INTRODUCTION

The 3:5-cAMP¹ signaling pathway can be regulated pharmacologically by many drugs that are of particular value in the treatment of various diseases, and therefore there is much current interest in identifying new agents acting on this pathway. Regulation of this pathway can be achieved through changes in the activities of 3:5-cAMP-phosphodiesterases (1), 3:5-cAMP-dependent protein kinases (2), or adenylyl cyclases. Adenylyl cyclase is a family of membrane-bound enzymes that catalyze the formation of 3:5-cAMP from 5ATP and is regulated by numerous neurotransmitters and hormones via cell surface receptors and guanine nucleotide-dependent regulatory proteins (G-proteins). In an isozyme-dependent manner, enzyme activity is also regulated directly, by forskolin, by Ca²⁺/calmodulin, and by certain adenosine derivatives via a site that is distinct from the catalytic site. Whereas numerous drugs have been developed that act on the cyclic nucleotide phosphodiesterases, those that act directly on adenylyl cyclases have been less well explored; the main class of such pharmacological agents comprises forskolin and its analogs (3).

The domain through which inhibition of mammalian adenylyl cyclases occurs with adenosine derivatives, excepting the enzyme from sperm, is referred to as the P-site from an evident requirement for a purine moiety (4, 5, 6, 7, 8, 9, 10, 11). Key structural requirements for P-site ligands include a requirement for an intact adenine moiety, enhanced inhibitory potency with 2-deoxy- and especially 2,5-dideoxy-ribosyl moieties, and a notably strong preference for a 3-phosphate (4, 5, 6, 7, 8). The most potent ligands have been 2,5-dideoxyadenosine 3-monophosphate (2, 5-dd-3AMP), the naturally occurring 3AMP, and 2-d-3AMP (8, 12), and recently we described the effects of the 2,5-dd-3ADP and 2,5-dd-3ATP on brain adenylyl cyclases (13). Noncompetitive inhibition kinetics with these ligands (13, 14, 15) and irreversible inactivation studies with P-site-selective covalent affinity probes (16)² are consistent with inhibition occurring at a site that is distinct from the catalytic site. As a part of our effort (17) to investigate the structure and properties of this inhibitory site on adenylyl cyclases, we describe here a short and efficient synthesis of a number of adenosine 3-polyphosphates and the potent inhibition of adenylyl cyclase that these 3-nucleotides exert. Although the biology and biochemistry of nucleoside 3-polyphosphates are essentially unexplored in eukaryotic systems, inhibition of adenylyl cyclases by this class of compound may well imply roles as intracellular regulators of this transmembrane signaling system.

EXPERIMENTAL PROCEDURES

Both detergent-solubilized and particulate preparations of adenylyl cyclase from rat and bovine brains were prepared and assayed as described previously (8, 15, 16). Lubrol-PX was filtered through alumina (Neutral, AG7, from Bio-Rad) to remove peroxides. Bovine brain adenylyl cyclase was purified as described by Pfeuffer et al. (18). Inhibition kinetics were determined on enzyme assayed with concentrations of divalent cation fixed in excess of the 5ATP concentration as described previously (19).

Nucleotides were quantified after HPLC as areas under peaks determined with a Waters 996 photo-diode array detector and the accompanying Millennium software (v.2.10). Chromatography was on a DEAE-5PW column (Altex, 5 µm, 7.5 × 75 mm) developed with sequential step gradients of triethylammonium bicarbonate, pH 8.5, to separate the nucleoside 3-mono-, 3-di-, and 3-triphosphates and 2:3-cAMP and 3:5-cAMP.

Recombinant type I enzyme was generously supplied by Drs. R.Taussig and A. G. Gilman. Membranes and membrane extracts were from fall army worm ovarian (Sf9) cells infected with a type I adenylyl cyclase encoding baculovirus. THF was distilled from benzophenone ketyl before use. NMR spectra were recorded with a Bruker AC250 at 250 MHz for proton spectra and at 101 MHz for ³¹P spectra, with a 85% solution of H₃PO₄ as external standard. FAB mass spectra were recorded with a glycerol matrix.

