Screening for new ligands of the MB327-PAM-1 binding site of the nicotinic acetylcholine receptor.

Intoxications with organophosphorus compounds (OPCs) effect a severe impairment of cholinergic neurotransmission that, as a result of overstimulation may lead to desensitization of nicotinic acetylcholine receptors (nAChRs) and finally to death due to respiratory paralysis. So far, therapeutics, that are capable to address and revert desensitized neuromuscular nAChRs into their resting, i.e. functional state are still missing. Still, among a class of compounds termed bispyridinium salts, which are characterized by the presence of two pyridinium subunits, constituents have been identified, that can counteract organophosphate poisoning by resensitizing desensitized nAChRs. According to comprehensive modeling studies this effect is mediated by an allosteric binding site at the nAChR termed MB327-PAM-1 site. For MB327, the most prominent representative of the bispyridinium salts and all other analogues studied so far, the affinity for the aforementioned binding site and the intrinsic activity measured in ex vivo and in in vivo experiments are distinctly too low, to meet the criteria to be fulfilled for therapeutic use. Hence, in order to identify new compounds with higher affinities for the MB327-PAM-1 binding site, as a basic requirement for an enhanced potency, two compound libraries, the ChemDiv library with 60 constituents and the Tocriscreen Plus library with 1280 members have been screened for hit compounds addressing the MB327-PAM-1 binding site, utilizing the [2H6]MB327 MS Binding Assay recently developed by us. This led to the identification of a set of 10 chemically diverse compounds, all of which exhibit an IC50 value of ≤ 10 µM (in the [2H6]MB327 MS Binding Assay), which had been defined as selection criteria. The three most affine ligands, which besides a quinazoline scaffold share similarities with regard to the substitution pattern and the nature of the substituents, are UNC0638, UNC0642 and UNC0646. With binding affinities expressed as pKi values of 6.01 ± 0.10, 5.97 ± 0.05 and 6.23 ± 0.02, respectively, these compounds exceed the binding affinity of MB327 by more than one log unit. This renders them promising starting points for the development of drugs for the treatment of organophosphorus poisoning by addressing the MB327-PAM-1 binding site of the nAChR.

New Resensitizers for the Nicotinic Acetylcholine Receptor by Ligand-Based Pharmacophore Modeling.

INTRODUCTION: Irreversible inhibition of the acetylcholinesterase upon intoxication with organophosphorus compounds leads to an accumulation of acetylcholine in the synaptic cleft and a subsequent desensitization of nicotinic acetylcholine receptors which may ultimately result in respiratory failure. A direct intervention at the nicotinic acetylcholine receptor (nAChR) was proposed as an alternative therapeutic approach to the treatment with atropine and oximes. METHODS: The bispyridinium compound MB327 has been found to recover functional activity of nAChR thus representing a promising starting point for the development of new drugs for the treatment of organophosphate poisoning. Recent solid-supported membrane-based electrophysiological experiments have identified symmetrically substituted bispyridinium compounds e.g. MB327, MB583, and PTM0001 that are able to resensitize nAChR of Torpedo californica. In addition, six compounds have been found not to show any resensitizing potential and were thus classified as inactive. This set of active and inactive bispyridinium compounds was taken to develop a pharmacophore model and in silico screening of a virtual database of bispyridinium compounds to identify new compounds that are able to restore the functional activity of desensitized nAChR. RESULTS: Screening of a virtual compound database of symmetrically substituted bispyridinium compounds with the derived pharmacophore yielded several promising compounds which satisfy the pharmacophore and ought to have the same or even better resensitizing effect on nAChR as the parent compound MB327.

Synthesis of a Series of Non-Symmetric Bispyridinium and Related Compounds and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor.

