis(Me‐SATE)

2′,5′‐dd‐2‐fluoro‐adenosine‐3′‐(acetyl‐2‐thioethyl)‐phosphate

MANT‐5′‐GTPγS

3′‐(2′)‐O‐N‐methylanthraniloyl‐guanosine‐5′‐[γ‐thio]triphosphate

MANT‐5′‐ITPγS

3′‐(2′)‐O‐N‐methylanthraniloyl‐inosine‐5′[γ‐thio]triphosphate

MANT‐5′ATP

3′‐(2′)‐O‐N‐methylanthraniloyl‐5′‐ATP

MANT‐5′GTP

3′‐(2′)‐O‐N‐methylanthraniloyl‐5′‐GTP

3′‐MANT‐2′‐d‐5′‐ATP

3′‐O‐N‐methylanthraniloyl‐2′‐deoxy‐5′‐ATP

3′‐7M4AMC‐2′‐d‐5′‐ATP

3′‐(7‐methoxy‐4‐aminomethylcoumarinn)‐2′‐deoxy‐5′‐ATP

3′‐Dansyl‐2′‐d‐5′‐ATP

3′‐(dansyl)‐2′‐deoxy‐5′‐ATP

2′,5′‐dd‐3′‐ADP‐(β‐7M4AMC)

2′,5′‐dideoxyadenosine‐{β‐(7‐methoxy‐4‐aminomethyl‐coumarin)}‐3′‐diphosphate

2′,5′‐dd‐3′‐ATP‐(γ‐7M4AMC)

2′,5′‐dideoxyadenosine‐{γ‐(7‐methoxy‐4‐aminomethyl‐coumarin)}‐3′‐triphosphate

2′,5′‐dd‐3′‐ATP‐(γ‐7A4AMC)

2′,5′‐dideoxyadenosine‐{γ‐(7‐amino‐4‐aminomethyl‐coumarin)}‐3′‐triphosphate

Synonyms

Adenylyl cyclase (preferred); Adenylate cyclase; Adenyl cyclase (original); ATP:pyrophosphate lyase; Cyclizing (E.C.4.6.1.1.)

Definition

Adenosine 3′:5′‐monophosphate (cAMP) regulates effects in all eukaryotic cells, principally through activation of cAMP‐dependent protein kinase (PKA), but also through cAMP‐gated ion channels (CNGs) and select guanine nucleotide exchange factors (Epacs) (Fig. 1). Cellular levels of cAMP levels reflect the balance of activities of adenylyl cyclases (AdCy: 5′‐ATP ⎨ cAMP + PPi) and cAMP phosphodiesteras​es (PDE: cAMP ⎨ 5′‐AMP). Adenylyl cyclases occur throughout the animal kingdom and play diverse roles in cell regulation [1].
https://static-content.springer.com/image/prt%3A978-3-540-38918-7%2F1/MediaObjects/978-3-540-38918-7_1_Part_Fig1-3_HTML.jpg
Adenylyl Cyclases. Figure 1

Synthesis, degradation, and actions of cAMP.

Basic Characteristics

Classes of Adenylyl Cyclases

Adenylyl cyclases belong to the larger class of purine nucleotide cyclases. These have been divided into classes I–VI [2]. Class I cyclases include those in gram negative bacteria, e.g. Escherichia coli and Yersinia pestis, in which cAMP levels respond to external nutrient levels and mediate effects on transcription factors and gene expression. Class II cyclases comprise the extracellular soluble toxins of certain pathogens (see below under “Bacterial and other adenylyl cyclases”), e.g. those of Bacillus anthracis, Bordetella pertussis, and Pseudomonas aeroginosa. Class III cyclases (with subclasses IIIa–IIId) include most adenylyl and guanylyl cyclases (Fig. 2). These enzymes respond to changes in the extracellular environment. In mammals this occurs via hormones, neurotransmitters, odorants, or tastes. In lower organisms influences may be via changes in ionic factors, glucose, bicarbonate, or serum factors, and through osmoregulation, chemotaxis, phototaxis, or pH, in various bacteria. Class IV–VI cyclases are tentative assignments with few members each, but contain soluble and smallest adenylyl cyclases (∼180 amino acids). The Class IV adenylyl cyclase of Y. pestis has been crystallized and its structure determined. The enzyme in Prevotella ruminicola was designated as a Class V cyclase [2]. And the enzyme found in the nitrogen‐fixing bacterium Rhizobium etli was designated as a Class VI adenylyl cyclase.
https://static-content.springer.com/image/prt%3A978-3-540-38918-7%2F1/MediaObjects/978-3-540-38918-7_1_Part_Fig2-3_HTML.jpg
Adenylyl Cyclases. Figure 2

Domain organization of several class III adenylyl cyclases. Domains are abbreviated: CHD: cyclase homology domains for subclasses (IIIa – IIId), predicted transmembrane domains are indicated as vertical blue bars, BLUF: blue light receptor with tightly bound flavin, CHASE: (Cyclase/histidine kinase‐associated sensing extracellular) domain, GAF switch domain: cGMP‐binding phosphodiesterases, Adenylyl cyclases, and E. coli transcription factor FhlA, Rec: receiver domain; HisK: histidine kinase domain, 2Fe‐2S: two iron‐two sulfur cluster domain, HAMP: tandem amphoteric α‐helices present in Histidine kinases, Adenylyl cyclases, Methyl‐accepting chemotaxis proteins, and Phosphatases, AI: auto‐inhibitory domain, leucine‐rich repeats are indicated a vertical grey bars, PP2C: protein phosphatase type 2C catalytic domain, RAS, RAS‐associating domain, and CAP: cyclase activating protein. This figure was modified from [2].