2,5-Dideoxyadenosine 3-O-[(2,2,2-Tribromoethyl)-morpholinophosphonate] 3a

To a suspension of 2,5-dideoxyadenosine 1a (20) (1.18 g, 5 mmol) in a mixture of THF (60 ml) and pyridine (10 ml) was added dropwise at room temperature a 0.9 M solution of tert-butyl-magnesium chloride (5.55 ml, 5 mmol) in THF. After stirring for 5 min, 2,2,2-tribromoethyl phosphoromorpholinochloridate 2 (2.25 g, 5 mmol) was added, and stirring was continued for 1.5 h. The mixture was then concentrated in vacuo, diluted with EtOAc (30 ml), quenched with water (60 ml), and then extracted with EtOAc (30 ml × 2). The combined organic layers were washed with a saturated solution of NaCl, were dried over Na₂SO₄, and then were evaporated to give a gum. The crude product was subjected to silica gel chromatography. Elution with CH₂Cl₂: Et₂O (95:5) followed by CH₂Cl₂:MeOH (95:5) afforded a mixture of the two diastereoisomers of the adduct 3a (1.61 g, 57%): R_f 0.45 and 0.34 (CH₂Cl₂-MeOH, 95:5). ¹H NMR (CDCl₃, the more polar diastereoisomer)  1.23 (d, 3H, J = 6.9 Hz, H-5), 2.71-2.80 (m, 1H, H-2"), 2.99-3.10 (m, 1H, H-2), 3.26 (m, 4H, morpholine), 3.69 (m, 4H, morpholine), 4.40 (m, 1H, H-4), 4.59-4.76 (m, 2H, CH₂CBr₃), 5.01 (m, 1H, H-3), 6.02 (s, 2H, NH₂), 6.35 (t, 1H, J = 6.7 Hz, H-1), 7.92 (s, 1H, H-2), 8.31 (s, 1H, H-8); ¹H NMR (CDCl₃, the less polar diastereoisomer)  1.40 (d, 3H, J = 6.6 Hz, H-5), 2.71-2.81 (m, 1H, H-2"), 2.97-3.08 (m, 1H, H-2), 3.06 (m, 4H, morpholine), 3.64 (m, 4H, morpholine), 4.32 (m, 1H, H-4), 4.55-4.73 (m, 2H, CH₂CBr₃), 4.97 (m, 1H, H-3), 6.30 (t, 1H, J = 6.6 Hz, H-1), 6.41 (s, 2H, NH₂), 7.89 (s, 1H, H-2), 8.25 (s, 1H, H-8).

5-O-(Dimethoxytrityl)-2-deoxyadenosine 3-O-[(2,2,2-tribromoethyl)-morpholinophosphonate] 3b

The title compound was prepared from 5-O-(dimethoxytrityl)-2-deoxyadenosine 1b (21) as described above for the synthesis of 2a, except the reaction was carried out in THF without adding pyridine, with a yield of 82%: R_f 0.50 and 0.46 (CH₂Cl₂-MeOH, 95:5); ¹H NMR (CDCl₃, mixture of the two diastereoisomers)  2.81-2.87 (m, 1H, H-2"), 3.01-3.04 (m, 1H, H-2), 3.16-3.26 (m, 4H, morpholine), 3.37-3.51 (m, 1H, H-5), 3.64-3.72 (m, 4H, morpholine), 3.75 (s, 6H, OCH₃), 4.43 (m, 1H, H-4), 4.52-4.74 (m, 2H, CH₂CBr₃), 5.28 (m, 1H, H-3), 6.24 (br s, 2H, NH₂), 6.44-6.52 (m, 4H, H-aryl), 7.20-7.27 (m, 9H, H-aryl), 7.94 and 7.96 (s, 1H, H-2), 8.24 and 8.26 (s, 1H, H-8).