The current standard therapy to counteract organophosphate intoxication is not effective in equal measure against all types of organophosphorus compounds (OPCs), as the outcome of oxime-induced reactivation of inactivated acetylcholinesterase (AChE) strongly depends on the particular OPC. In case the reactivation is insufficient, acetylcholine concentrations that rise to pathophysiological levels force the nicotinic acetylcholine receptor (nAChR) into a desensitized state and hence a functionally inactive state. As a consequence, neurotransmission is irreversibly disrupted at the neuromuscular junction. Previous electrophysiological studies identified the symmetric bispyridinium compound 1,1'-(propane-1,3-diyl)bis[4-(tert-butyl)pyridin-1-ium] diiodide (MB327) as a re-sensitizer of the desensitized nAChR. MB327 is thereby capable of restoring the functional activity. Very recently, in silico modeling studies suggested non-symmetric derivatives of MB327 as potential re-sensitizers with enhanced binding affinity and thus possible enhanced efficacy. In this study, 26 novel non-symmetric bispyridinium compounds and related derivatives were synthesized. For the synthesis of the highly polar target compounds in sufficient quantities, newly developed and highly efficient two-step procedures were used. Compounds were characterized in terms of their binding affinity toward the MB327 binding site at the nAChR using recently developed mass spectrometry (MS) Binding Assays. Regarding structure-affinity relationships at the MB327 binding site, the presence of two quaternary aromatic nitrogen centers as well as pyridinium systems with a tert-butyl group at the 4-position or a NMe2 group at the 3- or 4-positions appeared to be beneficial for high binding affinities.

Synthesis of a Series of Structurally Diverse MB327 Derivatives and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor.

A novel series of 30 symmetric bispyridinium and related N-heteroaromatic bisquaternary salts with a propane-1,3-diyl linker was synthesized and characterized for their binding affinity at the MB327 binding site of nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Compounds targeting this binding site are of particular interest for research into new antidotes against organophosphate poisoning, as therapeutically active 4-tert-butyl-substituted bispyridinium salt MB327 was previously identified as a nAChR re-sensitizer. Efficient access to the target compounds was provided by newly developed methods enabling N-alkylation of sterically hindered or electronically deactivated heterocycles exhibiting a wide variety of functional groups. Determination of binding affinities toward the MB327 binding site at the nAChR, using a recently developed mass spectrometry (MS)-based Binding Assay, revealed that several compounds reached affinities similar to that of MB327 (pKi =4.73±0.03). Notably, the newly prepared lipophilic 4-tert-butyl-3-phenyl-substituted bispyridinium salt PTM0022 (3 h) was found to have significantly higher binding affinity, with a pKi value of 5.16±0.07, thus representing considerable progress toward the development of more potent nAChR re-sensitizers.

Searching for putative binding sites of the bispyridinium compound MB327 in the nicotinic acetylcholine receptor.

Irreversible inhibition of the acetylcholine esterase upon intoxication with organophosphorus compounds leads to an accumulation of acetylcholine in the synaptic cleft and a subsequent desensitization of nicotinic acetylcholine receptors which may ultimately result in respiratory failure. The bispyridinium compound MB327 has been found to restore functional activity of nAChR thus representing a promising starting point for the development of new drugs for the treatment of organophosphate poisoning. In order to optimize the resensitizing effect of MB327 on nAChR, it would be very helpful to know the MB327 specific binding site to apply structure based molecular modeling. The binding site for MB327 at the nAChR is not known and so far goal of speculations, but it has been shown that MB327 does not bind to the orthosteric acetylcholine binding site. We have used docking calculations to screen the surface of nAChR for possible binding sites of MB327. The results indicate that at least two potential binding sites for MB327 at nAChR are present inside the channel pore. In these binding sites, MB327 intercalates between the γ-α and ß-δ subunits of nAChR, respectively. Both putative MB327 binding sites show an unsymmetrical distribution of surrounding hydrophilic and lipophilic amino acids. This suggests that substitution of MB327-related bispyridinium compounds on one of the two pyridinium rings with polar substituents should have a favorable effect on the pharmacological function.

Peptidase inhibitor 16 is a membrane-tethered regulator of chemerin processing in the myocardium.

A key response of the myocardium to stress is the secretion of factors with paracrine or endocrine function. Intriguing in this respect is peptidase inhibitor 16 (PI16), a member of the CAP family of proteins which we found to be highly upregulated in cardiac disease. Up to this point, the mechanism of action and physiological function of PI16 remained elusive. Here, we show that PI16 is predominantly expressed by cardiac fibroblasts, which expose PI16 to the interstitium via a glycophosphatidylinositol (-GPI) membrane anchor. Based on a reported genetic association of PI16 and plasma levels of the chemokine chemerin, we investigated whether PI16 regulates post-translational processing of its precursor pro-chemerin. PI16-deficient mice were engineered and found to generate higher levels of processed chemerin than wildtype mice. Purified recombinant PI16 efficiently inhibited cathepsin K, a chemerin-activating protease, in vitro. Moreover, we show that conditioned medium from PI16-overexpressing cells impaired the activation of pro-chemerin. Together, our data indicate that PI16 suppresses chemerin activation in the myocardium and suggest that this circuit may be part of the cardiac stress response.