Native ligands for α2A-AR are norepineprine and epinephrine, ... noradrenaline/adrenaline contribute to binding affinity and agonist's capability to activate ...
Molecular Mechanism for agonistpromoted alpha2A-adrenoceptor activation by norepinephrine and epinephrine
Tommi Nyrönen CSC - Scientific Computing Ltd.
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Molecular mechanism of agonist-promoted α2Aadrenoceptor activation by norepinephrine and epinephrine
“Ligand mutation” experiment H
H
O O H
H
O H
H
O H
N H
H
O
H O
H
N
H
O
H O
H
N
H
O
H
H
N H
O H
O
H
H
N H
N H
O H
O H
IDEA: examine a set of α2A-AR ligands that have different effects on α2AAR but differ little structurally and chemically
Collaboration between modeling, biological experiments and chemistry Modeling Interpret experiments and design models
Experiments Validate/Falsificate models
Synthetic Chemistry Ligands!
50µm
α2-ARs are found in both the central and peripheral nervous system, and located both pre- and post synaptically. α2A-AR is expressed e.g. in locus coerulus in the brain
Brain microvasculature
Blood-brain barrier
Extracellular side Cell membrane
G-Protein Brain vasculature pics are from Pardridge, Nature Drug Disc. 1 (2002) p.132 and p.133
Intracellular side
Intracellular loop (200 AA) Tommi Nyrönen 2002
Structure of the α2A adrenoreceptor (model) α2A -AR is a G-protein coupled receptor consisting of seven transmembrane (7TM) helices and loops connecting the helices α2A-AR is involved in hypertension, pain and depression Native ligands for α2A-AR are norepineprine and epinephrine, a.k.a. noradrenaline and adrenaline Long intracellular loop (IC) between TM5 and TM6 is important for α2A-AR’s interactions with G-proteins Key residue in catecholamine binding is an aspartate in TM3 X-ray structure of a first 7TM receptor rhodopsin appeared in PDB in Aug 2000 -> template for comparative protein modeling Two lower pictures display electrostatic surface of the α2A-AR model without the extracellular loops viewed from the extracellular surface R-epinephrine has been docked to α2A-AR binding site model. Interactions with R-epinephrine are mainly provided by amino acids from TM3, TM5, TM6 and TM7 Tommi Nyrönen 2001
α2A adrenoreceptor has different states Antagonists/agonist ligands can stabilize different conformational states The receptor can assume the G-protein activating conformation without the presence of an agonist Tommi Nyrönen 2001
Structural view of the machinery of the ligand-receptor complex Ligand point of view How chemical functional groups in natural ligands noradrenaline/adrenaline contribute to binding affinity and agonist’s capability to activate α2A-AR? Receptor point of view Which residues in the receptor binding site are interacting with an agonist ligand? meta-OH para-OH
OH
N-methyl:
OH N H
OH
Aromatic Ring
(Charged) Amine β−OH
Tommi Nyrönen 2001
Experiments A) model of α2A-AR based on rhodopsin structures (electron microscopy and bovine rhodopsin X-Ray str.) B) docking of small molecules using Autodock, GOLD, GRID, Superstar and Sybyl C) isolation of 9 enantiomerically pure phenethylamines and 3 aminoimidazolines D) receptor Cys to Ser, Ser to Cys mutants E) agonist (UK 14,304, high affinity state) and antagonist (RX821002, low affinity state) radioligand binding assays F) GTPγS functional assay
OH H2N
OH
OH H2N
OH
OH
OH
(R)-norepinephrine
OH H2N
OH
(R)-octopamine
OH Dopamine
(S)-octopamine
OH
H2N
OH
OH
H2N
OH
(R)-norpherephrine
N
UK 14,304
(R)-2-Amino-1-phenylethanol NH2
Cl
Cl N
N
N Br
OH
(S)-norphenephrine
N
N H
OH
OH
OH
N H
OH
(R)-epinephrine
H2N
H2N
N H
(S)-norepinephrine
OH H2N
OH
N H
N H
Cl
p-aminoclonidine
N H
N H
Clonidine
Cl
Results H O H
missing meta-OH group from the catechol ring
N H
(R)-Octopamine,
O H
OH
Assay results, compared to (R)-noradrenaline Antagonist c. -7 fold
Agonist c. -94 fold
H2N
OH OH
Activation (EC50)
Efficacy
-100 fold
48 %
H O
H
N H
O
(R)-Norphenephrine, missing para-OH group from the catechol ring
H
OH
Assay results, compared to (R)-noradrenaline
H2N
OH OH
Antagonist c.