Mammalian Adenylyl Cyclases

Of the ten known isozymes of mammalian adenylyl cyclase (AC1‐AC10; Table 1) all but one are membrane‐bound and are regulated via cell‐surface receptors linked to heterotrimeric (αβγ) stimulatory (Gs) and inhibitory (Gi) guanine nucleotide‐dependent regulatory proteins (G‐proteins) (Fig. 3, Table 2) [1]. These receptors are referred to as G‐protein coupled receptors, or GPCRs, and mediate effects of stimulatory and inhibitory hormones and neurotransmitters. Gαs stimulates all of these isozymes save the soluble AC10, and the plant‐derived diterpene, forskolin, stimulates all isozymes, save AC9 and AC10, by binding within the cleft formed by the enzyme’s two cytosolic domains (C 1 C 2 ; see below). The several isozymes differ more significantly in their responses to Gαi and Gβγ and in the physiological responses they control (Table 2). For example, Gαi inhibits some but not all isozymes and Gβγ inhibits AC1 and AC8, but significantly stimulates AC2, AC4, and AC7, and has reportedly different effects on AC5 and AC6 (Table 2).
https://static-content.springer.com/image/prt%3A978-3-540-38918-7%2F1/MediaObjects/978-3-540-38918-7_1_Part_Fig3-3_HTML.jpg
Adenylyl Cyclases. Figure 3

Membrane localization, topology, and regulation of mammalian adenylyl cyclases.

Adenylyl Cyclases. Table 1

Source, accession numbers, size, and gene loci for mammalian adenylyl cyclases

ADCY

Source

Reference accession numbers

Base pairs (mRNA)

Amino acids

Respective species chromosome

 

1

Bovine

NM_174229

3978

1134

4

 
 

Human

NM_021116

12499

1119

7p13‐p12

 
 

Mouse

NM_009622

12259

1118

11 1.25 cM

 

2

Rat

NM_031007

4008

1090

17p14

 
 

Human

NM_020546

6553

1091

5p15.3

 
 

Mouse

NM_153534

4211

1095

13 41.0 cM

 

3

Rat

NM_130779

4533

1144

6q14

 
 

Human

NM_004036

4342

1144

2p24‐p22

 
 

Mouse

NM_138305

3674

1145

12A‐B

 

4

Rat

NM_019285

3357

1064

15p13

 
 

Human

NM_139247

3320

1077

14q12

 
 

Mouse

NM_080435

3414

1077

14D3

 

5

Rat

NM_022600

4847

1262

11q22

 
 

Human

NM_183357

3842

1261

3q13.2‐3q21

 
 

Mouse

NM_001012765

7069

1262

16B‐5

 

6

Rat

NM_012821

6036

1166

7q36

 
 

Human

NM_015270 (1)

6594

1168

12q12‐q13

 
 

Human

NM_020983 (2)

5877

1115

12q12‐q13

 
 

Mouse

NM_007405

6038

1168

15F

 

7

Mouse

NM_007406 (1)

5199

1099

8 40.0 cM

 
 

Mouse

NM_001037723 (2)

4750

1099

8 40.0 cM

 
 

Mouse

NM_001037724 (3)

5938

1099

8 40.0 cM

 
 

Human

NM_001114

6138

1080

16q12‐q13

 

8A

Rat

NM_017142

4601

1248

7q33

 
 

Human

NM_001115

6005

1251

8q24.2

 
 

Mouse

NM_009623

5064

1249

15 37.5 cM

 

9

Mouse

NM_009624

4457

1353

16 2.0 cM

 
 

Human

NM_001116

7732

1353

16p13.3

 

10

Human

NM_018417

5061

1610

1q24

 

Sacy

Mouse

NM_173029

5211

1614

1 H2.3

 
 

Rat

NM_021684

5177

1608

13q23

 

Notes to Table 1:

(a) Adenylyl cyclases have been numbered in the order in which they were cloned and sequenced. In databases they are referred to as adcy#, with the exception of the soluble AC10, which is referred to variably as Sacy or Sac.

(b) Sources are those for which the database entries are given. The first source listed is the original source from which the isozyme was cloned. In some instances there are variant forms as indicated. For AC8 there are three splice variants; data are provided for variant 8A.

