State-dependent binding of cholesterol and an anionic lipid to the muscle-type Torpedo nicotinic acetylcholine receptor.

The ability of the Torpedo nicotinic acetylcholine receptor (nAChR) to undergo agonist-induced conformational transitions requires the presence of cholesterol and/or anionic lipids. Here we use recently solved structures along with multiscale molecular dynamics simulations to examine lipid binding to the nAChR in bilayers that have defined effects on nAChR function. We examine how phosphatidic acid and cholesterol, lipids that support conformational transitions, individually compete for binding with phosphatidylcholine, a lipid that does not. We also examine how the two lipids work synergistically to stabilize an agonist-responsive nAChR. We identify rapidly exchanging lipid binding sites, including both phospholipid sites with a high affinity for phosphatidic acid and promiscuous cholesterol binding sites in the grooves between adjacent transmembrane α-helices. A high affinity cholesterol site is confirmed in the inner leaflet framed by a key tryptophan residue on the MX α-helix. Our data provide insight into the dynamic nature of lipid-nAChR interactions and set the stage for a detailed understanding of the mechanisms by which lipids facilitate nAChR function at the neuromuscular junction.

Influence of lipid bilayer on the structure of the muscle-type nicotinic acetylcholine receptor.

The muscle-type nicotinic acetylcholine receptor is a transmitter-gated ion channel residing in the plasma membrane of electrocytes and striated muscle cells. It is present predominantly at synaptic junctions, where it effects rapid depolarization of the postsynaptic membrane in response to acetylcholine released into the synaptic cleft. Previously, cryo-EM of intact membrane from Torpedo revealed that the lipid bilayer surrounding the junctional receptor has a uniquely asymmetric and ordered structure, due to a high concentration of cholesterol. It is now shown that this special lipid environment influences the transmembrane (TM) folding of the protein. All five submembrane MX helices of the membrane-intact junctional receptor align parallel to the surface of the cholesterol-ordered lipids in the inner leaflet of the bilayer; also, the TM helices in the outer leaflet are splayed apart. However in the structure obtained from the same protein after extraction and incorporation in nanodiscs, the MX helices do not align to a planar surface, and the TM helices arrange compactly in the outer leaflet. Realignment of the MX helices of the nanodisc-solved structure to a planar surface converts their adjoining TM helices into an obligatory splayed configuration, characteristic of the junctional receptor. Thus, the form of the receptor sustained by the special lipid environment of the synaptic junction is the one that mediates fast synaptic transmission; whereas, the nanodisc-embedded protein may be like the extrajunctional form, existing in a disordered lipid environment.

Oleic Acid Could Act as a Channel Blocker in the Inhibition of nAChR: Insights from Molecular Dynamics Simulations.

The activation of the muscular nicotinic acetylcholine receptor (nAChR) produces the opening of the channel, with the consequent increase in the permeability of cations, triggering an excitatory signal. Free fatty acids (FFA) are known to modulate the activity of the receptor as noncompetitive antagonists, acting at the membrane-AChR interface. We present molecular dynamics simulations of a model of nAChR in a desensitized closed state embedded in a lipid bilayer in which distinct membrane phospholipids were replaced by two different monounsaturated FFA that differ in the position of a double bond. This allowed us to detect and describe that the cis-18:1ω-9 FFA were located at the interface between the transmembrane segments of α2 and γ subunits diffused into the channel lumen with the consequent potential ability to block the channel to the passage of ions.

MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor.

Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR, as well. In this study, UNC0642 has been utilized as a reporter ligand to establish new Binding Assays for this target. These assays follow the concept of MS Binding Assays for which by assessing the amount of bound reporter ligand by mass spectrometry no radiolabeled material is required. According to the results of the performed MS Binding Assays comprising saturation and competition experiments it can be concluded, that UNC0642 used as a reporter ligand addresses the MB327 binding site of the Torpedo-nAChR. This is further supported by the outcome of ex vivo studies carried out with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of UNC0646, an analog of UNC0642 with the highest binding affinity, in the recently proposed binding site of MB327 (MB327-PAM-1). With UNC0642 addressing the MB327 binding site of the Torpedo-nAChR, this and related quinazoline derivatives represent a promising starting point for the development of novel ligands of the nAChR as antidotes for the treatment of intoxications with organophosphorus compounds. Further, the new MS Binding Assays are a potent alternative to established assays and of particular value, as they do not require the use of radiolabeled material and are based on a commercially available compound as reporter ligand, UNC0642, exhibiting one of the highest binding affinities for the MB327 binding site known so far.

