View of Computational Design of a Truncated Javan Spitting Cobra (Naja sputatrix) Venom Cardiotoxin Analogue as a Promising Insulinotropic Agent

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BIOTROPIKA Journal of Tropical Biology

https://biotropika.ub.ac.id/

Vol. 11 | No. 3 | 2023 | DOI: 10.21776/ub.biotropika.2023.011.03.03

COMPUTATIONAL DESIGN OF A TRUNCATED JAVAN SPITTING COBRA (Naja sputatrix) VENOM CARDIOTOXIN ANALOGUE AS A PROMISING

INSULINOTROPIC AGENT

Ichda Arini Dinana^1)*, Widodo¹⁾, Nia Kurniawan¹⁾ ABSTRACT

Treatment options for diabetes may vary widely, however, around 50% of all diabetic patients do not reach the minimum glycemic target. Therefore, drugs that are able to lower glucose levels through unconventional targets are still needed for the treatment of type 2 diabetes. This study aimed to investigate the activity of cardiotoxins from Naja sputatrix towards Kv channels as potential targets for glucose level reduction. Molecular docking analysis was conducted using the ClusPro web server, and the resulting protein-ligand interactions were visualized using BioVia Discovery Studio and LigPlus v.4.5.3. The stability of the docking structures was further examined through molecular dynamics simulations. The CTX-1, CTX-3, CTX-4, CTX-5, and CTX-KJC3 sequences from N.

sputatrix were aligned and modeled, focusing on the 62-81 amino acid residues. The CTX- 3, CTX-4, and CTX-5 models demonstrated interactions with the binding site of the KcsA receptor. Additionally, substituting the Val73 residue in the CTX62-81 fragment with Lys73 resulted in reduced binding energy and mitigated the cytotoxic effects of CTX while maintaining its insulinotropic activity.

Keywords: cardiotoxin, diabetes, insulinotropic, Kv channels

INTRODUCTION

Diabetes, a disease divided into types I and II, is a condition that occurs due to insufficient endogenous insulin production and performance [1, 2]. Diabetes is classified as a metabolic disorder indicated by hyperglycemia, a high concentration of dissolved sugar in the blood, as well as metabolic dysfunction, which impairs the performance of several organs, leading to serious complications like chronic kidney illness [3, 4], visual impairment [5], heart problem [6], erectile dysfunction [7, 8], nerve damage [9] and immune system disorder [10, 11]. Diabetic patients are predominantly diagnosed with type II diabetes, which affects 90% of the population [12]. With a 70% increase since 2000, diabetes mellitus was listed by the World Health Organization (WHO) as one of the deadliest diseases in 2019, and by 2030, there will be 552 million individuals with diabetes worldwide, with the majority living in developing countries [13]. Patients with diabetes have access to a range of treatment options, including oral and injectable medications. More than 50% of diabetic patients did not meet the minimum glycemic target despite the availability of numerous treatment options [14, 15, 16, 17], thus the need for unconventional drugs for diabetes treatment remains high.

There are many active compounds in nature that possess insulinotropic activity, which is the ability to induce insulin production from pancreatic β- cells, specifically in the endocrine cells that detect

blood sugar levels [2, 18]. Several compounds showing insulinotropic potential have been isolated from nature, particularly from the venom of several animals [19, 20]. Venom is a compound rich in bioactive molecules such as peptides, proteins, and enzymes. As compounds that target receptors, membranes, and enzymes, venom compounds have much potential as drugs or therapeutic agents [21, 22]. The highly acknowledged venom of Leiurus hebraeus scorpions, charybdotoxin, has been proven by several previous research that it has the ability to block the voltage-dependent potassium channel (Kv) [20, 23], specifically Kv 1.2 and Kv 1.3 [24, 25]. The inhibition of Kv 1.3 improves insulin sensitivity in vitro [26]. However, scorpion venom is difficult to obtain since it is only produced in small quantities.

The venom of snakes, as previously mentioned by Koh et al. [27], can be utilized in the treatment of cancer, arrhythmia, diabetes, anticoagulation, antiviral, and antimicrobial purposes. Findings from [2] indicate that the presence of Cardiotoxin 1 (CTX-1), a member of the three-finger toxin (3FTX) protein family from the venom of Naja kaouthia, exhibits insulinotropic activity and has the potential to be used as a diabetes treatment, especially due to its lack of cytotoxic effects on INS-1E pancreatic β-cell cultures. In Indonesia, the use of snake venom for medicinal purposes is not uncommon. One example is the utilization of sun- exposed venom from the Javan spitting cobra (Naja sputatrix) as an oral treatment for cancer [28].

