Transcript TECNICHE DI BINDING RECETTORIALE E ANALISI DEI RISULTATI

BASIC METHODOLOGY
EFFECT
Criteria for hormone- and neurotransmittermediated events
Receptor must possess structural and steric specificity for a
hormone/neurotransmitter and for their close analogs as well.
Receptors are saturable and limited (i.e. there is a finite number of
binding sites).
Hormone/neurotransmitter-receptor binding is cell specific in
accordance with target organ specificity.
Receptor must possess a high affinity for the
hormone/neurotransmitter at physiological concentrations.
Once a hormone/neurotransmitter bind to the receptor, some
recognizable early chemical event must occur.
• Affinity: The “tenacity” by which a drug binds to its receptor.
• Intrinsic activity: Relative maximal effect of a drug in a particular
tissue preparation when compared to the natural, endogenous ligand.
– Full agonist = 1 (*equal to the endogenous ligand)
– Antagonist = 0
– Partial agonist = 0~1 (*produces less than the maximal response,
but with maximal binding to receptors.)
– Inverse agonist = <0
• Intrinsic efficacy: a drugs ability to bind a receptor and elicit a
functional response
– A measure of the formation of a drug-receptor complex.
• Potency: ability of a drug to cause a measured functional change.
Receptors have two major properties:
Recognition and Transduction
RECOGNITION:
the receptor protein must exist in a conformational state that allows
for recognition and binding of a compound and must satisfy the following criteria:
•Saturability – receptors exists in finite numbers.
•Reversibility – binding must occur non-covalently due to weak intermolecular
forces
•Stereoselectivity – receptors should recognize only one of the naturally occurring
optical isomers (+ or -, d or l, or S or R).
•Agonist specificity – structurally related drugs should bind well, while physically
dissimilar compounds should bind poorly.
•Tissue specificity – binding should occur in tissues known to be sensitive to the
endogenous ligand. Binding should occur at physiologically relevant concentrations.
the failure of a drug to satisfy any of these
conditions indicates non-specific binding to
proteins or phospholipids in places like blood
or plasma membrane components.
Receptors have two major properties:
Recognition and Transduction
TRANSDUCTION:
the second property of a receptor is that the binding of an agonist must be
transduced into some kind of functional response
(biological or physiological).
Different receptor types are linked to effector systems either directly or
through simple or more-complex intermediate signal amplification
systems. Some examples are:
• Ligand-gated ion channels – nicotinic Ach receptors
• Single-transmembrane receptors –insulin or EGF receptors
• 7-transmembrane GPCRs – opioid and muscarinic receptors
• Soluble steroid hormones – estrogen receptor
Cocktail of ingredients
ligand
tissue/cells
[in homologous curve]
The goal of the binding experiments
Receptor theory and receptor binding.
must obey the Law of Mass Action and follow
basic laws of thermodynamics.
Primary assumption:
a single ligand is binding to a homogeneous population of
receptors
LAW OF MASS ACTION
kon/k1
[ligand  receptor]
[ligand] + [receptor]
koff/k2
• kon = # of binding events/time (Rate of association) =
[ligand]  [receptor] kon = M-1 min-1
• koff = # of dissociation events/time (Rate of dissociation) =
[ligand  receptor] koff = min-1
• Binding occurs when ligand and receptor collide with the
proper orientation and energy.
• Interaction is reversible.
• Rate of formation [L] + [R] or dissociation [LR] depends
solely on the number of receptors, the concentration of
ligand, and the rate constants kon and koff.
At equilibrium, the rate of formation equals that of
dissociation so that:
[L]  [R] kon = [LR] koff
KD = k2/k1 = [L][R]
[LR]
*this ratio is the equilibrium dissociation constant or KD.
KD is expressed in molar units (M/L) and expresses the
affinity of a drug for a particular receptor.
KD is an inverse measure of receptor affinity.
KD = [L] which produces 50% receptor occupancy
Once bound, ligand and receptor remain
bound for a random time interval.
The probability of dissociation is the same at
any point after association.
Once dissociated, ligand and receptor should
be unchanged.
If either is physically modified, the law of
mass action does not apply (receptor
phosphorylation)
Ligands should be recyclable.
Studies of receptor number and
function
• We can directly measure the number
(or density) of receptors in the LR
complex.
• Ligand is radiolabeled (125I, 35S. or
3H).
• Saturation binding curve-occurs at
steady state conditions (equilibrium is
theoretical only).
Demonstrates the importance of saturability for any selective
ligand.
Provides information on receptor density and ligand affinity and
selectivity.
However, not every ligand is
radiolabeled…What to do?????
….a new cocktail of ingredients
tissue/cells
Radioligand
(single concentration)
competing agent
(multiple concentrations)
[in eterologous curve = in competition binding experiments]
Competition binding assays
 Allows one to determine a rough estimate of an
unlabeled ligand’s affinity for a receptor.