2,5-Dideoxyadenosine 3-triphosphate 4a

Phosphotriester 3a (0.89 g, 1.5 mmol) was added to a solution of pyridine (30 ml) containing activated zinc (22) (0.15 g) and bis(tri-n-butyl-ammonium)-pyrophosphate (15 mmol) under the exclusion of moisture. The mixture was stirred at room temperature during 2 days. The reaction mixture was then centrifuged, and the supernatant fraction was evaporated in vacuo and purified by QAE-Sephadex (HCO₃ form) with a linear gradient of triethylammonium bicarbonate (0.01-0.4 M). The appropriate fractions were lyophilized and then coevaporated several times with methanol, yielding 1.16 mmol (77%) of 2,5-dideoxyadenosine 3-triphosphate 4a. The nucleotide 4a was isolated as its sodium salt by the addition of 1 M sodium iodide in acetone to a methanol solution of the triethylammonium nucleotide. The precipitate was centrifuged and washed three times with cold acetone and dried in vacuo giving the sodium salt of 2,5-dideoxyadenosine 3-triphosphate. No impurities were noted on ion exchange HPLC: ¹H NMR (D₂O)  1.37 (d, 3H, J = 6.0 Hz, 3H-5), 2.72-3.00 (m, 2H, H-2 and H-2"), 4.40-4.47 (m, 1H, H-4), 6.43 (t, 1H, J = 6.9 Hz, H-1), 8.19 (s, 1H, H-2), 8.36 (s, 1H, H-8); ³¹P NMR (D₂O)  - 17.28 (dd, J = 18.8 Hz, P-2), -7.62 (dd, J_P-H = 7.7 Hz, J_P-P = 18.3 Hz, P-1), -1.33 (d, J = 18.8 Hz, P-3); FAB-MS 496 (M -2H + Na)⁺.

2-Deoxyadenosine 3-Triphosphate 4b

Phosphotriester 3b (2.37 g, 2.5 mmol) was added to a solution of pyridine (30 ml) containing activated zinc (0.25 g) and bis(tri-n-butylammonium)-pyrophosphate (25 mmol), under the exclusion of moisture. The mixture was stirred at room temperature during 2 days. Then the reaction mixture was centrifuged, and the supernatant was evaporated in vacuo and treated with 80% acetic acid at room temperature for 30 min. The medium was then neutralized with a cold solution of 0.5 M NaHCO₃, diluted to 2 liters, filtered, and then purified by chromatography on QAE-Sephadex as above, yielding 0.55 mmol (22%) of 2-deoxyadenosine 3-triphosphate 4b. This nucleotide was also isolated as its sodium salt as above. No impurities were noted on ion exchange HPLC: ¹H NMR (D₂O),  2.7-2.9 (m, 2H, H-2 and H-2"), 3.84 (s, 2H, 2 H-5), 5.08 (m, 1H, H-3), 6.47 (t, 1H, J = 7 Hz, H-1), 8.16 (s, 1H, H-2), 8.32 (s, 1H, H-8); ³¹P NMR (D₂O)  -15.66 (t, J = 19.1 Hz, P-2), -7.66 (dd, J_P-H = 8.0 Hz, J_P-P = 18.4 Hz, P-1), -0.99 (d, J = 13.0 Hz, P-3); FAB-MS 490 (M-H)⁺.

2,5-Dideoxyadenosine 3-Diphosphate 5a

Phosphotriester 3a (1.42 g, 2.5 mmol) reacted with mono(tri-n-butylammonium)-phosphate (30 mmol), as described above, yielding 1.35 mmol (54%) of 2,5-dideoxyadenosine 3-diphosphate 5a: ¹H NMR (D₂O)  1.30 (d, 3H, J = 6.6 Hz, 3H-5), 2.79-2.87 (m, 2H, H-2 and H-2"), 4.36-4.39 (m, 1H, H-4), 4.72-4.79 (m, 1H, H-3), 6.33 (t, 1H, J = 6.6 Hz, H-1), 8.06 (s, 1H, H-2), 8.25 (s, 1H, H-8); ³¹P NMR (D₂O)  -7.05 (dd, J_P-H = 7.6 Hz, J_P-P = 21.1 Hz, P-1), -1.77 (d, J = 21.1 Hz, P-2); FAB-MS 394 (M-H)⁺.

2-Deoxyadenosine 3-Diphosphate 5b

Phosphotriester 3b (0.55 g, 1 mmol) reacted with mono(tri-n-butylammonium)-phosphate (30 mmol), as described above, yielding 0.49 mmol (49%) of 2-deoxyadenosine 3-diphosphate 5a: ¹H NMR (D₂O)  2.57-2.62 (m, 2H, H-2 and H-2'), 3.49 (d, 2H, J = 4.75, Hz, 2H-5), 4.07-4.11 (m, 1H, H-4), 6.22 (t, 1H, J = 7.0 Hz, H-1), 7.96 (s, 1H, H-2), 8.10 (s, 1H, H-8); ³¹P NMR (D₂O)  -6.75 (dd, J_P-H = 7.4 Hz, J_P-P = 20.3 Hz, P-1), -1.35 (d, J = 20.3 Hz, P-2); FAB-MS 410 (M-H)⁺.

