Choline ester

The inhibitory effects of bisphenol A (BPA) and bisphenol S (BPS), which are common pollutants, especially in marine and freshwater, on the electric eel Acetylcholinesterase (AChE) activity were studied in vitro and in silico. Both produced full non-competitive inhibition, but the Ki value of BPA was half that of BPS. Molecular docking analyses revealed that both interact with residues W286, F297, Y337, F338 in the PAS and ABS regions in the middle and entrance of the active site gorge, and that BPS also has hydrogen bond with S203 of the catalytic triad. The surge at IC50 values of both compounds with an inflection point at pH: 8.2 suggested that Y124 and/or Y337 in the narrow gorge are primary structural factors in binding. Less effective inhibition of BPS, especially at 25-30 °C, the temperature at which enzyme activity peaks, was attributed to the conformation of the narrow gorge. Homology analyses for AChE initially revealed a significant degree of identity, particularly in the alpha/beta hydrolase domain, which also comprises the active site, with sequences from seven distinct teleost species of various environments. Finally, it was discovered for the first time that BPS, like BPA, is a significant inhibitor of AChE, and this was confirmed by in vitro and in silico analyses done at various pH and temperature levels. It was concluded that this effect might also apply to AChE of most other bony fish [1].

Acetylcholinesterase activity assay: assessing mode of inhibition [1]
Acetylcholinesterase (AChE) activity was measured using Ellman's method with some modifications (Ellman et al., 1961). Pure electric eel Acetylcholinesterase (AChE) was used, while acetylthiocholine iodide was employed as a substrate for the reaction. 5,5′-Dithio-bis(2-nitrobenzoic) acid (DTNB), which goes under reaction with thiol groups, is called Elman's reagent. Thiolate anion interacts with Ellman's reagent, and this reaction yields one mixed disulfide (R-S-TNB−) and 2-nitro-5-thiobenzoate anion (TNB2−), which absorbs light at 412 nm. 100 mM, pH: 8.0, phosphate buffer was used in the reaction, blank and control groups. 0.17 mM DTNB and three different acetylthiocholine iodide substrate concentrations (0.15, 0.25 and 0.35 mM) were tested in flat-bottom 96-well microplates to evaluate the inhibitory effect of BPA and BPS at their seven different concentrations ranging between 0.3 and 3.3 mM. Every trial of each concentration was prepared as triplicate (3 wells), and a specific blank was also designed and used as the fourth well. All those measurements were repeated independently four times, on different dates. Activity measurements were started as the enzyme was added, and enzyme activity was detected at 25 °C, at 412 nm for 10 min by measuring ABS at 30 s intervals. The specific activity values were calculated by using the following equation (Eq. (1)) [1]
where dA/dt is the rate of change in absorbance value measured at 412 nm, ε is extinction coefficient of acetylcholinesterase, whose value is 13.6 mM−1 cm−1 and DF is dilution factor. Specific activity values were submitted to the licensed software SigmaPlot 13.0 Enzyme Kinetics Module to detect the inhibition type. The mode of inhibition suggested by the software with the highest R2 value was accepted. Furthermore, enzyme kinetic plots, such as Michaelis-Menten, Lineveawer-Burk, Dixon, and so were used for analysis, demonstration and proof.

---

Homology survey for AcetylcholinesteraseAChE enzyme [1]
Acetylcholinesterase is an essential enzyme in the metabolism of living organisms and shares a common structure, especially in the same taxonomic units. All the defined and RefSeq qualified amino acid sequences of teleost species for this protein were derived from the NCBI protein database, and were all subjected to alignment by MEGA7 to evaluate the rate of similarity among those and to perform distance analysis. MEGA7 assignment yielded a phylogenic tree and stated the identical/similar protein structures/amino acid residues of the same enzyme from other bony fish species. Those phylogenetic tree and alignment reports were observed by Jalview 2.11.1.4 software. According to the cluster which contains the most identical/similar sequences, fish species in the cluster and their natural habitat were also discussed to make an inference on the possibility of similar inhibitory effect on Acetylcholinesterase/AChE of them all.

[1]. In vitro and in silico evaluation of inhibitory effects of bisphenol derivatives on acetylcholinesterase of electric eel (Electrophorus electricus L.).

The inhibitory effects of BPA and BPS on AChE were determined for seven different concentrations of these two xenobiotics applied in a wide range, and both the inhibition model and kinetic parameters were calculated with statistical accuracy. BPS caused smaller decrease in specific activity values, at all equal concentrations of xenobiotics, with respect to BPA. Moreover, the Ki value of BPA is about half of that of BPS (p < 0.05); which means the inhibitory effect of BPA is considerably higher concerning BPS. On the other hand, the proposed binding energies of both are very close to each other for the best docking position (Supplementary material Tables 1 and 2), which might explain the similarity of binding sites. [1]

