The Indonesian Biomedical Journal.
Vol.
No.
October 2025, p.
RESEARCH ARTICLE
Print ISSN: 2085-3297.
Online ISSN: 2355-9179 Design and Stability Evaluation of Active Peptides from Indonesian Echinozoa as Acetylcholinesterase (AChE) Inhibitors for AlzheimerAos Therapy Laelatul Afifah1,#.
Vania Uly Andyra1,#.
Marlyn Dian Laksitorini2.
Tri Rini Nuringtyas3,4,E Biotechnology Master Study Program.
Faculty of Graduate School.
Universitas Gadjah Mada.
Jl.
Teknika Utara.
Yogyakarta 55281.
Indonesia Faculty of Pharmacy.
Universitas Gadjah Mada.
Jl.
Sekip Utara.
Yogyakarta 55281.
Indonesia Faculty of Biology.
Universitas Gadjah Mada.
Jl.
Teknika Selatan.
Yogyakarta 55281.
Indonesia Research Center for Biotechology.
Universitas Gadjah Mada.
Jl.
Teknika Utara.
Yogyakarta, 55281.
Indonesia *Corresponding author.
Email: tririni@ugm.
Equally contributing as first authors Received date: Sep 26, 2025.
Revised date: Oct 20, 2025.
Accepted date: Oct 22, 2025 Abstract ACKGROUND: AlzheimerAos disease is characterized by cognitive decline resulting from decreased acetylcholine (AC.
levels due to excessive acetylcholinesterase (AChE) activity.
Current therapies, such as galantamine, have several side effects.
Bioactive peptides derived from marine Echinozoa .
ea urchins and sea cucumber.
have emerged as promising therapeutic agents owing to their structural diversity and diverse bioactivities.
Previous studies identified peptides from sea cucumbers and sea urchins collected along the southern coast of Gunung Kidul.
Yogyakarta (VLCAGDLR.
SWIGLK.
MNGKKITVRPR, and KTKDLK), which exhibit acetylcholinesterase (AChE) inhibitory activity.
However, the therapeutic use of these peptides is challenged by bloodAebrain barrier .
penetration and stability issues.
Therefore, this study was conducted to identify candidate peptides through in silico analysis and to evaluate their stability in phosphate-buffered saline (PBS) as potential AChE inhibitors.
METHODS: Molecular docking was conducted to evaluate peptide binding affinity to the active site.
The best candidate peptides were synthesized and tested in vitro for AChE inhibition using a colorimetric method.
Stability was assessed in PBS by monitoring aggregation through turbidity and Congo Red assays.
RESULTS: The sea cucumber peptide SWIGLK showed strong binding affinity (Ae10.
2 kcal/mo.
11% inhibition at 19 mM, while the sea urchin peptide KTKDLK exhibited Ae8.
2 kcal/mol and 11.
50% inhibition at 0.
19 mM.
Both peptides remained stable in PBS without aggregation for up to 48 h.
CONCLUSION: SWIGLK and KTKDLK demonstrate the most significant AChE inhibitory activity and maintained structural stability, hence supporting their potential as peptide-based candidates for AlzheimerAos therapy.
KEYWORDS: Alzheimer.
AChE inhibitor, holothuroidea, echinoidea, bioactive peptide, peptide stability Indones Biomed J.
: 493-502
Introduction More than 55 million people worldwide suffer from neurodegenerative diseases, with AlzheimerAos disease accounting for 60Ae70% of these cases.
Neurons play a crucial role in processing information and transmitting signals to target cells.
Damage to these neurons leads to symptoms such as memory loss, behavioral changes, and progressive cognitive decline over time.
Alzheimer's therapies recommended by the Food and Drug Administration (FDA) include donepezil, rivastigmine, and galantamine, which act as cholinesterase inhibitors, as well as memantine, which acts as a non-competitive inhibitor.