Connectivity between surface and interior in catalytic subunits of acetylcholinesterases inferred from their X-ray structures.

Catalytic activity and function of acetylcholinesterase (AChE; EC 3.1.1.7) have been recognized and studied for over a century and its quaternary and primary structures for about half a century, and its tertiary structure has been known for about 33 years. Clear understanding of relationships between the structure and the function is still pending for this enzyme. Hundreds of crystallographic, static snapshots of AChEs from different sources reveal largely one general backbone conformation with narrow entry into the active center gorge, tightly fit to accept one acetylcholine (ACh) molecule, in contrast to its high catalytic turnover. This short review of available X-ray structures of AChEs from electric ray Torpedo californica, mouse and human, finds some limited, yet consistent deviations in conformations of selected secondary structure elements of AChE relevant for its function. The observed conformational diversity of the acyl pocket loop of AChE, unlike the large Ω-loop, appears consistent with structurally dynamic INS data and solution-based SAXS experiments to explain its dominant role in controlling the size of the active center gorge opening, as well as connectivity between the immediate surroundings of the buried active Ser, and catalytically relevant sites on the AChE surface.

Identification of the native Torpedo californica nicotinic acetylcholine receptor's glycan composition after a multi-step sequential purification method using MALDI-ToF MS.

The Cys-loop pentameric ligand-gated ion channels comprise a dynamic group of proteins that have been extensively studied for decades, yielding a wealth of findings at both the structural and functional levels. The nicotinic acetylcholine receptor (nAChR) is no exception, as it is part of this large protein family involved in proper organismal function. Our efforts have successfully produced a highly pure nAChR in detergent complex (nAChR-DC), enabling more robust studies to be conducted on it, including beginning to experiment with high-throughput crystallization. Our homogeneous product has been identified and extensively characterized with 100% identity using Nano Lc MS/MS and MALDI ToF/ToF for each nAChR subunit. Additionally, the N-linked glycans in the Torpedo californica-nAChR (Tc-nAChR) subunits have been identified. To study this, the Tc-nAChR subunits were digested with PNGase F and the released glycans were analyzed by MALDI-ToF. The MS results showed the presence of high-mannose N-glycan in all native Tc-nAChR subunits. Specifically, the oligommanose population Man8-9GlcNac2 with peaks at m/z 1742 and 1904 ([M + Na]+ ions) were observed.

Benocyclidine (BTCP) as Non-labelled Reporter Ligand for MS Binding Assays for the PCP Ion Channel Binding Site of the Desensitized Torpedo Nicotinic Acetylcholine Receptor (nAChR).

In this study we present MS Binding Assays for the PCP ion channel binding site of Torpedo californica nicotinic acetylcholine receptor (nAChR) as an alternative to radioligand binding assays. As MS Marker Benocyclidine (BTCP) was employed, found to be more affine (Kd of 84.2 nM) than the radioligands, e. g. [3 H]PCP, used so far in respective binding assays. Based on a highly sensitive and fast LC-ESI-MS/MS method for quantification of BTCP samples, BTCP MS Binding Assays for the PCP ion channel binding site of Torpedo nAChR could be established comprising saturation, kinetic and competition experiments. The affinities obtained in competitive BTCP MS Binding Assays for ligands addressing the PCP ion channel binding site of Torpedo nAChR were in excellent accord with those reported from radioligand experiments. Thus, the new BTCP MS Binding Assays represent a potent and reliable alternative to radioligand binding assays used so far for the characterization of ligand binding to the PCP ion channel binding site of the nAChR.

Structural fractal analysis of the active sites of acetylcholinesterase from various organisms.

