Abstract 14: Small Molecule Inhibitors of the CD40-TRAF6 Interaction Reduce Atherosclerosis by Inducing Hypo-inflammatory Myeloid Cells
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Abstract
The co-stimulatory CD40-CD40L dyad is well known for its pro-inflammatory role in atherosclerosis. Inhibition of CD40(L) in hyperlipidemic mice drastically reduces atherosclerosis. However, long term inhibition of CD40(L) results in immunosuppression and/or thromboembolic events. Therefore more targeted inhibition of the CD40L-CD40 dyad is required. To elicit intracellular signalling upon activation, CD40 needs to recruit adaptor proteins: the tumour necrosis factor receptor-associated factors (TRAFs). Using mice with a mutation in the CD40-TRAF binding site, we previously showed that CD40-TRAF6, and not CD40-TRAF2/3/5, are pivotal in atherosclerosis.
To discover small drug-like molecules that inhibit the CD40-TRAF6 interaction an in silico approach of virtual ligand screening was used; this resulted in the identification of 40.000 compounds. Analysis of the top 800 compounds identified 7 compounds that dose-dependently reduced NFκB activation and IL1β and IL6 expression in RAW cells and CD40-stimulated bone marrow-derived macrophages, respectively. Surface plasmon resonance experiments confirmed direct binding of the compounds to the TRAF6 C-domain.
To analyze the effects of the top two compounds on atherosclerosis, Apoe-/- mice were daily treated with compounds 6877002 or 6860766 for 6 weeks. Compound treatment reduced atherosclerosis in the aortic arch by 47.1% (6877002) and 66.8% (6860766). The number of plaque monocytes/macrophages was decreased by 41.4% (6877002) and 53.0% (6860766), and granulocytes by 62.5% (6877002) and 41.5% (6860766). Intravital microscopy showed that the compounds reduced monocyte recruitment to the endothelium by 40.1% (6877002) and 51.2% (6860766), neutrophil adhesion was reduced by 40.2% (6877002) and 51.2% (6860766). In accordance, expression of CCL2-CCR2 and CCL5-CCR5 was remarkably reduced in compound treated macrophages. Moreover, compound treatment abolished CD40-induced expression of TNFα, IL1β, IL6, IL10, and IL12.
These results establish the importance and possibilities of long-term therapeutic inhibition of CD40-TRAF6 interactions in atherosclerosis and other inflammatory diseases and harbor the potential to overcome the current limitations of CD40(L) blocking therapies.
- © 2013 by American Heart Association, Inc.
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- Abstract 14: Small Molecule Inhibitors of the CD40-TRAF6 Interaction Reduce Atherosclerosis by Inducing Hypo-inflammatory Myeloid CellsTom Seijkens, Barbara Zarzycka, Oliver Soehnlein, Marten A. Hoeksema, Linda Beckers, Esther Smeets, Svenja U. Meiler, Marion J. Gijbels, Jochen Grommes, Roy Schrijver, Louis Boon, Tilman M. Hackeng, Gert Vriend, Sander B. Nabuurs, Norbert Gerdes, Menno P.J. de Winther, Christian Weber, Gerry A.F. Nicolaes and Esther LutgensArteriosclerosis, Thrombosis, and Vascular Biology. 2013;33:A14, originally published October 20, 2015
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- Abstract 14: Small Molecule Inhibitors of the CD40-TRAF6 Interaction Reduce Atherosclerosis by Inducing Hypo-inflammatory Myeloid CellsTom Seijkens, Barbara Zarzycka, Oliver Soehnlein, Marten A. Hoeksema, Linda Beckers, Esther Smeets, Svenja U. Meiler, Marion J. Gijbels, Jochen Grommes, Roy Schrijver, Louis Boon, Tilman M. Hackeng, Gert Vriend, Sander B. Nabuurs, Norbert Gerdes, Menno P.J. de Winther, Christian Weber, Gerry A.F. Nicolaes and Esther LutgensArteriosclerosis, Thrombosis, and Vascular Biology. 2013;33:A14, originally published October 20, 2015