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

Abstract

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, www.rcsb.org.

References

  1. 1.

    Haeggstrom, J. Z. & Funk, C. D. Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease. Chem. Rev. 111, 5866–5898 (2011).

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Radmark, O., Werz, O., Steinhilber, D. & Samuelsson, B. 5-Lipoxygenase, a key enzyme for leukotriene biosynthesis in health and disease. Biochim Biophys. Acta 1851, 331–339 (2015).

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Serhan, C. N. Pro-resolving lipid mediators are leads for resolution physiology. Nature 510, 92–101 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Shimizu, T. et al. Characterization of leukotriene A4 synthase from murine mast cells: evidence for its identity to arachidonate 5-lipoxygenase. Proc. Natl Acad. Sci. USA 83, 4175–4179 (1986).

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Dixon, R. A. F. et al. Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis. Nature 343, 282–284 (1990).

    CAS  PubMed  Article  Google Scholar 

  6. 6.

    Ferguson, A. D. et al. Crystal structure of inhibitor-bound human 5-lipoxygenase-activating protein. Science 317, 510–512 (2007).

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Vickers, P. J., Deluca, C., Wong, E. & Abramovitz, M. The effect of 5-lipoxygenase-activating protein (FLAP) on substrate utilization by 5-lipoxygenase. Adv. Exp. Med Biol. 400A, 145–151 (1997).

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    Abramovitz, M. et al. 5-lipoxygenase-activating protein stimulates the utilization of arachidonic acid by 5-lipoxygenase. Eur. J. Biochem 215, 105–111 (1993).

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Evans, J. F., Ferguson, A. D., Mosley, R. T. & Hutchinson, J. H. What’s all the FLAP about?: 5-lipoxygenase-activating protein inhibitors for inflammatory diseases. Trends Pharm. Sci. 29, 72–78 (2008).

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Werz, O., Gerstmeier, J. & Garscha, U. Novel leukotriene biosynthesis inhibitors (2012-2016) as anti-inflammatory agents. Expert Opin. therapeutic Pat. 27, 607–620 (2017).

    CAS  Article  Google Scholar 

  11. 11.

    Pettersen, D., Davidsson, O. & Whatling, C. Recent advances for FLAP inhibitors. Bioorg. Med Chem. Lett. 25, 2607–2612 (2015).

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Funk, C. D., Chen, X. S., Johnson, E. N. & Zhao, L. Lipoxygenase genes and their targeted disruption. Prostaglandins Other Lipid Mediat. 68-69, 303–312 (2002).

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Schneider, C., Pratt, D. A., Porter, N. A. & Brash, A. R. Control of oxygenation in lipoxygenase and cyclooxygenase catalysis. Chem. Biol. 14, 473–488 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Brash, A. R. Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate. J. Biol. Chem. 274, 23679–23682 (1999).

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Neau, D. B. et al. Crystal structure of a lipoxygenase in complex with substrate: the arachidonic acid-binding site of 8R-lipoxygenase. J. Biol. Chem. 289, 31905–31913 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Newcomer, M. E. & Brash, A. R. The structural basis for specificity in lipoxygenase catalysis. Protein Sci. 24, 298–309 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Gilbert, N. C. et al. The structure of human 5-lipoxygenase. Science 331, 217–219 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Bokoch, G. M. & Reed, P. W. Evidence for inhibition of leukotriene A4 synthesis by 5,8,11,14-eicosatetraynoic acid in guinea pig polymorphonuclear leukocytes. J. Biol. Chem. 256, 4156–4159 (1981).

    CAS  PubMed  Google Scholar 

  19. 19.

    Safayhi, H., Sailer, E. R. & Ammon, H. P. Mechanism of 5-lipoxygenase inhibition by acetyl-11-keto-beta-boswellic acid. Mol. Pharm. 47, 1212–1216 (1995).

    CAS  Google Scholar 

  20. 20.

