How 7 Nicotinic Acetylcholine Receptors Work

Allosteric Modulation of Nicotinic Acetylcholine Receptors - A New Therapy Option for the Treatment of Nerve Agent Poisoning?

The resulting cholinergic syndrome becomes life-threatening if not adequately treated. The current standard therapy, consisting of a competitive mAChR antagonist (e.g. atropine) and an oxime to reactivate the AChE (e.g. obidoxime, pralidoxime, HI-6) is not sufficient for poisoning with soman or tabun . Therefore, alternative therapeutic options are urgently needed. An innovative approach involves the use of active ingredients that interact selectively with the nAChR. More precisely, the use of positive allosteric modulators (PAM), which increase the population of the open and, in principle, metastable state of the nAChR, could be promising. MB327 (1,1 ’- (propane-1,3-diyl) bis (4-tert-butylpyridinium) diiodide) is able to restore muscle strength in tissue preparations of various species (including human respiratory muscle preparations) after soman poisoning. It was recently revealed that the mode of action of MB327 is receptor-mediated: the activity of previously desensitized nAChR could be restored, a typical property of so-called type II PAM. However, MB327 does not show high effectiveness and selectivity. As a result, more efficient and innovative active ingredients based on the optimal pharmacophore must be developed.

For the development of new antidotes, which can be used in particular in the therapy of soman or tabun poisoning, the elucidation of structure-effect relationships is necessary. Since the first results are promising, this challenge should be taken up with emphasis.


Nerve agents, poisoning, therapeutic approach, nicotinic acetylcholine receptors, desensitization, positive allosteric modulators, pharmacological studies, active ingredients, drug design


Nerve agents, poisoning, therapeutic approach, nicotinic acetylcholine receptors, desensitisation, positive allosteric modulators, pharmacological investigations, drugs, drug design


Last but not least, the use of nerve warfare agents against the civilian population in Syria at the beginning of April 2017 shows that chemical warfare agents are still a serious threat to both military personnel and the civilian population. In particular, the relatively easy production of highly toxic organophosphorus compounds, be it nerve agents or certain pesticides, underscores the need for effective medical treatment.

The decisive toxic mechanism of organophosphorus compounds is based on the inactivation of acetylcholinesterase (AChE, EC, a serine hydrolase, due to phosphorylation or phosphonylation of the hydroxyl group of serine, which is located in the active center of the enzyme [1, 2]. The AChE terminates the intrinsic activity of the neurotransmitter on the postsynaptic membrane in the neuron or the neuromuscular endplate. If AChE is inhibited, the hydrolytic degradation of acetylcholine is no longer catalyzed. Since acetylcholine continues to be released from the presynapse, the neurotransmitter accumulates in the synaptic gap [3] and the function of cholinergic receptors, on the one hand muscarinic (mAChR) and on the other hand nicotinic acetylcholine receptors (nAChR), is restricted.

The resulting cholinergic syndrome manifests itself with peripheral nervous (e.g. bronchorrhea, bronchospasm, changed heart rates, muscular paralysis, uncontrolled urination and defecation) and central nervous effects (e.g. tremors, convulsions, respiratory arrest) [4,5]. Ultimately, death occurs through peripheral and central respiratory arrest [6, 7].

Current therapy for nerve agent poisoning

Since the syndrome progresses rapidly and there is a threat of paralysis of the respiratory muscles, treatment must be initiated immediately, usually by first aiders or, in the military sector, as part of self-help and help from fellow soldiers [8]. Standard treatment includes atropine, which is a competitive antagonist for mAChR, and oximes (e.g. obidoxime, pralidoxime, HI-6), which reactivate acetylcholinesterase [9, 10]. In addition, anticonvulsants are appropriate to reduce the cholinergic effects in the central nervous system [11].

