N.J. Cusack

The structure-activity relationships of ectonucleotidases and of excitatory P2-purinoceptors: evidence that dephosphorylation of ATP analogues reduces pharmacological potency

The dephosphorylation of adenine nucleotides and their analogues by ectonucleotidases on the guinea-pig urinary bladder was studied using HPLC. The rate of dephosphorylation of each analogue was compared with its pharmacological potency at causing contraction. ATP, ADP and AMP were rapidly dephosphorylated, and substitution on the purine ring did not affect the rate of breakdown. The ectonucleotidases showed stereoselectivity towards the ribose moiety and towards the polyphosphate chain. In general, methylene isosteres of the nucleotides, and analogues in which one of the oxygen atoms on the terminal phosphate had been replaced, were resistant to degradation. None of the analogues that were readily dephosphorylated are more potent than ATP, and most but not all of the analogues resistant to degradation are more potent than ATP, suggesting that while resistance to degradation does not in itself confer high potency, susceptibility to degradation does limit the potency of ATP and its degradable analogues.

ATP analogues and the guinea-pig taenia coli: a comparison of the structure-activity relationships of ectonucleotidases with those of the P2-purinoceptor

The dephosphorylation of adenine nucleotides and their analogues by ectonucleotidases on the guinea-pig taenia coli was studied using HPLC. The rate of dephosphorylation of each analogue was compared with its pharmacological potency relative to ATP. ATP, ADP and AMP were rapidly dephosphorylated, and substitution on the purine ring had no effect upon the rate of breakdown. The ectonucleotidases showed stereoselectivity towards the ribose, the unnatural L enantiomers of nucleotides being dephosphorylated more slowly. Analogues in which one of the oxygen atoms on the terminal phosphate had been replaced were resistant to degradation. Phosphorothioate analogues in which a sulphur was attached to the penultimate phosphorus were degraded stereoselectively. Methylene isosteres of ATP and ADP resisted degradation, except for homo-ATP which was dephosphorylated at the same rate as ATP. Overall, no correlation was found between the potency of an analogue and its rate of degradation.

ADENOSINE 5′-DIPHOSPHATE ANTAGONISTS AND HUMAN PLATELETS: NO EVIDENCE THAT AGGREGATION AND INHIBITION OF STIMULATED ADENYLATE CYCLASE ARE MEDIATED BY DIFFERENT RECEPTORS

1 Adenosine 5′‐diphosphate (ADP) induces human platelet aggregation and noncompetitively inhibits stimulated human platelet adenylate cyclase; it has been suggested that these two effects are mediated by separate ADP receptors on the platelet surface. 2 Adenosine 5′‐triphosphate and seven adenine nucleotide analogues were tested as inhibitors of both effects of ADP on human platelets, and were found to be competitive. 3 pA2 values were calculated for each antagonist for inhibition of both effects of ADP, and a good correlation (correlation coefficient 0.87; P < 0.01) was found between the pA2 values for inhibition of ADP‐induced aggregation and the pA2 values for inhibition of the effect of ADP on stimulated adenylate cyclase. 4 Such a correlation does not support the suggestion that ADP‐induced aggregation and the inhibition by ADP of stimulated adenylate cyclase are mediated by two separate receptors.

5′-N-ETHYLCARBOXAMIDOADENOSINE: A POTENT INHIBITOR OF HUMAN PLATELET AGGREGATION

1 5′‐N‐ethylcarboxamidoadenosine (NECA) is an adenosine analogue which is 22,900 times more potent than adenosine as a vasodilator. Adenosine and some of its analogues are also inhibitors of human platelet aggregation. NECA was tested for its effects on human platelets. 2 NECA (1 μm) inhibited human platelet aggregation induced by adenosine 5′‐diphosphate (ADP), adrenaline, 5‐hydroxytryptamine (5‐HT) and thrombin more powerfully than adenosine. NECA was 5 to 10 times more potent than adenosine at inhibiting ADP‐ and adrenaline‐induced aggregation. 3 NECA, like adenosine, caused dose‐dependent increases in levels of platelet adenosine 3′,5′‐cyclic monophosphate (cyclic AMP), which were competitively inhibited by theophylline, an adenosine antagonist. 4 These effects of NECA, like those of adenosine, were completely stereospecific as the l‐enantiomer of NECA was inactive. 5 NECA did not interfere with the inhibition by ADP of prostaglandin E1 (PGE1)‐stimulated adenylate cyclase. 6 NECA is the most potent analogue of adenosine tested so far on human platelets, and is the first example of a 5′ modification to retain affinity for the platelet adenosine receptor.

