New Synthesized Tri-Peptide as Inhibitor of Krait (Bungarus Sindanus) Venom Acetylcholinesterase

In the current study, the cyclopeptide alkaloid discarine D-derived tri-peptides fragments were synthesized and then investigated for their inhibitory potential against krait (Bungarus sindanus) venom acetylcholinesterase (AChE) enzyme. The tri-peptides L-Leu- threo-D-Pheser-L-Phe and L-Leu- threo-L-Pheser-L-Phe were chemically synthesized by a conventional method using the benzyloxycarbonyl group for the alpha-amino (α-amino) safety and the methyl esters an amino acids derivative used for the safety of carboxyl group. The present paper described that the general synthetic strategy of tri-peptide allows the tri-peptide sequence to be acquired with the N-terminal extreme protected. Kinetic studies using the Lineweaver Burk plot indicated that tri-peptides fragments cause an un-competitive type of inhibition i.e. both Km and Vmax values decreased with the increase of tri-peptides fragment concentration (13.5–22.5 µM). The estimated Ki and IC50 for krait venom AChE were found to be 17.5 µM and 19.5 µM, respectively. Thus the present paper, clarified that the freshly produced tri-peptides fragment can be deliberated as a beneficial mediator for the inhibition of krait venom AChE.


Background
The acetylcholinesterase (AChE; E.C.3.1.1.7) enzyme or true cholinesterase is a serine hydrolase existing in both synaptic and non-synaptic tissue (Silman and Sussman, 2005). The main sources of AChE are the brain, muscles, erythrocyte, and cholinergic neurons where its function is revealed in monitoring the various biological measures by a neurotransmitter acetylcholine (ACh) hydrolysis (Chatonnet and Lockridge, 1989;Massoulie et al., 1993;Milatovic and Dettbarn, 1996;Silman and Sussman, 2005).
In nonsynaptic tissue, it is present in the snake's venom where its function is unknown (Frobert et al., 1997). In snakes, venom is mainly found in the poisonous snake family of the family Elapidae (Frobert et al., 1997). The Elapidae snakes are well known for the extremely toxic constituents in their venom. The Elapidae snake's venom has numerous toxins, some are enzymatic (enzymes) in nature and others are non-enzymatic (toxin, nerve growth factors, and inhibitors) which are extremely noxious to man, and causing death throughout the world, mainly in Asian countries (Bawaskar and Bawaskar, 2004). The krait (Bungarus) genera belong to the family Elapidae which has acetylcholinesterase (AChE) in its venom (Frobert et al., 1997). The AChE enzyme is present in high quantity in the venom of Bungarus snake, approximately 8 mg/g of dried snake venom (0.8% w/w) having 747,000 Ellman's units/g of dry snake venom of AChElike action (Frobert et al., 1997). Interestingly, this composition varies interspecifically (Fry et al., 2008;Tasoulis and Isbister, 2017), as well as intraspecifically, with many factors influencing this diversity including age (Dias et al., 2013), gender (Zelanis et al., 2016), location (Gonçalves-Machado et al., 2016), diet (Barlow et al., 2009), and season (Gubensek et al., 1974).
In the venom of krait (Bungarus sindanus) the AChE is existing in a monomeric, non-amphiphilic, soluble, form and has a 67 KDa molecular weight (Cousin et al., 1996a). Amino acid sequence analysis indicates that krait venom AChE has very close homology with Torpedo 1 3 154 Page 2 of 8 maramorata, using the same "catalytic triad" of His, Ser, and for ACh hydrolysis the Glu in the active site (Cousin et al., 1996a). At least 36 distinct proteins belonging to 8 toxin protein families were identified in krait snake venom. Three-finger toxin (3FTx), phospholipase A2 (including β-bungarotoxin A-chains), and Kunitz-type serine protease inhibitor (KSPI) were the most abundant, constituting ~ 95% of total krait venom proteins. The other toxin proteins of low abundance are snake venom metalloproteinase (SVMP), L-amino acid oxidase (LAAO), acetylcholinesterase (AChE), vespryn and cysteine-rich secretory protein (CRiSP) (Oh et al., 2018).
Alzheimer's disease (AD) is a neurodegenerative common type of dementia characterized by memory lapses and mental weakening. It is most common in the old population due to cholinergic neuron loss in selected brain areas due to irreversible deficiency in the formation of the neurotransmitter acetylcholine (ACh). Nowadays therapeutic strategies via inhibition of AChE are commonly used for AD treatment to increase the amount of ACh which is necessary for normal body function. The amino acid sequence study of venom AChE from different sources shows very close homology. They used the same "catalytic triad" of Ser, Glu, and His in the active site of enzyme for substrate (ATC) hydrolysis (Chhajlani et al., 1989;Cousin et al., 1996b). Thus the finding of an inhibitor can be used for multipurpose (Ahmed et al., 2006) based on quaternary structure, AChE is classified into (a) asymmetric and (b) globular form which contains several sub-units depending on species to species except for snake venom. The asymmetric form contains two (A8), three (A12), or one (A4) tetrameric associations of enzyme subunits disulfide that bonded to a single strand of triple-helical collagenic structure subunit. The globular forms (G1, G2, and G4), comprises one, two, or four catalytic subunits, and exhibits similar catalytic property (Silman and Futerman, 1987;Massoulié et al., 1999). The present study was conducted to synthesize short-chain peptides which can be evaluated as a therapeutic agent for inhibition of krait venom AChE.
Melting points (mp) were determined by using the MQAPF-301 apparatus. 1 H and 13 C NMR spectra (nuclear magnetic spectra) were documented on a Bruker Dpx-400 spectrometer operative at the 400.13 MHZ for 1 H and 100.62 MHZ for 13 C, using tetramethylsilane (TMS) as the interior standard and CDCl 3 as solvent. On pre-coated TLC plates, the thin-layer chromatography (TLC) was accomplished (Merck, silica gel 60 F-254). One or more of the following methods were used for the detection of spots: UV (254 nm), o-toluidine, ninhydrin (0.1% in ethanol), and dragendorff's reagent. The compound's purification chromatographic was carried out using a column packed with silica gel 60 (230-400 mesh) acquired from Merck Co. in system solvents selected by R f separation in TLC.
All amino acids and substances used, except threo-β-Phenylserine acquired conferring to Shaw and Fox (Kenneth et al., 1953), possess L-configuration, and were commercially available by Aldrich/Fluka, and were utilized without further purification.

