Adenylyl Cyclase

Adenosine 3':5'-monophosphate (cAMP) modifies cell function in all eukaryotic cells, principally through the activation of cAMP-dependent protein kinase (PKA), but also through cAMP-gated ion channels and guanine nucleotide exchange factors directly activated by cAMP. Cellular levels of cAMP reflect the balance between the activities of adenylyl cyclase (ATP: pyrophosphate lyase, cyclizing; EC 4.6.1.1), that catalyzes formation of cAMP from 5'-ATP, and cAMP phosphodiesterases, that catalyze conversion of cAMP to 5'-AMP.

Adenylyl cyclases occur throughout the animal kingdom and play diverse roles in cell regulation. In bacteria, the enzyme may be regulated in response to nutrients or it may constitute a toxic factor in mammals, as with adenylyl cyclases of B. pertussis, B. anthracis, P. aeruginosa, or Y. pestis.

In mammals, the family of adenylyl cyclases is central to one of the most important signal transduction pathways and includes at least ten isozymes. A soluble form (type X) is regulated by HCO₃^– , whereas the others are membrane-bound and are regulated physiologically through cell-surface receptors linked via heterotrimeric (αβγ) stimulatory (Gα_s) and inhibitory (Gα_i) guanine nucleotide-dependent regulatory proteins (G-proteins). Most isozymes are activated by Gα_s, but differ in their regulation by Gα_i and in the effects of Gβγ. These adenylyl cyclases exhibit a putative topology with two tandem repeats of a 6 membrane spanning region and a ~40 kDa cytosolic region. The two cytosolic domains (C₁ and C₂) share large conserved regions that interact to form a cleft forming the catalytic active site. N-terminal domains are variable and serve regulatory roles. Gα_s activates through interaction with the C₂ domain yielding the active enzyme: GTP•α_s •C. Inhibition by G-proteins occur by a direct effect of Gα_i with the C₁ domain or by the recombination of βγ with Gα_s.

Adenylyl cyclase activity is altered by numerous agents of physiological and biochemical interest. These include agents that act indirectly, by effects on hormone receptors, on Gα_s (e.g. cholera toxin) or Gα_i (e.g. pertussis toxin), and agents that act directly on the enzyme, in an isozyme-selective manner. All adenylyl cyclases are inhibited by oxidants and are protected by thiols. Most isozymes are stimulated by forskolin. But only select isozymes are activated by Ca²⁺-calmodulin or regulated by Ca²⁺ ions. Others are inhibited by nitric oxide (types I and VI) or proteins associated with myc, and several are regulated by protein kinases A and C.

The cleft formed by adenylyl cyclase C₁•C₂ domains binds both substrate and forskolin. The active site shares topology and reaction mechanism with guanylyl cyclases, with which there is considerable homology, and with oligonucleotide polymerases. Each catalyzes a cation-dependent attack of the 3'-OH on the α-phosphate of an NTP, with PP_i as leaving group. Adenylyl cyclases exhibit a reversible bireactant sequential mechanism in which free divalent cation and cation-5'-ATP serve as substrates and cAMP, metal-PP_i, and free divalent cation are products.

Although agents that indirectly activate or inhibit adenylyl cyclases are commonly used in the treatment of disease, e.g. β-adrenoceptor blockers, drugs acting directly on the enzyme have only recently been explored. The main classes of which are derivatives of either forskolin or adenine nucleosides. Adenylyl cyclases are inhibited competitively by substrate analogs, the best of which are β-L-2',3'-dd-5’-ATP (IC₅₀ ~24 nM) and, unexpectedly, MANT-5’-GTPγS. Most are also inhibited by adenine nucleoside 3'-polyphosphates, the most potent of which are 2',5'-dd-3’-ATP (IC₅₀~40 nM) and 2’,5’-dd-3’-A4P (IC₅₀ ~7 nM). These latter compounds belong to a class of inhibitors historically called P(purine)-site ligands, which inhibit via a non-competitive, dead-end, post-transition state mechanism. Inhibition by these ligands occurs with varying sensitivity in all isozymes, save those of bacteria and sperm, and they provide an exquisite means for inhibition of this signal transduction pathway.