RESULTS

The synthesis of nucleoside 3-polyphosphates has received considerably less attention than have those of the corresponding 5-polyphosphates because the former are more difficult to synthesize and because their biological roles are not as well described (23, 24, 25, 26, 27, 28, 29, 30, 31). To our knowledge the only reported chemical synthesis of 2-deoxy-nucleoside 3-triphosphates was that by Josse and Moffatt (28) and proceeded by homologation of the corresponding nucleoside 3-monophosphate by use of phosphoromorpholidate. Other nucleoside 3-polyphosphates have also been prepared by homologation of the respective 3-monophosphates (23, 24, 25, 26, 29). The major drawback with this method is that the synthesis of the phosphoromorpholidates requires easy accessibility to the corresponding nucleoside 3-monophosphate. Van Boom and colleagues prepared nucleoside 3-diphosphates in the thymidine and guanosine series by a phosphotriester approach that involved the use of an activated morpholinylphosphonate (27, 30, 31). Unfortunately, such a procedure cannot be applied when the base of the nucleotide is adenine, the 6-amino group being more reactive toward phosphorylating agents than is the 3-hydroxyl of the nucleoside moiety (32, 33). Recently Uchiyama et al. (33) reported that a facile 3-O-phosphorylation of N-unprotected nucleosides could be achieved via magnesium alkoxide formation. We wish to report here that sequential treatment of N-unprotected 2-deoxyadenosine derivatives 1 (20, 21) with an equimolar amount of tert-butylmagnesium chloride and tribromoethyl phosphoromorpholinochloridate 2 (34) in THF at 20 °C afforded the phosphotriesters 3 in good yields (57 and 82%) (Scheme 1). The phosphotriester derivative 3a was reacted with bis-(tri-n-butylammonium) pyrophosphate in the presence of activated zinc (22), affording 2,5-dd-3ATP 4a in 77% yield. 2-d-3ATP 4b was prepared similarly from 3b with an additional detritylation step in 22% yield. This method can also be applied to the synthesis of nucleoside 3-diphosphates, since 2,5-dd-3ADP 5a and 2-d-3ADP 5b also were prepared by reaction of 3a with tributylammonium phosphate in 54% and 49% yield, respectively.

In conclusion, the utility and efficiency of the method of the alkoxide activation combined with the use of the phosphorylating agent 2 has been demonstrated by its application to the syntheses of various adenosine 3-polyphosphates in only two steps, avoiding additional steps generally required for such syntheses (protection and deprotection of the exocyclic amino group and condensation of morpholine with the nucleoside monophosphate).

The adenosine 3-polyphosphates and the deoxy derivatives (Fig. 1) are a family of inhibitors of adenylyl cyclase in which potency increased with the number of 3-phosphates and with the removal of the 2-OH and 5-OH groups (Fig. 2). IC₅₀ values for inhibition of rat brain adenylyl cyclase by the three series of adenosine 3-phosphates are presented in Table I and compared with those of the parent nucleosides and with three cyclic nucleotides. The cyclic nucleotides were poor inhibitors. For each parent nucleoside, inhibitory potency increased with the number of 3-phosphates. Inhibitory potency of the 3-phosphates followed the order Ado < 2d-Ado < 2,5-dd-Ado. The greatest effects were those caused by the removal of the 2-hydroxyl group and by the sequential addition of phosphates at the 3 position, each increasing potency many-fold. The combined effects of the removal of the 2- and 5-hydroxyl groups and the addition of the 3-polyphosphate are incorporated in 2,5-dd-3ATP, which exhibited an IC₅₀ ~40 nM (Table I and Ref. 13). This potency is almost 2 orders of magnitude greater than that of previous ligands and makes 2,5-dd-3ATP the most potent nonprotein regulator of adenylyl cyclases thus far described. It approaches the potency of the stimulatory effect of rG_s on the type I adenylyl cyclase and the stimulatory and inhibitory effects of G on _s-activated-types II and I, respectively (35). The observation that adenosine and 2-deoxyadenosine 3-polyphosphates are also very potent inhibitors of adenylyl cyclase is important in that these 3-nucleotides are known to occur naturally, whereas the 2,5-dideoxyadenosine 3-polyphosphates are not. Consequently, the adenosine and 2-deoxyadenosine 3-polyphosphates are potentially important and unexplored intracellular regulatory nucleotides. The rank order presented in Table I was maintained with the purified native bovine type I and with the recombinant wild type I adenylyl cyclases, though with these enzymes each of the 3-nucleotides was noticeably less potent than with the enzyme in the cruder detergent-dispersed rat brain preparation (13). This is consistent with the loss of potency of 2-d-3AMP and 2,5-dd-Ado we had previously noted upon purification of the bovine brain adenylyl cyclase (8). This may reflect the loss of membrane phospholipid important for tertiary structure or loss of calmodulin in the purified preparations, because Ca²⁺/calmodulin enhances sensitivity to P-site ligands (36).

