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