Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages.
Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 µM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-α, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88- and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-β-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-β knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 µM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-α and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 µM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor κB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Perla Villani
PV
Borges
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Katelim Hottz
KH
Moret
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Clarissa Menezes
CM
Maya-Monteiro
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Franklin
F
Souza-Silva
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Carlos Roberto
CR
Alves
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Paulo Ricardo
PR
Batista
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Ernesto Raúl
ER
Caffarena
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Patrícia
P
Pacheco
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Maria das Graças
Md
Henriques
Laboratory of Applied Pharmacology, Institute of Drug Technology (P.V.B., K.H.M., P.P., M.d.G.H., C.P.), Computational Science Program, Computational Biophysics and Molecular Modeling Group (P.R.B.; E.R.C.), and Center for Technological Development in Health (M.G.H., C.P.), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; and Laborator of Immunopharmacology (C.M.M.-M.) and Molecular Biology and Endemic Diseases (F.S.S., C.R.A.), Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil cpenido@cdts.fiocruz.br.
Carmen
C
Penido
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D015232:Dinoprostone / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D036701:Limonins / Q000378:metabolism / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000737:chemistry / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D017472:Receptors, Interleukin-1 / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects*; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors* / Q000502:physiology
D016207:Cytokines; D018841:HSP90 Heat-Shock Proteins; D036701:Limonins; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D017472:Receptors, Interleukin-1; C499045:TIRAP protein, mouse; D051197:Toll-Like Receptor 4; C106014:gedunin; D015232:Dinoprostone