Atropine antagonizes the effects on the mAChR caused by the accumulated acetylcholine, but not on the nAChR, which are particularly important for muscle function. The nicotinic effects are currently being treated indirectly: Oximes dephosphylate the inhibited AChE and thereby restore its catalytic activity. The resulting breakdown of the excess acetylcholine and the return to the normal range mean that both nAChR and mAChR function “normally” again [12]. To date, however, there is no broad-spectrum oxime available that can be used universally as the only antidote for the treatment of all types of nerve agent poisoning [13]. Therefore the combination of several oximes with complementary and systematic effects seems to be a useful concept [14].

However, the use of oximes is limited if the AChE can no longer or only insufficiently be reactivated. This may be because the enzyme-nerve warfare agent complex in the human body is either generally not accessible for reactivation, as in the case of tabun [15], or reactivation is prevented by subsequent chemical processes, such as dealkylation. This so-called “aging” of the enzyme-organophos (phon) ate complex can take place within a few minutes in Soman [16 - 18]. In these cases, the neuromuscular transmission that has come to a standstill - caused by the nAChR dysfunction - cannot be remedied with medication. The resulting peripheral respiratory arrest is a life-threatening problem that is to be remedied in the future with a new therapeutic approach. Specifically, the goal is to restore neuromuscular transmission, especially in the respiratory muscles [19].

Thus, drugs that selectively interact with the nAChR are of therapeutic interest. In particular if the endogenous cholinergic tone is controlled by these active ingredients despite the acetylcholine excess, i. H. can be restored by dose and application time [20].

Nicotinic acetylcholine receptors

Fig. 1: Schematic representation of some nAChR subtypes in top view: A) human α7 (h5α7-nAChR), B) human muscle type (h2α1β1δε-nAChR), C) torpedo muscle type (t2αβγδ-nAChR); yellow circles: orthosteric binding sites, e.g. B. the agonist acetylcholine.
Nicotinic acetylcholine receptors are cation-selective, pentameric ion channels that are controlled by neurotransmitters. They are widespread in the brain, in ganglia of the sympathetic and parasympathetic nervous system, in skeletal muscle, in epithelial and immune cells [21] and are localized on both the pre- and post-sided membrane of the synaptic cleft [22, 23]. They play a crucial role in cholinergic neurotransmission in both the central (CNS) and the peripheral nervous system (PNS) and are found in numerous diseases of the CNS, such as B. Alzheimer's, Parkinson's, schizophrenia, tobacco addiction and PNS, z. B. congenital myasthenic syndrome, myasthenia gravis, is involved [24, 25].

The nAChR consists of five subunits (denoted by the Greek letters α, β, γ, δ and ε), each of which consists of an extracellular domain, a transmembrane region and an intracellular domain. The five subunits are not covalently linked to one another and can either be identical (homomeric, e.g. 5α7) or different (heteromeric, e.g. 2α1β1δε) [26]. They are arranged with one another in such a way that a pore is formed in the middle.

The so-called muscle type nAChR is a heteromeric subtype and consists of the subunits 2α1β1δε together [27]. This subtype is located exclusively at the neuromuscular synapse and converts a chemical signal into an electrical signal. In pathophysiological situations (e.g. atrophy due to immobilization), homomeric 5α7-nAChR are highly expressed in muscles, which are then involved in neurotransmission; it is probably a backup system [28, 29].

The endogenous agonist acetylcholine binds in binding pockets that are located in the extracellular domain and are each formed by two subunits. This so-called orthosteric binding site is found in the heteromeric muscle type 2α1β1δε-nAChR in the α / δ and the α / ε interface. With the homomeric 5α7-nAChR, 5 -orthosteric binding sites are theoretically possible (one for each α / α interface), but the occupation of two binding sites is sufficient for activation [30]. The nAChR is relatively highly conserved. For example, the muscle type nAChR of the electric ray (2αβγδ) shows a high degree of homology to the human muscle type nAChR (2α1β1δε) to [31] (Figure 1).

