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Vanessa Delfosse Groupe Récepteurs Nucléaires et Corégulateurs
Centre de Biochimie Structurale – Montpellier
Synergistic Activation of Human Xenobiotic Receptor by Binary Cocktails of Pharmaceutical and
Environmental Compounds
≈150,000 synthetic chemicals used in consumer products
Dissemination into the environment
Adverse effects on the endocrine system endocrine disrupting chemicals
(EDCs) interaction with nuclear receptors
Nuclear hormone receptors The main target of environmental pollutants
HRE Hormone
Response Element
Simplified view of hormone action through nuclear receptors A highly controlled process through fine spatio-temporal tuning of hormonal signal
Nuclear hormone receptors Biological function
HRE Hormone
Response Element
EDC Endocrine Disrupt ing
Chemical
X
Misregulation of the nuclear hormone receptor activity = cancers, infertility, diabetes, obesity, ...
Simplified view of hormone action through nuclear receptors A highly controlled process through fine spatio-temporal tuning of hormonal signal
Nuclear hormone receptors Biological function
Drugs
Food
Phthalates
Perfluorinated compounds
Bisphenols
Natural compounds
Pesticides
The cocktail effect Additive, antagonistic or synergistic ?
Obesity
Infertility
Malformations
Cancers
NUCLEAR RECEPTORS
Drugs
Food
Phthalates
Perfluorinated compounds
Bisphenols
Natural compounds
Pesticides
The cocktail effect Additive, antagonistic or synergistic ?
Individuals are chronically exposed to cocktail of exogenous substances that may have harmful effects
XRE
The cocktail effect Our target: PXR, the xenobiotic sensor
Xenobiotic and steroid metabolism
CYP3A4, 5, 7 CYP2B6 CYP2C8, 9, 10 UGT1A2 GSTA1 MRP2, 3 OATP
PXR = Pregnane X Receptor, or Steroid X Receptor Up-regulates major detoxification genes (redox reactions, conjugation, excretion)
Xenobiotics (drugs, pesticides,
chemicals, …)
XRE
The cocktail effect Our target: PXR, the xenobiotic sensor
Xenobiotic and steroid metabolism
CYP3A4, 5, 7 CYP2B6 CYP2C8, 9, 10 UGT1A2 GSTA1 MRP2, 3 OATP
Xenobiotics
PXR = Pregnane X Receptor, or Steroid X Receptor Up-regulates major detoxification genes (redox reactions, conjugation, excretion)
PXR has a large ligand binding pocket
- Drug-drug interactions
- Deregulation of natural hormones homeostasis
- Chemo-resistance (+++)
- Cancer aggressiveness (+++)
Hyper-activation of PXR
The cocktail effect Strategy, 1 = screening and in vitro studies
Medium-throughput screening on HG5LN-PXR reporter cell line
Gal4REx5 β globine LucF SV40 NeoR
Gal4
PXR
RXR
Gal4
PXR ±Transcription
HG5LN
xenobiotics
DNA binding
domain
Ligand binding domain
Luciferase reporter gene stably transfected
Delfosse et al., 2015
Nature Communications
The cocktail effect Strategy, 1 = screening and in vitro studies
Medium-throughput screening on HG5LN-PXR reporter cell line
First screen with 40 molecules (drugs, pesticides, industrial products, etc… )
= 780 binary mixtures
Gal4REx5 β globine LucF SV40 NeoR
Gal4
PXR
RXR
Gal4
PXR ±Transcription
HG5LN
xenobiotics
DNA binding
domain
Ligand binding domain
Luciferase reporter gene stably transfected
ethinylestradiol (EE2, contraceptive)
trans-nonachlor (TNC, pesticide)
Together, these compounds produced more than an additive effect
+
EE2
TNC EE2+TNC
Delfosse et al., 2015
Nature Communications
The cocktail effect Strategy, 1 = screening and in vitro studies
Synergistic activation of PXR by EE2 and TNC in different human cell lines
Hepatocellular carcinoma
Colon adenocarcinoma
Theoretical additivity curve
Delfosse et al., 2015, Nature Communications
The cocktail effect Strategy, 1 = screening and in vitro studies
Cocktail effect on CYP3A4 expression and activity
Primary human
hepatocytes
(PHHs)
Primary human
hepatocytes
(PHHs)
Liver samples from adult patients
Freshly isolated PHHs
Colon adenocarcinoma
Delfosse et al., 2015, Nature Communications
The cocktail effect Strategy, 2 = biophysical studies
Theoretical mass = 38 217.7 Da
PXR LBD
Mass spectrometry under native conditions
No ligand added
(M = 38 212.5 ± 0.7 Da)
*
*acetate adducts
Delfosse et al., 2015
Nature Communications
The cocktail effect Strategy, 2 = biophysical studies
Theoretical mass = 38 217.7 Da
PXR LBD
Mass spectrometry under native conditions
No ligand added
TNC
EE2
+
EE2
TNC
(M = 38 212.5 ± 0.7 Da)
*
*
*
* *
*
*acetate adducts
(296.4 g.mol-1)
(444.2 g.mol-1)
(740.6 g.mol-1)
Delfosse et al., 2015
Nature Communications
The cocktail effect Strategy, 2 = biophysical studies
Theoretical mass = 38 217.7 Da
PXR LBD
Mass spectrometry under native conditions
No ligand added
TNC
EE2
+
EE2
TNC
(M = 38 212.5 ± 0.7 Da)
*
*
*
* *
*
*acetate adducts
(296.4 g.mol-1)
(444.2 g.mol-1)
(740.6 g.mol-1)
Delfosse et al., 2015
Nature Communications
The cocktail effect Strategy, 2 = biophysical studies
EE2 affinity
x 60