The use of these drugs still causes various side effects, including diarrhea, nausea, vomiting, dizziness, and muscle cramps, and is unable to halt the progression of the disease.
In recent decades, most clinical AlzheimerAos disease drugs have failed to Copyright A 2025 The Prodia Education and Research Institute.
This work is licensed under a Creative Commons Attribution-NonCommercial 4.
0 International (CC-BY-NC) License.
DOI: 10.
18585/inabj.
be developed due to limited effectiveness or adverse side FDA approval of several new types of drugs, as well as the long-term effectiveness and safety of these drugs, still need to be further validated.
In Alzheimer's patients, damage to brain neurons causes the acetylcholinesterase (AChE) enzyme in the synaptic cleft to break down acetylcholine too quickly, disrupting communication between neurons and resulting in memory loss.
Therefore, it is necessary to treat Alzheimer's disease patients with therapy that restores cholinergic deficits, in this case through the AChE .
Research has developed an AlzheimerAos drug compound that can inhibit the aggregation of amyloidbeta (A).
AChE, and beta-site amyloid precursor protein cleaving enzyme 1 (BACE.
derived from the breakdown of heterocyclic amines, and showed significant inhibitory activity in enzyme inhibition.
Marine organisms from the Echinozoa class .
uch as sea urchins and sea cucumber.
produce various bioactive peptides with broad therapeutic potential, including antioxidant, anticancer, anti-fatigue, antidiabetic, antihypertensive, antimicrobial, and memory-enhancing activities through various molecular pathways that is related to the regulation of oxidative stress, energy metabolism, and nerve function.
Peptides are able to efficiently cross the blood-brain barrier, reaching the affected areas of the brain to provide therapeutic effects.
In general, peptides have low toxicity and can be modified to increase their stability and resistance to degradation.
Peptides can modulate various molecular targets, such as enzymes, inflammatory pathways, and synaptic function, thereby potentially addressing various aspects of Alzheimer's pathology.
In targeting AChE for treatment, the ability of biomolecules to cross the b must be considered.
The main obstacle currently being focused on is the failure of drug delivery to penetrate the blood brain barrier .
A total of 98% of biomolecular drugs cannot penetrate the b, and 100% of biological drugs cannot penetrate the b.
Echinozoa peptides have the potential to be used as therapeutic agents due to their diversity and effectiveness in treating neurodegenerative diseases, including targeting the AChE enzyme.
Bioactive peptides have great potential as therapeutic agents because most bodily functions result from amino acid interactions and they have fewer side effects compared to large proteins.
Peptidebased inhibitors have several advantages, including high specificity, low toxicity, rapid clearance, and the potential for de novo design to precisely tailor their properties.
Additionally, the diversity of side-chain structures Design and Stability of Echinozoa Peptides for AlzheimerAos (Afifah L, et al.
Indones Biomed J.
: 493-502
in peptides allows for selective recognition of various molecular targets, more controlled duration of action, and more consistent therapeutic effects.
The stability and efficacy of peptides must be maintained during drug Peptide instability can lead to peptide inactivation, thereby reducing therapeutic effects.
Previous research has explored active peptides from the Echinozoa (Stomopneustes variolaris and Holothuria pardali.
originating from sea cucumbers and sea urchin gonads on the southern coast of Gunung Kidul.
Yogyakarta.
These peptides have been identified as potential candidates and are to be tested for their activity in inhibiting acetylcholinesterase (AChE) enzymes in vitro.
Previous research has predominantly concentrated on identifying peptide sequences and evaluating their initial inhibitory effects on AChE, without addressing their stability or the underlying molecular mechanisms.
In contrast, the present study not only examines the AChE inhibitory potential of peptides derived from Echinozoa but also emphasizes their stability through both computational and experimental Given that peptide instability and degradation can significantly diminish therapeutic efficacy by limiting their ability to reach the target site, it is essential to develop innovative strategies to enhance peptide stability.
However, studies specifically focusing on this stability and inhibitory potential of Echinozoa-derived peptides against AChE remain limited.