When closed, the 5 subunits are twisted together so that the narrowest point is in the transmembrane region. If acetylcholine binds to the two binding sites, the ion channel "untwists" and widens its pore [32], whereby cations are passed through: sodium and calcium ions inwards and potassium ions outwards [33]. The influx of sodium leads to a depolarization of the postsynaptic membrane, which in turn sets in motion further cascades, ultimately muscle contraction in the neuromuscular system [34]. The nAChR can also be activated by other agonists such as carbamoylcholine, nicotine or epibatidine, which also bind to the orthosteric binding sites. The orthosteric binding sites can also be addressed by competitive antagonists such as α-bungarotoxin or conotoxins, so that the intrinsic effect is suppressed [35].

In addition to the orthosteric binding sites, the nAChR has numerous so-called allosteric binding sites. If ligands bind there, the activation of the nAChR is influenced either in a positive or negative way. Consequently, these substances are referred to as positive allosteric modulators (PAM) or negative allosteric modulators (NAM) [36].

Activation states

Fig. 2: Simplified representation of the various activation states of the nAChR; these are state probabilities that are taken depending on the acetylcholine concentration. The states "active" and "desensitized - fast onset" are metastable.
Four different activation states are known: the closed ("resting") state, the open ("active") state and the two desensitized states, of which there is a shallower ("fast - onset") and a deeper (" slow - onset "). These are probability states that are assumed by the nAChR depending on the agonist concentration in the synaptic gap. In the normal state, the closed state is most likely. The binding of the agonist acetylcholine causes the ion channel to open briefly - comparable to a “stuttering” water tap. In the case of pathophysiological conditions that are accompanied by an accumulation of acetylcholine in the synaptic gap (e.g. in the case of nerve agent poisoning), the desensitized state ("desensitized - slow onset") is most likely. The flatter, desensitized state (“desensitized - fast onset”) is only taken with a short-term excess of agonists; possibly this is a physiological protective function. Consequently, the “active” and the “desensitized - almost onset” state are metastable. However, the transitions between the individual states are fluid [37] (Figure 2).

In the desensitized state, the agonist is in the orthosteric binding site, but there is no activation. As already mentioned, this is the case with an excess of agonists. Obviously, however, further (allosteric) binding sites are occupied, which then stabilize the desensitized state. These substances called “desensitisers” or silent allosteric modulators (SAM) can not only be agonists in excess, but also various other ligands. The underlying mechanism is still unclear. One hypothesis is that the passage of the cations can be blocked in two places, on the one hand at the “resting” gate and on the other hand at the “desensitization” gate. If the nAChR is activated, these two gates must open in concert. In the case of desensitization, the “desensitization” gate obviously remains closed, so that the ion channel is no longer completely open, and consequently the intrinsic effect does not occur [38]. The surrounding membrane also seems to play a role in determining which functional state is stabilized: phosphatidylcholine-rich membranes stabilize the desensitized (closed, no longer activatable) state, while cholesterol-rich membranes stabilize the “resting” (closed, activatable) state [39].

Allosteric modulation of the nAChR

As already mentioned above, the nAChR not only has the orthosteric binding sites for agonists or antagonists, but also allosteric binding sites. By definition, the effect of allosteric modulators is linked to the effect of an orthosteric agonist: its effect is either increased or weakened. Positive allosteric modulators (PAM) increase the effect (affinity and / or activity) of an agonist, while negative allosteric modulators (NAM) weaken the effect of an agonist [40].

PAM are divided into two categories: Type I PAM increase the effect of an orthosteric agonist, but have no effect on the desensitization of the receptor. Type II PAM reduce the likelihood of desensitization and are able to restore the intrinsic activity of previously desensitized receptors [41, 42].