(c) Accession numbers are for the Reference Sequence (RefSeq) collection of data from the National Center for Biotechnology Information (NCBI). Values pertinent to the mammalian isozymes of adenylyl cyclases are compiled here. The link is: http://​www.​ncbi.​nlm.​nih.​gov/​

These numbers link to a “…comprehensive, integrated, nonredundant set of sequences, including genomic DNA, transcript (RNA), and protein products for several major research organisms. RefSeq standards serve as the basis for medical, functional, and diversity studies; they provide a stable reference for gene identification and characterization, mutation analysis, expression studies, polymorphism discovery, and comparative analyses. RefSeqs are used as a reagent for the functional annotation of some genome sequencing projects, including those of human and mouse.”

Adenylyl Cyclases. Table 2

Regulatory characteristics of mammalian adenylyl cyclases

AdCy

Effect of Gαi

Effect of Gβγ

Effects of Ca2+and/or calmodulin

Effects of protein kinases

Tissue distribution

Physiological functions

 

1

No Δ PKA

Brain (neuron), adrenal (medulla)

Neurotransmission, synaptic plasticity, LTP, memory,circadian rhythm

 

2

No Δ

No Δ PKA ↑ PKC

Brain, lung, skeletal muscle

Synaptic plasticity, arrest of cell proliferation

 

3

no Δ

↑ (In vitro)

 

Olfactory epithelium, brain, adrenal, adipose, pancreas

Olfactory response to odorants

 

4

 

No Δ

 

ubiquitous

  

5

↑↓

↓ (No CaM)

↓ PKA ↑ PKCα/ζ

Heart, brain (striatum)

Cardiac function, Ca2+‐dependent regulation

 

6

↑↓

↓(No CaM)

↓ PKA ↓ PKC

Heart, kidney, Brain, liver, widespread

Cardiac function, Ca2+‐dependent regulation, hormonal regulation of gluconeogenesis, cell proliferation, coincidence detector for NO

 

7

 

No Δ

↑ PKC

Brain, platelets, heart, spleen, lung

Ethanol dependency

 

8

 

 

Brain, lung

Neurotransmission, LTP, synaptic plasticity, memory

 

9

  

↓ (Ca2+/calcineurin)

↓ PKC

Skeletal muscle, heart, brain, pancreas

Neurotransmission

 

10

No Δ

No Δ

↑ (no CaM)

 

Testes (germ cells), widespread

HC03 - sensor; defect associated with absorptive hypercalciuria

 

Notes for Table 2:

(a) Empty cells imply that no information was available. Effects of additions on adenylyl cyclase activity are as indicated: up (↑) arrow: increase, down (↓) arrow: decrease, or “no Δ” (tested, but no effect on activity seen).

(b) Effects of Gαi or Gβγ are on enzyme stimulated by either Gαs or forskolin. In some instances differences were noted in the effects of different isoforms of β or γ. These are not distinguished here. For AC5 and AC6 both stimulation and inhibition have been reported, the difference being conditionally dependent on stimulation and whether or not full‐length enzyme has been expressed.

(c) For effects of Ca2+ and/or calmodulin, stimulation of adenylyl cyclase by Ca2+ usually requires calmodulin, except in the case of AC10 [Sacy; no calmodulin (CaM)]. All adenylyl cyclases are inhibited by high (mM) concentrations of Ca2+, through competition with divalent cation required for catalysis (cf. Figs. 6 and 7). The inhibition indicated here occurs with low (<μM) concentrations of Ca2+, without calmodulin, but with AC9 Ca2+ inhibition is with calcineurin.

(d) Ca2+/calcineurin has been observed to inhibit mouse AC9 but not human AC9.

(e) AC10 (Sacy/Sac), a soluble adenylyl cyclase discovered in testes is widely distributed and functions as a HC03 -ion sensor. It is also stimulated by Ca2+, independently of calmodulin.

(f) LTP: long term potentiation in neuronal function, PKA: cAMP‐dependent protein kinase, PKC: protein kinase C, CaM: calmodulin.

Stimulation and inhibition of the enzyme by the GPCR‐G‐protein cycle occur by analogous mechanisms. Agonists induce hormone receptors to increase a Gα‐GDP‐GTP exchange and subsequent Gαβγ dissociation (GDP•αxβγ + GTP ⎨ GTP–αx + βγ + GDP) (Fig. 4). Consequently, agents that affect either the dissociation of either Gi or Gs, or the association of their respective αs, αi, or βγ subunits with adenylyl cyclase could affect rates of cAMP formation in enzyme preparations or in intact cells and tissues. There are several important examples. Gαs is stably activated by poorly hydrolyzable analogs of GTP, e.g. GTPγS or GPP(NH)P, and it activation is hindered by GDPβS. A less obvious example is fluoride. It activates most mammalian adenylyl cyclases, indirectly through its AlF4 complex with Gαs•GDP. Another example includes the ADP‐ribosyltransferase activities of bacterial toxins. The toxin of Vibrio cholerae catalyzes the ADP‐ribosylation from NAD of GTP•Gαs, and that of Bordetella pertussis similarly ADP‐ribosylates αi of GDP•αiβγ, preventing its dissociation. In both cases the effect is elevated adenylyl cyclase activity and contributes to the pathophysiology of these bacteria. Of therapeutic relevance, of course, are agents acting as agonist or antagonist on GPCRs coupled to adenylyl cyclase, with the prominent example being antagonists of β‐adrenergic receptors (i.e. β‐blockers).
https://static-content.springer.com/image/prt%3A978-3-540-38918-7%2F1/MediaObjects/978-3-540-38918-7_1_Part_Fig4-3_HTML.jpg
Adenylyl Cyclases. Figure 4

