• Release of Signal
• Binding of Ligand toReceptor
• Signal Transductionand Amplification
• Cellular Response
• Response of Organ and Organism
Communication in the Organism
Principles in intercellular communication:Release of Signals
GAP-Junction Receptor mediated
Secreted molecules Membrane boundligand
Hormone
granulocytes/endothel. cells via selectineT-cell receptor/MHC complex
autocrineparacrineendocrineEicosanoideNeurotrans-mitter, NO
Interleukine
A B
3
Model ofGap-Junction:
6 TM-Proteins/cellform Connexon
GAP-Junctions
4
Cell‐Cell Contact
5
Principles in intercellular communication
Endocrine pathway
Cell of Gland
Hormone receptor
Targetcell
HormoneBlood stream
paracrine
autocrine
Principles in intercellular communication
…synapse
Receptor Binding
7
Describes how and where the ligand interacts with the receptor!
8
Signal Transduction
Affinity and Activity
[c4,C7] Chem9 derivates
-12 -10 -8 -6
0
50
100
150
Chemerin-9
cp5 cp7
cp2log c [M], peptide
Res
pons
e [%
]concentration/response
versusdose/response
• Frequently no linear correlation: semilogarithmic scale
• Effect correlates with occupied receptors, but frequently more, because of second messenger: maximal effect achieved before all receptors are occupied.
Dose/Concentration-Response Curves
11
Emax
Emax2
EC50
Efficacy = Maximal effect, correlates with intrinisic activity
EC50 = concentration of half maximal activity
EC80, EC20 = concentration with 80% or 20 % max. effectPotency = pEC50 = negative Log of molar EC50-concentration
Intrinsic Activity = Efficacy
Definition
Conc. Ligand
Effe
ct
12
Important Values
13
Full/Partial Agonist
• A und B full agonists with different affinity• A is a partial agonist• Different slope, different binding mechanism
Efficacy:C<A=B
EC50:C<A<B
Potency:C>A>B
14
Antagonist Effect
Competitive antagonist• Competes with the agonist • Shifts concetration-response curve of agonist• Has no influence on maximal activity of agonist• Effect can be reverted with high concentrations of agonist
Receptor Antagonist Complex
noagonistbinding
15
Antagonist Effect
Receptor Agonist Complex
noeffect
Non competitive antagonist•Different binding site of agonist and antagonist •Lowers maximal effect of agonist, EC50 is maintained•Curve gets less steep•Effect cannot be reverted with high concentrations of agonist
Antagonist
16
Inverse Agonists
17
Introduction in Receptor Theory
Induced Fit: Receptor conformation is changed by the bindingof a ligand (agonist), which leads tosignal transduction. Antagonists bind, but do not lead to a conformational change
Conformational Selection: There exists an equilibrium ofdifferent receptor conformations, agonistsshift the equilibrium to the active conformation,inverse agonists to the inactive,antagonists don‘t change the equilibrium.
18
R
R
Agonist:Stabilizes active conformation
EffectorInverse Agonist:Stabilizes inactiveconformation
Conformational Selection
Antagonist: stabilisation of equilibrium
Constitutively activereceptors
R*
R*
L
KAKA*
R*
19
Advanced Pharmacology
Methods to Study Receptors
Cell System
Ligand-Receptor InteractionAgonist-Antagonist-inverse Agonist
BindingSignal Transduction
Receptor Mutagenesis
Protein-Protein InteractionReceptor DimerisationInteracting MoleculesReceptor Trafficking
Cell Systems
• Tissue, which is rich of endogenously expressedreceptors, e. g. homogenized brain, rat liver, rabbit kidney….
• Cell lines that endogenously express the receptorImmortilized cells (tumor or induced immortality)
ATCC (SK-NM-C, MCF7, etc.)Primary cells
• Transfected cell lines (stabile or transcient)
Cell line Origin Properties
CHO hamster Stable, transcient
BHK hamster Stable, transcient
HEK 293 human Stable, transcient
COS monkey SV40 antigene,Only transcient
Expression vector(plasmid)
Origin of replication
Beta-Lactamase (resistance gene E. coli)
Eukaryotic transcription unit:
Receptor
Selection marker
mutant cell
Defect is complemented by plasmid
Cells die as theyMiss something
Minimal media
transfectionRezessiv marker
normal cell
detoxification by plasmid
Cells die in toxicmilieu
toxic media
transfectiondominant marker
Frequently used genes for selection marker
Lipofection
defect cell
endocytosis
Mix Lipid solution
slow degradation offoreign DNA
Assays to test receptors
Affinity: Binding assays: Kd-Value, BmaxAutoradiography: Receptor distribution
Activity: functional assaysAgonistsAntagonistsSuperagonistsinverse Agonists
Activity without ligand: constitutive activity
30
Receptor Binding Assay
Receptor Binding
Cell lineCell line
centrifugationorfiltration
32
Radioactive labelling of hormones
Why?• Tracer for binding studies and autoradiography• Biodistribution and pharmacokinetics (stability)
How?• Direct iodination of tyrosine• Bolton‐Hunter (like) reaction of lysine
33
Direct iodination of tyrosine
Autoradiography of brain slices with125I nociceptin
