Synthesis and biological evaluation of novel MB327 analogs as resensitizers for desensitized nicotinic acetylcholine receptors after intoxication with nerve agents.

Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising MB327 analogs with a 4-aminopyridinium ion partial structure (PTM0056, PTM0062, and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new analogs of the aforementioned compounds with a 4-aminopyridinium ion partial structure (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90-0012 and PTMD90-0015) designed to substitute entropically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research.

Identification of ligands binding to MB327-PAM-1, a binding pocket relevant for resensitization of nAChRs.

Desensitization of nicotinic acetylcholine receptors (nAChRs) can be induced by overstimulation with acetylcholine (ACh) caused by an insufficient degradation of ACh after poisoning with organophosphorus compounds (OPCs). Currently, there is no generally applicable treatment for OPC poisoning that directly targets the desensitized nAChR. The bispyridinium compound MB327, an allosteric modulator of nAChR, has been shown to act as a resensitizer of nAChRs, indicating that drugs binding directly to nAChRs can have beneficial effects after OPC poisoning. However, MB327 also acts as an inhibitor of nAChRs at higher concentrations and can thus not be used for OPC poisoning treatment. Consequently, novel, more potent resensitizers are required. To successfully design novel ligands, the knowledge of the binding site is of utmost importance. Recently, we performed in silico studies to identify a new potential binding site of MB327, MB327-PAM-1, for which a more affine ligand, UNC0646, has been described. In this work, we performed ligand-based screening approaches to identify novel analogs of UNC0646 to help further understand the structure-affinity relationship of this compound class. Furthermore, we used structure-based screenings and identified compounds representing four new chemotypes binding to MB327-PAM-1. One of these compounds, cycloguanil, is the active metabolite of the antimalaria drug proguanil and shows a higher affinity towards MB327-PAM-1 than MB327. Furthermore, cycloguanil can reestablish the muscle force in soman-inhibited rat muscles. These results can act as a starting point to develop more potent resensitizers of nAChR and to close the gap in the treatment after OPC poisoning.

A novel binding site in the nicotinic acetylcholine receptor for MB327 can explain its allosteric modulation relevant for organophosphorus-poisoning treatment.

Organophosphorus compounds (OPCs) are highly toxic compounds that can block acetylcholine esterase (AChE) and thereby indirectly lead to an overstimulation of muscarinic and nicotinic acetylcholine receptors (nAChRs). The current treatment with atropine and AChE reactivators (oximes) is insufficient to prevent toxic effects, such as respiratory paralysis, after poisonings with various OPCs. Thus, alternative treatment options are required to increase treatment efficacy. Novel therapeutics, such as the bispyridinium non-oxime MB327, have been found to reestablish neuromuscular transmission by interacting directly with nAChR, probably via allosteric mechanisms. To rationally design new, more potent drugs addressing nAChR, knowledge of the binding mode of MB327 is fundamental. However, the binding pocket of MB327 has remained elusive. Here, we identify a new potential allosteric binding pocket (MB327-PAM-1) of MB327 located at the transition of the extracellular to the transmembrane region using blind docking experiments and molecular dynamics simulations. MB327 forms striking interactions with the receptor at this site. The interacting amino acids are highly conserved among different subunits and different species. Correspondingly, MB327 can interact with several nAChR subtypes from different species. We predict by rigidity analysis that MB327 exerts an allosteric effect on the orthosteric binding pocket and the transmembrane domain after binding to MB327-PAM-1. Furthermore, free ligand diffusion MD simulations reveal that MB327 also has an affinity to the orthosteric binding pocket, which agrees with recently published results that related bispyridinium compounds show inhibitory effects via the orthosteric binding site. The newly identified binding site allowed us to predict structural modifications of MB327, resulting in the more potent resensitizers PTM0062 and PTM0063.