Regulation of adenylyl cyclases by G‐proteins. Abbreviations: Hs, Hi, Rs, and Ri denote hormones and receptors that lead to stimulation or inhibition, respectively, of adenylyl cyclases, Ca and Ci are active and inactive configurations of adenylyl cyclase, Fo: forskolin binding site, Gs and Gi are GTP‐dependent regulatory proteins comprising their respective αs, αi, and βγ subunits.

Activities of all isozymes are affected by Ca2+. At higher concentrations (mM) Ca2+ is inhibitory through competition with divalent cation required for catalysis (see below). At lower concentrations (<μM), Ca2+ regulates activity physiologically. This can be through: (i) a direct effect at the catalytic active site, increasing activity of AC10, or decreasing activity of AC5 and AC6; (ii) as a Ca2+/calmodulin complex, activating ACs 1,3,8; (iii) with calcineurin to inhibit AC9; or (iii) indirectly through activation of PKC. Phosphorylation of adenylyl cyclase varies among the isozymes and is determined by differences in their primary sequences and is catalyzed by cAMP‐dependent protein kinase (PKA) or protein kinase C (PKC) (Protein Kinase C) (Table 2). Activity of adenylyl cyclases can be indirectly influenced by the specific phosphorylation of hormone receptors or of G‐proteins.

Membrane‐bound forms of mammalian adenylyl cyclases exhibit a putative topology with twelve membrane‐spanning regions and two largely homologous ∼40 kDa cytosolic domains (C 1 and C 2 ) (cf. Fig. 2). Differences in N‐terminal and other domains are significant and influence regulation by a variety of agents as noted above (cf. Table 2). AC5(C 1) and AC2(C 2 ) domains have been separately expressed, recombined, and the resulting structure was solved in complex with GTP•Gαs (Fig. 5) [3]. αs•GTP activates the enzyme through interaction with C 2 , yielding the active enzyme: Inhibition of adenylyl cyclase may occur by interaction of Gαi with the C 1 domain of adenylyl cyclase, yielding GTP•αiC, or by the recombination of βγ with Gαs. The structure obtained with β‐L‐2′,3′‐dd‐5′‐ATP allowed the demonstration that the pseudo symmetric cleft formed by the C 1 C 2 domains binds 5′‐ATP, forskolin, and cation at two sites [4]. The active site shares topology and reaction mechanism with guanylyl cyclases, with which there is considerable homology, and with oligonucleotide polymerases.
https://static-content.springer.com/image/prt%3A978-3-540-38918-7%2F1/MediaObjects/978-3-540-38918-7_1_Part_Fig5-3_HTML.jpg
Adenylyl Cyclases. Figure 5

Catalytic cleft and active site of a chimeric mammalian adenylyl cyclase. The cleft is formed by the pseudosymmetric interaction of enzyme cytosolic domains, C1 and C2. Panel A: part of the crystal structure of the chimeric adenylyl cyclase AC5C1•AC2C2 with Gαs, indicating binding sites for substrate (5′ATP) and forskolin (FSK). The Switch II domain of Gαs interacts with the C2 domain of adenylyl cyclase. Panel B: the catalytic active site modeled with 5′‐ATP and amino acids involved in catalysis; based on structure with β‐L‐2′,3′‐dd‐5′‐ATP (C). Panel C: structure with β‐L‐2′,3′‐dd‐5′‐ATP and loci for two metal sites, A and B. Panel D: enlargement of C with Zn2+ (metal A) and Mn2+ (metal B) used in forming the crystal. Catalysis occurs with the metal catalyzed attack of the ribosyl 3′‐OH group of the substrate α‐phosphate. Adapted from [3].

Although there is substantial homology among the membrane‐bound forms of the mammalian adenylyl cyclases, the striking differences in the character and extent of regulation by a variety of agents imply that primary and secondary structural characteristics are important determinants in the interactions of the enzyme with cell constituents and hence will regulate enzyme activity, the rate of formation of cAMP, and the downstream effects that this will have. All the studies on mammalian adenylyl cyclases notwithstanding it is uncertain if all forms and variants of the enzyme have been identified, whether all modes of regulation have been determined, when during development, cell life cycles, and cell–cell interactions that specific isozymes are expressed, and how these processes are regulated. Perhaps because of this, the enzyme family continues to be a focus of much research and even as targets for drug discovery.