Some pharmacological and biochemical interactions of the enantiomers of adenylyl 5′‐(β, γ‐methylene)‐diphosphonate with the guinea‐pig urinary bladder

1 Adenosine 5′‐triphosphate (ATP) and adenylyl 5′‐(β,γ methylene)‐diphosphonate (AMP‐PCP) both contracted the guinea‐pig urinary bladder, but the response to AMP‐PCP was much greater. We synthesized the enantiomer of AMP‐PCP, L‐adenylyl 5′‐(β,γ‐methylene)‐diphosphonate (L‐AMP‐PCP), and tested it on the guinea‐pig bladder. 2 L‐AMP‐PCP contracted the guinea‐pig bladder, and was more potent than AMP‐PCP and much more potent than ATP. The potential breakdown product of L‐AMP‐PCP, L‐adenosine, unlike adenosine (the breakdown product of AMP‐PCP), did not inhibit contractions of the guinea‐pig bladder. 3 ATP and its enantiomer L‐adenosine 5′‐triphosphate (L‐ATP) were rapidly degraded by the muscle, and AMP‐PCP was also degraded, but more slowly. L‐AMP‐PCP, however, was completely resistant to degradation. 4 L‐AMP‐PCP would appear to be a useful ATP analogue, as it is potent and resistant to degradation, and its potential breakdown product, L‐adenosine, is inactive.

Pharmacological effects of isopolar phosphonate analogues of ATP on P2-purinoceptors in guinea-pig taenia coli and urinary bladder.

1 Isopolar methylene phosphonate analogues of adenosine triphosphate (ATP) were synthesized and tested on the guinea‐pig isolated taenia coli (where ATP causes relaxation) and urinary bladder (where ATP causes contraction), to see if restoration of the electronegativity of the methylene linkage would enhance pharmacological potency. The compounds used were the dichloromethylene and difluoromethylene analogues of adenosine 5′‐(β,γ‐methylene)triphosphonate (AMP‐PCP), l‐adenosine 5′‐(β,γ‐methylene)triphosphonate (l‐AMP‐PCP) and 2‐methylthioadenosine 5′‐(β,γ‐methylene)‐triphosphonate (2‐methylthio‐AMP‐PCP). 2 The order of potency of the analogues depended on the tissue, and was independent of the nature of the purine or ribose moieties. None of the analogues was degraded by ectonucleotidases on either tissue. 3 In the taenia coli the order of potency for relaxation was difluoromethylene > dichloromethylene > methylene, and this reflected the order of electronegativity of the analogues. The isopolar analogues of l‐AMP‐PCP were inactive in the taenia coli. 4 In the bladder the order of potency for contraction was difluoromethylene > methylene > dichloromethylene, suggesting that electronegativity is of lesser importance here. The isopolar analogues of l‐AMP‐PCP were active in this tissue. 5 The differences between the two tissues in the order of potency for these non‐degradable analogues supports suggestions that P2‐purinoceptors in the taenia coli (P2Y) are different from those in the bladder (P2X). The isopolar analogues of l‐AMP‐PCP, like l‐AMP‐PCP itself, were selective agonists at the P2X‐purinoceptor.

EFFECTS OF RP AND SP DIASTEREOISOMERS OF ADENOSINE 5′‐O‐(1‐THIODIPHOSPHATE) ON HUMAN PLATELETS

1 RP and SP diastereoisomers of adenosine 5′‐O‐(1‐thiodiphosphate) ((R)‐ADP‐β‐S and (S)‐ADP‐β‐S), an adenosine 5′‐diphosphate (ADP) analogue, were tested on intact human platelets. 2 Each diastereoisomer induced aggregation, (S)‐ADP‐α‐S being 5 times more potent than (R)‐ADP‐α‐S but they achieved only 75% of the maximal effect of ADP. 3 Aggregation induced by each diastereoisomer was competitively inhibited by ATP (50 μm) 4 Simultaneous addition of each diastereoisomer inhibited aggregation induced by ADP but not by 11α, 9α‐epoxymethano prostaglandin H2, a stable endoperoxide analogue. Both diastereoisomers are therefore partial agonists at the ADP receptor mediating aggregation. 5 Unlike ADP, neither diastereoisomer inhibited prostaglandin E1 (PGE 1‐stimulated adenylate cyclase, but each competitively inhibited the effect of ADP, with (S)‐ADP‐α‐S again being 5 times more potent than (R)‐ADP‐α‐S. 6 These are the first reported examples of ADP analogues to induce platelet aggregation without inhibiting PGE 1‐stimulated adenylate cyclase.

