Discovery of Guanidine Derivatives from Buthus martensii Karsch with Metal-Binding and Cholinesterase Inhibition Properties

Two rare guanidine-type alkaloids, Buthutin A (1) and Buthutin B (2), along with two other compounds (3, 4), were isolated from Buthus martensii Karsch, and determined using extensive spectroscopic data analysis and high resolution-mass spectrometry. Compound 1 showed the most potent inhibition on AChE and BChE with IC50 values of 7.83 ± 0.06 and 47.44 ± 0.95 μM, respectively. Kinetic characterization of compound 1 confirmed a mixed-type of AChE inhibition mechanism in accordance with the docking results, which shows its interaction with both catalytic active (CAS) and peripheral anionic (PAS) sites. The specific binding of compound 1 to PAS domain of AChE was also confirmed experimentally. Moreover, compounds 1 and 3 exhibited satisfactory biometal binding abilities toward Cu2+, Fe2+, Zn2+ and Al3+ ions. These results provide a new evidence for further development and utilization of B. martensii in health and pharmaceutical products.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and memory loss. With the acceleration of the aging process of the world population, the incidence of AD increases year by year, and it is estimated that the number of AD patients worldwide will exceed 100 million by 2050.
Although AD pathogenesis has not been fully identified, it is confirmed to be due to the deficit of acetylcholine. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can catalyze the hydrolysis of acetylcholine. Previous structural studies [1,2] have been shown to characterize the overall structure of AChE into several subsites: the active site (CAS) consisting of the catalytic triad, anionic subsite and acyl binding pocket, and the peripheral anionic site (PAS) at the mouth of the gorge leading to the active site. AChE inhibitors may inhibit AChE via a competitive mechanism, by interacting with CAS of the enzyme, via a non-competitive mechanism, by binding with the peripheral anionic site (PAS), or via both mechanisms, by exerting a dual binding AChE inhibition [2]. Among the existing medications, galantamine is a strictly competitive inhibitor of AChE, while donepezil is a mixed competitive/noncompetitive inhibitor that interacts with CAS as well as with PAS of the AChE [3]. Compared with AChE, BChE plays a supportive role in the cholinergic neurotransmission. However, during the progression of AD, level of AChE in the patient brain decreases while the level and activity of BChE significantly increases. Potent BChE inhibitors may provide greater efficacy in late AD when BChE activity becomes dominant [4]. Another advantage of BChE is that, unlike AChE, it is not expressed in the peripheral and parasympathetic autonomous nervous systems, and

Results
The 85% methanolic extract of B. martensii was fractioned with ethyl acetate. The aqueous fraction was chromatographed on silica gel, ODS, and Sephadex LH-20 to obtain two novel compounds (1 and 2) designated as Buthutin A and Buthutin B, respectively, as well as the known compounds trigonelline (3) and 3-methylbuthyl hydrodisulfide (4) (Figure 1). Potent BChE inhibitors may provide greater efficacy in late AD when BChE activity becomes dominant [4]. Another advantage of BChE is that, unlike AChE, it is not expressed in the peripheral and parasympathetic autonomous nervous systems, and then inhibiting BChE may not cause the adverse side-effects of AChE specific inhibitors. Therefore, the synergistic inhibition of both AChE and BChE, like rivastigmine, may be one more valuable approach to positively improve the course of AD [5].
Furthermore, the recent literature has shown that the abnormally high levels of metal ions in the brain promote the formation of Aβ plaques and catalyze the production of reactive oxygen species (ROS), which further elicit oxidative stress contributing to the AD pathogenesis [6]. Therefore, metal-binding agents are useful materials for the treatment of AD due to their beneficial effects in prevention of oxidative damage caused by free radicals [7]. Reportedly, a large proportion of active compounds and medicines currently used for central nervous system disorders are of natural origin or are modified from such compounds [8,9]. Based on the above considerations, researchers are seeking new AChE and BChE inhibitors with the multifunctional characteristics from edible and natural sources.
To date, it is well-known that alkaloidal compounds of natural origin are a great source of cholinesterase inhibitors. Scorpions are one of the oldest known groups of arthropods on earth, and over 2400 scorpion species are now widely distributed on all continents except Antarctica. Two scorpion species have been reported to have an inhibitory effect on acetylcholinesterase activity [10,11], and several alkaloids [12][13][14] have been obtained from scorpions. Consequently, the tracing of new scorpion alkaloids piqued our interest due to their possible applications for either finding or improving treatments against AD. The scorpion Buthus martensii Karsch is widely distributed in China, and has long been used as a tonic food for human health benefits, such as scorpion wine and fried foods for thousands of years. Recently, it has been developed as a kind of canned foods with its perfect protein and unique flavor [14][15][16]. As a Chinese traditional medicine, it is also recommended for treating convulsion, epilepsy, apoplexy, facial paralysis, hemiplegia, rheumatism and relieving pain [17,18]. Nowadays, a few small molecules including two alkaloids were investigated from the B. martensii Karsch [13,14,19]. As part of our ongoing investigations on the cholinesterases inhibition by natural products, we now describe the detailed bioactivities and inhibition kinetics of active alkaloids from the whole body of B. martensii Karsch.

