Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both α7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing α7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca²⁺ rise, but inhibited the acetylcholine-evoked Ca²⁺ rise with IC₅₀ 9 to 12 μM. In the A549 lung cancer cells, where α7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with K_i = 16.7 ± 1.5 μM and αK_i = 51.4 ± 4.1 μM for AChE and K_i = 70.5 ± 6.3 μM and αK_i = 214 ± 17 μM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.

Cholinergic dysfunction in Alzheimer’s disease (AD) can be mediated by the neuronal α7 nicotinic acetylcholine receptor (α7nAChR). Beta-amyloid peptide (Aβ) binds to the α7nAChR, disrupting the receptor’s function and causing neurotoxicity. In vivo not only Aβ but also its modified forms can drive AD pathogenesis. One of these forms, iso-Aβ (containing an isomerized Asp7 residue), shows an increased neurotoxicity in vitro and stimulates amyloidogenesis in vivo. We suggested that such effects of iso-Aβ are α7nAChR-dependent. Here, using calcium imaging and electrophysiology, we found that iso-Aβ is a more potent inhibitor of the α7nAChR-mediated calcium current than unmodified Aβ. However, Asp7 isomerization eliminated the ability of Aβ to decrease the α7nAChR levels. These data indicate differences in the interaction of the peptides with the α7nAChR, which we demonstrated using computer modeling. Neither Aβ nor iso-Aβ competed with ¹²⁵I-α-bungarotoxin for binding to the orthosteric site of the receptor, suggesting the allosteric binging mode of the peptides. Further we found that increased neurotoxicity of iso-Aβ was mediated by the α7nAChR. Thus, the isomerization of Asp7 enhances the inhibitory effect of Aβ on the functional activity of the α7nAChR, which may be an important factor in the disruption of the cholinergic system in AD.

Nicotinic acetylcholine receptors (nAChRs) present as many different subtypes in the nervous and immune systems, muscles and on the cells of other organs. In the immune system, inflammation is regulated via the vagus nerve through the activation of the non-neuronal α7 nAChR subtype, affecting the production of cytokines. The analgesic properties of α7 nAChR-selective compounds are mostly based on the activation of the cholinergic anti-inflammatory pathway. The molecular mechanism of neuropathic pain relief mediated by the inhibition of α9-containing nAChRs is not fully understood yet, but the role of immune factors in this process is becoming evident. To obtain appropriate drugs, a search of selective agonists, antagonists and modulators of α7- and α9-containing nAChRs is underway. The naturally occurring three-finger snake α-neurotoxins and mammalian Ly6/uPAR proteins, as well as neurotoxic peptides α-conotoxins, are not only sophisticated tools in research on nAChRs but are also considered as potential medicines. In particular, the inhibition of the α9-containing nAChRs by α-conotoxins may be a pathway to alleviate neuropathic pain. nAChRs are involved in the inflammation processes during AIDS and other viral infections; thus they can also be means used in drug design. In this review, we discuss the role of α7- and α9-containing nAChRs in the immune processes and in pain.

A series of 14 new analogs of α-conotoxin PnIA Conus pennaceus was synthesized and tested for binding to the human α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine-binding proteins (AChBP) Lymnaea stagnalis and Aplysia californica. Based on computer modeling and the X-ray structure of the A. californica AChBP complex with the PnIA[A10L, D14K] analog [1], single and multiple amino acid substitutions were introduced in α-conotoxin PnIA aimed at compounds of higher affinity and selectivity. Three analogs, PnIA[L5H], PnIA[A10L, D14K] and PnIA[L5R, A10L, D14R], have high affinities for AChBPs or α7 nAChR, as found in competition with radioiodinated α-bungarotoxin. That is why we prepared radioiodinated derivatives of these α-conotoxins, demonstrated their specific binding and found that among the tested synthetic analogs, most had almost 10-fold higher affinity in competition with radioactive α-conotoxins as compared to competition with radioactive α-bungarotoxin. Thus, radioiodinated α-conotoxins are a more sensitive tool for checking the activity of novel α-conotoxins and other compounds quickly dissociating from the receptor complexes.

Human protein SLURP-1 is an endogenous neuromodulator belonging to the Ly-6/uPAR family and acting on nicotinic acetylcholine receptors. In the present work, the gene of SLURP-1 was expressed in E....