    Sailer, E. R., Schweizer, S., Boden, S. E., Ammon, H. P. & Safayhi, H. Characterization of an acetyl-11-keto-beta-boswellic acid and arachidonate-binding regulatory site of 5-lipoxygenase using photoaffinity labeling. Eur. J. Biochem 256, 364–368 (1998).

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Poeckel, D. & Werz, O. Boswellic acids: biological actions and molecular targets. Curr. Med. Chem. 13, 3359–3369 (2006).

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Abdel-Tawab, M., Werz, O. & Schubert-Zsilavecz, M. Boswellia serrata: an overall assessment of in vitro, preclinical, pharmacokinetic and clinical data. Clin. Pharmacokinet. 50, 349–369 (2011).

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Sturner, K. H. et al. A standardised frankincense extract reduces disease activity in relapsing-remitting multiple sclerosis (the SABA phase IIa trial). J. Neurol. Neurosurg. Psychiatry 89, 330–338 (2017).

  24. 24.

    Werz, O. & Steinhilber, D. Development of 5-lipoxygenase inhibitors–lessons from cellular enzyme regulation. Biochem Pharm. 70, 327–333 (2005).

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Kemal, C., Louis-Flamberg, P., Krupinski-Olsen, R. & Shorter, A. L. Reductive inactivation of soybean lipoxygenase 1 by catechols: a possible mechanism for regulation of lipoxygenase activity. Biochemistry 26, 7064–7072 (1987).

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Mitra, S., Bartlett, S. G. & Newcomer, M. E. Identification of the substrate access portal of 5-lipoxygenase. Biochemistry 54, 6333–6342 (2015).

  27. 27.

    Schexnaydre, E. E. et al. A 5-lipoxygenase-specific sequence motif impedes enzyme activity and confers dependence on a partner protein. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1864, 543–551 (2018).

  28. 28.

    Ericsson, U. B., Hallberg, B. M., Detitta, G. T., Dekker, N. & Nordlund, P. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal. Biochem 357, 289–298 (2006).

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Eek, P. et al. Structure of a calcium-dependent 11R-lipoxygenase suggests a mechanism for Ca2+ regulation. J. Biol. Chem. 287, 22377–22386 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Rakonjac Ryge, M. et al. A mutation interfering with 5-lipoxygenase domain interaction leads to increased enzyme activity. Arch. Biochem Biophys. 545, 179–185 (2014).

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Werz, O. Inhibition of 5-lipoxygenase product synthesis by natural compounds of plant origin. Planta Med. 73, 1331–1357 (2007).

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Gerstmeier, J., Weinigel, C., Barz, D., Werz, O. & Garscha, U. An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis. Biochim Biophys. Acta 1840, 2961–2969 (2014).

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Werner, M. et al. Targeting biosynthetic networks of the proinflammatory and proresolving lipid metabolome. FASEB J. 33, 6140–6153 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Siemoneit, U. et al. On the interference of boswellic acids with 5-lipoxygenase: mechanistic studies in vitro and pharmacological relevance. Eur. J. Pharm. 606, 246–254 (2009).

    CAS  Article  Google Scholar 

  35. 35.

    Surette, M. E., Palmantier, R., Gosselin, J. & Borgeat, P. Lipopolysaccharides prime whole human blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen. J. Exp. Med 178, 1347–1355 (1993).

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Werz, O. et al. Human macrophages differentially produce specific resolvin or leukotriene signals that depend on bacterial pathogenicity. Nat. Commun. 9, 59 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  37. 37.

    Deng, B. et al. Maresin biosynthesis and identification of maresin 2, a new anti-inflammatory and pro-resolving mediator from human macrophages. PLoS ONE 9, e102362 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  38. 38.

    Carion, T. W. et al. Immunoregulatory role of 15-lipoxygenase in the pathogenesis of bacterial keratitis. FASEB J. 32, 5026–5038 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39.