Cell permeable inhibitors of adenylyl cyclases comprise nucleosides, their derivatives, and recently described pro-nucleotides. The former are effective in the low micromolar range, whereas pro-nucleotides function as prodrugs, with IC₅₀ values in the nanomolar range. These compounds have been used to lower cellular cAMP levels and to alter function in numerous studies with both isolated cells and intact tissues.

Activators

• Forskolin (F6886) - adenylyl cyclase activator
  
• NKH 477 (N3290) - adenylyl cyclase activator (water-soluble)
  
• PACAP-27 (A9808) - neuropeptide that stimulates adenylate cyclase
• PACAP-38 (A1439) - neuropeptide that stimulates adenylate cyclase
  
• Adenylyl Cyclase Toxin from Bordetella pertussis (A0847)- converts host ATP to cyclic AMP (cAMP)
  

Inhibitors

• NB001 (SML0060) - inhibitor of adenylyl cyclase 1 (AC1)
• 9-Cyclopentyladenine monomethanesulfonate (C4479) - stable, cell-permeable, non-competitive adenylyl cyclase inhibitor
• SQ 22,536 (S153) - cell-permeable adenylyl cyclase inhibitor
• MDL-12,330A hydrochloride (M182)- adenylyl cyclase inhibitor
• 2′,5′-Dideoxyadenosine (D7408) - cell-permeable adenylyl cyclase inhibitor
• 2′,5′-Dideoxyadenosine 3′-triphosphate tetrasodium salt (D0939) - potent inhibitor of adenylyl cyclase
• MANT-GTPγS (M6317) - potent and competitive adenylyl cyclase inhibitor
• 2′,3′-Dideoxyadenosine (D1285) - specific adenylyl cyclase inhibitor
• NKY80 (N2165) - selective adenylyl cyclase-V inhibitor
• KH7 (K3394) - selective inhibitor of soluble adenylyl cyclase
  

Other Products

• 1,9-Dideoxyforskolin (D3658) - biologically inactive forskolin analog, useful as a negative control for forskolin