TNC affinity
x 40
The cocktail effect Strategy, 2 = biophysical studies
EE2 affinity
x 60
TNC affinity
x 40
EE2 and TNC bind cooperatively to PXR
The binary cocktail has a nanomolar affinity
Delfosse et al., 2015, Nature Communications
The cocktail effect Strategy, 3 = structural studies
Fo-Fc omit maps (3σ)
EE2
TNC
PDB id = 4X1G res = 2.25 Å R / Rfree = 0.174 / 0.218
H9
H2’
H11
H12
H1
H4
H5
S4
S1
S3
H3
H8
S2
S1’
H3’
H6
H7
Delfosse et al., 2015, Nature Communications
The cocktail effect Strategy, 3 = structural studies
M246
M243
H6
H2’
H11
H12
H7
H5
H3
S4
S1
EE2
M323
H327
W299 Y306 F288
F281
C284
M425
L411
I414
L206
R410
E321
S247
TNC
S3
S247
The cocktail effect Strategy, 3 = structural studies
M246
M243
H6
H2’
H11
H12
H7
H5
H3
S4
S1
EE2
M323
H327
W299 Y306 F288
F281
C284
M425
L411
I414
L206
R410
E321
S247
TNC
S3
S247
TNC
+
EE2
Supramolecular ligand
The cocktail effect Strategy, 3 = structural studies
M246
M243
H6
H2’
H11
H12
H7
H5
H3
S4
S1
EE2
M323
H327
W299 Y306 F288
F281
C284
M425
L411
I414
L206
R410
E321
S247
TNC
S3
S247
Delfosse et al., 2015, Nature Communications
1st screening with 40 molecules
(receptor = PXR)
ethinylestradiol (EE2, contraceptive)
transnonachlor (TNC, pesticide)
Cooperative binding (ITC) (EE2 TNC+EE2)
Kd ÷ 60
EE2 TNC + EE2
Mutual stabilisation in the LBP
res = 2,25 Å
Kd = 10,8 µM Kd = 0,17 µM
Synergy observed in human cell lines
Transactivation Hepatocellular
carcinoma
HepG2
add
syn
TNC EE2
The cocktail effect Proof of concept
Conclusion
The dose makes the poison All substances are poisonous There is none which is not a poison The right dose differentiate a poison and a remedy
Paracelsus (1493-1541)
… The association makes the poison
Conclusion The association makes the poison
Conclusion
The dose makes the poison All substances are poisonous There is none which is not a poison The right dose differentiate a poison and a remedy
Paracelsus (1493-1541)
… The association makes the poison
Supramolecular ligand: molecular assembly consisting of two or more compounds that interact with each other inside the LBP of a receptor, resulting in the creation of a new entity with improved functional characteristics in regard to those of its individual components.
Conclusion The association makes the poison
Conclusion
The dose makes the poison All substances are poisonous There is none which is not a poison The right dose differentiate a poison and a remedy
Paracelsus (1493-1541)
… The association makes the poison
Huge chemical/size diversity of xenobiotics + high structural plasticity of LBPs, the mechanism defined for PXR is likely to occur also with other NRs.
Supramolecular ligand: molecular assembly consisting of two or more compounds that interact with each other inside the LBP of a receptor, resulting in the creation of a new entity with improved functional characteristics in regard to those of its individual components.
Conclusion The association makes the poison
Conclusion
The dose makes the poison All substances are poisonous There is none which is not a poison The right dose differentiate a poison and a remedy
Paracelsus (1493-1541)
… The association makes the poison
Huge chemical/size diversity of xenobiotics + high structural plasticity of LBPs, the mechanism defined for PXR is likely to occur also with other NRs.
Broad reaching implications for the fields of endocrine disruption, toxicology, and chemical risk assessment.
Supramolecular ligand: molecular assembly consisting of two or more compounds that interact with each other inside the LBP of a receptor, resulting in the creation of a new entity with improved functional characteristics in regard to those of its individual components.
Conclusion The association makes the poison
Assess the degree of synergy in vitro
Native MS
Fluo. aniso.
Elucidate the mechanism of action of cocktails at atomic level
RX
Identify synergistic cocktails
HT screening in luciferase reporter system
Platform PCC – IRCM
Environnemental compounds
(200)
Drugs (1280)
Natural products (320)
In situ and in vivo validations
Larval systems (xenopus and zebrafish) V. Laudet (OOB, Banyuls) B. Demeinex (MNHM, Paris)
Human cell lines, tissues, xenografts P. Balaguer (IRCM, Mtplr) J.-M. Pascussi (IGH, Mtplr)
The cocktail effect Integrative approach
CBS (Montpellier) Structural and biophysical analyses
Tiphaine Huet Jean-Luc Pons Gilles Labesse William Bourguet
2013 2015
Marina Grimaldi Abdelhay Boulahtouf
IRCM (Montpellier) Screening & functional studies
Patrick Balaguer
Béatrice Dendele
IRB (Montpellier) Hepatocytes
Sabine Gerbal
Martine Daujat NovAliX (Illkirch) Mass spectrometry & ITC
Dominique Roecklin Christina Muller Valérie Vivat
IGF (Montpellier) Functional studies
Bertrand Beucher Jean-Marc Pascussi
Theoretical additivity curve
The cocktail effect Variations on a theme
Delfosse et al., 2015, Nature Communications