Classical procedure
separation
Chloramine T
34
Bolton‐Hunter (like) reaction of lysine
125I
Lys
ON
O
O
O3H3H
3H
3H 3HTritiation by propionic acid NHS
NHS N‐hydroxy succinimide, Lys lysine, SFB = Succinimidyl‐fluorobenzoate
18F
O
ON
O
O
[18F]‐SFB
[18F]‐Fluoride
18F
NH
O
Peptid
[18F]‐Fluorobenzoyl‐Peptide
(Cyclotron)
+ Peptid
Affinity: specific binding at receptor
Specific bindingBinding of compound to receptor,saturable
Non specific binding1. Binding of ligand to other binding sites (same receptor, other
enzyme, transporter, etc. ), saturable2. Binding to non-receptor components of the tissue, membrane,
uptake in cells, or vesicle, non-saturable3. Unbound ligand that could not be separated from the bound ligand,
non-saturable
RBC3 - 36
total binding: Variation of concentration of radioligand
non-specific binding: Variation of concentration of radioligand in the presence of unlabelled ligand (c > 100-1000-fold Kd)
Kd represents affinity
Bmax receptor number per cell
Saturation Experiment, Kd-Value
Kd
Bmax
[L] Occupation of receptor
0 0%
1 x Kd 50%
4 x Kd 80%
9 x Kd 90%
99 x Kd 99%
Bmax – all receptors are occupied with ligand
Kd – conentration of ligand required for 50 % occupied receptors
• Specific binding of ligand and receptor is saturable and goes along classic kinetics
L + R LRkon
koff
LR
LRRLLR
RL max
on
offd
kkK• equilibrium:
bindingspec.L
LR[LR]d
max
K
kon: constant of association velocitykoff: constant of dissociation velocity
• Kd : equilibirum dissociation constant for a specific ligand L
… direct test to test binding of a radioligand to the specific receptor, determination of Kd and Bmax
max
Saturation Experiment, Kd-Value
Receptor Binding: Melatonin
RBC3 - 39
Typical values Bmax 10-1000 fmol binding sites per milligram of protein, Kd between 10 pM and 100 nM.Determination of Bmax and Kd:Past: Scatchard plot, today:fit data to the equation using nonlinear regression.
This analysis is based on these assumptions:• Binding follows the law of mass action and has equilibrated.• There is only one population of receptors.• Only a small fraction of the radioligand binds so that the free
concentration is essentially identical to the concentration added.• There is no cooperativity. Binding of a ligand to one binding site does
not alter the affinity of another binding site. In other words,the Kd is constant during the experiment.
Receptor Binding
RBC3 - 40
max
Receptor Analysis of Scatchard
-1/Kd
41
Binding Assays
Radiolabelled ligand; Saturation curve3H oxytocine, 125I insuline
Competition Binding
Constant amount oftracerVarying concentrationof unlabeled competitor
RBC3 - 43
Competition Binding , KI- and IC50-Values
Relevant to determine dissociation constants of unlabelled ligands that compete with the radiotracer for the same binding site
• Constant amount of radiotracer L ([L]), usually lower than KD• Variation of concentration of unlabelled ligand/inhibitor
IC50 : Concentration of Inhibitor/Ligand, required to displace 50 % of specific binding of tracer
Log IC50
0,5B0+NS
B0+NS
NS[L][I]1
[L]
ID
maxI
KK
BB
• Specific binding in absence of the inhibitors B0:
• if [L] = IC50, then BI = 0,5B0
• After rearrangement Cheng-Prusoff-Gleichung
[L][L]
Dmax0
K
BB
[L][L]0,5
[L]IC1
[L]D
max
I
50D
maxIC50
K
B
KK
BB
DK
K[L]1
IC50I
Competition Binding , KI- and IC50-Values
Concentration of tracerand KD of tracer
Competition binding [3H]DHA and β2-adrenergic ligands for human β2-adrenoceptors expressed in the CHO cells
David A. Sykes et al. Mol Pharmacol 2014;85:608-617
DHA:[3H]-dihydroalprenololantagonist
46
Competition Binding Curve
Fixed concentration of tracer: 1 nM 3H cannabinoid
Cannabinoid receptor
47
Competition Binding Curve
More advanced….two binding sites and Hill slope
• Heterogeneous receptors. The receptors do not all bind the unlabeled drug with the same affinity. This can be due to the presence of different receptor subtypes, or due to hetero-geneity in receptor coupling to other molecules such as G proteins.
• Negative cooperativity. Binding sites are clustered (perhapsseveral binding sites per molecule) and binding of the unlabeled ligand to one site causes the remaining site(s) to bind the unlabeled ligand withlower affinity.
• Curve fitting problems.
48
Radiobinding versus Fluorescence
49
Novel attempts: Fluorescently labelled ligands+ no radioctivity‐ High unspecific binding‐ Autofluorescence of cells‐ Sensitivity‐ Fluorescence bleaching‐ Effect affinity due to size
Fluorescene polarisation
Radiobinding versus Fluorescence
+ Solubile receptors (TF)+ Extracellular domain (RTK)- Membrane bound receptors
(GPCR, ion channel)
Tamra(tetramethylrhodamin)
50
51
Tb-labeled SNAP-CB2R monitored by HTRFScheme of the homogenous HTRF-based binding technique.
Eva Martínez-Pinilla et al. J Pharmacol Exp Ther 2016;358:580-587
Binding ligandNon binding ligand