Despite the high availability, the venom of the

Submitted : July, 8 2023 Accepted : January, 19 2024

Authors affiliation:

1) Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Indonesia

Correspondence email:

*[email protected]

How to cite:

Dinana, IA, Widodo, Kurniawan N. 2024.

Computational design of a truncated javan spitting cobra (Naja sputatrix) venom cardiotoxin analogue as a promising insulinotropic agent. Biotropika: Journal of Tropical Biology 11 (3): 140-155.

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Javan spitting cobra exhibits largely unexplored potential as a source of bioactive compounds. The venom of the Javan spitting cobra and the monocled cobra (N. kaouthia) has identical compositions, percentages, and dominance to other cobra species' venoms, which might also share the same insulinotropic activity [29, 30], providing a new solution to diabetes problems worldwide.

Regarding those problems mentioned, this study aimed to evaluate the potential of the Javan Spitting Cobra Cardiotoxin (CTX) protein in connection to its insulinotropic activity through a bioinformatics approach.

METHODS

Data collection. The data used in this study include the protein sequence of CTX-1 from N.

kaouthia venom (UniProt: P60305) and Charybdotoxin (CHT) (UniProt: P13487, PDB:

2CRD), which have been confirmed to have insulinotropic activity through the Potassium Channel Blocking mechanism, as a control [2, 20].

The tested sequences consist of CTX proteins from N. sputatrix, including CTX-1 (UniProt: O93471), CTX-3 (UniProt: P60302), and CTX-4 (UniProt:

O93473) as P-type CTX, and S-type CTX represented by CTX-5 (UniProt: O73857) and CTX-KJC3 (UniProt: P60311). The sequences were then downloaded in FASTA format from the UniProt (https://www.uniprot.org/) database and then aligned using ClustalW integrated with MEGA X software [31].

Protein preparation. The physicochemical properties of the truncated protein sequences were then analysed using the Expasy ProtParam tool (https://web.expasy.org/protparam/) [32] to compute various physical and chemical properties of the used peptide. The aligned CTX protein sequences were then modeled using PEP-FOLD3 (https://bioserv.rpbs.univ-paris-

diderot.fr/services/PEP-FOLD3/), a web server for peptide modeling [33]. All obtained 3D structures were saved in PDB format. Structural homology was examined using the Biovia Discovery Studio visualization application. The 3D structure of the Potassium Channel KcsA (PDB: 2A9H) will serve as the receptor protein during the docking process.

The protein preparation was conducted using BioVia Discovery Studio 2019 to eliminate water molecules and native ligands from the receptor protein. All the prepared protein and ligand structures were saved in PDB format for further analysis.

Molecular docking and dynamic. Molecular docking analysis was performed using ClusPro (https://cluspro.bu.edu/), a web server designed explicitly for protein-protein or protein-peptide

docking [34]. The KcsA protein structure in PDB format was set as the receptor, and the modeled CTXs were set as the ligand. The docking results were selected based on the lowest binding energy displayed in the final result. Protein-ligand interactions were visualized using Biovia Discovery Studio. Following the docking results, the analysis proceeded with Molecular Dynamics (MD) to see the flexibility of the docked structure and estimated the interacting chain movement during simulation. MD analysis was performed using the CABS-flex 2.0 web server (http://biocomp.chem.uw.edu.pl/CABSflex2) with 100 cycles, and other settings followed the server's standard parameters [35]. The simulation results were then downloaded in .csv format for further visualisation and analyzed using Microsoft Excel.

RESULTS AND DISCUSSION

Peptide modeling. The protein used as the target receptor in this study is KcsA, a type of potassium channel modelled from the bacterium Streptomyces lividans. Despite having similar activity as eukaryotes’ potassium channel, this receptor has a simpler structure. Previous studies [19, 36] have shown its compatibility as a receptor in the specific receptor design process targeting potassium channels in eukaryotes. One example of its application is in determining specific inhibitors for Kv channels for the treatment of type II diabetes [19]. Considering that modulation of Kv channels, including Kv1.3, Kv1.4, Kv2.1, and KCa such as KCa3.1, can be found in pancreatic β-cells [36].

KcsA has a homo-tetrameric protein structure with four chains, namely A, B, C, and D (Figure 1), forming a pore (channel) (Figure 1b). Both KcsA receptor and other potassium channel receptors have a conserved receptor binding site (RBS) located at amino acid residues Thr75, Val76, Gly77, Tyr78, and Gly79 (Figure 1c), which form bonds with potassium ions. The success of receptor performance inhibition is determined by forming bonds between the inhibitor and these residues in all four pairs of protein chains [37].