Structures of the family of adenosine 3-polyphosphates.

Inhibition of rat brain adenylyl cyclase by several adenosine 3-phosphates.

Table I. IC50 values for inhibition of rat brain adenylyl cyclase by adenosine 3-phosphates Other activities were determined in the presence of 100 µM ATP, 5 mM MnCl2, and 100 µM forskolin. 3-Phosphate NUCLEOSIDE Ado 2-d-Ado 2,5-dd-Ado (µM) None 82a 15a 2.7a 3~P 8.9a 1.2a 0.46b 3~PP 3.9 0.14 0.10b 3~PPP 2.0 0.09 0.04b 3.5cP >300a >300a 2:3cP 250 a Values from Ref. 8. b Values from Ref. 13.

Because inhibition by this class of compound occurred with purified enzyme, inhibition is an effect on the enzyme directly. We reported previously that 2,5-dd-3ATP and 2,5-dd-3ADP elicited linear noncompetitive inhibition of both purified and crude, detergent-extracted preparations of adenylyl cyclases (13). This behavior was fully consistent with inhibition occurring at the P-site (15). Not surprisingly, the 2-deoxyadenosine 3-polyphosphates also caused noncompetitive inhibition (Fig. 3). Thus, all of the adenine 3-nucleotides synthesized and tested here constitute a class of substantially more potent P-site inhibitors of adenylyl cyclases.

Double-reciprocal plot of effects of 2-d-3ADP on rat brain adenylyl cyclase.

3-Nucleotide Stability

Typical adenylyl cyclase preparations contain a number of phosphohydrolyses and substantial degradation of the adenine nucleoside 3-polyphosphates might be expected (37, 38). However, when these 3-nucleotides were incubated with a detergent-dispersed adenylyl cyclase from rat brain under conditions known to elicit excellent sensitivity to P-site-mediated inhibition, with the exception of 3ATP, there was no significant breakdown of any of the adenosine or 2-deoxyadenosine 3-polyphosphates or of 2,5-dd-3ATP (Table II).³ Notably, there was some conversion of 3ADP to 3ATP (as well as to breakdown products of 3ATP) and of 2,5-dd-3ADP to 2,5-dd-3ATP. This phosphorylation of 3ADP and 2,5-dd-3ADP was found to be due to contaminating kinase(s) in the incubation and did not occur in the absence of enzyme. 3ATP breakdown led to the formation of 3AMP and 2AMP in variable ratios, and this occurred also in the absence of enzyme. The hydrolysis of 3ATP implies its potency (IC₅₀ ~2 µM) was likely an underestimation. In the absence of the adenylyl cyclase preparation, the principal product of 3ATP hydrolysis was 2:3-cAMP (Table III) and was found to be catalyzed by divalent cation alone. It occurred with Mn²⁺, Mg²⁺, and to a slightly lesser extent with Ca²⁺ (Table III). Because the inclusion of the adenylyl cyclase preparation led to the formation of 3AMP or 2AMP (cf. Table II) but to no measurable 2:3-cAMP, the sequence 3ATP  2:3-cAMP  3AMP + 2AMP is suggested. The latter step would be catalyzed by enzymes in the rat brain extract. This reaction sequence was obviously not possible with the 2-deoxyribosides, and with them significant breakdown did not occur and the consequences of the little breakdown of the 3-polyphosphates that does occur could be ignored.

