Muscarinic acetylcholine receptors (mAChRs) comprise five distinct subtypes denoted M₁ to M₅. The antagonism of M₂ subtype could increase the release of acetylcholine from vesicles into the synaptic cleft and improve postsynaptic functions in the hippocampus via M₁ receptor activation, displaying therapeutic potentials for Alzheimer's disease. However, drug development for M₂ antagonists is still challenged among different receptor subtypes. In this study, by optimizing a scaffold from virtual screening, we synthesized two focused libraries and generated up to 50 derivatives. By measuring potency and binding selectivity, we discovered a novel M₂ antagonist, ligand 47, featuring submicromolar IC₅₀, high M₂/M₄ selectivity (~30-fold) and suitable lipophilicity (cLogP = 4.55). Further study with these compounds also illustrates the structure-activity relationship of this novel scaffold. Our study could not only provide novel lead structure, which was easy to synthesize, but also offer valuable information for further development of selective M₂ ligands.

A series of new dibenzodiazepinone-type muscarinic receptor ligands, including two homo-dimeric compounds, was prepared. Sixteen representative compounds were characterized in equilibrium binding studies with [(3)H]N-methylscopolamine ([(3)H]NMS) at the muscarinic receptor subtype M2, and seven selected compounds were additionally investigated at M1, M3, M4 and M5 with respect to receptor subtype selectivity. The side chain of the known M2 preferring muscarinic receptor antagonist DIBA was widely varied with respect to chain length and type of the basic group (amine, imidazole, guanidine and piperazine). Most of the structural changes were well tolerated with respect to muscarinic receptor binding, determined by displacement of [(3)H]NMS. Compounds investigated at all subtypes shared a similar selectivity profile, which can be summarized as M2>M1≈M4>M3≈M5 (46, 50, 57, 62-64) and M2>M1≈M4>M3>M5 (1, 58). The homo-dimeric dibenzodiazepinone derivatives UNSW-MK250 (63) and UNSW-MK262 (64) exhibited the highest M2 receptor affinities (pIC50=9.0 and 9.2, respectively). At the M2 receptor a steep curve slope of -2 was found for the dimeric ligand 63, which cannot be described according to the law of mass action, suggesting a more complex mechanism of binding. In addition to equilibrium binding studies, for selected ligands, we determined pEC50,diss, an estimate of affinity to the allosteric site of M2 receptors occupied with [(3)H]NMS. Compounds 58 and 62-64 were capable of retarding [(3)H]NMS dissociation by a factor >10 (Emax,diss >92%), with highest potency (pEC50,diss=5.56) residing in the dimeric compound 64. As the monomeric counterpart of 64 was 100 times less potent (62: pEC50,diss=3.59), these data suggest that chemical dimerization of dibenzodiazepinone-type M receptor ligands can enhance allosteric binding.

Muscarinic receptor (mAChRs) subtypes are viable targets for the design of novel agents for use in a number of central and peripheral disorders. In vitro isolated tissue functional assays for muscarinic receptor subtypes have played an invaluable role in basic research and drug discovery. The availability of biological assays for generation of quantitative estimates of affinity and potency of ligands allows evaluation of the contribution of a given mAChR to the functional end organ response and also enables drug discovery by facilitating the iterative process of screening and optimization of chemical leads. This unit describes isolated tissue functional assays for the quantification of ligand affinity and efficacy at the M(1), M(2), M(3), M(4), and M(5) muscarinic receptor subtypes in tissues expressing the native receptor using organ bath techniques.