Results
The 85% methanolic extract of B. martensii was fractioned with ethyl acetate. The aqueous fraction was chromatographed on silica gel, ODS, and Sephadex LH-20 to obtain two novel compounds (1 and 2) designated as Buthutin A and Buthutin B, respectively, as well as the known compounds trigonelline (3) and 3-methylbuthyl hydrodisulfide (4) ( Figure 1).    Figure S1), indicating that the molecule has six degrees of unsaturation. Based on the representative MS +2 spectrum ( Figure S2) of compound 1, the proposed fragmentation pathways of compound 1 are shown in Figure 2. As a result, the fragmentation of the molecular ion of compound 1 at m/z 251.1514 led to the predominant product ion at m/z 121.0293, arising from cleavage of the amide linkage. The ion at m/z 234.1232 could be produced by loss of an ammonia molecular. In addition, the ion at m/z 192.1035 could be derived from terminal loss of the neutral guanidine molecule.  Figure S1), indicating that the molecule has six degrees of unsaturation. 89 Based on the representative MS +2 spectrum ( Figure S2) of compound 1, the proposed 90 fragmentation pathways of compound 1 are shown in Figure 2. As a result, the 91 fragmentation of the molecular ion of compound 1 at m/z 251.1514 led to the predominant 92 product ion at m/z 121.0293, arising from cleavage of the amide linkage. The ion at m/z 93 234.1232 could be produced by loss of an ammonia molecular. In addition, the ion at m/z 94 192.1035 could be derived from terminal loss of the neutral guanidine molecule.

Cholinesterase Inhibition Activities
Inhibitory activities on AChE and BChE in comparison to the reference compound galanthamine were determined. The crude extract, ethyl acetate soluble fraction, and aqueous fraction of B. martensii Karsch were tested for AChE inhibitory activity, and the aqueous fraction possessed particular inhibitory activity with its IC 50 value of 56.8 ± 3.72 µg/mL. Accordingly, we screened the cholinesterase inhibitory activities of the isolated compounds from this fraction. The IC 50 values of tested compounds and their selectivity indexes (SI) for AChE over BChE are listed in Table 1. In the AChE assay, compound 1 displayed the most potent inhibitory activity with IC 50 value of 7.83 ± 0.06 µM, and compounds 2 and 4 showed moderate inhibitory activity, with IC 50 values of 61.45 ± 2.34 and 40.93 ± 3.21 µM, respectively, while compound 3 had a weaker inhibitory effect. In terms of inhibitory activity against BChE, compounds 1-4 showed higher IC 50 values than those for AChE inhibition with selectivity index ranging from 1.99 to 6.05, indicating that all compounds 1-4 acted as selective AChE inhibitors. The significantly high inhibition of compound 1 than compound 2 indicated that substitution of the hydroxyl group at C-4 and electron deficient pyridine ring could be the indelible factors could be an indelible factor for AChE and BChE inhibitory activities.

Propidium Iodide Displacement Assay
Propidium iodide is a known specific inhibitor of peripheral anionic site (PAS) of AChE. The binding abilities of compounds 1-4 to PAS site was determined by competitively displacing the propidium iodide. The results obtained in Table 1 showed that compounds 1 (18.29 ± 0.53%) and 2 (17.95 ± 0.98%) highly displaced propidium iodide from the propidium iodide-AChE enzyme complex, which are comparable with those of donepezil (18.50 ± 1.13%). Compounds 3 and 4 appeared to have considerably less capable in displacing the propidium iodide from AChE. These results indicated that compounds 1 and 2 could efficiently bind to the PAS site of AChE.