Due to their wide range of pharmacological diversity, allosteric modulators offer many possibilities for therapeutic approaches [43]. A decisive advantage is that only the effect of the orthosteric ligands is modulated; consequently, this interaction and its effect on the organism are no longer given if one of the partners fails. This “use-dependent” effect is particularly interesting when there is a risk that the positive effect will turn into negative with increasing dosage. With nAChR, this would be the case with antagonists, for example. The population of desensitized nAChR would be decreased and that of the activatable but closed (“resting”) states would be increased. If the concentration of antagonists was too high, the population would be shifted towards the “resting” states to such an extent that there would hardly be any open states. With regard to the intrinsic effect, there is no difference between antagonization and desensitization: in both cases there is no passage of cations. In the case of a desensitization of the nAChR induced by nerve warfare agents, the administration of antagonists would only be “the lesser evil”, and an extremely narrow therapeutic range would be expected. PAM would therefore be a far better alternative [36].

nAChR modulators as a new therapeutic approach

In order to close the therapeutic gap for the treatment of poisoning with soman or tabun, active ingredients with a modulating effect on the nAChR function are required. In particular, the “freezing” (also described as “open channel block”) of the open receptor state, which is actually metastable, is gaining in importance [44, 45]. Basically, this is nothing more than the effect of PAM on the nAChR.

Earlier attempts with the bispyridinium compound SAD-128 (1,1'-oxydimethylene bis (4-tert-butylpyridinium) dichloride) showed a therapeutic effect against soman in vitro and in vivo [46-49]. Since this compound does not have an oxime group and it has also been experimentally demonstrated that the -Soman-AChE complex was not reactivated by SAD-128, it was suggested that the positive therapeutic effect was probably due to a direct interaction with nAChR [50 ].

In the case of MB327, a compound similar in structure to SAD-128 (1,1 ‘- (propane-1,3-diyl) bis (4-tert-butylpyridinium) diiodide), it has been proven that the restoration of neuromuscular transmission does not take place via reactivation of the inhibited enzyme AChE, but rather via receptors [51 - 53]. On muscle type (2αβγδ) -nAChR, which is derived from the electrical organ of the Californian electric ray (Torpedo californica) and are very similar to the human subtype, it could be shown that MB327 does not interact directly with the orthosteric binding site, but does influence the binding of orthosteric ligands such as the agonist epibatidine [54, 55]. The increase in affinity of the agonist observed here indicated an allosteric effect of the MB327.Using the same nAChR-containing material, functionality measurements were carried out with a special bilayer-based electrophysiology (so-called "cell-free electrophysiology") developed at InstPharmToxBw. MB327 was able not only to amplify the signals of the agonist carbamoylcholine, but also to reverse carbamoylcholine-induced desensitization [56]. This proved that MB327 interacts directly with the nAChR and unfolds its pharmacological effect as PAM.

However, MB327 has numerous disadvantages: Firstly, the effectiveness is only in the micromolar range, so that high doses have to be applied, which makes practical use more difficult. Second, the effect of MB327 is relatively unspecific [57]. For example, MB327 interacts with the α7 nAChR in the same way as it does with the muscle type nAChR. Under certain circumstances, this can also be disadvantageous, especially if the nAChR of the respiratory muscles are to be addressed separately. The antagonistic effect in muscarinic systems is also remarkable. MB327 showed a relaxing effect on the smooth muscle, whereby the concentrations involved restored muscle function in respiratory muscle preparations poisoned with nerve agents [58].

Rational drug design

Even though MB327 has too low a potency, a substance was identified that acts as a type II PAM, i.e., previously desensitized nAChR is restored to a functional state ("resensitized"). If active ingredients could be developed on this basis that have a sufficiently high potency for a drug and also meet the criteria required for therapeutic use (“drug-like properties”), a way could be found to fill the therapeutic gap the treatment of poisoning, especially with soman or tabun.

This was the starting point for a medicinal chemistry project (E / U2AD / CF514 / DF561) in collaboration with the working group of Prof. Dr. Wanner, Chair for Pharmaceutical / Medicinal Chemistry in the Department of Pharmacy - Center for Pharmaceutical Research at LMU Munich, in which potent and selective modulators of the nAChR are to be developed using rational drug design.

This is an iterative process of synthesis, affinity studies with mass spectrometric detection (MS binding studies), molecular modeling, and electrophysiological and physiological investigations (Figure 3).