The concept that polyamines may represent a universal template in the receptor recognition process is embodied in the design of ligands for different biological targets. As a matter of fact, the insertion of different pharmacophores onto the polymethylene tetraamine backbone can tune both affinity and selectivity for any given receptor. The application of this approach provided a prospect of modifying benextramine (1). structure to achieve specific recognition of muscarinic receptors that led to the discovery of methoctramine (2). which is widely used as a pharmacological tool for muscarinic receptor characterization. In turn, appropriate structural modifications performed on the structure of methoctramine led to the discovery of new polyamines endowed with high affinity and selectivity for (a). muscarinic receptor subtypes, (b). G(i) proteins, and (c). muscle-type nicotinic receptors. Thus, polyamines tripitramine (9) and spirotramine (33), among others, were designed, which were shown to be highly selective for muscarinic M(2) and M(1) receptors, respectively. Several polyamines have been discovered, which inhibit noncompetitively a closed state of the nicotinic receptor. These ligands, such as 66, resulted in important tools for elucidating the mode and site of interaction of polyamines with the ion channel. It was discovered that reducing the flexibility of the diaminohexane spacer of methoctramine led to polyamines, such as 70, which are endowed with a biological profile significantly different from that of the prototype. Most likely, tetraamine (70) is a potent activator of G(i) proteins. Finally, the universal template approach formed the basis for modifying benextramine (1) structure to the design of ligands, which display affinity for acetylcholinesterase and muscarinic M(2) receptors. Thus, these polyamines, such as caproctamine (78), could have potential in the investigation of Alzheimer disease.

1 Despite the growing social interest in human urinary tract disorders, the aetiology of detrusor instability remains poorly understood. Myogenic and neural impairment of detrusor activity caused by CNS or autonomic injuries can results in dysfunctions of normal voiding of the bladder such as urinary incontinence. 2 The contractility of human detrusor smooth muscle is critically dependent on acetylcholine-induced muscarinic receptor activation. Biochemical and functional in vivo and in vitro studies suggest the presence of an heterogeneous population of muscarinic receptor subtypes (M1-M4) localized at muscular and neutral sites. There is increasing evidence on the prejunctional auto- and hetero-regulation of acetylcholine release from parasympathetic nerve endings in modulating detrusor muscle contraction during micturition. 3 Activation of P2X purinoreceptors closely associated with the parasympathetic varicosities seems to be implicated to varying extent in the contractility in normal or instable human detrusor. Interestingly, P2X(1) subtype expression on smooth muscle increases considerably in the symptomatically obstructed bladder. A striking absence of P2X(3) and P2X(5) subtypes was observed in the cholinergic innervation of detrusor from patients with urgent incontinence. Thus, it is likely that alteration of the neural acetylcholine control can play a critical role in pathological states. 4 If the failures in storage and voiding can be recognized urodynamically, considerable difficulties remain in investigating the underlying functional changes especially because the study of the pathophysiology requires techniques that can be justified in animals but not in humans. 5 Recently, to solve this problem an alternative technique using human smooth muscle cells in culture has been developed. Human cell lines may be relevant in investigating the molecular pathways in physiological and pathological conditions. 6 The potential development of novel molecular therapeutic strategies such as gene therapy and tissue engineering is also discussed.

In our search for M2-selective muscarinic receptor antagonists, we synthesized 1,3-disubstituted indenes. The effects of different basic moieties with regard to binding and selectivity towards the five distinct muscarinic receptor subtypes were investigated. The results show that the quinuclidine series afforded the most promising compounds in terms of both receptor affinity and M2-subtype selectivity.

The identity of the muscarinic receptor subtype in the chick ileum was investigated in functional and binding studies. Preliminary studies [Choo, L.-K., Mitchelson, F., Napier, P. 1988. J. Auton. Pharmacol. 8, 259-266] suggested apparent avian and mammalian family differences in the muscarinic receptor profile of ileal smooth muscle. In the current study, further characterisation was undertaken using a greater range of antagonists exhibiting high affinity for specific muscarinic receptor subtypes. Dissociation constants from functional and binding experiments were compared with published values for antagonists at each of the five muscarinic receptor subtypes. Linear regression and correlation analyses revealed the receptor initiating the contractile response was most likely of the muscarinic M(3) receptor subtype as the slope of the linear regression was 1.01+/-0.14 and the corresponding correlation coefficient (r) was 0.95. The mammalian muscarinic M(5) receptor subtype also showed a high correlation with the data giving a slope of 0.89+/-0.27 and r value of 0.76. These findings were in direct contrast to those from binding experiments in which the single binding site detected was of the muscarinic M(2) receptor subtype. The slope of the linear regression was 1.14+/-0.24 with an r value of 0.87. Thus, these results suggest that there exists a high proportion of the muscarinic M(2) receptor subtype within the tissue that does not contribute to the functional response.