Enzyme Kinetic Analyses against AChE
To gain further insight into the mechanism of AChE enzyme inhibition and to understand the dual-binding site character, enzyme kinetic analyses were performed on active compounds 1 and 2. The results from Figure S29 exhibited that compounds 1 and 2 are reversible inhibitors, as in the presence of different concentrations of compounds, plots of the initial velocity versus enzyme concentration gave a series of straight lines, all of which passed through the origin.
The kinetic characterization of compounds 1 and 2 against AChE was also carried out by measuring the enzyme's activity at different concentrations of substrate. The results in Figure 4 showed that compounds 1 and 2 had a family of straight lines with different slopes but they intercepted one another in the first and second quadrant, respectively. All of these lines had no intersection on the horizontal or vertical axis, indicating that compounds 1 and 2 cause a mixed type of inhibition, thus supporting the dual binding character of compounds 1 and 2 that bind, in all likelihood, to both catalytic active site (CAS) and PAS of the enzyme. Dixon and Cornish-Bowden plots ( Figure 5

Metal-Binding Studies
The ability of the synthesized compounds in binding metals would be an added a vantage in the treatment of AD. Herein, compounds 1-4 were studied for their bindi abilities toward Cu 2+ , Fe 2+ , Zn 2+ and Al 3+ , using UV spectrophotometer with wavelengt ranging from 200 nm to 400 nm. As shown in Figure 6, the spectra of compounds 1 and were significantly changed upon the addition of CuCl2, FeCl2, ZnCl2 and AlCl3. The d matic decreases in absorbance indicated the possible interactions between these biomet and compounds 1 and 3. The potent metal binding ability of compounds 1 and 3 could due to the contribution of hydroxyl group and the donation of carboxyl anion, respe tively.

Metal-Binding Studies
The ability of the synthesized compounds in binding metals would be an added a vantage in the treatment of AD. Herein, compounds 1-4 were studied for their bindi abilities toward Cu 2+ , Fe 2+ , Zn 2+ and Al 3+ , using UV spectrophotometer with wavelengt ranging from 200 nm to 400 nm. As shown in Figure 6, the spectra of compounds 1 and were significantly changed upon the addition of CuCl2, FeCl2, ZnCl2 and AlCl3. The d matic decreases in absorbance indicated the possible interactions between these biomet and compounds 1 and 3. The potent metal binding ability of compounds 1 and 3 could due to the contribution of hydroxyl group and the donation of carboxyl anion, resp tively.

Metal-Binding Studies
The ability of the synthesized compounds in binding metals would be an added advantage in the treatment of AD. Herein, compounds 1-4 were studied for their binding abilities toward Cu 2+ , Fe 2+ , Zn 2+ and Al 3+ , using UV spectrophotometer with wavelengths ranging from 200 nm to 400 nm. As shown in Figure 6, the spectra of compounds 1 and 3 were significantly changed upon the addition of CuCl 2 , FeCl 2 , ZnCl 2 and AlCl 3 . The dramatic decreases in absorbance indicated the possible interactions between these biometals and compounds 1 and 3. The potent metal binding ability of compounds 1 and 3 could be due to the contribution of hydroxyl group and the donation of carboxyl anion, respectively. ranging from 200 nm to 400 nm. As shown in Figure 6, the spectra of compounds 1 an were significantly changed upon the addition of CuCl2, FeCl2, ZnCl2 and AlCl3. The d matic decreases in absorbance indicated the possible interactions between these biome and compounds 1 and 3. The potent metal binding ability of compounds 1 and 3 could due to the contribution of hydroxyl group and the donation of carboxyl anion, resp tively.

