J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Original Research

Volume 21, March 2023, pages 62-71

Biomolecular interactions and inhibition kinetics of human soluble epoxide hydrolase by tetrapeptide YMSV

Figure 1. Effect of YMSV on the activity of soluble epoxide hydrolase (sEH). (a) Structure of peptide YMSV; (b) plots of the product (6M2N) formed at constant [S] (5 µM PHOME) over time at different concentrations of the inhibitor, YMSV; (c) concentration-dependent sEH inhibitory activity of YMSV; (d) Lineweaver-Burk double reciprocal plot of sEH at different YMSV concentration.

Figure 2. Plots of the product (6M2N) formed by human soluble epoxide hydrolase at the substrate (PHOME) concentrations of 1.25–20 µM in the presence of (a) 250 µM (b) 200 µM (c) 150 µM (d) 100 µM (e) 50 µM (f) 25 µM inhibitor (YMSV).

Figure 3. Plots of P_∞ against varying concentrations of the inhibitor, YMSV with changing [S] (PHOME = 1.25−20 µM).

Figure 4. Plots of ln([P]_∞ − [P]) vs. time to determine the apparent inactivation rate constant of soluble epoxide hydrolase in the presence of (a) 25 µM YMSV, and (b) 250 µM YMSV.

Figure 5. Plots of the reciprocal of apparent inactivation rate constant (A) vs. [S] in the absence and presence of different concentrations of inhibitor (25 and 250 µM YMSV).

Figure 6. Circular dichroism spectra of soluble epoxide hydrolase in the absence (sEH control) and presence (sEH+YMSV) of the inhibitor peptide.

Figure 7. Molecular docking simulation showing (a) 2D and (b) 3D binding interaction of YMSV peptide and the active site of human soluble epoxide hydrolase (sEH), (c) 2D and (d) 3D binding interaction of YMSV peptide and human sEH upon blind docking.

**Table 1.** Kinetics parameters of sEH catalyzed reaction in the absence and presence of different concentrations of the YMSV peptide
YMSV (µM)	V_max (μM/min)	K_m (μM)
K_m, Michaelis constant in the absence and presence of the inhibitor; V_max, maximum reaction velocity in the absence and presence of the inhibitor; ns, not significant (p ≥ 0.05); , significant at 0.01 > p > 0.05); , significant at 0.01 > p > 0.001; , significant at 0.001 > p > 0.0001; **, significant at p < 0.0001 compared to control sEH (at 0 µM inhibitor).
0	110.30 ± 0.36	3.514 ± 0.02
25	89.31 ± 4.55^***	2.928 ± 0.11^ns
50	90.06 ± 1.29^***	3.216 ± 0.05^ns
100	91.42 ± 1.54^***	4.648 ± 0.17^ns
150	103.60 ± 0.90^ns	7.375 ± 0.14^**
200	98.61 ± 0.66^*	11.037 ± 0.16^****
250	128.43 ± 9.92^***	24.830 ± 2.80^****

Table 1. Kinetics parameters of sEH catalyzed reaction in the absence and presence of different concentrations of the YMSV peptide

YMSV (µM)

V_max (μM/min)

K_m (μM)

K_m, Michaelis constant in the absence and presence of the inhibitor; V_max, maximum reaction velocity in the absence and presence of the inhibitor; ns, not significant (p ≥ 0.05); *, significant at 0.01 > p > 0.05); **, significant at 0.01 > p > 0.001; ***, significant at 0.001 > p > 0.0001; ****, significant at p < 0.0001 compared to control sEH (at 0 µM inhibitor).