Catalytic Mechanism

Catalysis by adenylyl cyclases involves cation‐mediated attack of the 3′‐OH on the α‐phosphate of 5′‐ATP, with PPi as leaving group. It is a reversible bireactant sequential mechanism with free cation and cation •5′‐ATP as substrates and cAMP, cation•PPi, and free cation as products (Fig. 6; transition state is depicted as EE*). Cation participation in catalysis through two sites was predicted from enzyme kinetics and was later confirmed in the solved enzyme structure (Fig. 5) [3]. Available data suggest that for some isozymes substrate binding and product release are ordered and for others random. Typically, reaction velocities are considerably greater with Mn2+ as cation than with Mg2+. Maximal velocities observed with various ATP analogs follow the order: 2′‐d‐5′ATP > ATP > ATPγS > APP(NH)P > APP(CH2)P. Km values for rat brain cyclase are: KMnATP, ∼9μM; KMn 2+, ∼4μM; KMgATP, ∼60μM; and KMg 2+, ∼860μM. Notably, activation of adenylyl cyclases by hormones or by Gαs, via the active enzyme configuration GTP•αsC, causes a reduction in KMg 2+ of more than an order of magnitude to ∼50μM, without a change in KMgATP.
https://static-content.springer.com/image/prt%3A978-3-540-38918-7%2F1/MediaObjects/978-3-540-38918-7_1_Part_Fig6-3_HTML.jpg
Adenylyl Cyclases. Figure 6

Adenylyl cyclase catalytic cycle. Points during the catalytic cycle of adenylyl cyclases at which inhibition by competitive and noncompetitive nucleotides occur; E* represents the catalytic transition state.

Miscellaneous Observations

Since its first description, adenylyl cyclase has been an intensely investigated enzyme family. Consequently, numerous observations have been made of agents that affect its activity, principally in isolated membranes, but also of purified enzyme. Some of these effects would be of importance for investigators intending to work with the enzyme. First, typical enzyme preparations, whether from native or recombinant sources, are of membranes or membrane extracts that contain enzyme activities that can alter concentrations of substrate or product of adenylyl cyclases. These include activities of cyclic nucleotide phosphodiesterases, ATPases, among others, that must be taken into consideration in assays of adenylyl cyclase activities. In addition, it has been universally observed that the enzyme is protected by thiols, with β‐mercaptoethanol, 2,3‐dimercaptopropanol, and dithiothreitol being the most commonly used. Conversely, adenylyl cyclases are generally susceptible to oxidants, e.g. H2O2, (IC50 ∼3μM) and benzoquinone (IC50 ∼3μM), and alkylating agents, e.g. N‐ethylmaleimide (IC50 ∼100μM), p‐aminophenylarsenoxide (IC50 ∼40μM), p‐aminophenyldichloroarsine (IC50 ∼80μM), or o‐iodosobenzoate (IC50 ∼10μM for AC1 against calmodulin stimulation). Not surprisingly, the crude membrane‐bound enzyme is susceptible to thermal inactivation (e.g. 50% inactivation at 35° in 10 min) and purified enzyme is more labile, but protection is afforded by forskolin, substrate, P‐site ligands, Ca2+/calmodulin (e.g. with AC1), and by GTPγS•Gαs. Proteases also elevate adenylyl cyclase activity. For example, acrosin, trypsin, and thrombin can cause 5–10‐fold activation, and these exhibit some isozyme selectivity (AC2 > AC3 >> AC5). The basis for this activation in each case is not clear, though serine proteases are known to cleave Gαi, and this could lead to indirect effects on adenylyl cyclase activity.

Bacterial and Other Adenylyl Cyclases

Adenylyl cyclases are found throughout the animal kingdom and serve a variety of roles. Structures of enzyme from but a few of these sources have been determined, although amino acid sequences and domain structures have been deduced for an ever increasing number. Available evidence indicates that there is little sequence homology between these adenylyl cyclases and the membrane‐bound mammalian form. As is evident from the varied domain structure just of class III adenylyl cyclases (Fig. 2) [2], although the enzyme is principally membrane bound in metazoan species, it may or may not be in lower organisms. Furthermore, those forms that are membrane bound are more often than not regulated by means quite different from that described above for mammalian systems.

A comprehensive summary of these enzyme families is beyond the scope of this chapter, but a few examples are worth emphasis. The Class I adenylyl cyclases of the enterobacteria Salmonella typhimurium, Yersinia pestis, and Escherichia coli are membrane bound yet sequences do not give ready evidence of typical transmembrane domains. The enzymes comprise two principal domains, with the catalytic domain being N‐terminal to a glucose‐sensing regulatory domain; the enzyme is inhibited the presence of glucose. Its regulation is coordinated with that of carbohydrate permeases by the phosphoenolpyruvate:sugar phosphotransferase system. This is important for bacterial responses to changes in nutrient levels. In other bacteria, the enzyme may be regulated in response to nutrients and/or it may constitute a toxic factor in mammals, as with Class II forms of adenylyl cyclase of Bordetella pertussis, Bacillus anthracis, Pseudomonas aeruginosa, or Yersinia pestis. These enzymes constitute the ‘toxin class’ of adenylyl cyclases. The well‐studied adenylyl cyclases of Bordetella pertussis and Bacillus anthracis are both soluble, Ca2+/calmodulin‐dependent, but G‐protein independent enzymes that are exported from the respective bacteria. (The adenylyl cyclase of P. aeruginosa is not calmodulin‐dependent.) Because these enzymes are then transported into infected cells, adenylyl cyclase actually constitutes a virulence and toxic factor in mammals. The B. pertussis adenylyl cyclase is a large (1706 amino acids) bifunctional enzyme, the N‐terminal end constituting the adenylyl cyclase activity fused to a C‐terminal end exhibiting hemolytic activity and its capacity for being secreted into external medium. The B. anthracis adenylyl cyclase (800 amino acids), also known as ‘edema factor’ (EF), exhibits four domains, a signal peptide essential for protein secretion, a docking domain allowing binding to the protective antigen (PA), the adenylyl cyclase catalytic site, and a fourth region of unknown function. The B. anthracis adenylyl cyclase has been crystallized and its structure determined.

