2008 Receptor Theory

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Receptor Theory&

Toxicant-Receptor Interactions

Richard B. Mailman



Some examples of receptors

1

E2

R E1

ligand

β γ

β γ

α α

2Ion

R R

ligand

ligand

nucleus

R

R

3 ligand

E

R R

4

R R

ATP

ADP

P

ATP

ADP

PP

P



What is a receptor?

• To a neuroscientist

– A protein that binds a neurotransmitter/modulator• To a cell biologist or biochemist

– A protein that binds a small molecule

– A protein that binds another protein

– A nucleic acid that binds a protein• To a toxicologist

– A macromolecule that binds a toxicant

• Etc.



Definitions

• Affinity:

– the “tenacity” by which a ligand binds to its receptor• Intrinsic activity (= “efficacy”):

– the relative maximal response caused by a drug in a tissue preparation. A full

agonist causes a maximal effect equal to that of the endogenous ligand (or

sometimes another reference compound if the endogenous ligand is not

known); a partial agonist causes less than a maximal response.

– Intrinsic efficacy (outmoded): the property of how a ligand causes biological

responses via a single receptor (hence a property of a drug).

• Potency:

– how much of a ligand is needed to cause a measured change (usuallyfunctional).



Radioactivity Definitions

• Curie: 1 Ci = 2.22 x 1012 disintegrations/min (dpm)

• Becquerel: 1 Bq = 60 disintegration/min– The Bq has replaced Ci in the SI system

• Efficiency: the percentage of dpm that are actually captured (cpm)

– What affects this?

• Specific activity: how many moles of radioactive atom are on each

radioactive molecule

– Usually expressed in radioactivity units per unit of mole

– Why is this important to a toxicologist?



Radioactivity Principles

• Specific activity depends ONLY on half-life, and is totally

independent of mode or energy of decay.

• When decay occurs for all of the biologically important isotopes

(14C; 3H; 32P; 35S; 125I; etc.), the decay event changes the chemical

identity of the decaying atom, and in the process, destroys themolecule on which the atom resided.

– e.g., 3HHe

– Do NOT adjust the specific activity of your radiochemical based on decay – for

every decay, there is a loss of the parent molecule.



Drug-Receptor Interactions

Ligand + ReceptorLgand-Receptor

Complex

Response(s)



Bimolecular Interactions:

Foundation of Most Studies

Ligand + Receptor Ligand-Receptork on

k off

[Ligand] [Receptor] k [Ligand Receptor] kon off ⋅ ⋅ = ⋅ ⋅

Rearrange that equation to define the equilibrium dissociation constant KD.

[Ligand] [Receptor]

[Ligand Receptor]

k

kKoff

on

D

⋅

⋅= =

At equilibrium:

Ligand + ReceptorLigand-Receptor

Complex Response(s)



Saturation Equations

F+K

B*F

D

max= B

Fractional occupancy

[Ligand]

[Ligand] K D= +

D D K

B B

K F

B max1+−=

Michealis-Menten form

Scatchard form



Linear & Semilog

0 20 40 60 80 100

Free

Linear Plot

0

0.2

0.4

0.6

0.8

1

B o u

n d

-2 -1 0 1 2log [Free]

Semi-Log Plot

0

0.2

0.4

0.6

0.8

1

B o u n d




F+K

B*F

D

max

= B

Fractional occupancy [Ligand][Ligand] K D

=+

D D K B B

K F B max1 +−=


Scatchard form



Calculations from Basic Theory (I)

log [competing ligand] (M)

S p e c i f i c B i n d i n g

( % )

10-9 10-8 10-7 10-6 10-5 10-4 10-3

0

25

50

75

100

91%

9%

100-fold

Commit this to memory!!!!!



Saturation Radioreceptor Assays

unbound labeled drug +

unbound test drug

drug-receptorcomplex

radiolabeled

drug

receptor

preparation

FiltrationBeta

Counter

Tissue

Preparation



Characterizing Drug-Receptor Interactions:

Saturation curves

0 2 4 6 8 10 12 14 16 18

Radioligand Added (cpm x 1000)

A m o

u n

t B

o u n

d Specific Binding! (calculated)

Non-Specific

Total Binding

800

600

400

200

0




F+K

B*F

D

max= B

Fractional occupancy[Ligand]

[Ligand] K D

=+

D D K

B BK F

Bmax

1+−=


Scatchard form





Competition Radioreceptor Assays

unbound labeled drug +

unbound test drug

drug-receptorcomplex

radiolabeled

drug

receptor

preparation

test

drug

FiltrationBeta

Counter

Tissue

Preparation



Competition Curve

log [ligand] (nM)

0

10

20

30

40

50

60

70

80

90

100

0.10.01 1.0 10 100

T o

t a l

B i n d

i n g

( d p

m * 1 0

, e

. g . )

IC50

Top

Bottom

Specific

Binding

NSB



Calculations from Basic Theory (I)


S p e c i f i c B i n d i n g

( % )

10-9

10-8

10-7

10-6

10-5

10-4

10-3

0

25

50

75

100

90%

10%

81 Fold



Calculations from Basic Theory (II)


S p e c

i f i c B i n d i n g

( % )

10-9

10-8

10-7

10-6

10-5

10-4

10-3

0

25

50

75

100

91%

9%

100-fold

Commit this to memory!!!!!



Competition Curves

Log [ligand] (nM)

0

10

20

30

40

50

60

70

80

90

100

0.10.01 1.0 10 100 1000

S p

e c

i f i c B i

n d i n

g ( %

)

B

A



Concentration (nM)

0

10

20

30

40

50

60

70

80

90

100

0.10.01 1.0 10 100 1000

S p

e c i f i c

B i n

d i n

g ( %

)

A DCB



Schild Analysis: Functional effects & antagonists

Log Agonist Concentration (M)

0

0.2

0.4

0.6

0.8

1.0

-10-11 -9 -8 -7 -6

R e s p

o n

s e ( F

r a c t i o

n o

f m

a x i m

a l )

Control

(agonist with no

antagonist)

+ Increasingconcentrations

of antagonist B

Raw Data



More Advanced Concepts of Receptor Theory



Spare receptors and “full agonists”

αD1

E1

β γ

E2

α

RE1

β γ

cAMP stimulation

????

????

D1 D1



Full & Partial Agonists

Concentration (nM)

0

20

40

60

80

100

Full agonist

Partial agonist

( % s t i

m u

l a t i o n

r e l a t i

v e

t o d

o p

a m

i n e

)

c A M

P s y n

t h e s i s

1 10 100 1000 10000 100000



Normal Agonist F.S. Drug

D2R

G-protein α

βγ

No activation

A B

C D

Functional

Complex

#1

Functional

Complex

#2

Ligand #1

Typical Agonist

Ligand #2

Functionally Selective Agonist

α

βγ

Functional Selectivity



Therapeutic consequences:

Traditional Drug

•TherapeuticEffect

•SideEffect



Therapeutic consequences:

Functionally Selective Drug

•TherapeuticEffect

•SideEffect



Functional selectivity:

JPET, January 2007

tissue or organism. Besides the heuristically interesting natureof functional selectivity, there is a clear impact on drug discov-ery, because this mechanism raises the possibility of selectingor designing novel ligands that differentially activate only asubset of functions of a single receptor, thereby optimizingtherapeutic action. It also may be timely to revise classic con-



August 2007

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2008 Receptor Theory