Table II. Stability of adenosine 3-polyphosphates during reaction with detergent-extracted adenylyl cyclase from rat brain Values are percentages normalized from the areas of the eluted peaks from anion exchange HPLC for each nucleotide added. Nucleotides were exposed for 15 min at 30 °C with normal adenylyl cyclase reaction mixture, including 50 mM triethanolamine · HCl, pH 7.5, 5 mM MnCl2, 100 µM forskolin, 1 mM 3-isobutyl-1-methylxanthine, 1 mM dithiothreitol, 1 mg of bovine serum albumin/ml, 0.1% (w/v) Lubrol-PX, 2 mM creatine phosphate, and 100 µg of creatine kinase/ml, but absent 5ATP.3 Extracted rat brain protein was 21 µg/tube. Nucleotide Nucleoside 3~P 3~PP 3~PPP % 2d3AMP 2.6 97.4 2d3ADP 1.2 3.0 95.8 2d3ATP 1.4 2.0 5.3 91.3 2,5dd3AMP 21.4 78.6 2,5dd3ADP 1.7 7.8 <0.6 89.9 2,5dd3ATP 1.4 <0.1 <0.1 98.6 3ADP 2.7 2.0 73.1 22.2 3ATP 2.3 72.9 6.1 18.8

Table III. Cation-induced hydrolysis of 3ATP 3ATP was incubated for 15 min at 30 °C with a reaction mixture containing 1 mM dithiothreitol, 0.1% Lubrol-PX, 2 mM creatine phosphate, 100 µg of creatine kinase/ml, 100 µM forskolin, triethanolamine · HCl, pH 7.5. Approximate retention times are given in parentheses for the several products that were identified. The values are percentages normalized from the areas of the eluted peaks from HPLC. Cation (5 mM) Nucleoside (3.7 min) 2:3cAMP (10 min) 3AMP (17 min) 3ADP (22 min) 3ATP (25 min) Unknown (26 min) % None 1.4 4.2 <0.1 6.8 87.6 <0.1 Mn2+ 1.9 84.4 <0.1 4.9 5.8 3.1 Mg2+ 1.8 81.3 <0.1 5.3 8.2 3.5 Ca2+ 1.8 72.5 <0.1 5.1 20.7 <0.1

Because possible products of nucleoside 3-polyphosphate hydrolysis would include inorganic pyrophosphate (PP_i) or inorganic triphosphate (PPP_i), we determined the potency and nature of inhibition of adenylyl cyclase by these agents (Fig. 4). PP_i (IC₅₀ = ~1.4 mM) was less potent than either PPP_i or PPPP_i (IC₅₀ = ~55 µM for both). For PP_i kinetics of inhibition was mixed (15), and for PPP_i inhibition was competitive with respect to substrate. Thus, inhibition by the adenosine 3-polyphosphates conformed to established behavior of classical P-site ligands, and inhibition could not be accounted for by either potency or inhibition kinetics of the minimal hydrolytic products formed in the adenylyl cyclase incubations.

Related to the question of 3-nucleotide stability was the possibility that 3ATP or 2-d-3ATP might also serve as substrates of adenylyl cyclase for the formation of 3:5-cAMP or 2-d-3:5-cAMP, respectively. In three experiments with purified type I adenylyl cyclase from bovine brain, with substrate concentrations ranging from 100 to 500 µM, no 3:5-cAMP was detected from 3ATP nor 2-d-3:5-cAMP from 2-d-3ATP. Because an optically based detection system was used, it remains possible that by use of a radioactively labeled substrate and the greater inherent sensitivity it would allow, some small amount of 3:5-cAMP or 2-d-3:5-cAMP might have been detected. This caveat notwithstanding, the data argue that the adenylyl cyclases catalyze the hydrolysis of pyrophosphate and the formation of the cyclic 3:5-diester solely from 5ATP at the catalytic site and that this does not also occur from the adenosine 3-triphosphates at the P-site.