Isozymea (source)	I (bovine)	II (rat)	III (rat)	IV (rat)	V (rat)
Structuresb amino acids:	1134	1090	1144	1064	1262
accession:	M25579	M80550	M55075	M80633	M96159
Chromosome	7p12 (human) 11A2 (mouse)	5p15 (human) 13C1 (mouse)	2p22-24 (human) 12A-B (mouse)	14q11.2 (human) 14D3 (mouse)	3q13.2-q21 (human) 16B5 (mouse)
Tissue Expressionc	Brain Adrenal	Brain Lungs Skeletal muscle	Olfactory Brain Adrenal Adipose Pancreas	Ubiquitous	Heart Brain
Physiological Functiong	Neurotransmission Synaptic plasticity Memory Circadian rhythm	Not known	Olfactory response to oderants	Not Known	Cardiac function
Disease Relevance	Adenylyl cyclases mediate signal transduction in all tissues and changes in cellular cAMP levels occur in disease states or may be induced therapeutically in the treatment of disease. Some bacterial toxins raise cAMP levels. Regulation of type V enzyme may be important in cardiomyopathies or of type VI in mitigating aspects of diabetes. Inhibition of adenylyl cyclases may be a useful strategy in combination therapy for treatment of some cancers and for treatment of yeast and a broad range of fungal infections and some cancers.
G-αs	↑	↑	↑	↑	↑
G-αi	↓	↓	↓	↓d	↓
G-βh	↓	↑	No change	↑	↓
Forskolin Derivatives	Rank order of stimulatory potency: 7-deacetyl-forskolin (D3533) ≈ 6-[3-(dimethylamino)propionyl]forskolini ≈ 6-[N-(2-isothiocyanatoethyl)aminocarbonyl]forskolini ≈ 5,6-dehydroxy-7-deacetyl-7-nicotinoylforskolini > 6-acetyl-7-deacetyl-forskolin > 7-deacetyl-7-O-hemisuccinyl-forskolin >> 1,9-dideoxy-forskolin (D3658)i
Ca2+/ Calmodulin	↑	No change	(↑↓)	No change	↓ (no CaM)
PKC/PKA	↑ (PKC)	↑+++ (PKC)	↑ (PKC)	↓ (PKC)	↑ (PKC) ↓ (PKA)
Competitive Inhibitorsj	Rank-order of inhibitory potency: β-L-2',3'-dd-5'-ATP ≈ MANT-5’-GTPγS > MANT-5’-ITPγS > β-D-2',3'-dd-5'-ATP ≈ MANT-5’-ATP >> β-L-5'-ATP > 5'-AP(CH2)PPk
Non-competitive Inhibitors (P-site ligands)k,l	Rank-order of inhibitory potency: 2',5'-dd-3'-A4P > 2'-d-3'-A4P > 2',5'-dd-3'-ATP (D0939) > 2',5'-dd-3'-ADP (D0814) ≥ 2'-d-3'-ATP (D6500) > 2',5'-dd-3'-AMP (D0689) ≥ 2'-d-3'-ADP (D6000) ≥ 2’,5’-dd-2F-Ado > 2',5'-dd-Ado (D7408) > 2'-d-2F-Ado ≥ 9-Xyl-Ade > 2'‑d-3'-AMP > 3'-ADP > 3'-AMP > 2'-d-Ado (D7400) = 3'-d-Ado (C3394) > 5'-d-Ado (D1771) > 9-Ara-Ade (A5762) = 9‑THF-Ade (SQ 22,536) (S153) ≥ 9-CP-Adem (C4479) > Ado (A9251)
Pro-nucleotide Inhibitorsn	2’,5’-dd-3’-AMP-bis(Me-SATE) > 2’,5’-dd-3’-AMP-bis(t-Bu-SATE) > 2’,5’-dd-3’-AMP bis(Ph-SATE)
Other Inhibitors	Rank-order of inhibitory potency: PMEApp = PMEAp(NH)p > polyadenylate > 2',3'-dd-Ado (D1285) = 2-Cl-2',3'-dd-Ado > PMEA > 2‑amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80) (N2165) > MDL-12,330A (M182)k