Molecular Docking Studies
To investigate the binding pattern of compound 1 with AChE, molecular dock studies were performed using Discovery Studio ( Figure 7A). In the CAS, a conventio hydrogen bond was formed between the guanidine group and hydroxyl of Ser203. T guanidine group and Trp86 also interacted with π-cation. In addition, the carboxyl Glu202 residue, which plays an important role in molecular recognition and binding specific ligands to the catalytic triad [24], interacted with the guanidine group by a s bridge and a conventional hydrogen bond. For another, the phenyl moiety of compou 1 was located at the PAS, and formed two π-π stacked interactions with Trp286 a Tyr341. Its hydroxyl group also interacted with the benzene ring of Trp286 through a donor hydrogen bond. This interaction study demonstrated that compound 1 w strongly bound to both the binding sites CAS and PAS of AChE, which showed a c sistent inhibitory pattern on AChE to what revealed by its kinetic study and propidi displacement test. As the PAS of AChE is involved in an increased Aβ aggregation ra dual interaction with two binding sites (CAS and PAS) would be especially advantageo for slowing the progression of AD [24,25].
The interaction of compound 1 with BChE was also carried out. As seen in Figure  compound 1 was bound to the residues Gly117 and Gly116 from the oxyanion h (OAH), Ser198 and His438 from CAS, Asp70 from PAS, and additionally to Thr120 a Asn83 residues, via two charge interactions, one π-cation interaction, one π-π T-shap interaction, four conventional hydrogen bonds, and one carbon hydrogen bond. Co pared to its extended conformation bound to AChE, compound 1 exhibited a somew

Molecular Docking Studies
To investigate the binding pattern of compound 1 with AChE, molecular docking studies were performed using Discovery Studio ( Figure 7A). In the CAS, a conventional hydrogen bond was formed between the guanidine group and hydroxyl of Ser203. The guanidine group and Trp86 also interacted with π-cation. In addition, the carboxyl of Glu202 residue, which plays an important role in molecular recognition and binding of specific ligands to the catalytic triad [24], interacted with the guanidine group by a salt bridge and a conventional hydrogen bond. For another, the phenyl moiety of compound 1 was located at the PAS, and formed two π-π stacked interactions with Trp286 and Tyr341. Its hydroxyl group also interacted with the benzene ring of Trp286 through a π-donor hydrogen bond. This interaction study demonstrated that compound 1 was strongly bound to both the binding sites CAS and PAS of AChE, which showed a consistent inhibitory pattern on AChE to what revealed by its kinetic study and propidium displacement test. As the PAS of AChE is involved in an increased Aβ aggregation rate, dual interaction with two binding sites (CAS and PAS) would be especially advantageous for slowing the progression of AD [24,25].
for slowing the progression of AD [24,25]. 233 The interaction of compound 1 with BChE was also carried out. As seen in Figure 7B, 234 compound 1 was bound to the residues Gly117 and Gly116 from the oxyanion hole 235 (OAH), Ser198 and His438 from CAS, Asp70 from PAS, and additionally to Thr120 and 236 Asn83 residues, via two charge interactions, one π-cation interaction, one π-π T-shaped 237 interaction, four conventional hydrogen bonds, and one carbon hydrogen bond. Com-238 pared to its extended conformation bound to AChE, compound 1 exhibited a somewhat 239 U-shaped conformation, which might partly explain its lower inhibitory potency. The interaction of compound 1 with BChE was also carried out. As seen in Figure 7B, compound 1 was bound to the residues Gly117 and Gly116 from the oxyanion hole (OAH), Ser198 and His438 from CAS, Asp70 from PAS, and additionally to Thr120 and Asn83 residues, via two charge interactions, one π-cation interaction, one π-π T-shaped interaction, four conventional hydrogen bonds, and one carbon hydrogen bond. Compared to its extended conformation bound to AChE, compound 1 exhibited a somewhat U-shaped conformation, which might partly explain its lower inhibitory potency.

Arthropod Material
Dried body of B. martensii Karsch were purchased from a Chinese medicine store in Anguo, Hebei of China, and identified by Prof. Yu-Ming Liu. A voucher specimen (HB-17-1203) was deposited at the Department of Pharmacy Engineering, Tianjin University of Technology.

Propidium Iodide Displacement Assay
The assay mixture of AChE (5U) was incubated with test compounds (final concentration 100 µM, 150 µL) for 6 h at 25 • C. After reaction, 50 µL Propidium iodide (1 µM) was added. After 10 min, the fluorescence intensity was observed at an excitation wavelength (λex) of 535 nm and an emission wavelength (λem) of 595 nm using a fluorescence plate reader (BioTek Instruments Inc., Winooski, VT, USA). The percentage of displacement was calculated by the following expression: 100 − (IF i /IF 0 × 100), where IF i and IF 0 are the fluorescence intensities with and without the test compounds, respectively [27,28].

