Structural and mechanistic insights into 5-lipoxygenase inhibition by natural products


Leukotrienes (LT) are lipid mediators of the inflammatory response that are linked to asthma and atherosclerosis. LT biosynthesis is initiated by 5-lipoxygenase (5-LOX) with the assistance of the substrate-binding 5-LOX-activating protein at the nuclear membrane. Here, we contrast the structural and functional consequences of the binding of two natural product inhibitors of 5-LOX. The redox-type inhibitor nordihydroguaiaretic acid (NDGA) is lodged in the 5-LOX active site, now fully exposed by disordering of the helix that caps it in the apo-enzyme. In contrast, the allosteric inhibitor 3-acetyl-11-keto-beta-boswellic acid (AKBA) from frankincense wedges between the membrane-binding and catalytic domains of 5-LOX, some 30 Å from the catalytic iron. While enzyme inhibition by NDGA is robust, AKBA promotes a shift in the regiospecificity, evident in human embryonic kidney 293 cells and in primary immune cells expressing 5-LOX. Our results suggest a new approach to isoform-specific 5-LOX inhibitor development through exploitation of an allosteric site in 5-LOX.

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Fig. 1: NDGA induces disorder at the active site of Stable-5-LOX.
Fig. 2: AKBA is wedged between the two domains of Stable-5-LOX.
Fig. 3: The AKBA- and NDGA- binding sites in 5-LOX.
Fig. 4: AKBA modulates the enzymatic activity of purified human 5-LOX in a cell-free assay.
Fig. 5: AKBA modulates LM formation in 5-LOX-expressing HEK293 cells.
Fig. 6: Effects of AKBA on LM formation in human neutrophils and M1-like MDM activated with E. coli.

Data availability

Coordinates and structure factors (6N2W, 6NCF) are available at the Protein Bank,


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This work was funded in part by grants to M.E.N. (nos. NIH HL107887 and AHA 16GRNT31000010, and the NIH P50AT002776 seed grant, and the Louisiana Governor’s Biotechnology Initiative) and O.W. (Deutsche Forschungsgemeinschaft (DFG, the German Research Foundation), project no. 316213987, SFB 1278 PolyTarget (project nos. A04 and C02), CRC 1127 ChemBioSys (project no. A04) and Free State of Thuringia and the European Social Fund (2016 FGR 0045)). J.G. received a Carl Zeiss postdoctoral stipend. Preliminary X-ray data were collected at the Center for Advanced Microstructures and Devices (Baton Rouge). We thank the staff at the Center for Advanced Microstructures and Devices for screening and data collection of macromolecular crystals at the Protein Crystallography beamline. The work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (grant no. P30 GM124165). The Eiger 16M detector on 24-ID-E beam line is funded by a NIH-ORIP HEI grant (no. S10OD021527). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.

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M.E.N. and O.W. designed the study. N.C.G. designed and executed the protein expression, purification, biochemical evaluation of enzyme stability and crystallization, as well as crystal structure phasing, map interpretation and refinement. In addition, N.C.G. prepared the 5-LOX variants and executed the corresponding enzyme assays. D.B.N. collected and processed the diffraction data. E.E.S. contributed the initial LM production and immunofluorescence studies in HEK293 cells, and preliminary enzyme activity assays. J.G. performed the LM analysis in stably transfected HEK293 cells, neutrophils and M1-like MDM, provided the data evaluation and statistics and prepared the graphs. F.B. performed the immunofluorescence experiments and the analysis of SPM in HEK293 cells. U.G. analyzed the LM profile of isolated 5-LOX. M.E.N. and O.W. wrote the manuscript and all authors contributed to data interpretation and manuscript preparation.

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Correspondence to Oliver Werz or Marcia E. Newcomer.

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Gilbert, N.C., Gerstmeier, J., Schexnaydre, E.E. et al. Structural and mechanistic insights into 5-lipoxygenase inhibition by natural products. Nat Chem Biol (2020).

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