Based on the in silico results, parent peptides were selected and the lowest binding affinity values, resulting in four candidate parent peptides for further in vitro testing.
Therefore, the present study was conducted to design and evaluate the stability of active peptides from Echinozoa as potential AChE inhibitors for AlzheimerAos therapy.
Methods Enzyme Inhibitory Assay of AChE The inhibitory activity of the peptides against AChE was evaluated using a colorimetric method based on the Acetylcholinesterase Inhibitor Screening Kit MAK324 (Sigma-Aldrich.
St.
Louis.
MO.
USA).
Four-hundred U/L Acetylcholinesterase solution (Cat.
No.
C3389.
Sigma-Aldric.
, peptides .
urity >95%.
WuHan Dangang Biological Technology.
Wuhan.
Chin.
, and galantamine hydrobromide (Cat.
No.
G1660.
Sigma-Aldric.
were prepared prior to the assay.
In a 96-well plate, 5 L of 40% dimethyl sulfoxide (DMSO) and 45 L of assay buffer were added for the no-enzyme control, whereas 5 L of 40% DMSO and 45 L of AChE were added for the no inhibitor The Indonesian Biomedical Journal.
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October 2025, p.
For the test wells, 5 L of peptide or galantamine was mixed with 45 L of AChE solution and incubated for 15 min.
A reaction mixture containing 154 L of assay buffer, 1 L of substrate, and 0.
5 L of 5,5A-Dithiobis2nitrobenzoic acid (DTNB) was prepared, and 150 L of this mixture was added to each well.
Absorbance was measured at 412 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Allsheng FlexA-200.
Allsheng.
Hangzhou.
Chin.
at 0 and 10 min.
The percentage inhibition was calculated using the following formula: Inhibition (%) = .
A Test Cp.
A no inhibito.
with iA test cpd = difference in absorbance of the test compound, and iA no inhibitor = difference in absorbance of the inhibitor-free in silico Analysis and Molecular Docking Three-dimensional models of the peptides as ligan were generated using Chimera .
ttps://w.
chimera/) by looking at the structure of amino acids forming a double helix or coil structure, while the human AChE structure (UniProt ID: P22.
was obtained from the Protein Data Bank .
ttp://rcsb.
org/) and modeled using SwissModel .
ttps://swissmodel.
org/).
Active sites of AChE were predicted using PrankWeb .
ttps:// cz/).
Molecular docking simulations were performed using PyRx with both blind and rigid docking approaches at the binding site interacting with galantamine.
The root mean square deviation (RMSD) values were calculated using PyMOL .
ttps://w.
org/) to evaluate conformational stability.
Interactions between peptides and AChE residues were visualized with BIOVIA Discovery Studio (Dassault Systymes.
Vylizy-Villacoublay.
Franc.
Based on unfavorable interactions, peptides were truncated into 3Ae4 amino acid fragments, which were further docked against AChE using the same protocol to compare binding affinity and binding interactions with the parent peptides.
Peptide Aggregation and Stability in PBS Phosphate-buffered saline (PBS) was prepared by dissolving 4 g NaCl, 0.
72 g NaCCHPOCE), 0.
1 g KCl, and 0.
12 g KHCCPOCE in distilled water up to 500 mL.
Peptides were dissolved at 1 mg/mL and diluted with PBS to a final volume of 100 L in 96-well plates.
To evaluate concentration-dependent stability, peptides were prepared at 0.
5, 0.
75, 1, and 1.
mg/mL in a final volume of 100 L with PBS.
Absorbance was measured at 350 nm using Allsheng FlexA-200 ELISA reader with indirect principal at 0 h, 30-min, 1, 2.
5, 2, 2.
3, 24, and 48 h.
Print ISSN: 2085-3297.
Online ISSN: 2355-9179 Congo Red Assay for Aggregation Evaluation A 20 M Congo Red solution was prepared from a 100 M stock by dissolving 1 mg of Congo Red in 14.