Acetylcholinesterase (AChE) is the object of many studies due to the fact that it plays an important role in the vital activity of organisms. In particular, when new AChE inhibitors are developed, much attention is paid to the specificity of their action. One of the approaches used to study the specificity is to compare AChE taken from various organisms. In this work, crystallographic data are used to investigate the active sites of AChE (ASAs) in the free (uncomplexed) state for the following five organisms: Homo sapiens (HS), Mus musculus (MM), Torpedo californica (TC), Electrophorus electricus (EE), and Drosophila melanogaster (DM). The structural fractal analysis (SFA) proposed by us earlier is used as a research method. This method is based on the calculation and comparison of the fractal dimensions of molecular structures. SFA demonstrates that there are no significant structural differences between the active sites of human AChE and other AChEs. However, differences are found for the MM/EE pair. Further analysis of individual AARs has revealed two different areas of active sites. Ser203, Trp236, Phe338, and Tyr341 are found to belong to a variable region, and the remaining AARs belong to a conservative region of the ASAs. The fraction of "variability" is low, 0.8%.

Fluorescence Studies of Nicotinic Acetylcholine Receptor and Its Associated Lipid Milieu: The Influence of Erwin London's Methodological Approaches.

Erwin London dedicated considerable effort to understanding lipid interactions with membrane-resident proteins and how these interactions shaped the formation and maintenance of lipid phases and domains. In this endeavor, he developed ad hoc techniques that greatly contributed to advancements in the field. We have employed and/or modified/extended some of his methodological approaches and applied them to investigate lipid interaction with the nicotinic acetylcholine receptor (nAChR) protein, the paradigm member of the superfamily of rapid pentameric ligand-gated ion channels (pLGIC). Our experimental systems ranged from purified receptor protein reconstituted into synthetic lipid membranes having known effects on receptor function, to cellular systems subjected to modification of their lipid content, e.g., varying cholesterol levels. We have often employed fluorescence techniques, including fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and of nAChR intrinsic fluorescence by nitroxide spin-labeled phospholipids, DPH anisotropy, excimer formation of pyrene-phosphatidylcholine, and Förster resonance energy transfer (FRET) from the protein moiety to the extrinsic probes Laurdan, DPH, or pyrene-phospholipid to characterize various biophysical properties of lipid-receptor interactions. Some of these strategies are revisited in this review. Special attention is devoted to the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the nAChR. The receptor protein was shown to organize its PA-containing immediate microenvironment into microdomains with high lateral packing density and rigidity. PA and cholesterol appear to compete for the same binding sites on the nAChR protein.

Subcellular metal partitioning as a novel tool in ecotoxicological elasmobranch assessments: The case of lesser numbfish (Narcine brasiliensis) affected by the Mariana dam disaster in Southeastern Brazil.

This study comprises a novel report on subcellular metal partitioning and metallothionein (MT) metal detoxification efforts in lesser numbfish (Narcine brasiliensis) electric ray specimens, as well as the first assessment on MT contents in any ray electric organ. Individuals sampled from an area in Southeastern Brazil affected by the Mariana dam rupture disaster were assessed concerning subcellular metal partitioning and MT metal-detoxification in the liver, gonads, electric organ and muscle of both adults and embryos. Yolk was also assessed when available. Relative total and heat-stable (bioavailable) metal and metalloid comparisons between adults and embryos in different developmental stages demonstrates maternal transfer of both total and bioavailable metals and significant MT associations demonstrate the detoxification of As, Ag, Mn, Ni, Cd, Co, Cu, Se and V through this biochemical pathway. Our findings expand the lacking ecotoxicological assessments for this near-threatened species and indicates significant ecological concerns, warranting further biomonitoring efforts.

Structural mechanism of muscle nicotinic receptor desensitization and block by curare.

Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state. We then determined the receptor structure bound to the agonist carbachol, which stabilizes an asymmetric, closed channel desensitized state. We find conformational changes in a peripheral membrane helix are tied to recovery from desensitization. To probe mechanisms of antagonism, we obtained receptor structures with the active component of curare, a poison arrow toxin and precursor to modern muscle relaxants. d-Tubocurarine stabilizes the receptor in a desensitized-like state in the presence and absence of agonist. These findings define the transitions between resting and desensitized states and reveal divergent means by which antagonists block channel activity of the muscle-type nicotinic receptor.