The candidate inhibitors used as ligands are peptides derived from the cardiotoxin (CTX) protein of Javanese Cobra, including CTX-1, CTX- 3, CTX-4, CTX-5, and CTX-KJC3. These CTX peptides are abundant in Javanese Cobra venom [30, 38]. As references, two controls were used, namely charybdotoxin (CHT) protein from the scorpion L. hebraeus venom and peptides from N.

kaouthia Cardiotoxin [19, 36].

The physicochemical properties of all the inhibitor candidates along with the control peptide were evaluated. The tested properties include the molecular weight (MW), theoretical pI, Formula,

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extinction coefficient, half-life, instability index (II), Aliphatic Index, and Grand average of hydropathicity (GRAVY). The isoelectric point (pI) is the pH condition at which the net charge of protein molecules is zero. When the pH is below the pI, proteins carry a positive charge, while above the pI, they carry a negative charge [39]. A pI value above 7 indicates the peptide being tested is basic in nature, whereas a pI value below 7 signifies an acidic nature of the tested peptide. The results of the physicochemical analysis of the peptides entirely demonstrate theoretical pI values above 7, indicating that all tested epitope candidates tend to possess a basic nature in vivo (Supplementary Material 1).

The other tested parameter is the estimated half- life of the inhibitor candidates. The half-life estimation is the time required for a compound to lose exactly half of its initial dose concentration in the body, meaning that within one half-life duration, 50% of the compound's concentration has been eliminated from the body system [40]. All the tested peptides have a long half-life of more than 20 hours in yeast and more than 10 hours in E. coli.

All the snake peptides analogue has a 100-hour half-life in mammalian reticulocytes, while the charybdotoxin peptide only has 4.4 hours. This result indicates that snake venom CTX analogue has better bioavailability than charybdotoxin. The results of the half-life analysis align with the instability index values, wherein all analogues of snake CTX proteins, whether from N. sputatrix or N. kaouthia, exhibit instability index values below 40. However, charybdotoxin displays a value of

56.17. The Instability index provides insight into the stability of the peptides in the test tube. Peptides with an instability index value above 40 are categorized as unstable, while those with a value below 40 are categorized as stable [41].

The thermal stability of the inhibitor candidates is indicated by the aliphatic index. All cobra peptides have good thermal stability with a high aliphatic index, while charybdotoxin only has 28.16 as the aliphatic index, indicating low thermal stability. This result aligns with previous research by Panda & Chandra [42] CTX from both cobra species, which possess good thermal stability and are rich in hydrophobic amino acids while exhibiting polar characteristics. The hydrophobicity of the tested peptides was indicated using the GRAVY value. Peptides with negative GRAVY values are hydrophilic, while the positive values are hydrophobic [43]. The hydrophilic compound is considered more favourable in drug design to facilitate the hydrophilic nature of the cell and support good compound absorbance.

Charybdotoxin is very hydrophilic, with a GRAVY value of -0.763. While the peptide analogue of CTX-1 from the N. kaouthia and CTX-5 from N.

sputatrix was originally hydrophilic even before the adjustment from Val73 to Lys73, after the alteration, both displayed lower GRAVY values.

All the other N. sputatrix CTX peptide analogues are originally hydrophobic with a positive GRAVY value. After adjustment by changing the Val73 to Lys73, the GRAVY values were all negative, indicating a hydrophilic nature.

Figure 1. KcsA Receptor Structure. a) Components of the closed-state potassium channel KcsA; b) Top view of the KcsA receptor; c) Receptor Binding Site (RBS) area of KcsA interacting with potassium; d) Example of the binding formed with one of the tested ligands (CTX3-N. sputatrix)

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The amino acid sequences of N. sputatrix CTX were downloaded and aligned using ClustalW integrated into the MEGA X application. The N.

sputatrix CTX sequences consist of 81 amino acid residues, except for the KJC3 sequence, which consists of 61 amino acid residues. Compared to CHT, which consists of only 37 amino acid residues, the 3D structures of CTX and CHT are obviously different. The modeled 3D structures of CTX show different β-sheet folds depending on the type. P-Type CTX has three hydrophobic β-sheet folds, while S-Type has only one hydrophobic β- sheet fold [44]. These β-sheet folds are formed at the N-terminus of CTX, specifically at amino acid residues 1-39 (in KJC3 of N. sputatrix and CTX-1 of N. kaouthia) or up to amino acid residue 59 in CTX-1, CTX-3, and CTX-4. Previous research by Nguyen et al. [2], which tested the ability of the N- terminus and C-terminus of CTX to induce INS-1E cell death using MTT and LDH assays, showed that the 1-39 amino acid fragment with β-sheet folds significantly influenced the survival and integrity of INS-1E cells, while the 41-60 amino acid fragment did not show significant cytotoxic effects.