    Sailer, E. R. et al. Acetyl-11-keto-beta-boswellic acid (AKBA): structure requirements for binding and 5-lipoxygenase inhibitory activity. Br. J. Pharm. 117, 615–618 (1996).

    CAS  Article  Google Scholar 

  40. 40.

    Gillmor, S. A., Villasenor, A., Fletterick, R., Sigal, E. & Browner, M. F. The structure of mammalian 15-lipoxygenase reveals similarity to the lipases and the determinants of substrate specificity. Nat. Struct. Biol. 4, 1003–1009 (1997); erratum 5, 242 (1998).

  41. 41.

    Choi, J., Chon, J. K., Kim, S. & Shin, W. Conformational flexibility in mammalian 15S-lipoxygenase: Reinterpretation of the crystallographic data. Proteins 70, 1023–1032 (2008).

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Kobe, M. J., Neau, D. B., Mitchell, C. E., Bartlett, S. G. & Newcomer, M. E. The structure of human 15-lipoxygenase-2 with a substrate mimic. J. Biol. Chem. 289, 8562–8569 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Mandal, A. K. et al. The membrane organization of leukotriene synthesis. Proc. Natl Acad. Sci. USA 101, 6587–6592 (2004).

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Mandal, A. K. et al. The nuclear membrane organization of leukotriene synthesis. Proc. Natl Acad. Sci. USA 105, 20434–20439 (2008).

    CAS  PubMed  Article  Google Scholar 

  45. 45.

    Gerstmeier, J. et al. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation. FASEB J. 30, 1892–1900 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Neau, D. B. et al. The 1.85 A structure of an 8R-lipoxygenase suggests a general model for lipoxygenase product specificity. Biochemistry 48, 7906–7915 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Murphy, R. C. & Gijon, M. A. Biosynthesis and metabolism of leukotrienes. Biochem J. 405, 379–395 (2007).

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Flamand, N., Luo, M., Peters-Golden, M. & Brock, T. G. Phosphorylation of serine 271 on 5-lipoxygenase and its role in nuclear export. J. Biol. Chem. 284, 306–313 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49.

    Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778–2786 (2011).

    CAS  PubMed  Article  Google Scholar 

  50. 50.

    Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Schweizer, S., Eichele, K., Ammon, H. P. & Safayhi, H. 3-Acetoxy group of genuine AKBA (acetyl-11-keto-beta-boswellic acid) is alpha-configurated. Planta Med. 66, 781–782 (2000).

    CAS  PubMed  Article  Google Scholar 

  56. 56.

    Zwart, P. H. et al. Automated structure solution with the PHENIX suite. Methods Mol. Biol. 426, 419–435 (2008).

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Dauter, Z., Li, M. & Wlodawer, A. Practical experience with the use of halides for phasing macromolecular structures: a powerful tool for structural genomics. Acta Crystallogr. D Biol. Crystallogr. 57, 239–249 (2001).

    CAS  PubMed  Article  Google Scholar 

  58. 58.

    Parsons, S. Introduction to twinning. Acta Crystallogr. D Biol. Crystallogr. 59, 1995–2003 (2003).

    PubMed  Article  Google Scholar 

  59. 59.

    Wang, C. K., Weeratunga, S. K., Pacheco, C. M. & Hofmann, A. DMAN: a Java tool for analysis of multi-well differential scanning fluorimetry experiments. Bioinformatics 28, 439–440 (2012).

    PubMed  Article  CAS  Google Scholar 

  60. 60.

    Fischer, L., Szellas, D., Radmark, O., Steinhilber, D. & Werz, O. Phosphorylation- and stimulus-dependent inhibition of cellular 5-lipoxygenase activity by nonredox-type inhibitors. FASEB J. 17, 949–951 (2003).

    CAS  PubMed  Google Scholar 

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Acknowledgements

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 16, 783–790 (2020). https://doi.org/10.1038/s41589-020-0544-7

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