Jeudis de convergence, UMPC
Ligand receptor interactions involved in chemical
Jérôme Golebiowski
Institut de Chimie de Nice,
UMR CNRS - Université de Nice 6001

Institut de Chimie de Nice
• UMR CNRS – Université de Nice
– 37 people
• Radioecology
• Surfaces chemistry
• Bioactive molecules
• Flavour and fragrance chemistry
• Molecular modeling and recognition
– 5 people
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Molecular modelling and recognition
• Chemical reactivity and properties
– Enzymatic catalysis
– Chiral molecules
• Ligand-Receptor interaction
– Protein-protein docking
– Chemical senses
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Above all: Nice: the better place to find collaborations
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The sweet smell of success
• France : The champion of luxury and perfurmes
– One of the few activities that produce a trade surplus
• Academic research
– Nobel price in 2004, L. Buck and R. Axel
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Smell, a so powerfull sense
« La madeleine »
Et tout d’un coup le souvenir m’est apparu. Ce goût, c’était celui du petit morceau de madeleine que le dimanche matin à Combray […], ma tante Léonie m’offrait après l’avoir trempé dans son infusion de thé. […] quand d’un passé ancien rien ne subsiste, après la mort des autres, après la destruction des choses, seules […] l’odeur et la saveur restent encore longtemps, comme des âmes, à se rappeler, à attendre, à espérer, sur la ruine de tout le reste, à porter sans fléchir, sur leur gouttelette presque impalpable, l’édifice immense du souvenir.
Marcel Proust - A la recherche du temps perdu
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The 1 st step: OBP chemoreception
• Lipocalin family
– water-soluble
– binds hydrophobic molecules
– low specificity
• Adapted to odorants
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2 nd step: OR chemoreception
• 349 active OR in human !
• 7 Transmembrane (TM) proteins
• Class A GPCR
– No exp. structure available
– Few site-directed mutagenesis exp.
• Broad specificity
• A single odorant can activate several
ORs
• A single OR can be activated by several odorants
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Combinatorial code of odours
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A
X
Olfactory image
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B
X
Olfactory image
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A
B
X
Olfactory image
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Projected Olfactory image
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Our objective
• Short time goal
– Deciphering the function / mechanism of these molecular machines
• Long time goal
– Providing a rationale to the perception of smell on a molecular basis
– New definition of structure / odour relationship
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To resume
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Our tool : Molecular modelling
• Aims to provide an atomic-level view of molecular phenonema
• Based on ‘first principles’
– Quantum mechanics
– Newton’s Laws
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What do we need ?
• A 3D structure
– X-Ray or NMR data
– Built in silico from the sequence
What do we do ?
• Model the atoms interactions (Force field)
• Model the environment (solvent)
• Solve the equations of motion
Protocol
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What do we get ?
• Capture the physics of the system
• Conformational sampling
• Statistics (distance, energy)
Boltzmann
Stat.
Free Energy
Sampling high energy state ?
We need to introduce a bias in the simulation !
Probability
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Computational microscope folding
Human behaviour
Bond stretching
Wide movements fs ps ns
µ s ms s
The ergodic hypothesis : sampling 1 molecule for a long time to gain statistics for 10 23
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Connection with experiment : the free energy
• experiment : affinity constant, K
• calculation ∆ G
∆ G = -RT.
ln K
• For a transformation A = B we can show that
∆ G
A → B
= -RT.< exp (– ∆ H/ k T)>
A
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st
Odorant – Odorant Binding Protein interaction

Mammals OBP: exp. vs computation
• Available data
– The protein sequence
– The experimental affinity (Isothermal Titration Calorimetry)
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We have a predictive tool
• Validation of our approach
– Structure building
– Ligand docking
– Free energy calculation
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sampling the binding event

The protein door
• the ligands are totally occluded from the solvent
– The protein door is unknown
?
?
?
?
?
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Tyrosine 82 acts as a door in lipocalins
Exp.
Predicted in 2007 : J. Golebiowski et al. PROTEINS, 2007
Experimentally confirmed in 2009 : P. Nagnan-Le Meillour et al. BBA, 2009
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nd
Odorant – Olfactory Receptors interaction