The Class III adenylyl cyclases are sometimes referred to as the ancestral form of the enzyme and include numerous variants (cf. Fig. 2). Among these there are a couple of noteworthy examples. The adenylyl cyclase of Saccharomyces cerevisiae was the first to have been cloned and sequenced and is a prototypical Class III enzyme, with a sequence in the catalytic domain distinct from those of Class I and Class II enzymes and with the catalytic core located at the C‐terminal part of the protein (Fig 2). In such yeast/fungi (e.g. Candida albicans) the enzyme is membrane bound and is regulated by a G‐protein, in these cases Ras. As in mammalian systems it is involved in metabolic control, in mating responses, but also constitutes a virulence factor. In C. albicans, for example, which contains only one form of the enzyme, the cAMP signaling pathway is essential for hyphae formation and hence virulence. Sequences have been deduced for a number of enzymes of this family, including Schizosaccharomyces pombe, Saccharomyces kluyveri, Trypanosoma brucei, and T. equiperdum, Neurospora crassa, and Dictyostelium discoideum, where the adenylyl cyclase generates the cAMP that provides the signal for aggregation into a multicellular organism and the development of fruiting bodies.

Given that in many of these systems additional proteins and cofactors participate in the regulation of adenylyl cyclase activity, the full elucidation of the roles in which this enzyme activity participates in their growth, development, and function, is a long way off. This notwithstanding, the fact that the mammalian adenylyl cyclases differ so substantially from those of numerous pathogens in which the enzyme is an essential virulence factor gives motive to the idea that new classes of small molecule inhibitors of the pathogen adenylyl cyclases may be discovered that do not interact with mammalian forms of the enzyme.

Drugs

Although agents which indirectly activate or inhibit mammalian adenylyl cyclases are common and are even used in the treatment of disease, especially drugs targeting G‐protein‐coupled receptors, drugs acting directly on the enzyme have been less well explored. And for most compounds acting directly on adenylyl cyclases, high selectivity for specific isozymes has not been demonstrated. The main classes of such agents are derivatives of forskolin and of adenine nucleosides. Adenosine and derivatives of it have long been known to inhibit adenylyl cyclases and it became clear early on that certain modifications afforded substantially increased inhibitory potency. Notable are the approximately threefold increase in potency seen with the 2‐fluorine substitution on adenine and the increases in potency seen with various modifications to the ribose moiety. The orientation of the ribose (α vs β) and the presence, orientation, or absence of hydroxyl groups clearly contribute to inhibitory potency (Table 3). For example, arabinose and xylose differ from ribose only in the orientation of the 2′‐ and 3′‐OH groups yet exhibit markedly different potencies. Whereas 9‐(tetrahydrofuryl)‐Ade (SQ 22,536) and 9‐(cyclopentyl)‐Ade are without hydroxyl groups and are less potent, they offer metabolic and biochemical stability useful for many types of studies. It is, however, the removal of two of the hydroxyl groups, that elicits the largest improvement in inhibitory potency, in particular the 2′,5′‐dideoxy‐ modification (Table 3). With these improvements in potency, these cell permeable compounds, in particular 2′,5′‐dd‐Ado, have become useful research tools and have been used to inhibit adenylyl cyclases and to lower cAMP levels and alter function in numerous studies in isolated cells or intact tissues.
Adenylyl Cyclases. Table 3

Nucleoside inhibitors of adenylyl cyclase. Assays were with a detergent‐dispersed adenylyl cyclase from rat brain and were with 100 μM 5′ATΠ and 5 mM MnCl2 as substrates

Nucleoside

IC50 (μM)

 

β‐Adenosine

82

 

α‐Adenosine

>300

 

9‐(arabinose)‐Ade

30

 

9‐(xylose)‐Ade

3.2

 

9‐(tetrahydrofuryl)‐Ade

20

 

9‐(cyclopentyl)‐Ade

100

 

β‐2′‐d‐Ado

15

 

β‐3′‐d‐Ado (cordycepin)

13

 

β‐2′‐d‐Xyl‐Ade

15.5

 

β‐2′‐d‐2‐F‐Ado

4.6

 

α‐2′‐d‐2′‐F‐Ado

>100

 

β‐2′,3′‐dd‐Ado

9

 

β‐2′,5′‐dd‐Ado

2.8

 

β‐2′,5′‐dd‐Xyl‐Ade

16.4

 