Molecular docking analyses support in vitro experiments and the predicted inhibition model of the full non-competitive type. BPA and BPS interact with amino acids midway through the narrow gorge, especially in the PAS and ABS regions. It has been proposed that PAS facilitates the transport of ACh toward the catalytic triad, hence improving the catalytic performance of Acetylcholinesterase/AChE; that is, ACh binds to PAS and then diffuses quickly down to the catalytic site. This notion is bolstered by the fact that AChE is one of the most efficient biocatalysts in terms of kinetics, and its action kinetics are mostly restricted by diffusion. A critical residue that should be emphasized in the PAS region is W286 (Pourshojaei et al., 2019). Hydrophobic interactions between bisphenol derivatives and the indole group of this tryptophan play a key role in the binding of these two xenobiotics to PAS. Besides, ABS has some vital functions in positioning ACh via some hydrophobic interactions of aromatic side chains of certain amino acids. Phenylalanines at the positions of 295/297/338 perform such a function in natural metabolism. The binding of relatively hydrophobic BPA and BPS to those hydrophobic residues was expected and clearly screened by molecular docking issues. Thus, inhibiting both PAS and ABS of acetylcholinesterase not only slows the rate-limiting phase (diffusion of ACh to the active site) but may also diminish the enzyme's turnover number. Consequently, it is possible that the investigated chemicals could function as blockers of the typical ionic substrate (ACh) entry into the active site gorge. The influence of pH on the inhibitory potencies of BPA and BPS was investigated to reveal the enzyme's ionizable group(s) that contribute to their binding. Both showed similar curves, but the amount of the shift was most significant for BPS, indicating that the binding strongly depends on the acid/base characteristics of a specific amino acid, whose side chain may interact directly with bisphenol derivatives. The measured transition for BPS corresponds to a pKa of 8.099 ± 0.002. Although identifying the ionizable groups responsible for such a change is difficult, the inflection point around pH: 8.1 reveals that a Tyr residue appears to aid in BPS binding. This residue, likely Y124 and/or Y337, might be the key structural component resulting in relatively tight binding. In addition to all these, the structural and functional integrity of the enzyme at different pH values, especially the narrow gorge, should also be taken into account. Ser (125/203), Glu (202/334), Asp (74), Arg (296), His (447) and Tyr (72/124/133/337) residues, which are located both in this channel and around the active site and are involved in directing the substrate to the catalytic triad, undergo changes that will change the binding of both the substrate and other substances in acidic and alkaline environments outside the physiological pH range. As expected, the use of modeling procedures and specialized algorithms that can account for structural changes that occur during inhibitor interaction, particularly in channel-shaped proteins and in enzymes with channel-shaped active sites, like Acetylcholinesterase/AChE, is critical for achieving more accurate findings.

The current study determined that the optimal temperature for the Acetylcholinesterase/AChE enzyme is between 25 and 30 °C. In addition, for both BPA and BPS, after this interval, a statistically significant decrease is observed in the IC50 values calculated at the applied temperature of 35 °C. It can be argued that this breaking point is due to a change in the three-dimensional structure of the enzyme. Because of the collective dynamics of conserved amino acids at the gorge, AChE transits between “open” and “closed” conformations. At room temperature, AChE's substantial molecular flexibility governs access to the active site and binding to ligands (Silva et al., 2020). BPS was detected to be affected by this situation more apparently because its IC50 values shifted more than BPA; and it can be suggested that the hydrophobic and pi-pi interactions of BPS with the residues in the catalytic triad and with the amino acids at the bottom of the 20 angstrom-long gorge, which are also occupied by the natural substrate ACh - such as S203, might be responsible.

Acetylcholinesterases are characterized by the presence of the α/β hydrolase domain, in addition to some other sites. α/β hydrolase family is a functionally diverse superfamily containing esterases, peroxidases, dehalogenases, epoxide hydrolases, lipases and proteases (Bauer et al., 2020). The catalytic apparatus usually consists of three residues: a serine, a glutamate or aspartate, and a histidine. The mechanism frequently includes a nucleophilic attack on a carbonyl carbon atom. COBALT submission of the sequences stated that there is about 100 % identity at α/β hydrolase domain for the enzymes of the species in the closest cluster (Supplementary material Tables 3 and 4, Supplementary material Figs. 1–17), which means the mode and the magnitude of the inhibition by BPA and BPS might be similar for those other Acetylcholinesterase/AChEs of fishes and electric eel. This shows that the monitoring of BPA and BPS concentrations and kinetic parameters expressing their inhibition on AChE could also be used for different fish species from different ecosystems so that the list contains fish species from tropical to subtropical regions, from North and South America to Asia, and, finally from freshwater to brackish and salt water. It also reveals that the argument that BPS is harmless enough to replace BPA must be reconsidered within the framework of different species, geography, climatic conditions and habitat.

BPA is a dangerous and common pollutant, which is found primarily in plastic materials, in almost all ecosystems of the world. BPS was presented as an environmentally-friendly alternative to BPA, and it has a wide usage area. This study was aimed to reveal the inhibitory action of those two chemicals on acetylcholinesterase of a teleost fish species: electric eel. in vitro and in silico analysis and calculations completed in the current project put forward the inhibitory effects of those xenobiotics on this particular enzyme. BPS couldn't be valued as a non-toxic alternative because of its comparatively close Ki and IC50 values with BPA. Moreover, they apply their inhibitory effect on the AChE/Acetylcholinesterase enzyme in the same mode (non-competitive reversible) and in similar ways; so that the interacting residues at the narrow gorge were stated as similar or the same, which was also supported by the almost equal binding energies for the best docking position of each. Further, when the AChE protein sequences of teleost fishes were aligned, very high percentages of identity at critical functional parts such as the narrow gorge in α/β hydrolase domain suggested observing possibly similar inhibitory effects of BPA and BPS on AChEs of most other teleost fish species from diverse ecosystems. Taking account of the fact that there were α/β hydrolase domains in some other enzymes and proteins in various living organisms, BPA and BPS inhibition becomes much more important in the evaluation of their harmful effects on biota. [1]

9000-81-1

White to off-white solid powder

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 100 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)