Conformational transitions and ligand-binding to a muscle-type nicotinic acetylcholine receptor.

Fast synaptic communication requires receptors that respond to the presence of neurotransmitter by opening an ion channel across the post-synaptic membrane. The muscle-type nicotinic acetylcholine receptor from the electric fish, Torpedo, is the prototypic ligand-gated ion channel, yet the structural changes underlying channel activation remain undefined. Here we use cryo-EM to solve apo and agonist-bound structures of the Torpedo nicotinic receptor embedded in a lipid nanodisc. Using both a direct biochemical assay to define the conformational landscape and molecular dynamics simulations to assay flux through the pore, we correlate structures with functional states and elucidate the motions that lead to pore activation of a heteromeric nicotinic receptor. We highlight an underappreciated role for the complementary subunit in channel gating, establish the structural basis for the differential agonist affinities of α/δ versus α /γ sites, and explain why nicotine is less potent at muscle nicotinic receptors compared to neuronal ones.

Protein-lipid interplay at the neuromuscular junction.

Many new structures of membrane proteins have been determined over the last decade, yet the nature of protein-lipid interplay has received scant attention. The postsynaptic membrane of the neuromuscular junction and Torpedo electrocytes has a regular architecture, opening an opportunity to illuminate how proteins and lipids act together in a native membrane setting. Cryo electron microscopy (Cryo-EM) images show that cholesterol segregates preferentially around the constituent ion channel, the nicotinic acetylcholine receptor, interacting with specific sites in both leaflets of the bilayer. In addition to maintaining the transmembrane α-helical architecture, cholesterol forms microdomains - bridges of rigid sterol groups that link one channel to the next. This article discusses the whole protein-lipid organization of the cholinergic postsynaptic membrane, its physiological implications and how the observed details relate to our current concept of the membrane structure. I suggest that cooperative interactions, facilitated by the regular protein-lipid arrangement, help to spread channel activation into regions distant from the sites of neurotransmitter release, thereby enhancing the postsynaptic response.

Sequential purification and characterization of Torpedo californica nAChR-DC supplemented with CHS for high-resolution crystallization studies.

Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification. We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of ß-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-ß-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-ß-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.

Toward Simple, Predictive Understanding of Protein-Ligand Interactions: Electronic Structure Calculations on Torpedo Californica Acetylcholinesterase Join Forces with the Chemist's Intuition.

Contemporary efforts for empirically-unbiased modeling of protein-ligand interactions entail a painful tradeoff - as reliable information on both noncovalent binding factors and the dynamic behavior of a protein-ligand complex is often beyond practical limits. We demonstrate that information drawn exclusively from static molecular structures can be used for reproducing and predicting experimentally-measured binding affinities for protein-ligand complexes. In particular, inhibition constants (Ki) were calculated for seven different competitive inhibitors of Torpedo californica acetylcholinesterase using a multiple-linear-regression-based model. The latter, incorporating five independent variables - drawn from QM cluster, DLPNO-CCSD(T) calculations and LED analyses on the seven complexes, each containing active amino-acid residues found within interacting distance (3.5 Å) from the corresponding ligand - is shown to recover 99.9% of the sum of squares for measured Ki values, while having no statistically-significant residual errors. Despite being fitted to a small number of data points, leave-one-out cross-validation statistics suggest that it possesses surprising predictive value (Q2LOO=0.78, or 0.91 upon removal of a single outlier). This thus challenges ligand-invariant definitions of active sites, such as implied in the lock-key binding theory, as well as in alternatives highlighting shape-complementarity without taking electronic effects into account. Broader implications of the current work are discussed in dedicated appendices.

Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System.

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 Ki = 16.7 ± 1.5 µM and αKi = 51.4 ± 4.1 µM for AChE and Ki = 70.5 ± 6.3 µM and αKi = 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.

Polyproline-rich peptides associated with Torpedo californica acetylcholinesterase tetramers.

Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the polyproline-rich peptide(s) in Torpedo californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo californica electric organ, converted to 300 kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the polyproline-rich peptide in the trypsin-treated TcAChE tetramers.

