Molecular Mechanism for agonist- promoted alpha2A

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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