Novel Sleep-Promoting Peptides from Sea Cucumbers Targeting the GABAa Receptor Identified by a Multitechnique Approach of Proteolysis Simulation, Molecular Docking, and Pharmacophore Modeling.

Alcalase and chymotrypsin were identified as optimal enzymes for preparing sea cucumber oligopeptides via proteolysis simulation.

• Proteolysis simulation was conducted on proteins that do not represent the primary structural components of the sea cucumber body wall.
• The selection was confirmed through computational proteolysis simulation prior to wet-lab experimentation.
• This in silico approach was used to narrow down enzyme candidates before physical peptide preparation.

LFA (Leu-Phe-Ala) significantly shortened sleep latency in PCPA-induced insomnia mice.

• LFA reduced sleep latency to 520.5 ± 75.81 seconds in PCPA-induced insomnia mice.
• Sleep latency was measured using a standard pentobarbital sodium-induced sleep model in mice with PCPA (p-chlorophenylalanine)-induced insomnia.
• LFA was identified through a combination of bioinformatics tools, molecular docking, and pharmacophore modeling.

TFW (Thr-Phe-Trp) significantly shortened sleep latency in PCPA-induced insomnia mice.

• TFW reduced sleep latency to 188.3 ± 48.63 seconds in PCPA-induced insomnia mice.
• TFW showed a numerically shorter sleep latency compared to LFA (188.3 s vs. 520.5 s).
• TFW was identified through the same multitechnique screening approach as LFA.

LFA alleviated anxiety-like behavior in PCPA-induced insomnia mice.

• Anxiety-like behavior was assessed in the PCPA insomnia mouse model.
• This anxiolytic effect was observed in addition to LFA's sleep-latency-shortening effect.
• TFW was not reported to share this anxiety-alleviating property in the abstract.

Both LFA and TFW ameliorated the imbalance of excitatory and inhibitory neurotransmitters in insomnia mice.

• Neurotransmitter imbalance is a key feature of the PCPA-induced insomnia model.
• Both peptides normalized the ratio of excitatory to inhibitory neurotransmitters.
• This effect is consistent with their proposed mechanism of action at the GABA-A receptor.

LFA-GABAAR and TFW-GABAAR complexes interact via hydrogen bonds and hydrophobic forces with great structural stability.

• Molecular docking was used to characterize the binding interactions of both peptides with the GABA-A receptor (GABAAR).
• Binding was mediated by hydrogen bonds and hydrophobic forces.
• The complexes showed 'great structural stability' based on in silico analysis.
• Pharmacophore modeling was also used alongside molecular docking to characterize receptor interactions.

A multitechnique in silico and in vivo approach combining proteolysis simulation, molecular docking, and pharmacophore modeling effectively and rapidly discovered sleep-promoting peptides from sea cucumber proteins.

• The pipeline screened peptides computationally before validating them in a mouse model, reducing the need for exhaustive wet-lab screening.
• The approach identified two novel oligopeptides (LFA and TFW) not previously reported as sleep-promoting.
• The study used PCPA-induced insomnia mice as the in vivo validation model.
• The authors conclude this provides 'a theoretical basis for identifying novel sleep-promoting constituents from natural proteins.'