COMPETITIVE INHIBITION BY ADENOSINE 5′-TRIPHOSPHATE OF THE ACTIONS ON HUMAN PLATELETS OF 2-CHLOROADENOSINE 5′-DIPHOSPHATE, 2-AZIDOADENOSINE 5′-DIPHOSPHATE AND 2-METHYLTHIOADENOSINE 5′-DIPHOSPHATE

1 Adenosine 5′‐diphosphate (ADP) induces human platelet aggregation and noncompetitively inhibits stimulated human platelet adenylate cyclase; these two effects are mediated by the same ADP receptor, at which adenosine 5′‐triphosphate (ATP) is a competitive antagonist. 2 Two ADP analogues, 2‐azidoadenosine 5′‐diphosphate (2‐azido‐ADP) and 2‐methylthioadenosine 5′‐diphosphate (2‐methylthio‐ADP) have been reported to be more potent as inhibitors of adenylate cyclase than they are as aggregating agents, but no evidence has been presented that these actions are mediated solely by the ADP receptor. 3 We therefore tested the ability of ATP to inhibit the actions of these compounds and of another ADP analogue, 2‐chloroadenosine 5′‐diphosphate (2‐chloro‐ADP). 4 2‐Chloro‐ADP, 2‐azido‐ADP and 2‐methylthio‐ADP each induced aggregation and inhibited stimulated adenylate cyclase. Both of these actions were competitively inhibited by ATP (50 μm) with pA2 values similar to those previously found for inhibition by ATP of these effects of ADP. 5 The reported greater potency of 2‐azido‐ADP and of 2‐methylthio‐ADP as inhibitors of adenylate cyclase than as aggregating agents is therefore due only to their greater efficacy for this effect, not to some extra actions elsewhere.

PARTIAL AGONIST BEHAVIOUR OF ADENOSINE 5′O‐(2‐THIODIPHOSPHATE) ON HUMAN PLATELETS

1 The effects of an adenosine diphosphate (ADP) analogue, adenosine 5′‐O‐(2‐thiodiphosphate) (ADP‐β‐S), in which a terminal phosphate oxygen has been replaced by sulphur, were studied on human platelets. 2 ADP‐/3‐S induced platelet aggregation and inhibited prostaglandin E 1 (PGE 1)‐stimulated adenylate cyclase but in both cases was less potent than ADP and did not achieve the same maximal effects. 3 Both actions of ADP could be inhibited by the simultaneous addition of ADP‐β‐S (50 μm). 4 Aggregation induced by 11α, 9α‐epoxymethano prostaglandin H2 (a stable endoperoxide analogue) was not inhibited by simultaneous addition of ADP‐β‐S (50 μm). 5 The behaviour of ADP‐β‐S towards human platelets was consistent with it being a partial agonist.

Specific but noncompetitive inhibition by 2-alkylthio analogues of adenosine 5'-monophosphate and adenosine 5'-triphosphate of human platelet aggregation induced by adenosine 5'-diphosphate.

1 Some 2‐alkylthio derivatives of adenosine 5′‐monophosphate (AMP), adenosine 5′‐monophosphorothioate (AMPS) and adenosine 5′‐triphosphate (ATP) were examined as inhibitors of human platelet aggregation. 2 2‐Methylthio‐AMP, 2‐ethylthio‐AMP, 2‐(pentan‐l‐yl)thio‐AMP, 2‐ethylthio‐AMPS, 2‐methylthio‐ATP and 2‐ethylthio‐ATP (100 μm) each inhibited aggregation induced by adenosine 5′‐diphosphate (ADP) but not by 11α,9α‐epoxymethano prostaglandin H2, a stable endoperoxide analogue. 3 Log dose‐response curves to ADP in the absence and presence of each inhibitor were not parallel and the inhibition could not be overcome by high concentrations of ADP. 4 The ATP analogues achieved greater inhibition of aggregation induced by ADP (5 μm) than did the AMP analogues. 5 The order of potency of the AMP analogues was 2‐ethylthio‐AMPS > 2‐ethylthio‐AMP > 2‐(pentan‐l‐yl)thio‐AMP > 2‐methylthio‐AMP, and 2‐methylthio‐ATP was more potent than 2‐ethylthio‐ATP. 6 These 2‐alkylthio substituted analogues of AMP and ATP are specific but noncompetitive inhibitors of ADP‐induced human platelet aggregation.