L-3,3',5-triiodothyronine and pregnenolone sulfate inhibit Torpedo nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (nAChR) is an excitatory pentameric ligand-gated ion channel (pLGIC), homologous to the inhibitory γ-aminobutyric acid (GABA) type A receptor targeted by pharmaceuticals and endogenous sedatives. Activation of the GABAA receptor by the neurosteroid allopregnanolone can be inhibited competitively by thyroid hormone (L-3,3',5-triiodothyronine, or T3), but modulation of nAChR by T3 or neurosteroids has not been investigated. Here we show that allopregnanolone inhibits the nAChR from Torpedo californica at micromolar concentrations, as do T3 and the anionic neurosteroid pregnenolone sulfate (PS). We test for the role of protein and ligand charge in mediated receptor inhibition by varying pH in a narrow range around physiological pH. We find that both T3 and PS become less potent with increasing pH, with remarkably similar trends in IC50 when T3 is neutral at pH < 7.3. After deprotonation of T3 (but no additional deprotonation of PS) at pH 7.3, T3 loses potency more slowly with increasing pH than PS. We interpret this result as indicating the negative charge is not required for inhibition but does increase activity. Finally, we show that both T3 and PS affect nAChR channel desensitization, which may implicate a binding site homologous to one that was recently indicated for accelerated desensitization of the GABAA receptor by PS.

Molecular dynamics simulations of the interaction of Mouse and Torpedo acetylcholinesterase with covalent inhibitors explain their differential reactivity: Implications for drug design.

Although the three-dimensional structures of mouse and Torpedo californica acetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different. Both agents inhibit the mouse enzyme effectively by covalent modification of its active-site serine. In contrast, whereas the Torpedo enzyme is effectively inhibited by benzenesulfonyl fluoride, it is almost completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than the Torpedo enzyme, suggesting that enhanced 'breathing motions' of the mouse enzyme relative to the Torpedo enzyme may explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in the Torpedo enzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approaches the active-site serine in both mouse and Torpedo acetylcholinesterase in such simulations, phenylmethylsulfonyl fluoride is able to approach the active-site serine of mouse acetylcholinesterase, but remains trapped above the bottleneck in the Torpedo enzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:2.

Revisiting fish toxicity of active pharmaceutical ingredients: Mechanistic insights from integrated ligand-/structure-based assessments on acetylcholinesterase.

The release of active pharmaceutical ingredients (APIs) into the environment is of great concern for aquatic ecosystem as many of these chemicals are designed to exert biological activity. Hence, their impact on non-target organisms like fish would not be surprising. In this respect, we revisited fish toxicity data of pharmaceuticals to generate linear and non-linear quantitative structure-toxicity relationships (QSTRs). We predicted fish lethality data from the validated QSTR models for 120 APIs with no experimental fish toxicity data. Toxicity of APIs on aquatic organisms is not fully characterized. Therefore, to provide a mechanistic insight for the assessment of API's toxicity to fish, the outcome of the derived QSTR models was integrated with structure-based toxicophore and molecular docking studies, utilizing the biomarker enzyme acetylcholinesterase originating from fish Torpedo californica (TcAChE). Toxicophore virtual screening of 60 chemicals with pT > 0 identified 23 hits as potential TcAChE binders with binding free energies ranging from -6.5 to -12.9 kcal/mol. The TcAChE-ligand interaction analysis revealed a good nesting of all 23 hits within TcAChE binding site through establishing strong lipophilic and hydrogen bonding interactions with the surrounding key amino acid residues. Among the chemicals passing the criteria of our integrated approach, majority of APIs belong noticeably to the Central Nervous System class. The screened chemicals displayed not only comprehensive toxicophore coverage, but also strong binding affinities according to the docking calculations, mainly due to interactions with TcAChE's key amino acid residues Tyr121, Tyr130, Tyr334, Trp84, Phe290, Phe330, Phe331, Ser122, and Ser200. Moreover, we propose here that binding of pharmaceuticals to AChE might have a potential in triggering molecular initiating events for adverse outcome pathways (AOPs), which in turn can play an important role for future screening of APIs lacking fish lethality data.