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 22.214.171.124), 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 (Gαs) and inhibitory (Gαi) guanine nucleotide-dependent regulatory proteins (G-proteins). Most isozymes are activated by Gαs, but differ in their regulation by Gαi 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. Gαs activates through interaction with the C2 domain yielding the active enzyme: GTP•αs •C. Inhibition by G-proteins occur by a direct effect of Gαi with the C1 domain or by the recombination of βγ with Gαs.
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 Gαs (e.g. cholera toxin) or Gαi (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 PPi 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-PPi, 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.
For this series, deprotection rates are in the order given.
3'-d-Ado: 3'-deoxyadenosine (cordycepin)
9-THF-Ade: 9-(tetrahydrofuryl)-adenine (SQ22,536)
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