Venom
The venom of both female and male snakes was manually squeezed, mixed, lyophilized, and then stored at a low temperature of − 20 °C for future use. All the female and male krait snakes were captured from the Thatta District of Sindh Province, Pakistan.

Protein Determination
By following the Bradford standard procedure protein was assayed using bovine serum albumin (BSA) as standard (Bradford, 1976).

Anti-AChE Analysis
The activity of AChE was evaluated by the standard procedure by (Ellman et al., 1961). The rate of hydrolysis rates (V) were determined at different concentrations (0.05-1 mM) of acetylthiocholine iodide (ATC) substrate (S) in 1 mL assay solutions with phosphate buffer (62 mM) at 7.5 pH, and DTNB [5,5´-dithiobis(2-nitro-benzoic acid)] (0.2 mM) at room temperature 25 °C. About 40 mL of diluted snake venom [6 mg of protein] were mixed with the reaction mixture and followed by incubation at 37 °C for 10 min. About 0.06 mM Ethopropazine (inhibitor of BChE) was used to inhibit BChE. For determining the inhibitory potential of various concentrations of the tri-peptides fragment (13.5-22.5 µM), the same method and condition were assumed. The reaction of enzyme-substrate (E-S) was started immediately after adding various substrate concentrations. The yellow color development after adding substrate is an AChE activity indicator. The absorbance was examined at 412 nm after 15 s for 2 min utilizing the Hitachi 2001 spectrophotometer. All sample solutions operated in replica and repeat at least three times.

Determinations of Kinetic
The IC 50 was estimated by the simple plot of % inhibition and % activity vs inhibitor concentrations; while the K i (inhibitory constant) values were obtained utilizing Cornish-Bowden plots of S/V vs.
[I] (Cornish-Bowden and Cárdenas, 1991). The value of K m (Michaels Manton constant) was obtained employing two different estimates, 1/V vs. 1/S (Lineweaver and Burk, 1934) and V vs. V/S. (Hofstee, 1952;Dowd and Riggs, 1965). The kinetics of the interaction of the tri-peptides fragment with snake venom enzyme (AChE) was obtained by using the Lineweaver Burk (Lineweaver and Burk, 1934), double reciprocal plot to find out the type of inhibition concerning different concentrations of substrate range from 0.05-1 mM. To the reaction mixture these different concentrations of ACh were added at room temperature 25 °C after the enzyme (AChE) the mixture was incubated at 37 °C for 10 min in the presence and absence of tri-peptides fragments ranging from 13.5-22.5 µM. All tasters were run in triplicate.

Statistical Analysis
Statistical analysis was accomplished through the Statistica software package (Stat Soft®, TULSA, OK, USA) using one-way ANOVA, which was further followed by post-hoc analysis (Duncan multiple range tests).

Results
In the present study, the Z-L-Leu-threo-L-Pheser-L-Phe-OMe (4) and its diastereomer Z-L-Leu-threo-D-Pheser-L-Phe-OMe (5) was synthesized as shown in Scheme 1. The diastereomeric tripeptide Z-L-Leu-threo-L-Pheser-L-Phe-OMe was found inactive while the tri-peptide fragment Z-L-Leu-threo-D-Pheser-L-Phe-OMe (Scheme 2) was found to inhibit the hydrolytic property of krait venom AChE. The IC 50 , which inhibits 50% of venom AChE was assessed at a fixed AcSCh concentration (0.5 µM) and was found to be 19.5 µM by plotting the % inhibition and % residual activity vs inhibitor concentration (Fig. 1). Furthermore, the result shows that tri-peptides with a threo-D-Pheser unit (Z-L-Leu-threo-D Pheser-L-Phe-OMe), inhibit snake venom AChE in a concentration-dependent mode (Fig. 2). Kinetic analyses indicated that tri-peptides fragment caused an uncompetitive type of inhibition against AChE (Fig. 3) i-e both V max and K m decrease with the increase of tri-peptides fragment concentration (Table 1) which is a clear indication of the un-competitive type of inhibition. Moreover, we found that the tri-peptides fragment (13.5-22.5 µM) decreased the K m values from 17-64% whereas 32 to 69.8% inhibition was observed in the V max values compared with control ( Table 1). The estimated K i value for snake venom AChE was determined by plotting the Cornish-Bowden plot of S/V vs.

