We developed novel methods for analyzing the concentrationresponse curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA i ), is most applicable for the analysis of responses at G protein-coupled receptors. RA i is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M 2 receptor, including stimulation of phosphoinositide hydrolysis through G Drug discovery often involves testing compounds in isolated tissues and high throughput assays to determine activity at target receptors. In the case of agonists, the measured parameters are usually EC 50 and E max , whereas the parameters of greater relevance to drug design are observed affinity and intrinsic efficacy.1 If the receptor is a ligand-gated ion channel, then receptor activation can be measured directly as whole cell current, and the EC 50 and E max are reasonable estimates of observed affinity and intrinsic efficacy provided that desensitization is not excessive. It would seem that, in most instances, the observed affinity and intrinsic efficacy of an agonist for a ligand-gated ion channel would be constant, regardless of the tissue or cell in which the receptor is expressed.The situation is more complex for a G protein-coupled receptor (GPCR). First, GPCRs are inactive in isolation and must interact with a G protein to elicit a response. It is possible that the G protein with which the receptor interacts selects for a receptor conformation having a unique agonist profile and that the observed affinity and intrinsic efficacy of the agonist-receptor complex may be G protein-specific or influenced by other proteins interacting with the receptor (Leff et al., 1997;Berg et al., 1998). Second, because it is difficult to measure receptor activation directly, most assays involve measuring a downstream response, and the corresponding EC 50 and E max values may vary, depending on the point in the signaling cascade at which the experimenter measures the response. Therefore, the magnitude of the response elicited by a GPCR is usually not proportional to receptor activation, and EC 50 and E max are not equivalent to observed affinity and intrinsic efficacy. Rather, EC 50 and 1 Here and throughout the article, we use the term "observed intrinsic efficacy" to refer to Furchgott's definition of "intrinsic efficacy" (Furchgott, 1966), which denotes the amount of activated receptors. As described previously (Ehlert, 2000), it is useful to discriminate between the latter definition of observed intrinsic efficacy and the ratio of agonist affinity constants for ground and active conformations of the receptor (intrinsic efficacy).

Antagonism of the human A(2A) receptor has been implicated as a point of therapeutic intervention in the alleviation of the symptoms associated with Parkinson's disease. This is thought to occur, at least in part, by increasing the sensitivity of the dopaminergic neurons to the residual, depleted levels of striatal dopamine. We herein describe a novel series of functionalized triazolo[4,5-d]pyrimidine derivatives that display functional antagonism of the A(2A) receptor. Optimization of these compounds has resulted in improvements in potency, selectivity, and the pharmacokinetic properties of key derivatives. These efforts have led to the discovery of 60 (V2006/BIIB014), which demonstrates strong oral activity in commonly used models of Parkinson's disease. Furthermore, this derivative has shown excellent preclinical pharmacokinetics and has successfully completed phase I clinical studies. This compound is presently undergoing further clinical evaluation in collaboration with Biogen Idec.

We measured the intrinsic relative activity (RA i ) of muscarinic agonists to detect possible selectivity for receptor subtypes and signaling pathways. RA i is a relative measure of the microscopic affinity constant of an agonist for the active state of a GPCR expressed relative to that of a standard agonist. Our results show that the RA i estimate is a useful receptordependent measure of agonist activity.

We investigated the contractile role of M 2 muscarinic receptors in mouse urinary bladder. When measured in the absence of other agents, contractions elicited to the muscarinic agonist oxotremorine-M exhibited properties consistent with that expected for an M 3 response in urinary bladder from wild-type and M 2 knockout (KO) mice. Evidence for a minor M 2 receptormediated contraction was revealed by a comparison of responses in M 3 knockout and M 2 /M 3 double knockout mice. Treatment of wild-type and M 2 knockout urinary bladder with N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP mustard) caused a large inhibition of the muscarinic contractile response. The residual contractions were much smaller in M 2 knockout bladder compared with wild type, suggesting that M 2 receptors rescue the muscarinic contractile response in wildtype bladder following inactivation of M 3 receptors with 4-DAMP mustard. When measured in the presence of prostaglandin F 2␣ and isoproterenol or forskolin, oxotremorine-M mediated a potent contractile response in urinary bladder from M 3 KO mice. This response exhibited an M 2 profile in competitive antagonism studies and was completely absent in M 2 /M 3 KO mice. Following 4-DAMP mustard treatment, oxotremorine-M elicited a contractile response in wild-type urinary bladder in the presence of KCl and isoproterenol or forskolin, and this response was diminished in M 2 KO mice. Our results show that the M 2 receptor mediates contractions indirectly in the urinary bladder by enhancing M 3 receptor-mediated contractions and inhibiting relaxation. We also show that it is difficult to detect M 2 receptor function in competitive antagonism studies under conditions where a simultaneous activation of M 2 and M 3 receptors occurs.Micturition is mediated through the actions of several neurotransmitters. Among those that directly influence the tone of urinary bladder smooth muscle, acetylcholine is important in contracting the reservoir and relaxing the outlet through activation of muscarinic receptors (de Groat and Yoshimura, 2001). Most evidence shows that it is the M 3 subtype of the muscarinic receptor that mediates the direct contractile response to acetylcholine in the urinary bladder. For example, the contractile response to muscarinic agonists exhibits an M 3 profile in competitive antagonism studies (Noronha-Blob et al., 1989;Longhurst et al., 1995;Choppin and Eglen, 2001), and these contractions are nearly absent in urinary bladder from M 3 muscarinic receptor knockout (M 3 KO) mice (Matsui et al., 2000). Male M 3 KO mice exhibit prominent urinary bladder distension in vivo, demonstrating the essential role of the M 3 receptor in micturition (Matsui et al., 2000). The small, direct contractile response that persists in urinary bladder from M 3 KO mice is completely lost in mice lacking both M 2 and M 3 muscarinic receptors (M 2 /M 3 KO mice), demonstrating that the M 2 receptor is capable of mediating very small contractions and that muscarinic recep-

The ability of forskolin and isoproterenol to inhibit the contractile action of the muscarinic agonist, oxotremorine-M, was investigated in smooth muscle from wild-type and M 2 muscarinic receptor knockout mice. Forskolin (5.0 M) caused a significant reduction in the contractile activity of oxotremorine-M in ileum, trachea, and urinary bladder from both wild-type and M 2 muscarinic receptor knockout mice. This reduction in contractile activity was characterized by decreases in potency or maximal response, but not always both. Similar results were obtained with isoproterenol (1.0 M). The relaxant effects of forskolin in ileum, trachea, and urinary bladder from M 2 receptor knockout mice were approximately 3-to 9-fold greater than those observed in the same tissues from wild-type mice. Similar results were obtained with isoproterenol in ileum and urinary bladder, although the differences between wild-type and M 2 receptor knockout tissues were less than those observed with forskolin. In contrast, there was no significant difference between the relaxant effect of isoproterenol in trachea from wild-type and M 2 receptor knockout mice. In contrast to the results observed with oxotremorine-M as the contractile agent, forskolin and isoproterenol did not exhibit greater relaxant activity against KCl-induced contractions in M 2 receptor knockout mice compared with wild-type mice. These results suggest that a component of the contractile response to muscarinic agonists in smooth muscle involves an M 2 muscarinic receptor-mediated inhibition of the relaxant effects of agents that increase cAMP levels.