1. The pharmacological characteristics of muscarinic receptors in rat isolated uterus were studied in ovariectomized (ov.) and sham operated (sh.) animals. 2. Competition radioligand binding studies, using uterine membranes and [3H]-NMS, were undertaken with several muscarinic receptor antagonists. Most of the antagonists indicated a one-site fit with apparent affinity estimates (pKi) unchanged by ovariectomy. The selective M2 antagonist, tripitramine revealed high (representing 33+/-8 and 38+/-2%) and low (67+/-8 and 62+/-2%) affinity binding sites in both sh. and ov. rat uterus, respectively. These sites likely represented muscarinic M2 and M3 receptors and the proportions were not significantly different in the two conditions. 3. Carbachol induced concentration-dependent contractions which were surmountably antagonized by several muscarinic receptor antagonists (pKB, sh.; ov.): zamifenacin (9.19; 9.18), p-F-HHSiD (8. 50; 9.06), tripitramine (7.23; 7.54), himbacine (7.21; 7.41), methoctramine (6.79; 7.49), pirenzepine (6.48; 7.21), AF DX 116 (6. 26; 6.61), MTx 3 (<7.00; <7.00) and PD 102807 (<7.00; <7.00). 4. The apparent affinity values obtained in functional studies using the uteri from both sh. and ov. animals correlated most closely with values reported at human recombinant muscarinic M3 receptors. This suggests that the muscarinic M3 receptor mediates contraction under both conditions. 5. Radioligand binding experiments indicate the presence of M2 receptors, in addition to M3 receptors, which probably explains the discrepancies between functional and binding affinities. These data further suggest that the pharmacological profile and proportions of the two populations of muscarinic receptors are unaffected by ovariectomy.

A variety of neurons in gastrointestinal and genitourinary smooth muscle express muscarinic auto- as well as heteroreceptors. These receptors are found on the soma and dendrites of many cholinergic, sympathetic and NANC neurons and on axon terminals. A given neuron may contain both excitatory and inhibitory presynaptic muscarinic receptors. The subtypes involved are species- and tissue-dependent, and neuronal M1 to M4 receptors have been shown to be expressed in smooth muscle tissues. In this study, the ability of several selective muscarinic receptor antagonists to inhibit the effect of arecaidine propargyl ester (APE) on prejunctional muscarinic receptors on sympathetic nerve endings in the rabbit anococcygeus muscle (RAM) was investigated to characterise the receptor subtype involved. Electrical field stimulation (EFS) resulted in a release of noradrenaline (NA) eliciting monophasic contractions due to stimulation of postjunctional alpha1-adrenoceptors. The selective muscarinic agonist APE did not reduce contractions to exogenous NA, but caused a concentration-related and N-methylatropine-sensitive inhibition of neurogenic responses. All muscarinic antagonists investigated failed to affect the EFS-induced contractions, but shifted the concentration-response curve of APE to the right in a parallel and surmountable fashion. Schild analysis yielded regression lines of unit slope, indicating competitive antagonism. The following rank order of antagonist potencies (pA2 values) was found: tripitramine (9.10) > AQ-RA 741 (8.26) > or = himbacine (8.04) > or = (S)-dimethindene (7.69) > pirenzepine (6.46) > or = p-F-HHSiD (6.27). A comparison of the pA2 values determined in the present study with literature binding and functional affinities obtained at native or recombinant M1 to M5 receptors strongly suggests that NA release from sympathetic nerve endings in RAM is inhibited by activation of prejunctional muscarinic M2 receptors.

