Probing the orientation of inhibitor and epoxy-eicosatrienoic acid binding in the active site of soluble epoxide hydrolase

Abstract

Soluble epoxide hydrolase (sEH) is an important therapeutic target of many diseases, such as chronic obstructive pulmonary disease (COPD) and diabetic neuropathic pain. It acts by hydrolyzing and thus regulating specific bioactive long chain polyunsaturated fatty acid epoxides (lcPUFA), like epoxyeicosatrienoic acids (EETs). To better predict which epoxides could be hydrolyzed by sEH, one needs to dissect the important factors and structural requirements that govern the binding of the substrates to sEH. This knowledge allows further exploration of the physiological role played by sEH. Unfortunately, a crystal structure of sEH with a substrate bound has not yet been reported. In this report, new photoaffinity mimics of a sEH inhibitor and EET regioisomers were prepared and used in combination with peptide sequencing and computational modeling, to identify the binding orientation of different regioisomers and enantiomers of EETs into the catalytic cavity of sEH. Results indicate that the stereochemistry of the epoxide plays a crucial role in dictating the binding orientation of the substrate.

Keywords: Computational simulation; Epoxyeicosatrienoic acid; Peptide sequencing; Photoaffinity tag; Photolabel; Soluble epoxide hydrolase.

Figure 1

A) The general scaffold of one of the leading series of sEH inhibitors; B) Structure of UC2389; C) The photoaffinity analog of UC2389 (7); D) The 14,15-EET-PAL (16) and; E) The 11,12-EET-PAL (17)

Figure 2

A) The optimum ratio of analog 7 to sEH is 10 to 1 based on intact mass spectrometric analysis; B) Majority of human sEH was labeled after 5 min of irradiation at 365 nm; C) Mass spectrometric analysis of intact sEH photolabeled by analog 7 and; D) MASCOT analyses (minimum ion score ≥18) identified residues Val and/or Cys within the peptide 422-440 (shown as yellow; ion score 70) was labeled by analog 7. The crystal structure (PDB code: 4OCZ) shown that the identified peptide was located on the right side of the binding pocket. The inhibitor UC2389 is shown as blue stick to indicate the location of the C-terminus active site of human sEH.

Figure 3

A) The rate of hydrolysis of 14,15-EET-PAL 16 and 14,15-EET by human sEH is similar; 3B) LC/MS analysis of the human sEH photolabeled by 11,12-EET-PAL 17; 3C) The identified peptides at C-terminus domain photolabeled by EET-PAL 16 and 17 based on MASCOT analyses of human sEH are highlighted in yellow with the residues labeled by 14,15-EET-PAL 17 shown as stick; 3D) The representative LC/MS-MS spectrum of one of the identified peptides (AVASLNTPFIPANPNMSPLESIK) photolabeled by 11,12-EET-PAL (587.25 Da) with the identified fragmented peak indicated and labeled. The identified fragmented peaks indicate that the 11,12-EET-PAL labels the residues between F, I and/or P. In addition, the corresponding precursor ion [M+H]²⁺ spectrum was also shown. The exact mass of the precursor ion (m/z = 1499.76) matches the predicted adduct mass ion (m/z = 1499.13).

Figure 4

A) The overlay between crystal structure (PDB code: 4OCZ) of human sEH with UC2389 (shown as cyan) and the model of human sEH bound with UC2389 (purple) with three catalytic residues: Y466, Y383 and D335 shown and labeled; B) The models of human sEH bound with 8R, 9S-EET (green), 11R, 12S-EET (yellow) and 14R,15S-EET (purple) with three catalytic residues: Y466, Y383 and D335 shown and labeled. The epoxides with same stereochemistry binds to human sEH in the same orientation; C) The models of human sEH bound with 8S, 9R-EET (green), 11S, 12R-EET (yellow) and 14S,15R-EET (purple) with three catalytic residues: Y466, Y383 and D335 shown and labeled. The epoxides with same stereochemistry binds to human sEH in the same orientation and; D) MASCOT analyses identified the two identical peptides (shown as yellow in the structure) that are labeled by both EET-PAL 16 and 17. The crystal structure (PDB code: 4OCZ) indicated that these two photolabeled peptides (as shown as yellow in structure) are located at both side of the binding pocket at the C-terminal domain of human sEH. The inhibitor 2389 (shown as blue stick) indicated the location of the binding site in the crystal structure.