β‐2′,5′‐dd‐2‐F‐Ado

0.89

 

α‐2′,5′‐dd‐2‐F‐Ado

>100

 

β‐2′,5′‐dd‐2,5′‐di‐F‐Ado

0.98

 

α‐2′,5′‐dd‐2,5′‐di‐F‐Ado

29

 
An early observation that 2′‐d‐3′‐AMP was a more potent inhibitor of adenylyl cyclases than 2′‐d‐Ado suggested that the enzyme would accept substitutions at the 3′‐ribose position and that phosphate was particularly well tolerated. This led to the generation of a family of 3′‐phosphoryl derivatives of 2′,5′‐dideoxyadenosine exhibiting ever greater inhibition with the addition of an increasing number of 3′‐phosphoryl groups, the most potent of which is 2′,5′‐dideoxyadenosine‐3′‐tetraphosphate (2′,5′‐dd‐3′‐A4P; Table 4) [5]. These constitute a class of inhibitors historically referred to as P‐site ligands that caused inhibition of adenylyl cyclase that was kinetically either noncompetitive or uncompetitive (cf. Fig. 6). This implied binding of the inhibitor with either a different locus or different configuration than substrate. As it developed, these are configuration selective inhibitors and they provide an exquisite means for inhibition of this signal transduction pathway. We know now that most membrane‐bound forms of the mammalian adenylyl cyclase are inhibited by adenine nucleosides and their 3′‐polyphosphates derivatives. Inhibition by these ligands is conserved with varying sensitivity in all isozymes, save AC10 and those of bacteria.
Adenylyl Cyclases. Table 4

Nucleotide inhibitors of adenylyl cyclase. Enzyme source and assay conditions were as for Table 3. Values obtained for 3′‐ATP are overestimations due to the formation of 2′:3′‐cAMP from 3′‐ATP that occurs nonenzymatically in the presence of divalent cation

Adenine nucleoside 3′‐phosphates (IC50s μM)

 

3′‐phosphate

Ado

2′‐d‐Ado

2′,5′‐dd‐Ado

 

None

82

15

2.7

 

3′ ∼P

8.9

1.2

0.46

 

3′ ∼PP

3.9

0.14

0.1

 

3′ ∼PPP

2

0.09

0.04

 

3′ ∼PPPP

0.011

0.0074

 

3′ ∼PS

3.1

0.6

 

Substrate analogs (IC50s μM)

 

β‐L‐5′‐AMP

200

 

β‐L‐2′,3′‐dd‐5′‐AMP

62

 

β‐D‐5′‐AP(CH2)PP

30

 

β‐L‐5′‐ATP

3.2

 

β‐D‐2′,3′‐dd‐5′‐ATP

0.76

 

β‐L‐2′,3′‐dd‐5′‐ATP

0.024

 

Acyclic 9‐substituted‐Adenines (IC50s μM)

 

PMEA

65

 

PMEApp

0.17

 

PMEAp(NH)p

0.18

 

PMPA

6.3

 

PMPApp

0.5

 

2′‐ and 3′‐Substituted‐5′‐NTPs (IC50s μM)

 

2′(3′)‐MANT‐5′‐GTPγS

0.02

 

2′(3′)‐MANT‐5′‐ITPγS

0.039

 

2′(3′)‐MANT‐5′‐ATP

0.064

 

3′‐MANT‐2′‐d‐5′‐ATP

0.14

 

3′‐d‐2′‐MANT‐5′ATΠ

0.26

 

3′‐7M4AMC‐2′‐d‐5′‐ATP

0.36

 

3′‐Dansyl‐2′‐d‐5′‐ATP

3.21

 

Fluorescent‐phoshoryl‐derivatives (IC50s μM)

 

2′,5′‐dd‐3′‐ATP‐(γ‐7A4AMC)

0.166

 

2′,5′‐dd‐3′‐ATP‐(γ‐7M4AMC)

0.88

 

2′,5′‐dd‐3′‐ADP‐(γ‐7M4AMC)

1.65

 
Probably all adenylyl cyclases are inhibited competitively by substrate analogs, which bind at the site and to the enzyme configuration with which cation‐ATP binds (cf. Fig. 4). One of the best competitive inhibitors is β‐L‐2′,3′‐dideoxyadenosine‐5′‐triphosphate (β‐L‐2′,3′‐dd‐5′‐ATP; Table 4) [4], which allowed the identification of the two metal sites within the catalytic active site (cf. Fig. 4) [3]. This ligand has also been labeled with 32P in the β‐phosphate and is a useful ligand for reversible, binding displacement assays of adenylyl cyclases [4]. The two inhibitors, 2′,5′‐dd‐3′‐ATP and β‐L‐2′,3′‐dd‐5′‐ATP, are comparably potent (Table 4), but inhibit adenylyl cyclase by conformationally distinct mechanisms (cf. Fig. 6) by binding within the catalytic cleft in unique structures (Fig. 7).
https://static-content.springer.com/image/prt%3A978-3-540-38918-7%2F1/MediaObjects/978-3-540-38918-7_1_Part_Fig7-3_HTML.jpg
Adenylyl Cyclases. Figure 7