Isozymea (source)	VI (rat)	VII (mouse)	VIII e (rat)	IX (mouse)	Xf (human) (soluble)
Structuresb amino acids:	1180	1099	1248(A), 1218(B), 1182(C)	1353	1610
accession:	M96160	U12919	L26986	U30602	AF176813
Chromosome	12q12-13 (human) 15F (mouse)	16q12-13 (human)	8q24 (human)	16p13.3 (human) 16 (mouse)	1q24 (human)
Tissue Expressionc	Heart Kidney Brain Liver	Retina Brain Platelets	Brain Lung	Skeletal muscle Heart Brain	Testes
Physiological Functiong	Hormonal regulation of gluconeogenesis Cell proliferation	Ethanol dependency	Neurotransmission Synaptic plasticity Memory	Not known	HCO3– sensor
Disease Relevance	Adenylyl cyclases mediate signal transduction in all tissues and changes in cellular cAMP levels occur in disease states or may be induced therapeutically in the treatment of disease. Some bacterial toxins raise cAMP levels. Regulation of type V enzyme may be important in cardiomyopathies or of type VI in mitigating aspects of diabetes. Inhibition of adenylyl cyclases may be a useful strategy in combination therapy for treatment of some cancers and for treatment of yeast and a broad range of fungal infections and some cancers.
G-αs	↑	↑	↑	↑	No change
G-αi	↓	Not known	↓	↓	No change
G-βh	↓	↑	Not known	Not known	No change
Forskolin Derivatives	Rank order of stimulatory potency: 7-deacetyl-forskolin (D3533) ≈ 6-[3-(dimethylamino)propionyl]forskolini ≈ 6-[N-(2-isothiocyanatoethyl)aminocarbonyl]forskolini ≈ 5,6-dehydroxy-7-deacetyl-7-nicotinoylforskolini > 6-acetyl-7-deacetyl-forskolin > 7-deacetyl-7-O-hemisuccinyl-forskolin >> 1,9-dideoxy-forskolin (D3658)i			No change	No change
Ca2+/ Calmodulin	↓ (no CaM)	No change	↑	No change	Not known
PKC/PKA	↑(PKC) ↓(PKA)	↑(PKC)	Not known	Not known	Not known
Competitive Inhibitorsj	Rank-order of inhibitory potency: β-L-2',3'-dd-5'-ATP ≈ MANT-5’-GTPγS > MANT-5’-ITPγS > β-D-2',3'-dd-5'-ATP ≈ MANT-5’-ATP >> β-L-5'-ATP > 5'-AP(CH2)PPk				Not known
Non-competitive Inhibitors (P-site ligands)k,l	Rank-order of inhibitory potency: 2',5'-dd-3'-A4P > 2'-d-3'-A4P > 2',5'-dd-3'-ATP (D0939) > 2',5'-dd-3'-ADP (D0814) ≥ 2'-d-3'-ATP (D6500) > 2',5'-dd-3'-AMP (D0689) ≥ 2'-d-3'-ADP (D6000) ≥ 2’,5’-dd-2F-Ado > 2',5'-dd-Ado (D7408) > 2'-d-2F-Ado ≥ 9-Xyl-Ade > 2'‑d-3'-AMP > 3'-ADP > 3'-AMP > 2'-d-Ado (D7400) = 3'-d-Ado (C3394) > 5'-d-Ado (D1771) > 9-Ara-Ade (A5762) = 9‑THF-Ade (SQ 22,536) (S153) ≥ 9-CP-Adem (C4479) > Ado (A9251)				Not known
Pro-nucleotide Inhibitorsn	2’,5’-dd-3’-AMP-bis(Me-SATE) > 2’,5’-dd-3’-AMP-bis(t-Bu-SATE) > 2’,5’-dd-3’-AMP bis(Ph-SATE)				Not known
Other Inhibitors	Rank-order of inhibitory potency: PMEApp = PMEAp(NH)p > polyadenylate > 2',3'-dd-Ado (D1285) = 2-Cl-2',3'-dd-Ado > PMEA > 2‑amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80) (N2165) > MDL-12,330A (M182)k				Not known