36 mL of sterile distilled water.
Peptide solutions were prepared to achieve a final concentration of 1 mg/mL in 100 L of assay volume.
Peptide samples were mixed with PBS and measured at 490, 540, and 660 nm as baseline values.
Subsequently, 20 L of Congo Red solution was added to each sample well and 5 mL Eppendorf tubes .
Absorbance was remeasured at the same wavelengths at 0, 30, 60, 120, 180 min, and 22 h overnight.
Morphological observations of peptide aggregates were performed using a light microscope at the same time points.
Statistical Analysis Statistical analyses were conducted using GraphPad Prism software (GraphPad Software Inc.
San Diego.
CA.
USA).
Differences among groups were assessed using one-way analysis of variance (ANOVA), followed by DunnettAos multiple comparison test to compare each treatment group with the control.
A p-value<0.
05 was considered statistically Results in silico Analysis Results Molecular docking showed that galantamine bound strongly to the active site of AChE with a binding affinity of Ae8.
6 kcal/mol.
The interaction involved residues Tyr103.
Asp105.
Tyr155.
Trp317.
Ser324.
Val325.
Phe326.
Arg327.
Phe328.
Tyr368.
Phe369, and Tyr372 (Table .
Among the test peptides.
CP-1 formed a conventional hydrogen bond with Tyr372, an important residue at the peripheral anionic site (PAS) of AChE.
CP-2 exhibited van der Waals interactions with Trp317, also located at the PAS, and Ser324, a component of the catalytic triad.
UP-1 interacted with Tyr103 via a conventional hydrogen bond, while UP-2 interacted with Trp317 through van der Waals forces, both residues being essential at the PAS (Figure .
Peptide Inhibition Activity The inhibition assay demonstrated that CP-1 had higher inhibitory activity .
11% at 0.
19 mM) compared with CP-2.
Similarly.
UP-1 showed stronger inhibition .
19 mM) than UP-2 (Table .
These findings were consistent with the molecular docking results, in which CP-1 and UP-1 exhibited more favorable .
binding affinity values compared with CP-2 and UP-2.
Design and Stability of Echinozoa Peptides for AlzheimerAos (Afifah L, et al.
Indones Biomed J.
: 493-502
DOI: 10.
18585/inabj.
Table 1.
Molecular docking results between AChE and galantamine .
ositive contro.
and peptides from Echinozoa.
Name of Peptide Sequence of Peptide Galantamine CP-1
SWIGLK
Binding Affinity RMSD
Amino Acid
Peptide Bond Type
Active Site
Residue of AChE
Carbon hydrogen
Carbon hydrogen
TYR155
TYR368
Carbon hydrogen
Pi-Sigma
ASP105
TYR372
SER1
Pi-Alkyl
Conventional hydrogen bond
TRP317
TYR372
TRP2
GLY4
Conventional hydrogen bond
Carbon hydrogen
TRP317
TRP317
Van der waals SER324
Conventional hydrogen bond
Unfavorable bond
Conventional hydrogen bond
Conventional hydrogen bond
Alkyl
conventional hydrogen bond
GLN322
TRP317
TYR103
TYR106
LEU320
SER324
unfavorable bond
conventional hydrogen bond
LEU320
HIS318
CP-2
VLCAGDLR
UP-1
KTKDLK
LYS1
THR2
LYS3
UP-2
MNGKKITVRPR
MET1
ASN2
CP : Sea cucumber peptide.
UP : Sea urchin peptide.
Peptide Aggregation and Stability One-Way ANOVA analysis was used to determine the effect of time on peptide aggregation with 0 minutes as the control.
Peptides were tested for aggregation in PBS buffer pH 7.
CP-2 underwent aggregation due to significant differences at 24 and 48 hours (Figure 2B).
CP-1 showed good stability up to 48 hours with no aggregation detected (Figure A).