For this purpose, numerous new bispyridinium compounds with different substitution patterns were synthesized and then pharmacologically characterized. In analogy to MB327, selected compounds have a 4-tert-Butylpyridinium basic structure and have an additional functional group. This allows the influence of the additional substituent to be analyzed in terms of the development of structure-activity relationships. In addition, a new, so far not described, efficient and regioselective method for the synthesis of 4-tert-Butylpyridine derivatives established, which serve as the basis for the preparation of new bispyridinium salts [59].

Fig. 3: Iterative process of synthesis, molecular modeling and application of test systems to develop a new potent active ingredient.
In order to enable the search for new active substances based on the mechanism of action of MB327, its binding to the nAChR is to be carried out using MS binding assays - a work carried out by Prof. Dr. Wanner newly developed methods - can be characterized, and these binding assays can then be used as an efficient tool for the screening of affinity constants of new, structurally related compounds. The binding affinities are an essential building block for the rational drug design. The MS binding assays developed as part of the research project and now established follow the principle of radioligand binding studies, but in contrast to these are based on mass spectrometric detection of a native, i.e. H. unlabeled reporter ligand, which is typically bound by LC-MS / MS[1]) is quantified with high sensitivity [60].

In order to enable a sufficiently sensitive and reliable quantification of MB327 in MS binding assays, a powerful LC-ESI[2])- MS / MS method established which uses a deuterated analogue of MB327 as an internal standard. The binding of MB327 to the muscle type nAChR was then characterized in saturation experiments that use a centrifugation step to separate bound and unbound marker. For this purpose, membrane preparations from the electrical organ of the Californian electric ray (Torpedo californica) because an adequate expression system for the human muscle type-nAChR is not yet available. The results indicate a saturable specific binding of MB327 and thus show for the first time the existence of binding sites for this bispyridinium salt. With this method it could also be confirmed that the affinity of MB327 is only in the micromolar range. On the basis of the saturation assays, competition assays were then developed which allow the newly synthesized substances to be screened for their affinity [61].

The structure of the torpedo- Muscle type nAChR taken as the basis, since this receptor subtype (2α1ß1γδ) is also used in the pharmacological tests. In the course of the current project it was possible to identify two possible, previously unknown binding sites for the first time. These results could also be achieved in homology models of the human adult muscle type nAChR. In addition, it was shown that the two possible binding sites could also be addressed very well by asymmetrically substituted bispyridinium compounds with polar substituents.

Even if the previously developed in silico Methods are already very efficient, so it is necessary to update them regularly with new results from the affinity determination (MS binding studies) and pharmacological investigations and thus to continuously improve them. The particularly meaningful models accessible in this way provide valuable services in the further optimization of the modulators, as well as in the identification of their target binding sites.

Pharmacological studies

The pharmacological tests were carried out at InstPharmToxBw. These were electrophysiological (automated patch-clamp technique on whole cells, bilayer-based electrophysiology using plasma membrane preparations with muscle type - nAChR) and physiological methods (muscle strength measurements on diaphragm preparations of the rat).

The patch-clamp experiments were carried out with CHO[3]) Cells stably expressing the human nAChR of the subtype α7. Even if the nAChR subtype is different from the muscle type nAChR, these experiments provide extremely valuable information on the intrinsic effect of new substances. Here, further compounds could be identified that maintained activation of the nAChR by stabilizing the active state and prevented the occurrence of the desensitization caused by nicotine excess by extending the opening time. These effects were only induced in the presence of the agonist nicotine, which indicates a positive allosteric modulation. In this system, too, the activities of the new substances were only in the micromolar range. Nonetheless, the first structure-activity relationships could be identified, which are essential in the context of other molecular and physiological methods (bilayer-based electrophysiology, force measurements on muscle biopsies) [62].

The diaphragmatic preparation of the rat was used as a tissue model for the physiological investigations of the new substances.