DISCUSSION

Presented here are the synthesis of a new family of adenosine 3-polyphosphates and the effects of these 3-nucleotides on adenylyl cyclase. The characteristics of inhibition conformed to those of ligands acting on adenylyl cyclases via the P-site. For the parent nucleosides and for their respective 3-phosphorylated derivatives, potency increased with removal of ribosyl-hydroxyl groups and exhibited the rank order of Ado < 2-d-Ado < 2,5-dd-Ado. These compounds exhibited increased inhibitory potency with the successive addition of phosphates to the 3-ribose position. Although the most potent inhibitor was 2,5-dd-3ATP (IC₅₀ ~40 nM), it is important that the adenosine and 2-deoxyadenosine 3-polyphosphates also exhibited potency in the micro- to submicromolar range, respectively. Of these 3-nucleotides it is probable that all but the 2,5-dideoxyadenosine derivatives are naturally occurring.

Although the tertiary structure of adenylyl cyclases is not known, the deduced primary sequence suggests a membrane topology exhibiting a repeated structure of six-membrane spanning regions followed by a large cytosolic domain (39). The two cytosolic domains (C1 and C2) are homologous with each other and with the established catalytic domain of guanylyl cyclases (40), supporting the idea that each contains a nucleotide binding region. It is easy to speculate that each of these two cytosolic domains binds one of the nucleotides, one site for catalysis (5ATP) and a distinct site for inhibition (3ATP), but this is uncertain. It is known that expression of either C1 or C2 domain alone is insufficient to catalyze effectively the formation of 3:5-cAMP and that co-expression of independently vectored cytosolic domains substantially improves the catalytic efficiency (41). In fact, the chimeric protein created by linking the C1 domain of the type I enzyme with the C2 domain of the type II enzyme yielded a soluble enzyme also capable of catalysis and of inhibition by 2-d-3AMP (44). These observations suggest that both C1 and C2 participate in or must interact for catalysis and possibly also for inhibition by 3-nucleotides. However, it remains to be established whether C1 and C2 each interact separately with 5ATP or the adenosine 3-polyphosphates, or whether C1 and C2 act together to form one or two nucleotide binding sites, possibly at the interface of C1 and C2. To date results from truncation, deletion, and site-directed mutation studies have not allowed clear identification of two binding domains (39, 41, 42, 43, 44).

Available evidence suggests that for most adenylyl cyclases, catalysis and inhibition by P-site ligands occur at distinct sites. This conclusion is supported principally by studies of irreversible inactivation with P-site targeted ligands and by enzyme kinetics (15, 16). Inactivation by 2,5-dd-3FSBAdo (16) occurred in the presence of 5ATP but not in the presence of 2,5-dd-Ado, a modestly potent P-site ligand. We suggested that this was consistent with P-site ligands binding at a site distinct from and independent of that of the substrate. This is nominally supported by inhibition kinetics, which have consistently exhibited straightforward noncompetitive behavior (4, 5, 6, 7, 8, 13, 14, 15), except for enzyme activated by pretreatment with GTPS·_s, when uncompetitive inhibition was noted (15). Both uncompetitive and noncompetitive inhibition typically imply inhibitor binding to the enzyme in the presence of substrate and at a site distinct from the catalytic site (45), but other mechanisms may lead also to this apparent kinetic behavior as we have discussed previously (15). The principal argument is that P-site ligand may form an inactive metal-PP_i-adenosine complex at the catalytic site through the binding of the P-site ligand at the region occupied by product 3:5-cAMP (15, 46). Though consistent with noncompetitive and even uncompetitive inhibition, this argument is at odds with the observation that PP_i did not affect the apparent K_i for 2-d-3AMP (15). The nucleoside 3-triphosphates, being substantially more potent inhibitors, present a different circumstance in that the inhibitor contains three phosphates, and the possible formation of a dead-end enzyme-inhibitor complex subsequent to enzyme activation cannot be dismissed. Examples of this exist with Ordered Bi Bi reaction mechanisms and in the example of aldehyde reductase crystallographic evidence places inhibitor in the catalytic domain, at the alcohol product site (47, 48). Adenylyl cyclases also conform to a bireactant sequential reaction mechanism with divalent cation and cation-ATP as substrates and some isozymes exhibit ordered rather than random behavior (19, 49). Enzyme is activated by divalent cation, either by Mn²⁺ directly or indirectly by activated Gs,⁴ and becomes more susceptible to inhibition by P-site ligands (4, 5, 6, 7, 8, 10, 11, 12, 14, 15, 19, 36, 37, 46, 49). Thus, key characteristics of catalysis and inhibition by P-site ligands are the roles of divalent cation and the similar nucleotide requirements for both processes. Each mechanism requires divalent cation; each is adenine-specific; for each the 2-deoxyadenosine derivative is a better ligand; and for each the nucleoside triphosphate is either required (catalysis) or preferred (inhibition). Thus, we suggest that catalysis and inhibition likely occur at very similar if not the same sites.