UP-1
in PBS pH 7 solution showed no significant difference in absorbance .
>0.
in tests from 0 to 48 hours, indicating that the peptide is stable (Figure 2C).
UP-2 showed no significant difference in absorbance .
>0.
between 0 and 30 minutes up to 24 hours, but at 48 hours, the p-value was 027, indicating a significant difference .
<0.
compared to 0 minutes.
Based on these results.
UP-2 showed instability within 48 hours (Figure 2D).
Figure 3 showed the stability of CP-1 was evaluated at concentrations ranging from 0.
5 to 1.
25 mg/mL over different incubation times.
Two-way ANOVA revealed no significant effect of either time or concentration on CP-1 stability .
>0.
UP-1 also exhibited no significant changes in absorbance across all tested concentrations .
>0.
These results indicated that CP-1 and UP-1 remained stable without aggregation in the phosphate buffer at all tested concentrations, suggesting long-term physical Figure 4 and 5 present the Congo Red staining result observed under light microscopy.
No aggregation was detected for CP-1 or CP-2, indicated by the absence of red spots or clumps after 22 h of incubation with hourly monitoring and overnight observation.
Similarly.
UP-1 showed no red staining up to overnight incubation.
UP-2 exhibited peptide aggregation, as indicated by the presence of red-stained aggregates after overnight Discussion This study shows that peptides from sea cucumbers and sea urchins have significant ability to inhibit AChE enzyme activity at different levels.
Sea cucumber peptide CP-1 has stronger activity because it is able to bind to the active site of the PAS enzyme based on binding affinity results.
CP-2 has lower activity because the bond formed is limited to van der Waals bonds, resulting in a lower percentage of inhibition.
The activity of sea urchin peptides shows that UP-1 has more significant activity than UP-2.
This is in line with the lower binding affinity results and the presence of unfavorable bonds, indicating the existence of unwanted bonds.
The binding affinity value .
G) is a parameter of conformational stability between ligands and macromolecules.
The binding affinity of peptides that do not bind to the target site is also lower than that of ligands that bind to important sites such as PAS.
Furthermore.
RMSD values of 0 or less than 3 yI suggest that the peptideAeAChE docking simulations possess a high The Indonesian Biomedical Journal.
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October 2025, p.
Print ISSN: 2085-3297.
Online ISSN: 2355-9179
Interactions Van der Waals Carbon hydrogen bond
Pi-Sigma
Pi-Pi stacked Pi-Alkyl
TRP317
TRP2 GLY4
SER1
LEU5
TYR372
Interactions Van der Waals Conventional hydrogen
Carbon hydrogen bond
Covalent bond
TYR103
TRP317
ASR106
LEU2
VAL1
GLY5
GYS3
ALA4
LEU107
SE6
TYR372
Interactions Van der Waals Conventional hydrogen
Covalent bond
HIS318
LEU320
SE6
LYS5
LYS4
THR7
SER324
Interactions Van der Waals Conventional hydrogen bond Unfavorable acceptor-acceptor Covalent bond Interactions Van der Waals Unfavorable bump Conventional hydrogen bond Carbon hydrogen bond Alkyl Covalent bond Figure 1.
Visualization of molecular docking between peptides and AChE in 3D and 2D.
The binding interaction of galantamine with AChE as the control B: The docking result of peptide CP-1 with AChE.
C: The interaction of peptide CP-2 with AChE.
The UP-1 with AChE.
The docking of peptide UP-2 with AChE.
The visualization highlights the orientation of each peptide in the PAS region of the enzyme, indicating their potential inhibitory activity.
Design and Stability of Echinozoa Peptides for AlzheimerAos (Afifah L, et al.
Indones Biomed J.
: 493-502
DOI: 10.
18585/inabj.
Recent studies have highlighted the neuroprotective potential of marine-derived peptides against AlzheimerAos Sea cucumber peptides improved memory performance and repaired cholinergic function in scopolamine-induced memory-impaired mice by increasing ACh levels and protecting hippocampal neurons.