Main difficulty : the lack of structure
P
R
• OR building protocol
– Homology with crystallized GPCRs hOR1G1 PKMLANIQIQSQAISYSGCLLQLYFFMLFVMLEAFLLAVMAYDCYVAICHPLHYIL-IMSP hOR1E1 PKLLQNMQNQDPSIPYADCLTQMYFFLLFGDLESFLLVAMAYDRYVAICFPLHYTA-IMSP hOR1D2 PKMLVNLQSHNKAISYAGCLTQLYFLVSLVALDNLILAVMAYDRYVAICCPLHYTT-AMSP hOR1A1 PKMLANHLLGSKSISFGGCLTQMYFMIALGNTDSYILAAMAYDRAVAISRPLHYTT-IMSP rOR-I7 PKMLAGFI-GGQLISFEACMTQLYFFLGLGCTECVLLAVMAYDRYVAICHPLHYPV-IVSS mOR42-3 PPMLFSFSTRFNRISLPRCLGQMFCIYFLIVFDCNILLVMALDRYVAICYPLRYPE-IVTG mOR192 PKMLSDMFVDKKVISFLGCAIQLCLFSQFVVTECFLLASMAYDRYVAICKPLLYTL-IMSQ mOR_EG PKMLVNLVTMNRGISFVGCLVQFFFFCTFVVTESFLLGVMAYDRFVAIRNPLLYTV-AMSQ mOR_S25 PIMLRGFLRKGTFIPVAGCVAQLCIVVAFGTSESFLLASMAYDRYVAICSPLLYST-QMSS
Rhodo TTTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMS-NFRFGEN
β 2 PFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQS-LLTK
β 1 PFGATLVVRGTWLWGSFLCELWTSLDVLCVTASIETLCVIAIDRYLAITSPFRYQS-LMTR
D2DR PWVVYLEVVGEWKFSRIHCDIFVTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSK
5H2A PVSMLTILYGRWPLPSKLCAVWIYLDVLFSTASIMHLCAISLDRYVAIQNPIHHS-RFNSR
A2AA PFSLANEVMGYWYFGKAWCEIYLALDVLFCTSSIVHLCAISLDRYWSITQAIEYNL-KRTP
ACM1 NLYTTYLLMGHWALGTLACDLWLALDYVASNASVMNLLLISFDRYFSVTRPLSYRA-KRTP
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protocol
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• Activation of the OR raises [Ca 2+ ] intracel.
• Identified with a fluorescent probe
Calcium imaging
• Provide a semi-quantitative measure of the affinity
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sampling the binding event

Randomly accelerated MD
• Applies an acceleration on the ligand
– increase the distance between ligand and receptor centers of mass
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• How OBP modulates the OR function ?
– Where occurs the interaction between an
OBP and an OR ?
– Does it influence the
OR recognition ability ?
Further steps
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A new definition of
Structure Odor Relationship

Adrostenol
(musk)
Musk ketone
(musk)
?
isopropyl-methylfuranone
(herbaceous) ethyl-dimethylfuranone
(spicy)
?
A new definition of S.O.R.
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Olfatcome involves organic chemists
• Synthesis of various odorants analogues
– Géosmine (natural product with one of the lowest detection threshold)
– Sandalwood odorants
– (Z)β -santalol derivatives
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Olfatcome involve perfumers
• Main difficulty : characterisation of the odours
– The human factor !
• Solution :
– perfumers
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Conclusion
• Sense of smell
– Highly interdisciplinary field of research
• Chemistry, physics, signal processing, biology, neurology, human sciences… and artists !
– The computational microscope helps in understanding the atomic-level properties of this chemical sense
• Application :
– bio-inspired sensors
– Control of odorants
» Contraception
» Diet
» Odour masking agents
» Pest control…. with pheromones
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See you in Nice !