Agonist c.
Activation (EC50)
Efficacy
-5 fold
-28 fold
-14 fold
29%
(R)-1-Amino-2-phenyl-ethanol, H
N H
O
missing both catecholic hydroxyls
H
OH
Assay results, compared to (R)-noradrenaline
H2N
OH OH
Antagonist c.
Agonist c.
Activation
Efficacy
-1.6 fold
-147 fold
inactive
0%
H O
H O
H
N H
Dopamine, missing β-OH group
OH
Assay results, compared to (R)-noradrenaline
H2N
OH OH
Antagonist repl. Agonist repl.
Activation
Efficacy
-2.9 fold
-90 fold
94%
-7 fold
(S)-noradrenaline compared to (R)-noradrenaline Antagonist repl. Agonist repl.
Activation
Efficacy
- 4.3 fold
-18 fold
89%
- 23 fold
=
[-5,5]-fold change
-
[5,10]-fold worse
--
[10,100]-fold worse
---
[100,1000] fold worse
---- not measurable
OH
Summary
H2N OH
H O
H
N H
OH
compared to (R)noradrenaline, noradrenaline, native ligand
= Antag.
= Agonist
= Activ.
100% Eff.
-
--
--
29%
-
--
---
48%
=
---
---- 0%
=
-
--
93%
=
--
--
89%
O H H O
H
N H
O H
H
N H
O H H O
H O
H
N H
OH H2N
OH OH
Structural model
meta-OH: Thr118 in TM3 para-OH: Cys201 in TM5
OH
N-methyl: Phe411, Phe412 in TM7
N H
OH
OH
Aromatic Ring: Tyr394, Phe391 in TM6 Val114 in TM3 Phe205 in TM5
(Charged) amine: Asp113 in TM3 β−OH: Asp113 in TM3 β−
Residues that have been experimentally shown to be in contact with agonist ligands include positions 5.41, 5.42, 5.43, 5.46 in TM5 (serines and cysteins). However, all these residues cannot directly be in contact with the ligand simulataneously.
Structural model
α2A-AR activation by phenethylamine agonist ligands may be coupled to a structural rearrangement which involves TM5
Conformation 1
Conformation 2 (model)
meta-OH: Thr118 in TM3 para-OH: Cys201 in TM5
OH
N-methyl: Phe411, Phe412 in TM7
N H
meta-OH: Thr118 in TM3, Ser204 in TM5 para-OH: Ser200, Cys201 in TM5
OH
OH
N-methyl: Phe411, Phe412 in TM7
OH
N H
Aromatic Ring: Tyr394, Phe391 in TM6 Val114 in TM3 Phe205 in TM5
OH
OH
(Charged) amine: Asp113 in TM3 β−OH: Asp113 in TM3 β−
(Charged) amine: Asp113 in TM3 β−OH: Asp113 in TM3 β−
Antagonist competition
Probes a different receptor conformation than
Agonist competition
Aromatic Ring: Tyr394, Phe391 in TM6 Val114 in TM3 Phe205 in TM5
Nyrönen, T., Pihlavisto, M., Peltonen, J. M., Hoffrén A.-M., Varis, M., Salminen, T., Wurster, S., Marjamäki, A., Kanerva, L., Katainen, E., Laaksonen, L., Savola, J.-M., Scheinin, M., Johnson, M. S. Molecular mechanism for agonist-promoted α2A-adrenoceptor activation by norepinephrine and epinephrine, Molecular Pharmacology, 59 (2001) 1343-1354
Thank you for your attention
Lab view from Finland