Similarly, other study identified nine novel oligopeptides (IGFH.
LGFH.
DWF, and FQF) from simulated gastrointestinal hydrolysates of sea cucumbers that exhibited significant CD38 inhibitory and anti-A aggregation These findings demonstrate the strong potential of sea cucumber peptides as natural agents for AlzheimerAos prevention and therapy.
This reinforces the ability of sea cucumber peptides to overcome Alzheimer's disease through certain mechanisms.
Pig bristles have potential in current treatments, including Alzheimer's, although studies on pig bristle peptides are still very limited.
Previous research showed that Echinochrome A can inhibit AChE in vitro through an irreversible/uncompetitive mechanism and exhibits antioxidant activity, indicating the therapeutic potential of echinoids in the context of acetylcholine-related .
The stability of CP-1 and UP-1 peptides is the best, characterized by no significant difference from the control and remaining stable across variations in peptide This may be influenced by the shorter amino acid length, which gives both peptides good stability.
Peptides with more than 5 consecutive amino acids tend to be at higher risk of aggregation, known as AggregationProne Region (APR), which is the initial core of aggregate formation with high hydrophobicity and low net charge.
Table 2.
Peptide inhibition percentage results.
Sample Concentration .
M) Percent
Inhibition CP-1
CP-2
UP-1
UP-2
degree of reliability and predictive accuracy.
Each peptide has a different amino acid length and amino acid residue sequence.
This illustrates how amino acid variation and sequence can influence the type of interaction and its inhibitory capacity.
Additionally, if ligands form hydrogen bonds outside the PAS domain .
utside the PAS binding pocke.
, these interactions will cause binding instability and reduce affinity.
Binding affinity and RMSD values are important parameters in docking analysis, as both are used to assess the ability of ligands to interact with receptors and the stability of the complexes formed.
The use of molecular docking in this study is in line with the approach used in the study of Bidens vulgaris leaf essential oil, where docking simulations were used to predict affinity and inhibition potential against target enzymes.
Bidens vulgaris leaf essential oil shows potential as a -lactamase inhibitor based on molecular docking results with iG values ranging from Ae4.
3 to Ae8.
kcal/mol.
Low binding affinity values correlate with the ability of ligands to effectively inhibit protein function, confirming that docking analysis is a reliable approach for predicting the biological activity of drug candidates.
Absorbance ( = 350 n.
Absorbance ( = 350 n.
Absorbance ( = 350 n.
Time .
Time .
Absorbance ( = 350 n.
5 2 2.
Time .
Time .
Figure Results parent peptide stability screening in PBS buffer at a concentration of 1 mg/mL.
A: CP-1.
B: CP-2.
C: UP-1.
D: UP-2.
Results are presented as meanASEM.
Analysis was performed using One-Way ANOVA with comparison to the control group .
*p<0.
05, **p<0.
***p<0.
001 compared to control .
Stability was monitored at 0 h-48 h.
The Indonesian Biomedical Journal.
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Print ISSN: 2085-3297.
Online ISSN: 2355-9179 Time .
Absorbance ( = 350 n.
Concentration .
g/mL) Absorbance ( = 350 n.
Time .
Concentration .
g/mL) .
Peptides with a hydrophobic amino acid sequence tend to have a higher probability of aggregation due to the possibility of attraction and the tendency to form spherical or ball-like aggregates to reduce the surface area of the aggregate in solution.
The aggregation process of the CIGB-814 peptide occurs in a gradual, multi-step manner and is significantly influenced by peptide concentration.
At micromolar concentrations, the peptides aggregate to form small oligomers of nanoscale dimensions, primarily due to interactions between the hydrophobic regions of the .
CP-1
CP-2
Figure 3.
The stability of CP-1 and UP-1 was tested at concentrations 5Ae1.
25 mg/mL with varying times up to 48 h.