Normal physiological voiding as well as generation of abnormal bladder contractions in diseased states is critically dependent on acetylcholine-induced stimulation of contractile muscarinic receptors on the smooth muscle (detrusor) of the urinary bladder. Muscarinic receptor antagonists are efficacious in treating the symptoms of bladder hyperactivity, such as urge incontinence, although the usefulness of available drugs is limited by undesirable side-effects. Detrusor smooth muscle is endowed principally with M2 and M3 muscarinic receptors with the former predominating in number. M3 muscarinic receptors, coupled to stimulation of phosphoinositide turnover, mediate the direct contractile effects of acetylcholine in the detrusor. Emerging evidence suggests that M2 muscarinic receptors, via inhibition of adenylyl cyclase, cause smooth muscle contraction indirectly by inhibiting sympathetically (beta-adrenoceptor)-mediated relaxation. In certain diseased states, M2 receptors may also contribute to direct smooth muscle contraction. Other contractile mechanisms involving M2 muscarinic receptors, such as activation of a non-specific cationic channel and inactivation of potassium channels, may also be operative in the bladder and requires further investigation. From a therapeutic standpoint, combined blockade of M2 and M3 muscarinic receptors would seem to be ideal since this approach would evoke complete inhibition of cholinergically-evoked smooth muscle contractions. However, if either the M2 or M3 receptor assumes a greater pathophysiological role in disease states, then selective antagonism of only one of the two receptors may be the more rational approach. The ultimate therapeutic strategy is also influenced by the extent to which pre-junctional M1 facilitatory and M2 inhibitory muscarinic receptors regulate acetylcholine release and also which subtypes mediate the undesirable effects of muscarinic receptor blockade such as dry mouth. Finally, the consequence of muscarinic receptor blockade in the central nervous system on the micturition reflex, an issue which is poorly studied and seldom taken into consideration, should not be ignored.

In many smooth muscle tissues a minor M3-muscarinic acetylcholine (mACh) receptor population mediates contraction, despite the presence of a larger M2-mACh receptor population. However, this is not the case for guinea-pig uterus where radioligand binding and functional studies exclude a dominant role for M3-mACh receptors. Using tissue from animals pre-treated with diethylstilboestrol, estimates of antagonist affinity were made before and after selective alkylation procedures, together with estimates of agonist affinity to characterise the mACh receptor population mediating carbachol-induced contraction of guinea-pig isolated uterus. Antagonist affinity estimates made at 'protected' receptors were not significantly different from those made in untreated tissues. However all estimations were significantly different from those reported in guinea-pig ileum and atria. The rank order of affinities were atropine>zamifenacin=tripitramine> methoctramine. Carbachol-induced contractions were insensitive to the M4-selective muscarinic toxin MTx-3, or PD102807 (0.1 microM) ruling out a role for M4-mACh receptors. The agonist affinity value for L-660,863, a putative 'M2-selective' agonist of 5.44+/-0.30 (n=6) was significantly different from that reported in guinea-pig atria. In contrast, the pKA value for carbachol (4.22+/-0.17 n = 8) agrees with that reported for guinea-pig ileum. Carbachol-induced contractions were insensitive to pertussis toxin although carbachol-induced inhibition of forskolin-stimulated cyclic AMP production was attenuated, ruling out the involvement of Gi-proteins in contraction. Radioligand binding studies revealed a KD for N-[3H]-methylscopolamine of 0.12+/-0.05 nM and a Bmax of 147+/-18 fmol mg protein(-1). Antagonist affinity estimates made using competition binding studies supported previous data suggesting the presence of a homogenous population of M2-mACh receptors. These data suggest a small population of mACh receptors with an atypical operational profile which can not be distinguished using radioligand binding studies may mediate carbachol-induced contraction of guinea-pig isolated uterus.