110.30 ± 0.36

3.514 ± 0.02

89.31 ± 4.55^***

2.928 ± 0.11^ns

90.06 ± 1.29^***

3.216 ± 0.05^ns

100

91.42 ± 1.54^***

4.648 ± 0.17^ns

150

103.60 ± 0.90^ns

7.375 ± 0.14^**

200

98.61 ± 0.66^*

11.037 ± 0.16^****

250

128.43 ± 9.92^***

24.830 ± 2.80^****

**Table 2.** *In silico* absorption, distribution, metabolism, and excretion (ADME) profile of the YMSV peptide and AUDA generated using SwissADME
	YMSV	AUDA
Abbreviations: Molecular Weight (MW) (g/mol); Number Of Rotatable Bonds (ROTB); Hydrogen Bond Donors (HBD); Hydrogen Bond Acceptors (HBA); Estimated Solubility (ESOL) (Delaney, 2004); Toxin Support Vector Machine (SVM) score (BIOPEP and ToxinPred) (Gupta et al., 2013); Topological Polar Surface Area (TPSA); Logarithm Of Compound Partition Coefficient Between N-Octanol And Water (ClogP); Bioavailability score, probability of F > 10% in rat (Martin, 2005); Lipinski Filter (Lipinski’s Rule-Of-5); Gastrointestinal Absorption (GIA); P-Glycoprotein Substrate, And Cytochrome P450 3A4 (CYP3A4) Inhibitor; Blood Brain Barrier (BBB) permeation.
Physicochemical properties	MW (g/mol)	498.59	392.58
ROTB (n)	17	15
HBA (n)	8	3
HBD (n)	7	3
ESOL (Log S)	0.23	- 4.98
Lipophilicity	TPSA (Å²)	216.38	78.43
CLogP (o/w)	−0.39	4.69
Drug-likeness and pharmacokinetics	Bioavailability score	0.17	0.56
Abbreviations: Molecular Weight (MW) (g/mol); Number Of Rotatable Bonds (ROTB); Hydrogen Bond Donors (HBD); Hydrogen Bond Acceptors (HBA); Estimated Solubility (ESOL) (Delaney, 2004); Toxin Support Vector Machine (SVM) score (BIOPEP and ToxinPred) (Gupta et al., 2013); Topological Polar Surface Area (TPSA); Logarithm Of Compound Partition Coefficient Between N-Octanol And Water (ClogP); Bioavailability score, probability of F > 10% in rat (Martin, 2005); Lipinski Filter (Lipinski’s Rule-Of-5); Gastrointestinal Absorption (GIA); P-Glycoprotein Substrate, And Cytochrome P450 3A4 (CYP3A4) Inhibitor; Blood Brain Barrier (BBB) permeation.
	Lipinski filter	No	Yes
P-glycoprotein substrate	Yes	No
CYP 3A4 inhibitor	No	Yes
BBB permeation	No	No

Table 2. In silico absorption, distribution, metabolism, and excretion (ADME) profile of the YMSV peptide and AUDA generated using SwissADME

YMSV

AUDA

Abbreviations: Molecular Weight (MW) (g/mol); Number Of Rotatable Bonds (ROTB); Hydrogen Bond Donors (HBD); Hydrogen Bond Acceptors (HBA); Estimated Solubility (ESOL) (Delaney, 2004); Toxin Support Vector Machine (SVM) score (BIOPEP and ToxinPred) (Gupta et al., 2013); Topological Polar Surface Area (TPSA); Logarithm Of Compound Partition Coefficient Between N-Octanol And Water (ClogP); Bioavailability score, probability of F > 10% in rat (Martin, 2005); Lipinski Filter (Lipinski’s Rule-Of-5); Gastrointestinal Absorption (GIA); P-Glycoprotein Substrate, And Cytochrome P450 3A4 (CYP3A4) Inhibitor; Blood Brain Barrier (BBB) permeation.

Physicochemical properties

MW (g/mol)

498.59

392.58

ROTB (n)

HBA (n)

HBD (n)

ESOL (Log S)

0.23

- 4.98

Lipophilicity

TPSA (Å²)

216.38

78.43

CLogP (o/w)

−0.39

4.69

Drug-likeness and pharmacokinetics

Bioavailability score

0.17

0.56

GIA

Low

High

Lipinski filter

Yes

P-glycoprotein substrate

Yes

CYP 3A4 inhibitor

Yes

BBB permeation