Discussion
In the present study, the Z-L-Leu-threo-L-Pheser-L-Phe-OMe (4) and its diastereomer Z-L-Leu-threo-D-Pheser-L-Phe-OMe (5) was synthesized as shown in Scheme 1. The diastereomeric tripeptide Z-L-Leu-threo-L-Pheser-L-Phe-OMe was found inactive while the tri-peptide fragment Z-L-Leu-threo-D-Pheser-L-Phe-OMe (Scheme 2) was found to inhibit the hydrolytic property of krait venom AChE in vitro using test tube assay based on Ellman's 15 methods with some modification (Ahmed et al., 2006;Ahmed et al., 2007). The IC 50 , which inhibits 50% of venom AChE was assessed at a fixed ATC concentration (0.5 µM) and was established to be 19.5 µM by plotting the % inhibition and % residual activity vs inhibitor concentration (Fig. 1). Furthermore, the result shows that tri-peptides with a threo-D-Pheser unit (Z-L-Leu-threo-D Pheser-L-Phe-OMe), inhibit snake venom acetylcholinesterase in a concentration-dependent mode (Fig. 2), while its stereoisomer with a threo-L-Pheser, was inactive. Kinetic analyses showed that the new tri-peptides fragment exhibited an un-competitive type against AChE inhibition (Fig. 3) i-e both V max and K m decrease with the increase of tri-peptides fragment concentration (Table 1) Scheme 2 Tri-peptides Z-L-Leu-threo-L-Pheser-L-Phe-OMe (4) and Z-L-Leuthreo-D-Pheser-L-Phe-OMe (5) synthesis Fig. 1 Snake venom acetylcholinesterase. A plot of % residual activity and % residual inhibition vs tri-peptides fragment concentration Fig. 2 Inhibition of Krait (Bungarus sindanus) AChE in the presence of various doses of the synthesized tri-peptides fragment. The rates of hydrolysis V were dignified at 412 nm by using a fixed ACh concentration of 0.5 mM (AcSCh) in 1 mL assay solutions with phosphate buffer (62 mM and pH 7.4) and 0.2 mM of DTNB followed by incubation at 37C˚ for 10 min before adding of the substrate. (n = 3), p < 0.05 which is a clear indication of the un-competitive type of inhibition. Moreover, we found that the tri-peptides fragment (13.5-22.5 µM) decreased the K m values from 17 to 64% whereas 32 to 69.8% inhibition was observed in the V max values compared with control (Table 1). The estimated K i value was calculated for snake venom AChE by using Cornish-Bowden plots of S/V vs. [I] ( Table 2). The interaction between tri-peptides and AChE primarily involves the following amino acid residues: Arg289, Trp279, Tyr334, Phe330, Phe331, Gly118, His440, Trp84, and Asn85. AChE has a peripheral anion site (PAS), which is composed of five residues Tyr 70, Asp 72, Tyr 121, Trp 279, and Tyr 334. PAS of AChE can participate in catalytic allosteric regulation of the active center and are targets of various anticholinesterase (Johnson and Moore, 2006;Yu et al., 2020) and AChE also has catalytic sites including Trp84, Gly118, Gly119, Ala201, Tyr130, Ser200, Glu327, Tyr330, Phe331 and His440, in particular, aromatic residues Trp279 and residue Trp84 play a key role. The tri-peptides were able to reach the PAS and catalytic sites in AChE, and generate interactions with Trp279, Tyr334, Trp84, Gly118, and His440. Tripeptide forms three conventional hydrogen bonds with Arg289, Trp84, and Asn85 residues of AChE, and forms five pi-alkyls with Tyr334, Phe330, and Phe331 residues of AChE. In addition, Trp279 binds to tripeptide CIK as a pication, Gly118 is connected with a carbon-hydrogen bond, and residue His440 forms a carbon-hydrogen bond and a salt bridge with a tripeptide (Sussman et al., 1991).

Conclusion
It is concluded from the present study that the easily synthesized new tri-peptides fragment can be considered as an inhibitor of krait venom AChE. Furthermore, this finding opens a new area of research to investigate the inhibitory action of this simple tri-peptide fragment against another source of AChE to consider this synthetic peptide as a therapeutic agent for AD treatment. The present outcomes suggested that the stereochemistry of the β-phenylserine residue in the tripeptide structure influences bioactivity.
Author Contributions MA, NM, NS, and AFM performed experiments, analyzed data, and wrote the paper. All authors read and approved the final manuscript. Fig. 3 Krait Venom AChE experiments in the absence and presence of various doses of the tri-peptides fragment as shown in the legend box. All experiments were repetitive at least four times and in all cases, comparable results were acquired