1. The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells. 2. Ascending concentrations of carbachol (3-300 microM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (Gmax) of 27.4+/-1.4 nS and a mean -log EC50 of 5.12+/-0.03 (mean+/-s.e.mean) (n=114). 3. Muscarinic antagonists with higher affinity for the M2 receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA2 values of 8.1, 8.0 and 9.1, respectively. 4. All M3 selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC50 for carbachol. At higher concentrations M3 antagonists shifted the agonist curve to the right, increasing the EC50, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in Gmax (M3 effect) and significant increase in the EC50 (M2 effect) in the same concentration range. 5. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPgammaS to the pipette (without application of carbachol) was unaffected. 6. The results support the hypothesis that carbachol activates M2 muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M3 receptors. As an increasing fraction of M3 receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M2 receptors are disabled.

1. The muscarinic receptor subtypes mediating contraction of the guinea-pig lung strip and inhibition of the release of acetylcholine from cholinergic vagus nerve endings in the guinea-pig trachea in vitro have previously been characterized as M2-like, i.e. having antagonist affinity profiles that are qualitatively similar but quantitatively dissimilar compared to cardiac M2 receptors. The present study sought to establish definitely the identity of these receptor subtypes by using the selective muscarinic receptor antagonist, tripitramine. Guinea-pig atria and guinea-pig trachea (postjunctional contractile response) were included for reference. 2. It was found that tripitramine antagonized methacholine-induced contractions of the guinea-pig lung strip with pKB value of 8.76 +/- 0.05. Both the parallel shifts of the concentration-response curves and the slope of the Schild plot begin not significantly different from unity (when antagonist preincubation was for 2 h) indicated the involvement of a single population of receptors in the contractile response. From the pKB values obtained with tripitramine and a range of other selective muscarinic receptor antagonists (cf. Roffel et al., 1993), this single population of receptors can only be classified as M2-like. 3. Tripitramine antagonized methacholine-induced chronotropic and inotropic responses in guinea-pig right and left atria with apparent pKB values of 9.4-9.6. However, such values were only obtained when antagonist preincubation was relatively long and/or antagonist concentration relatively high (e.g with 1 h at 100 or 300 nM but 3 h at 30 nM). It thus appears that low concentrations of tripitramine do not readily equilibrate with M2 receptors in guinea-pig atria nor with M2-like receptors in the guinea-pig lung strip. 4. Tripitramine increased electrical field stimulation-induced cholinergic twitch contractions in guinea-pig trachea in concentrations of 0.3-100 nM, by blocking prejunctional muscarinic inhibitory autoreceptors; with higher concentrations, twitch contractions were progressively diminished, as a result of blocking postjunctional M3 receptors (apparent pKB value 6.07 +/- 0.15). The pEC20 value (-log concentration that increases twitch by 20% maximum) was 8.29 +/- 0.08, which would suggest that M4 receptors are involved in this response. 5. Oxotremorine-induced inhibition of the release of prelabelled [3H]-acetylcholine from guinea-pig trachea, under conditions where there is no auto-feedback, was blocked by tripitramine (2 h preincubation) with a pKB value of 8.56 +/- 0.06. The slope of the corresponding Schild plot was not significantly different from unity, which together with the parallel shifts of the concentration-response curves indicated the involvement of a single muscarinic receptor subtype. 6. Since the pKB value for tripitramine at prejunctional receptors in guinea-pig trachea is in between the affinities towards M2 and M4 receptors, correlation plots were constructed to compare the pKB values obtained with tripitramine and a range of other selective muscarinic receptor antagonists (cf. Kilbinger et al., 1995) to reported affinities at M1-M4 receptors. This showed rather similar distribution patterns of the data points around the line of equality in the case of M2 and M4 receptor subtypes. However, the correlation coefficient was markedly better for M2 (0.9667) than for M4 (0.5976). Since recent evidence suggests that M4 receptors are not expressed in cholinergic nerves from guinea-pig trachea, it is concluded that prejunctional muscarinic autoinhibitory receptors in this tissue exhibit an atypical M2 type character, with a pharmacological profile distinct from cardiac M2 receptors.

