TY - JOUR
T1 - Allosteric Regulation of the Soluble Epoxide Hydrolase by Nitro Fatty Acids
T2 - a Combined Experimental and Computational Approach: Allosteric regulation of hsEH by nitro fatty acids
AU - Qiu, Qiongju
AU - Abis, Giancarlo
AU - Mattingly-Peck, Florence
AU - Lynham, Steven
AU - Fraternali, Franca
AU - Conte, Maria R.
N1 - Funding Information:
We thank Ms Simona Popova and Dr Yin-Biao Sun from the Randall Centre for Cell and Molecular Biophysics for the help with the HPLC experiments and Miss Maria Nefeli Papadopolou for her assistance in running inhibition assays. GA and MRC thank Philip Eaton for helpful discussions. GA was a BHF funded interdisciplinary PhD student (RE/08/003). FMP holds a PhD studentship funded by a BHF programme FS/4yPhD/P/20/34021. QQ is funded by a King's-China Scholarship Council PhD studentship. We thank the BHF Centre of Excellence RE/18/2/34213 for funding support. FF thanks BBSRC for their support BB/T002212/1. MRC, GA and FF ideated the study. GA performed protein expression and purification of recombinant hsEH CTD, SDS-PAGE, ESI-MS and enzymatic assays. GA and SL performed and analysed LC-MS/MS experiments. QQ performed the AlloSigMA analysis assisted by FMP. FMP performed Ohm analysis. MRC and GA performed analysis and data interpretation of experimental data. QQ, FMP, MRC and FF performed analysis and data interpretation of in silico data. All the authors contributed to the discussion and wrote the manuscript. MRC and FF obtained funding for this work. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funding Information:
GA was a BHF funded interdisciplinary PhD student (RE/08/003). FMP holds a PhD studentship funded by a BHF programme FS/4yPhD/P/20/34021. QQ is funded by a King’s-China Scholarship Council PhD studentship. We thank the BHF Centre of Excellence RE/18/2/34213 for funding support. FF thanks BBSRC for their support BB/T002212/1.
Publisher Copyright:
© 2022 The Authors
PY - 2022/9/15
Y1 - 2022/9/15
N2 - The human soluble epoxide hydrolase (hsEH) is a key regulator of epoxy fatty acid (EpFA) metabolism. Inhibition of sEH can maintain endogenous levels of beneficial EpFAs and reduce the levels of their corresponding diol products, thus ameliorating a variety of pathological conditions including cardiovascular, central nervous system and metabolic diseases. The quest for orthosteric drugs that bind directly to the catalytic crevice of hsEH has been prolonged and sustained over the past decades, but the disappointing outcome of clinical trials to date warrants alternative pharmacological approaches. Previously, we have shown that hsEH can be allosterically inhibited by the endogenous electrophilic lipid 15-deoxy-Δ12,14-Prostaglandin-J2, via covalent adduction to two cysteines, C423 and C522. In this study, we explore the properties and behaviour of three electrophilic lipids belonging to the class of the nitro fatty acids, namely 9- and 10-nitrooleate and 10-nitrolinoleate. Biochemical and biophysical investigations revealed that, in addition to C423 and C522, nitro fatty acids can covalently bind to additional nucleophilic residues in hsEH C-terminal domain (CTD), two of which predicted in this study to be latent allosteric sites. Systematic mapping of the protein mutational space and evaluation of possible propagation pathways delineated selected residues, both in the allosteric patches and in other regions of the enzyme, envisaged to play a role in allosteric signalling. The responses elicited by the ligands on the covalent adduction sites supports future fragment-based design studies of new allosteric effectors for hsEH with increased efficacy and selectivity.
AB - The human soluble epoxide hydrolase (hsEH) is a key regulator of epoxy fatty acid (EpFA) metabolism. Inhibition of sEH can maintain endogenous levels of beneficial EpFAs and reduce the levels of their corresponding diol products, thus ameliorating a variety of pathological conditions including cardiovascular, central nervous system and metabolic diseases. The quest for orthosteric drugs that bind directly to the catalytic crevice of hsEH has been prolonged and sustained over the past decades, but the disappointing outcome of clinical trials to date warrants alternative pharmacological approaches. Previously, we have shown that hsEH can be allosterically inhibited by the endogenous electrophilic lipid 15-deoxy-Δ12,14-Prostaglandin-J2, via covalent adduction to two cysteines, C423 and C522. In this study, we explore the properties and behaviour of three electrophilic lipids belonging to the class of the nitro fatty acids, namely 9- and 10-nitrooleate and 10-nitrolinoleate. Biochemical and biophysical investigations revealed that, in addition to C423 and C522, nitro fatty acids can covalently bind to additional nucleophilic residues in hsEH C-terminal domain (CTD), two of which predicted in this study to be latent allosteric sites. Systematic mapping of the protein mutational space and evaluation of possible propagation pathways delineated selected residues, both in the allosteric patches and in other regions of the enzyme, envisaged to play a role in allosteric signalling. The responses elicited by the ligands on the covalent adduction sites supports future fragment-based design studies of new allosteric effectors for hsEH with increased efficacy and selectivity.
KW - allostery
KW - covalent inhibitors
KW - docking
KW - epoxy fatty acids
KW - statistical mechanical models
UR - http://www.scopus.com/inward/record.url?scp=85129487953&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2022.167600
DO - 10.1016/j.jmb.2022.167600
M3 - Article
C2 - 35460669
AN - SCOPUS:85129487953
SN - 0022-2836
VL - 434
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 17
M1 - 167600
ER -