Enzyme Kinetic Analysis against AChE
In order to clarify whether inhibitors interact with the target enzyme via noncovalent bond (i.e., reversibility), the initial velocity (V) of substrate hydrolysis as measured by the change in OD 412 over the course of 2 min (after the addition of 7.5 mM ATCI) was calculated for five different concentrations of AChE (0.025, 0.04, 0.05, 0.08, and 0.10 U/mL). After enzyme activities were performed for 2 min, the inhibition type of the enzyme were analyzed by the Lineweaver-Burk plots at five different concentrations of ATCI (3.75, 5.00, 7.50, 10.00, and 15.00 mM) [27]. Inhibition constants for AChE were determined from Dixon plot (1/V i vs. (I)) and Cornish-Bowden transformation ((S)/V i vs. (I)) [29].

Metal-Binding Studies
The chelating studies were performed on an ultraviolet-visible spectrophotometry meter (HITACHI U-3900H, Tokyo, Japan). The UV absorption spectra of the test compound alone (20 µM, final concentration) or in the presence of CuCl 2 , FeCl 2 , ZnCl 2 and AlCl 3 (20 µM, final concentration) were recorded with the wavelength ranging from 200 to 400 nm after incubating for 30 min in methanol at room temperature [27,30]. The binding modes were generated by using the Discovery Studio CDOCKER software (2017R2, Accelrys, San Diego, CA, USA) [31]. The crystal structure of human AChE (hAChE) in complex with donepezil (PDB code 4EY7) was taken from the Protein Data Bank. For docking purposes, the hAChE structure was previously prepared by initially adding and orienting hydrogen atoms, as well as removing waters molecules, ions and any ligands, then its structure was protonated at pH 7.4. Parameters used include: a 10.0 Å radius sphere centered at x = −13.9849, y = −43.9747 and z = 27.8950 within the human AChE active site; heating steps and cooling steps set to 2000 and 5000, respectively, while the heating and cooling temperatures were set to 700, and 300, respectively. The cocrystallized donepezil ligand was extracted and redocked in the hAChE grid for validation of the docking parameters. The poses of the cocrystallized and redocked ligands were compared using a superposition tool, and the root-mean-square deviation (RMSD) value was found to be 0.79 Å ( Figure S28), indicating that the docking methods and parameters used in this study were approximate for the AchE system.

Molecular Docking of Compound 1 into BchE
Flexible docking was carried out using Discovery Studio 2017 R2 (Accelrys, San Diego, CA, USA) [32]. The crystal structure of BchE from Homo sapiens (PDB code 4BDS) was obtained from the Protein Data Bank. The simulation was performed as our described previously [27].

Conclusions
Two rare guanidine-type alkaloids, Buthutin A (1) and Buthutin B (2), including two known compounds (3,4), were extracted from the dried body of B. martensii. Trigonelline (3), as a vitamin B 3 homologue, has been reported as an important ingredient of fenugreek seeds and coffee beans [33], although it had never been identified in B. martensii Karsch. Trigonelline (3) has been reported to have a wide variety of biological activities including antioxidant, anti-inflammatory, hypoglycemic, hypolipidemic, anti-tumor, antimigraine, sedative, memory-improving, and neuroprotective ones [34]. In combination with the above biological activities reported in the literature, it could be concluded that trigonelline (3) is necessary to maintain the functional properties of B. martensii Karsch.
Although guanidine containing metabolites are quite rare in nature, natural guanidine derivatives have drawn continuous attention due to their potent antimicrobial, antiproliferative, antioxidant, analgesic, and anticoagulant activities [35,36]. The guanidine compounds are also known as cholinesterase inhibitors [37][38][39]. In this work, it is the first time to discover this kind of compound from the genus Buthus. Buthutin A (1) demonstrated the most potent inhibition to AChE and BChE with IC 50 values of 7.83 ± 0.06 µM and 47.44 ± 0.95 µM, respectively. Additionally, kinetic analysis showed that Buthutin A (1) was a mixed-type reversible inhibitor of AChE, simultaneously binding to the catalytic and peripheral anionic sites, which was verified by in silico docking studies. Furthermore, compounds 1 and 3 showed satisfactory metal-binding properties toward Cu 2+ , Fe 2+ , Zn 2+ and Al 3+ ions. Thus, the edible B. martensii Karsch could serve as a valuable natural source of multifunctional cholinesterase inhibitors with health benefits for potential application in functional food and medicine.