This finding underlies the truncation of the intact CTX sequence at the N-terminus and leaves the C- terminus. Ligand modeling was performed using peptides derived from the CTX41-60 or CTX62-81 amino acid fragments. However, in vitro tests by Nguyen et al. [36] showed that the ability to stimulate Ca²⁺ release using the truncated CTX41-60

fragment was significantly lower compared to using intact CTX. Therefore, a modification was made by changing the Val52 residue to Lys52, and this change was proven to restore the ability to stimulate Ca²⁺ release with or without glucose stimulation. Based on the sequence alignment, creating N. sputatrix CTX analogue proteins by replacing the Val73 residue with Lys73 in the CTX62-81 sequence also improved the similarity of the binding region with the CHT structure.

Modeling was then performed using the PepFOLD3 web server for the CTXKJC341-60 and CTX62-81 fragments and [Lys73] CTX-141-60 or CTX-162-81.

Molecular docking analysis. CHT is a venom from the scorpion L. hebraeus that has been shown to inhibit K+ channel activity by binding to the RBS of KcsA [19, 20, 45]. Related research on the utilization of venom as a potential drug for the treatment of type 2 diabetes mellitus through the inhibition of K+ channels has also been conducted on N. kaouthia venom, specifically the CTX-1 fragment, which showed positive results [2, 36].

The interactions of both controls indicate that the peptide structures form hydrogen bonds in the filter region of the KcsA K+ channel, particularly at the Tyr78 residue in all four chains (A, B, C, and D),

which are part of the RBS (Figure 1a). These bonds then close the KcsA channel, increase insulin production, and maintain depolarization of the pancreatic β-cell membrane [46]. The docking result for CHT shows a binding energy of -1246.74 kcal/mol, while the docking result for CTX-141-60 requires energy of -839.61 kcal/mol (Supplementary Material 2).

The docking analysis of the CTX-162-81 fragment from N. sputatrix as the ligand and KcsA as the receptor shows the same binding interaction as the controls, but no interaction is formed with Tyr78 (Figure 2b) as the center binding region. The docking result between N. sputatrix CTX-162-81

requires a binding energy of -782.84 kcal/mol.

Although nine of the 13 interacting amino acid residues are the same as those interacting with CHT, only the Ser5 residue interacts with the RBS residue Gly79(A). This condition is believed to contribute to the low inhibitory capacity of CTX, as reported by Nguyen et al. [36]. When the Val73 residue is changed to Lys73 to improve conformational similarity with CHT, the binding energy required by N. sputatrix [Lys73] CTX-1 decreases to -789.27 kcal/mol (Supplementary Material 2). The interacting amino acid residues increase to 17, and 15 of them interact with the same residues as CHT. Additionally, the Lys4 residue of [Lys73] CTX-1, located in chain E, interacts with the Tyr78 residue of the four KcsA chains (A, B, C, and D) by forming a hydrogen bond with its oxygen atom, thus closing the pore/filter in the channel (Figure 2b). Despite having higher binding energy than the two controls, the interaction between the CTX-1 fragment from N. sputatrix and KcsA still indicates a positive result (Supplementary Material 2).

Docking between KcsA and CTX-3 and CTX-4 of N. sputatrix (Figure 3) shows that CTX-4 has a lower average binding energy compared to CTX-3.

The CTX-3 fragment interacts with 12 amino acid residues of KcsA (Supplementary material 2), and 11 of them interact with the same residues as the control CHT (Supplementary material 2). The energy required for both to form bonds is -755.8 kcal/mol. The Lys4 residue of CTX-3 also interacts with the oxygen atom of the Tyr78 residue in the four chains of KcsA (Figure 3). In [Lys73] CTX-3, the binding affinity value shows a significant decrease to -853.81 kcal/mol. In addition to the decrease in binding affinity value, the number of interacting residues increases to 17, and all of them interact with the same residues as the control. The interaction between CTX-4 and KcsA also shows a similar pattern. Before the Val73 residue in CTX- 4 is changed to Lys73, the interaction between KcsA shows a binding energy of -774.12 kcal/mol, which significantly decreases to -898.46 kcal/mol

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after being changed to [Lys73] CTX-4. The amino acid residues that interact with CTX-4 are 14 in total, and 12 of them are the same as the control. In [Lys73] CTX-4, the interacting residues are 17 in

total, all of which are the same as the control. Both CTX-4 and [Lys73] CTX-4 show interactions between the Lys4 residue and the Tyr78 residues in each chain of KcsA (Figure 3).