Footnotes

1. Isozyme source is for structure information.
2. Structures available at http://www.ncbi.nlm.nih.gov
3. Tissue expression is for evidence of protein expression; this is not a complete listing.
4. Empty cells implies no information.
5. Type VIII exhibits three splice variants (A,B,C).
6. Type X adenylyl cyclase is stimulated by HCO₃^–.
7. All adenylyl cyclase isozymes save type X mediate transmembrane regulation of cell function by hormones through altered rates of cAMP formation. The functional consequences of this are thus dependent on tissue-specific receptors and on downstream effectors through which cAMP acts.
8. Effects of G-βγ are on Gα_s-stimulated enzyme.
9. 6-[3-(dimethylamino)propionyl]-forskolin (NHK477) exhibits enhanced selectivity for type V adenylyl cyclase; 6-[N-(2-isothiocyanatoethyl)aminocarbonyl]forskolin exhibits enhanced selectivity for type II adenylyl cyclase; 5,6-dehydroxy-7-deacetyl-7-nicotinoylforskolin exhibits enhanced selectivity for type III adenylyl cyclase; 1,9-dideoxy-forskolin does not stimulate.
10. Competitive inhibitors interact with the pre-transition configuration of the enzyme. Isozyme selectivity has not been determined; comparisons are from experiments with a detergent-extract from rat brain.
11. Inhibitory P-site ligands are characteristically adenine nucleosides or nucleoside phosphates that inhibit adenylyl cyclases by a noncompetitive or uncompetitive, dead-end- (post-transition-state) mechanism. This comparison is from experiments with a detergent-extract from rat brain. These ligands likely inhibit all isozymes, though potencies may vary; comparisons have been made only with types I, II, VI, VII, and VIII.
12. Of the cell-permeable 9-substituted adenine derivatives, 9-CP-Ade is the most stable chemically and metabolically.
13. Unprotected nucleotides are not cell-permeable whereas nucleosides, nucleoside analogs, and the protected (pro-) nucleotides are cell permeable. The 3'-bis(acyl-2'-thioethyl)phosphate derivatives of 2',5'-dd-Ado require intact cells for inhibition of cAMP formation and exhibited IC50 values in intact macrophages or adipocytes comparable to the IC50 values seen with the corresponding nucleoside-3'-triphosphate on isolated adenylyl cyclase from these cells. Rank order of inhibitory potency depends on relative rates of uptake, deprotection, and subsequent processing within cells. For this series, deprotection rates are in the order given.
14. Unprotected nucleotides are not cell-permeable whereas nucleosides, nucleoside analogs, and the protected (pro-) nucleotides are cell permeable. The 3'-bis(acyl-2'-thioethyl)phosphate derivatives of 2',5'-dd-Ado require intact cells for inhibition of cAMP formation and exhibited IC50 values in intact macrophages or adipocytes comparable to the IC50 values seen with the corresponding nucleoside-3'-triphosphate on isolated adenylyl cyclase from these cells. Rank order of inhibitory potency depends on relative rates of uptake, deprotection, and subsequent processing within cells.

For this series, deprotection rates are in the order given.

Abbreviations

Ado: adenosine
2'-d-Ado: 2'-deoxyadenosine
3'-d-Ado: 3'-deoxyadenosine (cordycepin)
5'-d-Ado: 5'-deoxyadenosine
2',5'-dd-Ado: 2',5'-dideoxyadenosine
2',3'-dd-Ado: 2',3'-dideoxyadenosine
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-3'-AMP: 2'-deoxyadenosine 3'-monophosphate
2'-d-3'-ADP: 2'-deoxyadenosine 3'-diphosphate
2'-d-3'-ATP: 2'-deoxyadenosine 3'-triphosphate
2',5'-dd-3'-AMP: 2',5'-dideoxyadenosine 3'-monophosphate
2',5'-dd-3'-ADP: 2',5'-dideoxyadenosine- 3'-diphosphate
2',5'-dd-3'-ATP: 2',5'-dideoxyadenosine 3'-triphosphate
2',5'-dd-3'-A4P: 2',5'-dideoxyadenosine 3'-tetraphosphate; 5'-APP(CH2)P, adenosine 5'-(βγ-methylene)-triphosphate
βL-5'-ATP: β-L-adenosine 5'-triphosphate; β-L-2',3'-dd-5'-ATP, β-L-2',3'-dideoxyadenosine 5'-triphosphate
PMEA: 9-(2-phosphonylmethoxyethyl)-adenine; PMEApp, 9-(2-diphosphorylphosphonylmethoxyethyl)-adenine
MDL-12,330A: (cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine·HCl; NKY80, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone
2',5'-dd-3'-AMP-bis(Me-SATE): 2',5'-dd-Ado-3'-(acetyl-2-thioethyl)-phosphate; 2',5'-dd-3'-AMP-bis(t-Bu-SATE), 2',5'-dd-Ado-3'-(pivaloyl-2-thioethyl)-phosphate
2',5'-dd-3'-AMP-bis(Ph-SATE): 2',5'-dd-Ado-3'-(phenhyl-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

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