. Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates amyloid processing and produces antipsychotic-like activity in rats. J Neurosci. 2008 Oct 8;28(41):10422-33. PubMed.


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  1. The difficulty in developing drugs for specific subtypes of some neurotransmitter receptors is that the transmitter binding site is normally highly conserved. One way around this is to design molecules that modulate a putative allosteric site(s) in the receptor. This is the take-home message of the paper of Jones et al., 2008, that describes some interesting effects in vitro and in vivo of TBPB, a novel allosteric activator of the M1 muscarinic receptor (mAChR). The authors show a high selectivity of TBPB for the rat M1 mAChR as compared to the human M2-M5 mAChR subtypes using one readout in vitro, namely agonist-evoked increases of intracellular calcium ions. Furthermore, TBPB, like other selective and non-selective M1 agonists, elevates α-APPs in cell-based assays that contain mainly the M1 mAChR (in this study, PC12 cells co-transfected with human M1 mAChR and APP Swedish mutation). In another interesting aspect of this paper, the authors describe the effects of TBPB in animal models that may have some predictive value for the treatment of symptoms associated with schizophrenia. While clearly this paper is an important step forward in our understanding of M1 mAChR activation, its allosteric site(s), and the important role of this receptor in affective disorders such as schizophrenia, several issues need to be addressed regarding this paper and development of M1 agonists for treatment of CNS diseases such as Alzheimer’s (AD) or schizophrenia, in general:

    1. We have succeeded over the years in developing functionally selective M1 agonists of the AF series (e.g., AF102B, AF150(S), AF267B, and more) with high specificity and ability to cross the blood-brain barrier (see attached papers). AF267B, for example, is orally available, penetrates the blood-brain barrier, and has an excellent pharmacokinetic profile both in preclinical and clinical studies, a high bioavailability, and a wide safety margin. AF267B (originally from our lab, >99.9 percent chemical and enantiomeric purity) was shown to reduce both Aβ and tau pathologies in the hippocampus and cortex, and to reverse cognitive deficits in 3xTg-AD mice (Caccamo et al., 2006). These effects of AF267B were observed without side effects at 1 and 3 mg/kg (ip, daily administration for two months), respectively—doses 45 or 15 times lower than those in which overt effects such as salivation were seen. This showed clearly that the compound has a wide safety margin and that the beneficial effects of AF267B can be attributed to M1 mAChR activation. Furthermore, in the same study, reduced M1 mAChR activation (by dicyclomine, a relatively selective M1 antagonist) increased Aβ production, tau hyperphosphorylation, and cognitive deficits in the same model.

    2. Different receptor reserves of mAChR subtypes in cell cultures and different experimental design can provide results that may differ from one lab to another. Given this caveat and the difficulty in predicting whether a putative M1 agonist, be it orthosteric or allosteric, will be solely selective for the M1 mAChR both in vitro and in vivo, one has to use several tests in order to obtain a valid estimate, and of course the final test will be in clinical studies. In this context, AF267B (originally from our lab, >99.9 percent chemical and enantiomeric purities) emerged in our studies as a highly selective functional M1 agonist as evidenced, inter alia, in cell cultures stably transfected with the human M1-M5 mAChR subtypes by mobilization of intracellular calcium ions (EC50 = 1.7 μM [almost full agonist vs. carbachol] with marginal effects [20-10 percent] on M3 and M5 mAChR and no effects on M2 and M4 mAChR subtypes when tested at 100 μM). AF267B emerged also as a selective M1 partial agonist vs. its effect on M3 and M5 mAChR as assayed by an increase in phosphoinositides turnover in the same cell cultures. Additionally, in cell cultures such as PC12 cells stably transfected with rat M1 mAChR, AF267B was a partial agonist (readout: phosphoinositides elevation or arachidonic acid release, but inactive on cAMP release mediated by the M1 mAChR). Moreover, AF267B was inactive as an agonist on cell cultures that lack a significant population of the M1 mAChR, but still have intrinsic M3 mAChR (e.g., PC12 or SH-SY5Y cells). For in vivo selectivity of AF267B toward M1 mAChR-mediated functions, see point 1 above.

    3. Regarding the allosteric agonist TBPB, the authors emphasize its value in the non-amyloidogenic processing of APP as mediated by M1 mAChR. This is an important finding that confirms studies with other muscarinic agonists, including the functionally selective M1 agonists from the AF series. However, effects of TBPB in cell culture or brain slices that contain several mAChR subtypes were not reported in the paper. Notably, in such preparations the M1 agonists of the AF series appeared even more potent than the non-selective agonist carbachol most probably due to their selective effects on the M1 mAChR.