Structures of potent inhibitors of adenylyl cyclase. Structures for 2′,5′‐dd‐3′‐ATP (IC50 ∼40 nM; noncompetitive inhibitor), β‐2′,3′‐dd‐5′‐ATP with Mg2+ and Zn2+ (IC50 ∼24 nM; competitive inhibitor), and 3′‐MANT‐GTP with Mn2+ (IC50 ∼90 nM; competitive inhibitor) are from coordinates obtained for these compounds in respective crystal structures with AC5C 1 •AC2C 2 . Divalent cations are indicated: Mn2+: purple, Mg2+: green, Zn2+: blue. The 3′‐MANT‐group fits into a hydrophobic pocket of the enzyme. Note the difference in contortion of the phosphate chains in these structures relative to positions for divalent cation.

It has been known for some time that the enzyme tolerated large substitutions to the 3′‐ribose position. This was taken advantage of with the development of 2′(3′)‐O‐MANT‐derivatives of nucleoside 5′‐triphosphates [6] (Table 4). It was surprising, though, that potent inhibition was seen with bases other than adenine, implying that base specificity is less stringent than had been generally assumed. Subsequently, fluorescent derivatives have been made with different fluorophores at 2′‐ and 3′‐positions. 3′‐Substitutions showed advantage over corresponding 2′‐substitutions and 2′(3′)‐O‐MANT‐substitutions were clearly preferable to coumarin and dansyl derivatives, but followed the order of guanosine ≥ inosine > adenosine (Table 4). Fluorescent phosphoryl derivatives were also well tolerated, in particular the 7‐amino‐coumarin. These fluorescent ligands have opened possibilities for investigations of adenylyl cyclase structure, activity, and interactions with other substances not heretofore possible.

Although the 3′‐ and 5′‐polyphosphate derivatives mentioned above exhibit exquisite inhibitory potency these compounds are not cell permeable. To take advantage of the potency of such derivatives for studies with intact cells and tissues, there are two possibilities. One is chemically to protect the phosphate groups from exonucleotidases that also allows the compound to transit the membrane intact. The other is to provide a precursor molecule that is cell permeable and is then metabolized into an inhibitor by intracellular enzymes. The general term for such a compound is prodrug; nucleotide precursors are also referred to as pronucleotides. Families of protected monophosphate derivatives were synthesized, based on β‐L‐ and β‐D‐2′,5′‐dd‐3′‐AMP, β‐L‐2′,3′‐dd‐5′‐AMP, and the acyclic 9‐substituted adenines, PMEA and PMPA. Protective substituents were: (i) ‐(S‐pivaloyl‐2‐thioethyl)= (t‐Bu‐SATE‐); (ii) ‐S‐acetyl‐2‐thioethyl)=(Me‐SATE‐); (iii) ‐(S‐benzyl‐2‐thioethyl)=(Ph‐SATE‐); (iv) ‐cyclosalicyl=(H‐Sal‐); and (v) ‐3‐methyl‐cyclosalicyl=(Me‐Sal‐). Although triphosphate forms of each of the precursor compounds inhibit isolated adenylyl cyclases with IC50s in the nanomolar range, only protected forms of 2′,5′‐dd‐3′‐AMP inhibited cAMP formation in intact cells [7]. Of these the SATE‐derivatives proved the most effective. None of the pronucleotide forms of 2′,5′‐dd‐3′‐AMP inhibited adenylyl cyclase per se, whether isolated from rat brain or OB1771 cells. Nor were identifiable extracellular metabolites of these agents responsible for the drugs’ blocking effects on intact cells. These compounds exhibit all the hallmarks of prodrugs. They are taken up, are deprotected, and are converted to extremely potent inhibitors of adenylyl cyclase, but only by intact cells and tissues. These prodrugs have been used to block cAMP formation in isolated cells and intact tissue and elicit functional effects (Table 5). For example, pretreatment of isolated rat atria with 1 μM 2′,5′‐dd‐3′‐AMP‐bis(t‐Bu‐SATE) completely blocked the positive chronotropic effects of 1 μM epinephrine. It is likely that pronucleotide inhibitors of adenylyl cyclases will find applications in many intact cell systems, as an additional upstream block of the adenylyl cyclase‐cAMP‐PKA signaling cascade, in biochemical, pharmacological, and potentially even therapeutic contexts.
Adenylyl Cyclases. Table 5

Prodrug inhibition of [ 3 H]cAMP formation in intact cells. Cells were prelabeled for 2 h with [3H]adenine before 50 μM forskolin and pronucleotides were added. After a 15 min incubation the newly formed [3H]cAMP was extracted and quantified as in (7)

Pronucleotide

OB‐1771 Preadipocytes

THP1 Monocytes

 

IC50 (nM)

 

2′,5′‐dd‐3′‐AMP‐bis(Me‐SATE)

6.7

260

 

2′,5′‐dd‐2F‐Ado‐3′‐P‐bis(Me‐SATE)

9.8

110

 

Copyright information

© Springer-Verlag Berlin Heidelberg New York 2008
Show all