 Introduction into the incubation mixture of a nonradioactive drug (e.g. drug B) that also binds to R will
result in less of R being available for binding with D*,
thus reducing the amount of [D*R] that forms. This
second drug essentially competes with D* for
occupation of R. Increasing concentrations of B result
in decreasing amounts of [D * R] being formed.
Competition binding assays
1.
2.
3.
4.
5.
6.
7.
Radioactive ligand
Tissue preparation
Blank
Competing drugs
Other addition
Incubation
Separation of bound radioactivity
1. Radioactive ligand: how to choice it?
•
•
•
•
•
High potency, specificity and stability (S.A.)
What chemical structure?
Labeled drug: 3H, 35S, 32P, 14C 45Ca ….and 125I
Commercial and “home made” ligands
Breakdown of ligand
THE CHOICE FOR
A COMMERCIAL LIGANDS
• High specific activity = greather stability in
diluite solution containing EtOH or
scavengers
• Greather stability for frozen solution (not for
3H: self-radiolysis)
• Greather stability under nitrogen or dark
SOME NOTES
Specific radioactivity of the radioligand
is expressed as Ci/mmol or as Bq/mmol
[1 Ci = 37.027 MBq]
####
Specific radioactivity is stated in terms of cpm
####
1 Ci = 2.22x1012 dpm
radionuclide
half life
specific
activity
(Ci/mmol)
3H
12.43 y
28.8
3He
18
125I
59.6 d
2176
125Te
--
32P
14.3 d
9131
32S
1710
35S
87.4 d
1494
35Cl
167
14C
5730 y
0.062
14N
156
decays to:
 energy
(keV)
RADIOACTIVE DECAY
Nt = No x e –Kdecay t
t 1/2 = ln(2)/Kdecay = 0.693/ Kdecay
WHAT CHEMICAL STRUCTURE?
2. TISSUE PREPARATION
• Simple omogenate
• Washed resuspension preparation
• Subcellular fraction enriched in plasma
membranes (frozen)
• Cells
• Membranes from cells
3H-glutamic acid
bound [fmol]
PROTEIN DEPENDENCE OF 3HGLUTAMMIC ACID BINDING
75
50
R2 = 0.98
25
0
0
25
50
75 100 125 150
protein [g]
[personal data]
the concentration of tissue in the assay
should be such that
less than 10% of added radioactivity is bound
3. BLANK
membranes
+
hot ligand
Total bound
membranes
+
cold ligand
+
hot ligand
Non specific bound
the concentration of cold ligand
in blank tubes should usually be
about 100 times the concentration
required to inhibit specific bound
by 50%
4. COMPETING DRUGS
Drug screening program
(activity and potency)
Ki
IC50
COMPETITION CURVES
Hm2 [ 3H]-NMS (IC50)
carbacolo
100
%B/Bo
(+)-AA
75
(+)-BB
50
25
0
-8
-7
-6
-5
log dose [M]
-4
-3
[personal data]
COMPETITION CURVES
RAT BRAIN [3H]-NMS
NMS
OXO
%B/Bo
100
Antago 1
Antago 2
50
0
-10.0
-7.5
-5.0
LOG DOSE [M]
-2.5
[personal data]
5. OTHER ADDITIONS
• Preservation of ligands or competing drugs
or receptors
• Elimination of unwanted binding sites
the addition doesn’t affect the assay
6. INCUBATION
• Volume of incubation mixture (0.05-2 ml)
• Incubation temperature (4°-37°C)
• pH and composition of incubation buffer
TIME AND TEMPERATURE
DEPENDENCE OF SPECIFIC 3H-AVP
BINDING
[Maggi et. al, 1988]
EFFECTS OF CATION ON BINDING
OF (-)-3H-D888
[Reynolds et. al, 1986]
LAW OF MASS ACTION
7. HOW TO SEPARATE
BOUND FROM FREE
• Filtration
– Slow Koff, very low Bns
• Centrifugation
– Rapid Koff, low affinity ligands, increase Bns
• Equilibrium dialysis or gel filtration
– Degradation or sticking of receptor or ligand
– Too much time to obtaine the equilibrium
Filtration
BINDING STUDIES
tissue
cells
buffer
membranes
competing drug
radioligand drug
STEPS IN RECEPTOR BINDING
Binding of radioactive ligand (+ nonradioactive
ligand) to the receptor
Separation of bound from free
Measurement of the amount of radioactivity
bound
ANALYSIS OF THE RESULTS
MOST COMMONLY USED
EXPERIMENTAL PROTOCOLS
Saturation binding experiments (EQUILIBRIUM)
increasing concentrations of hot ligand
fixed concentrations of cold ligand
Competitive binding experiments (EQUILIBRIUM)
fixed concentrations of hot ligand
increasing concentrations of displacing ligand
Kinetic experiments (VARING TIME)
fixed concentrations of hot ligand
fixed concentrations of hot ligand
EXPERIMENTAL PROTOCOLS