Figure 2. Visualization of KcsA and ligand docking results. A) Control; B) CTX-162-81 and [Lys73] CTX- 162-81 from N. sputatrix

Figure 3. Visualization of KcsA and ligand docking results. A) CTX-362-81 and [Lys73] CTX-362-81 from N. sputatrix; B) CTX-4 62-81 and [Lys73] CTX-4 62-81 from N. sputatrix. Both are P-Type CTXs

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N. sputatrix Cardiotoxin S-Type is represented by CTX-5 and KJC3 (Figure 4). The interaction between KcsA and N. sputatrix CTX-5 requires a binding energy of -793.39 kcal/mol. It interacts with 12 amino acid residues on KcsA, three of which are different from the control, while the rest bind to the same residues as CHT (Table 1). On the other hand, [Lys73]CTX-5 shows a significant decrease in binding energy, reaching -924.98 kcal/mol, and interacts with 15 amino acid residues, two of which are different from the control.

CTX-KJC3 is the most abundant CTX in N.

sputatrix, accounting for 22.66% of the total cardiotoxin content in its venom [47]. The interaction between CTX KJC3 and KcsA shows the highest binding affinity among all tested ligands, with a value of -713.31 kcal/mol.

Additionally, KJC3 does not interact with the RBS of KcsA. Apart from not binding to the RBS, the amino acid residues of KJC3 also do not interact with chain B of KcsA at all. The docking results between KJC3 and KcsA reveal 14 amino acid interactions, with only one being different from the control. When the Val73 residue in KJC3 is changed to Lys73, the required binding energy

significantly decreases to -908.58 kcal/mol (Table 1). In addition to the decrease in binding energy, changing the Val73 residue to Lys73 in the KJC3 fragment also affects the interaction between its residues. [Lys73]KJC3 interacts with the oxygen atom of Tyr78 residue present in all KcsA chains, forming a hydrogen bond that closes the pore in the channel, indicating inhibitory activity (Figure 4b).

Compared to the control [Lys27] CTX-1 from N. kaouthia, all CTX fragments from N. sputatrix in which the Val73 residue has been changed to Lys73 show a significant decrease in binding energy, particularly in the CTX S-Type group. The low binding energy indicates that CTX from N.

sputatrix forms bonds with KcsA more easily than N. kaouthia. The change of Val73 residue to Lys73 in the CTX-1 fragment not only decreases the binding energy in silico analysis but also triggers insulin production in the study by Nguyen et al.

[36], regardless of glucose stimulation. The insulin production induced by [Lys73] CTX in the absence of glucose can reach levels equivalent to a 20 mM glucose stimulation. Furthermore, cleaving CTX into CTX41-60/62-81 fragments also eliminates the β-sheet folding, thereby reducing its cytotoxic activity [36].

Figure 4. Visualization of KcsA and ligand docking results. A) CTX-562-81 and [Lys73] CTX-562-81 from N. sputatrix; B) CTX-KJC341-60 and [Lys73] CTX-KJC341-60 from N. sputatrix. Both are S-Type CTXs

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Molecular dynamics analysis. A molecular dynamics analysis (MD) was performed on the docking results between KcsA as the receptor and the Ligand. The fragment [Lys27] CTX-1 from N.

kaouthia was used as the control ligand. Kumar et al. [48] stated that RMSF values between 1-3 Å indicate the stability of the complex during the simulation period. RMSF fluctuates over those threshold values, indicating movements and flexibility of the tested residues. The MD results of CTX and [Lys73]CTX showed some fluctuations in the RMSF values. The fluctuations mainly appear in the residues with significant roles in the opening and closing of the channel pore. The graph of the MD simulation results with the CTX ligand from N. sputatrix before the Val73 residue was changed to Lys73 (Figure 5) showed higher fluctuations and higher average RMSF values.

Additionally, residues in chain E, which are part of the ligand, also exhibited instability, with the majority of RMSF values still above 4 Å. However,

alteration of the Val73 residue to Lys73 residue significantly lowers the graph fluctuation with all ligand graph values under 3 Å except for the analogue of CTX3 of N. sputatrix and CTX I of N.

kaouthia.

The MD results of the CTX fragment from N.

sputatrix used as the ligand, which has been changed from Val73 to Lys73, are shown in Figure 6, indicating RMSF values with a lower average compared to before the alteration to Lys73.

Although there are still some fluctuations with RMSF values above 3Å, these RMSF values have decreased. The highest peak has an RMSF value of 6.603 Å, which comes from the control ([Lys27]

CTX-1 N. kaouthia). The peptide chain E, which is part of the CTX peptide group as the ligand, also indicates stability in its structure with RMSF values below the threshold. The highest RMSF value in chain E is 4.731 Å, which belongs to the control, followed by 3.377 Å from [Lys73] CTX-3 N. sputatrix.

