Background The conventional identification of bioactive peptides from protein hydrolysates, such as goat casein, via peptide isolation followed by mass spectrometry is time-consuming, costly, and yields suboptimal results. Peptides obtained from hydrolysate screening typically exhibit low activity and stability. Rational design approaches can develop novel peptides with enhanced activity and improved stability. Purpose This study aimed to identify and optimize α- glucosidase inhibitory peptides derived from goat milk (Capra hircus) casein using in silico digestion and molecular docking approaches to enhance inhibitory activity. Methods Peptides were generated in silico from hydrolysis using PeptideCutter. The peptides were then screened, and their interactions with Î±-glucosidase (PDB ID: 3AXH) were analyzed via molecular docking using HADDOCK. The rational design targeted initial active peptide structure using optimization via amino acid substitutions guided by alanine scanning, followed by evaluation of binding potential to α-glucosidase using Pepsite2 and molecular docking. The modified peptide exhibiting the lowest binding affinity and favorable interactions with α-glucosidase was synthesized, and its inhibitory potency was assessed in vitro through determination of IC50, as well as kinetic characterization and inhibition mode analysis. Results Peptide IPIQY exhibited a binding affinity of − 9.0 kcal/mol and formed a hydrogen bond with the catalytic residue Asp352, with an IC50 value of 83.25 ± 4.69 µM. Subsequent alanine scanning and residue substitution led to the design of a modified peptide, IPIEQ, which demonstrated improved binding affinity (− 9.7 kcal/mol), maintained interaction with Asp352, and successfully enhanced inhibitory activity, with an IC50 value of 62.62 ± 2.42 µM. Kinetic analysis revealed that the peptide IPIEQ exhibits mixed inhibition, with both competitive and uncompetitive characteristics, with Vmax of 0,43 mM/min and Km of 31,66 mM. Conclusion These findings demonstrate the effectiveness of in silico strategies for the rational design and optimization of α-glucosidase inhibitory peptides from IPIQY to IPIEQ, offering a promising approach for the development of functional food ingredients or therapeutic agents for glycemic control.

Rational Design of α-Glucosidase Inhibitor Peptides Derived from Goat (Capra hircus) Milk Casein

期刊论文
发表日期：

2026年2月4日

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Ika Kurniasari,
Respati Tri Swasono,
Tri Joko Raharjo

摘要

Background

The conventional identification of bioactive peptides from protein hydrolysates, such as goat casein, via peptide isolation followed by mass spectrometry is time-consuming, costly, and yields suboptimal results. Peptides obtained from hydrolysate screening typically exhibit low activity and stability. Rational design approaches can develop novel peptides with enhanced activity and improved stability.

Purpose

This study aimed to identify and optimize α- glucosidase inhibitory peptides derived from goat milk (Capra hircus) casein using in silico digestion and molecular docking approaches to enhance inhibitory activity.

Methods

Peptides were generated in silico from hydrolysis using PeptideCutter. The peptides were then screened, and their interactions with Î±-glucosidase (PDB ID: 3AXH) were analyzed via molecular docking using HADDOCK. The rational design targeted initial active peptide structure using optimization via amino acid substitutions guided by alanine scanning, followed by evaluation of binding potential to α-glucosidase using Pepsite2 and molecular docking. The modified peptide exhibiting the lowest binding affinity and favorable interactions with α-glucosidase was synthesized, and its inhibitory potency was assessed in vitro through determination of IC₅₀, as well as kinetic characterization and inhibition mode analysis.

Results

Peptide IPIQY exhibited a binding affinity of − 9.0 kcal/mol and formed a hydrogen bond with the catalytic residue Asp352, with an IC₅₀ value of 83.25 ± 4.69 µM. Subsequent alanine scanning and residue substitution led to the design of a modified peptide, IPIEQ, which demonstrated improved binding affinity (− 9.7 kcal/mol), maintained interaction with Asp352, and successfully enhanced inhibitory activity, with an IC₅₀ value of 62.62 ± 2.42 µM. Kinetic analysis revealed that the peptide IPIEQ exhibits mixed inhibition, with both competitive and uncompetitive characteristics, with V_max of 0,43 mM/min and K_m of 31,66 mM.

Conclusion

These findings demonstrate the effectiveness of in silico strategies for the rational design and optimization of α-glucosidase inhibitory peptides from IPIQY to IPIEQ, offering a promising approach for the development of functional food ingredients or therapeutic agents for glycemic control.