A: CP-1.
B: UP-1.
Two-Way ANOVA analysis was used to determine the significant effect of time or concentration on the stability of CP-1 .
>0.
This was further validated by Congo Red staining, which showed that peptides with longer amino acid sequences were more prone to aggregation.
This was evident in the clearer aggregation observed in UP-2.
Based on the results of the study, it was found that the morphology and ability of peptides are influenced by the balance of hydrophilic and hydrophobic amino acids arranged in the peptide chain.
Peptides with many hydrophobic residues tend to form -sheet structures, which lead to the formation of aggregates or fibrils.
The amyloid fibril structure is not clearly observable because the microscope Figure 4.
Observation of sea cucumber peptide aggregation using Congo Red staining under a light from 0 h to 22 h.
Peptide aggregation was not detected at this magnification.
Black bar: 20 AAm.
Design and Stability of Echinozoa Peptides for AlzheimerAos (Afifah L, et al.
Indones Biomed J.
: 493-502
DOI: 10.
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Figure 5.
Observation peptide aggregation using Congo Red monitored from 0 h to 22 h.
The black arrow in UP-2 .
indicates Black bar: 20 AAm.
UP-1
UP-2
is limited to a resolution of 200 nm, while the CR-absorbed peptide aggregates are too small, too dispersed, or too amorphous to be clearly seen directly as distinct structures.
Therefore, a Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) microscope is needed to show changes in the size and morphology of the aggregates over time, from monomers to plaques, in order to observe aggregates with micrometer sizes.
To substantiate the computational findings, in vitro assays can be employed for the most promising peptide The Ellman method can be utilized to verify their AChE inhibitory activity, while the Congo Red assay can evaluate peptide stability and aggregation by detecting -sheet structures associated with amyloid formation.
These experimental assessments will furnish essential evidence supporting the computational predictions and enhance the potential of Echinozoa-derived peptides as stable and effective AChE inhibitors for Alzheimer's therapy.
The results of this study confirm that Echinozoa peptides have the potential as AChE inhibitors with distinct characteristics of inhibition, stability, and aggregation.
This provides insight into the relationship between amino acid composition and the biological functions as well as the physical properties of peptides.
The limitation of this study lies in the fact that it is still at the in vitro testing stage as an initial step in designing peptides that can meet the requirements of biomolecules to cross the b.
Therefore, further investigations are needed, including peptide fragmentation, toxicity analysis, and more detailed enzyme kinetics assays.
Future in vivo investigations are also advised to confirm the inhibitory efficacy and neuroprotective properties of the peptides, thereby reinforcing their potential as therapeutic agents for AlzheimerAos disease.
Additionally, molecular dynamics simulations should be utilized to evaluate the dynamic stability of peptideAeAChE complexes under physiological conditions and to elucidate key residue interactions that influence binding affinity and overall inhibitory performance.
Conclusion Two peptide candidates from sea cucumbers (SWIGLK) and sea urchins (KTKDLK) show the most significant activity in inhibiting the AChE enzyme through binding to the PAS side of the enzyme compared to other candidates.
Stability testing shows that both peptides do not undergo aggregation, remain stable in PBS, and do not interact with Congo Red Hence, this initial identification provides an important basis for selecting the most active peptide candidates that have the potential to be developed as Alzheimer's therapies.
Acknowledgments The authors gratefully acknowledge the financial support provided by the PTM BIMA program 2025 from the Ministry of Research and Education of the Republic of Indonesia (Kementerian Riset dan Pendidikan Republik Indonesi.
Authors Contribution LA and VUA performed the experiments, analyzed data and writing the manuscript draft.
TRN designed, supervised the research and revise the manuscript.
MDL provided input on experimental design and manuscript writing.
All authors discussed the results and approved the final version of the The Indonesian Biomedical Journal.
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No.
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Conflict of Interest The authors declare no conflicts of interest or competing interests related to the content of this manuscript.
References