Figure 5. Results of molecular dynamic analysis of KcsA and Ligand docking before modification

Figure 6. Results of molecular dynamic analysis of KcsA and ligand docking. The CTX N. sputatrix CTX fragment residue has been modified from Val73 to Lys73

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Mechanism of action. Upon glucose stimulation, insulin secretion initiates when glucose enters the β-pancreatic cells and undergoes metabolism to produce ATP, resulting in a higher ATP-to-ADP ratio. This difference in ratio then activates KATP, leading to the closure of ATP- dependent/sensitive potassium channels and triggering membrane depolarization, simultaneously activating voltage-gated dependent calcium channels (VDCC). The VDCC pathway facilitates the entry of calcium ions from the extracellular space into the cell. As a result, the intracellular calcium ion (Ca²⁺) concentration increases, which subsequently triggers insulin exocytosis. Meanwhile, the inhibition of voltage- gated potassium channels (Kv) can keep the VDCC channels open and ensure the entry of Ca²⁺ ions into the cell, thereby sustaining insulin production.

The closure of Kv channels facilitates insulin production in the presence or absence of glucose.

Inhibition of Kv channels can prolong the action potential and maintain VDCC channels open, thereby enhancing glucose-induced insulin release.

The closure of Kv channels maintains a negative resting membrane potential to provide a source of energy for the entry of Ca²⁺ through VDCC channels [46].

CONCLUSION

CTX-1, CTX-3, CTX-4, CTX-5, and CTX- KJC3 were among the most abundant groups of N.sputatrix CTX. The analogue of these CTXs was then modeled by taking fragments from amino acid residues of 62-81. The docking analysis between the CTX62-81 fragment of N. sputatrix and the KcsA receptor revealed that the Lys4 residue of CTX-3, CTX-4, and CTX-5 interacts with the KcsA RBS at the Tyr78 residue, forming a hydrogen bond. On the other hand, CTX-1 and CTX-KJC3 do not bind to the RBS at all. Modeling the analogue protein of CTX by replacing the Val73 residue in the CTX62-81 fragment with Lys73 can decrease the binding energy of the KcsA-CTX interaction and reduce the cytotoxic effects of CTX while still maintaining its insulinotropic ability. After changing Val73 to Lys73, all tested ligands show interactions with the RBS at the Tyr78 residue in all four KcsA chains.

The MD results also indicate that the Val73 to Lys73 mutation can enhance structural stability.

The CTX62-81 fragment in N. sputatrix has the potential for insulinotropic activity by inhibiting K⁺ channels, stimulating an increase in intracellular Ca²⁺ ions, and inducing insulin production.

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Supplementary Material 1. Physicochemical properties of the peptide analogue used in the study

Peptide Molecular

weight Theoretical pI Formula Extinction

coefficients: Estimated half-life Instability index:

Aliphatic

index GRAVY

Charybdotoxin 4390.06 9.34 C179H291N59O56S7 7365

4.4 hours (mammalian reticulocytes, in vitro).

>20 hours (yeast, in vivo).

>10 hours (Escherichia coli, in vivo).

56.17 28.16 -0.763

Naja kaouthia CTX-1 41-60 2272.70 8.51 C94H158N28O29S4 1740

100 hours (mammalian reticulocytes, in vitro)

9.68 82.50 -0.025

Naja sputatrix CTX-1 62-81 2245.67 8.51 C93H157N27O29S4 1740

19.30 82.50 0.110

9.68 68.00 -0.125

Naja sputatrix CTX-KJC3 41-60 2230.62 8.51 C91H152N28O29S4 1740

9.68 68.00 -0.125

[Lys]Naja kaouthia CTX-1 41-

60 2301.74 8.89 C95H161N29O29S4 1740 100 hours (mammalian

reticulocytes, in vitro) 9.68 68.00 -0.430

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Peptide Molecular

weight Theoretical pI Formula Extinction

coefficients: Estimated half-life Instability index:

Aliphatic

index GRAVY

[Lys]Naja sputatrix CTX-3 62-

81 2274.71 8.89 C94H160N28O29S4 1740

19.30 68.00 -0.295

81 2274.71 8.89 C94H160N28O29S4 1740

19.30 68.00 -0.295

81 2259.66 8.89 C92H155N29O29S4 1740

9.68 53.50 -0.530

81 2274.71 8.89 C94H160N28O29S4 1740

19.30 68.00 -0.295

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Supplementary Material 2. Details of the interaction between KcsA and Control, as well as KcsA and N.

sputatrix CTX.