Second messengers are typically viewed as mediating the effects of extracellular signals to regulate specific intracellular activities, thereby serving to coordinate functions of individual cells with events elsewhere in the organism. By contrast, adenosine 3-polyphosphates, formed from precursors within cells, may be viewed as cell-initiated censors or attenuators, effectively minimizing the influence of extracellular signals, specifically of those that activate the adenylyl cyclase-3:5-cAMP-protein kinase A cascade. Levels of adenosine 3-polyphosphates may conceivably be modulated by intracellular events or possibly other extracellular signals via cascades in which the formation of these attenuators is a critical function, that is, to shut down the 3:5-cAMP-protein kinase A pathway, thereby allowing other cell functions to occur. Because there are few if any adenine nucleotides that interact with only one protein or protein type, we suspect that adenosine 3-polyphosphates will be found to interact with a number of proteins or enzymes other than adenylyl cyclases and will thereby induce a family of physiological responses.

Important in this conjecture, though, is whether adenosine 3-polyphosphates occur in mammalian cells and to what extent their levels may change and be regulated. Although the natural occurrence of ribo- and 2-deoxyribo-nucleoside 3-di- and 3-triphosphates per se have been known for several decades, they have received relatively little attention in mammalian systems (50, 51, 52, 53, 54, 55, 56, 57, 58). Nucleoside 3-polyphosphates have been of interest in the regulation or prokaryotes because guanosine 3-diphosphate was identified during early investigations of guanosine 5-di- and 5-triphosphate-3-diphosphate (magic spot; ppGpp⁵ and pppGpp) (50, 51, 52, 53, 54). The analogous adenosine polyphosphates, pppAppp⁵, pppApp, and ppApp, were also reported to be synthesized by ribosomes from sporulating but not vegetative bacteria (55, 56) and as part of the stringent response (57). This has suggested that in these organisms adenosine 3,5-bis-polyphosphates may be involved in the regulation of development. For guanosine 3-polyphosphates, the likely precursors are the respective 3,5-bis-polyphosphates. By analogy and given the prevalence of 5-nucleotidases, the likelihood that adenosine 3-polyphosphates may also occur naturally is suggested. Although these more rigorous studies have been done in bacterial systems, the reported presence of both guanosine and adenosine 3,5-bis-polyphosphates in cultured mammalian cells suggests that they may be involved in comparable responses also in these more complex systems (58). The concentration of pppAppp was estimated in WI-38 cells to be 6-7% that of 5GTP, or in the low micromolar range, and similar results were obtained with Chinese hamster ovary and BHK-21 cells (58). This could result in 3ATP levels consistent with the IC₅₀ for inhibition of adenylyl cyclase. Thus, an increase in the levels of adenosine or 2-deoxyadenosine 3-polyphosphates would precipitate P-site-mediated inhibition of adenylyl cyclase, lowered cellular levels of 3:5-cAMP, and all the downstream effects this would cause. The effectiveness of this form of regulation may differ among tissues, reflecting differential expression of adenylyl cyclase isozymes and their respective sensitivity to P-site ligands.⁶

Cell-permeable P-site ligands induce a number of responses in cells and tissues. We and others have used 2,5-dd-Ado with epididymal fat cells (9), isolated hepatocytes (59), primary cultures of thyroid follicles (60), dorsal root ganglion neurons (61), bone organ cultures (62), platelets (6), cortical collecting tubules (63), and cultured mouse fibroblasts (64) to name but a few. End points included changes in water conductance (63), action potential after-hyperpolarization (61), PTH-stimulated bone resorption (62), glycerol production (9), altered enzyme activities (59), DNA synthesis and cell growth (60), and cell differentiation (64). The effects of 2,5-dd-Ado on cell function and on cellular 3:5-cAMP levels have been uniformly consistent with P-site-mediated inhibition of adenylyl cyclase. It is not the effect of 2,5-dd-Ado per se that is important, though, but rather the natural intracellular regulatory processes and ligands that it may mimic.