Here, diaphragmatic hemispheres were fixed in a multi-organ bath system on myographs and made to contract by means of indirect electrical field stimulation (20, 50, and 100 Hz). Muscle strength was increased before and after administration of soman as well as after application of various bispyridinium compounds (1 - 300 µM[4])) measured and evaluated as a time-force integral.

Soman, in a concentration of 3 µM, completely inhibited the muscles. Even after washing out the organophosphate, the development of muscle strength could not be restored. With MB327 at the low stimulation frequency of 20 Hz at a concentration of 300 µM a restoration of muscle strength by about 30% could be observed. Other bispyridinium compounds were also able to induce muscle contractions again after Soman poisoning. As with the patch-clamp experiments, this depended heavily on the substitution pattern. However, in this model too, the effect of the bispyridium compounds could only be observed at relatively high concentrations (100-300 µM). At higher stimulation frequencies and thus an increased release of the neurotransmitter acetylcholine, their effectiveness again decreased very sharply.

Overall, relatively clear correlations to the electrophysiological methods (patch clamp, bilayer-based electrophysiology) were observed [63]. All the knowledge gained here about structure-activity relationships also completed the data situation for the in silico Screening.


The application of active ingredients that restore the activity of desensitized nAChR despite accumulated acetylcholine in the synaptic gap could be a possible therapeutic approach in the case of nerve agent poisoning conventionally untreatable with atropine and oximes. Substances with type II PAM properties, which were first detected for MB327 in various pharmacological studies, are capable of doing this. Recently, it was shown in MS binding studies that the binding of MB327 to the nAChR can be saturated, i.e. that there are explicit binding sites for MB327. With the help of in silico Computer models were able to identify two possible binding sites and calculate optimal pharmacophores for addressing these binding sites.

The next step is to synthesize substances that are designed on the basis of the theoretically determined pharmacophores and then introduced into the various pharmacological tests. The results obtained from this continue to complete the data situation for comprehensive structure-activity relationships.

The constant broadening of the database with regard to the relationships between chemical structure and biological activity will allow the generated computer models to be used for -Ligand- and structure-based design, for the detection of the -target binding sites and for in silico Significantly improve screening. This will make a decisive contribution to the identification and design of new drug derivatives and drug classes.

Since groundbreaking results have already been achieved for this, this development should definitely be pursued further with the aim of finding more potent and more selective active ingredients.


Although nAChR play an important role in nerve agent poisoning, they have so far been neglected as possible target structures for therapeutic intervention. This was probably also due to the fact that only conventional nAChR antagonists were considered, but with which there is a risk of a narrow therapeutic range.

A promising approach could be type II PAM, which restore the desensitized nAChR to a functional state - i.e. act as a "resensitiser" - whereby the effect is not self-sufficient, but dependent on the agonist.

The bispyridinium compound MB327 was identified as such a type II PAM, but still has too low an effective strength and selectivity. Nevertheless, this substance represents a valuable basis for the development of more potent active ingredients. Because, based on MB327 as the lead structure, new active ingredient candidates can be synthesized and pharmacologically characterized. The recent identification of possible binding sites of the bispyridinium salt MB327 at the nAChR can now be used to refine the pharmacophore and offers the opportunity to open up entirely new perspectives in antidote research. In view of the recent events in Syria, the therapeutic gap in the treatment of soman or taboo poisoning weighs heavily. It is all the more important that the path taken with this research project is pursued at full speed.


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For the authors:

Head pharmacist Karin V. Niessen

Institute for Pharmacology and Toxicology of the Bundeswehr

Neuherbergstrasse 11, 80937 Munich

Email: [email protected]


LC-MS / MS = liquid chromatography coupled with tandem mass spectrometry


[2]ESI = electrospray ionization


CHO = Chinese Hamster Ovary; Versatile cell culture line obtained from the ovaries of the Chinese hamster species Cricetulus griseus was won


[4]) µM = µmol / l


Date: 01.06.2017

Source: Military Medical Monthly 2017/5