Ligand Interacting Residue Cathegory

Binding energy (Kcal/Mol)

Charybdotoxin (Control 1)

E: ARG825 – C: ASP80 Hydrogen Bond;Electrostatic

-1246,74 E: ARG825 – C: ASP64 Electrostatic

E: ARG825 – C: ASP64 Electrostatic E: ARG834 – D: ASP80 Hydrogen Bond B: TYR82 – E: CYS807 Hydrogen Bond C: TYR82 – E: GLY826 Hydrogen Bond D: ALA58 – E: TYR836 Hydrogen Bond D: TYR82 – E: ARG825 Hydrogen Bond E: LYS811 – C: GLY56 Hydrogen Bond E: TRP814 – C: TYR82 Hydrogen Bond E: LYS827 – D: TYR78 Hydrogen Bond E: LYS827 – B: TYR78 Hydrogen Bond E: LYS827 – C: TYR78 Hydrogen Bond E: LYS827 – A: TYR78 Hydrogen Bond E: LYS827 – B: TYR78 Hydrogen Bond E: ASN830 – A: ASP80 Hydrogen Bond E: LYS831 – A: GLY56 Hydrogen Bond E: LYS831 – A: ALA57 Hydrogen Bond E: ARG834 – D: ASP80 Hydrogen Bond E: ARG834 -A: TYR82 Hydrogen Bond E: ARG834 -A: TYR82 Hydrogen Bond E: TYR836 – D: ASP80 Hydrogen Bond E: SER837 – D: GLY56 Hydrogen Bond E: ARG825 – C: ASP80 Hydrogen Bond E: ARG825 -D: TYR82 Hydrogen Bond E: ARG834 – D: ASP80 Hydrogen Bond E: SER837- D: TYR82 Other

E: MET829 -A: TYR82 Hydrophobic D: TYR82 – E: TYR836 Hydrophobic B: TYR82 – E: CYS807 Hydrophobic B: TYR82 – E: CYS828 Hydrophobic E: TRP814 – C: ALA58 Hydrophobic E: TRP814 – C: ALA58 Hydrophobic E: TYR836 – D: ALA57 Hydrophobic E: TYR836 – D: ALA58 Hydrophobic

[Lys27] CTX1 N. kaouthia (Kontrol 2)

E: VAL1 – D: GLY56 Hydrogen Bond

-839,61 E: VAL1 – D: GLY56 Hydrogen Bond

E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – B: TYR78 Hydrogen Bond E: LYS4 – B: TYR78 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: ASN5 – A: ASP80 Hydrogen Bond E: ASN15 – D: GLY56 Hydrogen Bond E: ASN15 – D: ALA57 Hydrogen Bond E: ASN5 – A: GLY79 Hydrogen Bond D: ALA58 – E: ASN15 Hydrogen Bond D: ASP80 – E: ASN5 Hydrogen Bond B: TYR82 – E: LEU8 Hydrophobic

CTX1 N. sputatrix

E: LYS4 – A: ASP64 Hydrogen Bond;Electrostatic

-782,84 E: LYS4 – A: ASP80 Hydrogen Bond;Electrostatic

E: LYS4 – A: ASP64 Hydrogen Bond;Electrostatic E: ARG18 – C: ASP80 Electrostatic

E: SER5 – A: ASP80 Hydrogen Bond

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Binding energy (Kcal/Mol) E: SER5 – A: GLY79 Hydrogen Bond

E: SER5 – A: ASP80 Hydrogen Bond C: TYR82 – E: VAL1 Hydrogen Bond E: SER5 – A: ASP80 Hydrogen Bond D: ALA58 – E: CYS13 Hydrophobic B: TYR82 – E: PRO3 Hydrophobic B: TYR82 – E: LYS4 Hydrophobic C: TYR82 – E: VAL1 Hydrophobic

[Lys74] CTX-1 N. sputatrix

E: LYS12 – A: ASP80 Electrostatic

-789,27 E: LYS4 – D: GLY79 Hydrogen Bond

E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – B: TYR78 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: SER5 – D: ASP80 Hydrogen Bond E: SER5 – D: ASP80 Hydrogen Bond E: TYR11 – B: GLY56 Hydrogen Bond E: LYS12 – A: ASP80 Hydrogen Bond E: PRO3- D: TYR82 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond E: SER5 – A: TYR82 Hydrogen Bond C: ALA58 – E: VAL1 Hydrophobic D: TYR82 – E: PRO3 Hydrophobic

CTX3 N. sputatrix

E: LYS4 – B: TYR78 Hydrogen Bond

-755,8 E: LYS4 – C: TYR78 Hydrogen Bond

E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond E: SER5 – A: ASP80 Hydrogen Bond D: ASP80 – E: CYS2 Hydrogen Bond C: TYR82 – E: TYR11 Other