A new method for estimation of agonist-affinity (KA) and relative efficacy was introduced. This method afforded a procedure by which relative efficacy may be estimated while the actual KA values of agonist-receptor complexes are unknown. The relative efficacy may be estimated by employing a newly defined drug parameter, namely the eES value. The eES value is related to drug efficacy and is defined in such a manner that an isolated eES is a meaningful quantity which may indicate whether or not spare receptors are present in an agonist-effector system. The estimation of eES was based on the fact that fixed agonist-competitive antagonist combinations mimic partial agonists and mediate submaximal concentration-effect curves. However, for the practical estimation of eES one may employ data acquired from agonistic concentration-effect curves determined in the absence and presence of increasing concentrations of a competitive antagonist. This procedure was illustrated by utilizing theoretical concentration-effect curves and applied practically by estimating eES and KA values acquired from sets of carbachol and salbutamol curves. The sets of carbachol and salbutamol concentration-effect curves were determined in the absence and presence of their respective competitive antagonists, namely tripitramine and pindolol.

In longitudinal muscle of guinea-pig ileum, activation of muscarinic receptors causes contraction antagonised by M3 receptor subtype antagonists despite a preponderance of M2 receptor subtype binding sites. Experiments on single smooth muscle cells under voltage-clamp described here show that the cationic current evoked by carbachol which normally causes depolarization of the muscle is inhibited competitively by M2 antagonists with affinities typical of antagonism at a M2 receptor. However, M3 antagonists strongly reduced the maximum cationic current which could be evoked by carbachol in a non-competitive manner with affinities typical for an action at M3 receptors. Thus cation channels are gated by M2 receptor activation but strongly modulated by activation of M3 receptors.

Methods are presented which enables the dissociation constant of agonist-receptor complexes (KA) to be estimated without knowledge of the relative intrinsic efficacies. The dissociation constants are estimated from fixed agonist-competitive antagonist concentration combinations which simulate partial agonists. In general, estimating of KA by employing these methods require phi and KB to be known; where phi = [antagonist]/[agonist] and KB is the dissociation constant of the competitive antagonist-receptor complex. Some of these methods require only knowledge of phi, while KB may be unknown. A method is also described to estimate KA without knowing the value of KB and the competitive antagonist concentration. In addition to the estimation of KA values, a new method for estimation of the dissociation constants of competitive antagonist-receptor complexes (KB) is reported. One of the new methods was exemplified practically by using sets of experimental agonist concentration-effect curves determined in the absence and presence of increasing concentrations of competitive antagonists. From this pharmacological data the apparent KA value for carbachol (in muscarinic M3 receptors of the guinea-pig ileum) was determined. This action illustrated that the apparent affinity values (KA) and height of the stimulus curve of an agonist may be determined from published pharmacological data. This procedure affords a possibility to establish whether or not spare receptors are present in a particular system. It was also shown that relative affinity values provide more reliable information than does isolated KA values.

Muscarinic receptors are composed of a family of four subtypes each of which can be distinguished pharmacologically and structurally. The physiological role of each subtype in the central and peripheral nervous systems remains to be clarified, due, in part, to a lack of agonists and antagonists with adequate subtype selectivity. Nonetheless, several agonists with functional selectivity for M1 receptors are now in advanced clinical evaluation for Alzheimer's disease, while selective M1/M3 antagonists may prove useful in the treatment of disorders of smooth muscle function. These novel compounds thus provide an advance over earlier therapeutics with which the clinical efficacy was compromised by the side effect profile. This review attempts to assess novel, selective agonists and antagonists, both in terms of their use in defining muscarinic receptor subtypes and their potential clinical utility.