    4. By binding to allosteric sites on the receptor, allosteric modulators can sometimes be more selective than orthosteric agonists for the M1 vs. M2-M5 mAChR. However, paradoxically one cannot disregard the scenario that such an allosteric agonist will bind or modulate other G protein-coupled receptors (GPCR) in spite of its excellent selectivity for the M1 mAChR. While the orthosteric site is the target that dictates the selectivity of a given neurotransmitter for a receptor including its subtypes, the exact orthosteric site is not found in other GPCRs. However, we lack enough compelling information for whether the allosteric site(s) for one receptor (e.g., the M1 mAChR) is found exclusively in this particular receptor and cannot be found in some other GPCR. Thus, while the orthosteric agonist AF267B was shown to bind specifically only to the mAChR subtypes when tested on a plethora of GPCRs and other receptors and enzymes, it is not clear from the paper whether TBPB is solely specific for the M1 mAChR. In fact, in the range of its full effects on the M1 mAChR, TBPB also binds to the D2 receptors in the μM range.

    5. Finally, the clinical value of a selective M1 agonist, be it orthosteric or allosteric, is dictated by its pharmacokinetic profile and bioavailability. In this context, AF267B has an excellent pharmacokinetic profile producing the metabolite, N-des-methyl analog, a highly selective partial M1 agonist, in vitro and in vivo. The pharmacokinetic profile of TBPB was not mentioned, and no effects of low doses were reported in in vivo studies. Notably, based on the lesson learned with xanomeline (highly selective in vitro, yet bad pharmacokinetics and selectivity in vivo) selectivity for a mAChR subtype in vitro is not a guarantee for a good selectivity in vivo or a promising bioavailability/pharmacokinetic profile. Definitively, more studies are required to substantiate the practical value of TBPB and to show whether it has a preclinical profile that can match a good M1 candidate for AD treatment.


    . M1 muscarinic agonists target major hallmarks of Alzheimer's disease--the pivotal role of brain M1 receptors. Neurodegener Dis. 2008;5(3-4):237-40. PubMed.

    . Cholinergic treatments with emphasis on m1 muscarinic agonists as potential disease-modifying agents for Alzheimer's disease. Neurotherapeutics. 2008 Jul;5(3):433-42. PubMed.

    . M1 receptors play a central role in modulating AD-like pathology in transgenic mice. Neuron. 2006 Mar 2;49(5):671-82. PubMed.

    View all comments by Abraham Fisher
  2. The paper by Jones et al. describes TBPB, a novel compound that represents a new generation of highly specific drugs. The study is important for the AD field because this drug will allow the role of the M1 muscarinic receptor in AD to be more clearly defined, including its potential for AD therapeutics. There is a long and well-known history of research on the cholinergic system in AD that led to the development of cholinesterase inhibitors as approved therapies. However, the therapeutic utility of cholinesterase inhibitors is modest, and because these drugs lead to non-specific activation of many different subtypes of muscarinic and nicotinic receptors, side effects are frequent and tolerability suboptimal. For these reasons, development of selective agonists has been a long sought goal for AD treatment, dating back to the discovery of the cholinergic deficiency in AD. Indeed, a wealth of preclinical and clinical data have supported the prediction that highly specific M1 agonists would be more efficacious for cognitive and behavioral symptoms of AD. Such drugs would also be expected to be better tolerated with fewer cholinergic side effects than cholinesterase inhibitors. However, the development of selective drugs has been an elusive goal despite decades of intensive efforts by big pharma.

    There has been a resurgence of interest in M1 receptor-based therapeutics for AD with the findings of LaFerla's and Fisher's groups in 2006 that AF267B (Caccamo et al., 2006), a purported M1 agonist, reduced amyloid and tau pathologies in the triple transgenic AD mouse model. The current study is important as it represents a true breakthrough in the pharmacology of the muscarinic cholinergic system, with the development of a truly selective M1 agonist termed TBPB. As the authors show, TBPB has anti-amyloidogenic effects on cells, and activates central M1 receptors to produce expected behavioral, cognitive, and neurophysiological benefits. Moreover, the study demonstrates that AF267B is not as selective for M1 as previously believed. Conn's group carefully assesses the specificity of AF267B and shows that this compound also activates M3 receptors—perhaps even better than M1. While this might be a potential beneficial property for anti-amyloid effects, it makes it impossible to tease out the role of M1 vs. M3 from previous studies, and its potential as a drug could be limited by M3 side effects (which are likely a major source of the peripheral cholinergic side effects plaguing current drugs, particularly GI, and can limit dosing). Thus, TBPB and other M1 selective drugs may have improved efficacy and tolerability.

    The pharmacological breakthrough comes from targeting the receptor to a different binding site than has been conventionally targeted for cholinergic drugs. TBPB binds to an allosteric binding site, which is more highly divergent among the five closely related muscarinic receptor family members. Other drugs acting at this site have been recently described by Acadia Pharmaceuticals. With this knowledge in hand, one might expect newer generations of highly selective drugs for M1 and the other muscarinic receptor subtypes. Additional studies of these drugs will be necessary to determine if they will be advantageous for clinical applications, including neuroprotection for AD.


    . M1 receptors play a central role in modulating AD-like pathology in transgenic mice. Neuron. 2006 Mar 2;49(5):671-82. PubMed.

    View all comments by Allan Levey

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