E: TYR11 – C: TYR82 Hydrophobic D: ALA58 – E: CYS2 Hydrophobic E: TYR11 – C: ALA58 Hydrophobic A: TYR82 – E: PRO3 Hydrophobic

[Lys74] CTX3 N. sputatrix

E: LYS4 – D: TYR82 Hydrogen Bond

-853,81 E: LYS4 – A: TYR78 Hydrogen Bond

E: LYS4 – B: TYR78 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond E: LYS12 – A: TYR82 Hydrogen Bond E: LYS12 – A: PRO55 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS12 – A: GLY56 Hydrogen Bond E: LYS12 – A: TYR82 Hydrogen Bond A: ALA57 – E: LEU8 Hydrophobic D: ALA58 – E: VAL9 Hydrophobic A: TYR82 – E: LEU8 Hydrophobic C: TYR82 – E: VAL1 Hydrophobic

CTX4 N. sputatrix

E: LYS4 – A: GLY79 Hydrogen Bond

-774,12 E: LYS4 – A: ASP80 Hydrogen Bond

E: LYS4 – B: TYR78 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond

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Binding energy (Kcal/Mol) E: LYS4 – A: TYR78 Hydrogen Bond

E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – B: TYR78 Hydrogen Bond E: PRO3 – A: ASP80 Hydrogen Bond E: PRO3- A: ALA57 Hydrogen Bond E: VAL1 – A: VAL84 Hydrophobic A: ALA57 – E: PRO3 Hydrophobic A: ALA58 – E: PRO3 Hydrophobic C: TYR82 – E: LEU8 Hydrophobic

C: TYR82 – E: VAL1 Hydrogen Bond

E: LYS4 – B: TYR78 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: SER5 – D: TYR82 Hydrogen Bond E: LYS12 – A: TYR82 Hydrogen Bond E: PRO- C: TYR82 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – B: TYR78 Hydrogen Bond E: LYS12 – A: GLY56 Hydrogen Bond A: TYR82 – E: LEU8 Hydrophobic B: TYR82 – E: LEU7 Hydrophobic C: TYR82 – E: VAL1 Hydrophobic E: TYR11 – A: ALA58 Hydrophobic

CTX5 N. sputatrix

E: CYS2 – A: TYR82 Hydrogen Bond

E: LYS4 – B: TYR78 Hydrogen Bond E: LYS4 – B: TYR78 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: ASN5 – B: ASP80 Hydrogen Bond E: ASN5 – B: GLY79 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – B: TYR78 Hydrogen Bond A: ALA57 – E: PRO3 Hydrophobic A: VAL84 – E: VAL1 Hydrophobic

E: LYS12 – D: ASP80 Hydrogen Bond;Electrostatic

-924,98 C: TYR82 – E: ASN5 Hydrogen Bond

E: VAL1 – B: GLY56 Hydrogen Bond E: CYS2 – B: TYR82 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – B: TYR78 Hydrogen Bond E: LYS12 – A: TYR82 Hydrogen Bond A: PRO55 – E: TYR11 Hydrogen Bond E: LYS4 – B: TYR78 Hydrogen Bond A: GLY56 – E: TYR11 Other

B: ALA58 – E: VAL1 Hydrophobic

KJC3 N. sputatrix

E: LYS10 – A: ASP80 Hydrogen Bond;Electrostatic

-713,31 C: TYR82 – E: ARG18 Hydrogen Bond

E: LYS4 – C: ASP80 Hydrogen Bond E: LYS4 – C: TYR82 Hydrogen Bond

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Binding energy (Kcal/Mol) E: ASN5 – D: ASP80 Hydrogen Bond

E: ASN5 – A: TYR82 Hydrogen Bond E: SER6 – A: GLY79 Hydrogen Bond E: ALA7 – A: TYR82 Hydrogen Bond E: LYS10 – A: ASP80 Hydrogen Bond E: ARG18 – C: GLY56 Hydrogen Bond E: ARG18- C: GLY56 Hydrogen Bond E: ASN5 – D: ASP80 Hydrogen Bond A: TYR82 – E: ALA7 Hydrophobic D: TYR82 – E: LYS4 Hydrophobic

[Lys74] KJC3 N. sputatrix

C: TYR82 – E: CYS2 Hydrogen Bond

-908,58 E: LYS4 – C: TYR78 Hydrogen Bond

E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – A: TYR78 Hydrogen Bond E: LYS4 – B: TYR78 Hydrogen Bond E: ASN5 – A: GLY79 Hydrogen Bond E: SER6 – D: TYR82 Hydrogen Bond E: LYS12 – A: ALA57 Hydrogen Bond D: GLY792 – E: ASN5 Hydrogen Bond E: LYS4 – C: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond E: LYS4 – D: TYR78 Hydrogen Bond