Receptor theory

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Receptor theory

• First postulated by John Langley (1878)
• Established after his experiments using nicotine
  and curare analogues on muscle contraction.
• Isolated muscle fibers pilocarpine (contraction)
  and atropine (inhibition).
• Two compounds competing for a third, but unknown
  substrate.
• Furthered by Paul Ehrlich (1854-1915)
• Demonstrated that stereoselectivity was
  imperative in drug-receptor signaling.

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John Langley

• In 1901, Langley challenged the dominant
  hypothesis that drugs act at nerve endings by
  demonstrating that nicotine acted at sympathetic
  ganglia even after the degeneration of the
  severed preganglionic nerve endings.
• That year, Langley also discovered for himself a
  tool in the form of renal extract (containing
  adrenaline) which produced sympathomimetic
  responses when applied to tissues exogenously.
• But it was not until 1905 that Langley published
  the results of the decisive experiments using
  systemic injections of curare and nicotine given
  to chicks. It was through these experiments that
  Langley concluded the existence of a receptive
  substance in striated muscle.

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Nerve/Muscle Endings
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John Langley

• Langley concluded that a protoplasmic "receptive
  substance" must exist which the two drugs compete
  for directly. He further added that the effect of
  combination of the receptive substance with
  competing drugs was determined by their
  comparative chemical affinities for the substance
  and relative dose.

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Intercellular Signaling
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Classes of cell-surface receptors
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Criteria for hormone-mediated events

• Receptor must possess structural and steric
  specificity for a hormone and for its close
  analogs as well.
• Receptors are saturable and limited (i.e. there
  is a finite number of binding sites).
• Hormone-receptor binding is cell specific in
  accordance with target organ specificity.
• Receptor must possess a high affinity for the
  hormone at physiological concentrations.
• Once a hormone binds to the receptor, some
  recognizable early chemical event must occur.

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• Affinity The tenacity by which a drug binds to
  its receptor.
• Discussion a very lipid soluble drug may have
  irreversible effects is this high-affinity or
  merely a non-specific effect?
• Intrinsic activity Relative maximal effect of a
  drug in a particular tissue preparation when
  compared to the natural, endogenous ligand.
• Full agonist IA 1 (equal to the endogenous
  ligand)
• Antagonist IA 0
• Partial agonist IA 01 (produces less than
  the maximal response, but with maximal binding to
  receptors.)
• 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.

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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 (H-bonding, van
  der Waal 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.

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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.
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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 RTKs like
  insulin or EGF receptors
• 7-transmembrane GPCRs opioid receptors
• Soluble steroid hormones estrogen receptor

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Predicting whether a drug will cause a response
in a particular tissue

• Factors involving the equilibrium of a drug at a
  receptor.
• Limited diffusion
• Metabolism
• Entrapment in proteins, fat, or blood.
• Response depends of what the receptor is
  connected to.
• Effector type
• Need for any allosteric co-factors THB on
  tyrosine hydroxylase.
• Direct receptor modification phosphorylation

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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

NH3
COO-
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  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.

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• At equilibrium, the rate of formation equals that
  of dissociation so that
• L ? R kon LR koff
• KD k2/k1 LR
• 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

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• 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.

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Receptor occupancy, activation of target cell
responses, kinetics of binding

• Activation of membrane receptors and target cell
  responses is proportional to the degree of
  receptor occupancy.
• However, the hormone concentration at which half
  of the receptors is occupied by a ligand (Kd) is
  often lower than the concentration required to
  elicit a half-maximal biological response (ED50)

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Receptor Fractional Occupancy

• F.O. LR____ LR___
  now substitute the KD equation.
• Total Receptor Rf LR
• R KD LR ? F.O. Ligand
• L Ligand KD
• Use the following numbers
• L KD 50 F.O.
• L 0.5 KD 30 F.O.
• L 10x KD 90 F.O.
• L 0 0 F.O.

100
Fractional Occupancy
50
0
Ligand Concentration
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Assumptions of the law of mass action.

• All receptors are equally accessible to ligand.
• No partial binding occurs receptors are either
  free of ligand or bound with ligand.
• Ligand is nor altered by binding
• Binding is reversible
• Different affinity states?????

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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).
  Selection of proper radioligand
• Agonist vs. antagonist (sodium insensitive)
• Higher affinity for antagonists
• Longer to steady state binding
• 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.

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Scatchard transformation

• Y-axis is Bound/Free (total radioligand-bound)
• X-axis Bound (pmol/mg protein)
• Straight lines are easier to interpret.

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• The amount of drug bound at any time is solely
  determined by
• the number of receptors
• the concentration of ligand added
• the affinity of the drug for its receptor.
• Binding of drug to receptor is essentially the
  same as drug to enzyme as defined by the
  Michelis-Menten equation.

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However, not every ligand is radiolabeledWhat to
do?????
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Competition binding assays

• Allows one to determine a rough estimate of an
  unlabeled ligands affinity for a receptor.
• Competitive or non-competitive.
• Introduction into the incubation mixture of a
  non-radioactive 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 DR 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.
• Method
• Single concentration of labeled ligand
• Multiple (log-scale) concentrations of the
  unlabeled/competing ligand.

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Competition binding assays

• The concentration of inhibitor which displaces
  50 of the radiolabeled ligand is known as the
  IC50 for that drug.
• IC50 cannot be viewed as the KD of the
  inhibitor because it is just an estimate.
• Ki the equilibrium inhibitor dissociation
  constant.
• It is the concentration of the competing ligand
  that would bind to 50 of sites in the absence of
  the radioligand.
• Ki can only be determined after the IC50 is
  known.
• Uses the equation of Cheng and Prusoff.

Ki IC50 1 radiolabeled
ligand Kd
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Example Find Ki of morphine in a preparation
with 3H-diprenorphine.   IC50 100 nM Ki 25
nM L 3 nM KD 1 nM
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Dose-response experiments.

• Measures the functional response of a drug, which
  is an indirect assessment of receptor binding.
• Can be in vitro, in vivo, or ex vivo.
• Is response directly proportional to receptor
  occupancy????
• Clarkes Theory the effect of a drug is
  proportional to the fraction of receptors
  occupied by the drug and maximal response occurs
  when all receptor are bound. Is this true????
• Actually, more is not necessarily better.

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Fractional response

• Equation for fraction response for Drug A
• Rf is the fractional response for any
  concentration of agonist.
• The dose producing the maximum effect (Emax) is
  termed the maximum effective dose, whereas the
  concentration of agonist producing the
  half-maximal response is termed the EC50.
• If the agonist concentration is expressed in log
  terms then the resultant dose-response curve is
  sigmoid shaped.
• A concentration of agonist 10 fold higher than
  its EC50 would produce a response that is 90 of
  Emax whereas a concentration of agonist 100 fold
  higher than its EC50 would produce a response 99
  of Emax.

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However, not all agonists acting at the same
receptor produce the same maximal response.
Dose nM
A. Three drugs with presumably different B.
Inverted U-shaped curve. receptor affinities and
potencies. -Same maximal effect.
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Partial agonists

• Some agonists never elicit a maximal response
  (compared to the endogenous agonist) even when
  nearly all of the receptors are occupied.
• However, the EC50 for these are remarkably close
  to full agonists
• Similar potency, but lower efficacy Intrinsic
  activity 01
• High efficacy drug need to occupy fewer
  receptors to produce a response than one with
  lower efficacy.
• Why????
• several conformation changes can occur by
  different agonists.
• Similarly, partial agonists will elicit a very
  low or no measurable functional response even
  when a significant number of receptor are
  occupied.

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Partial agonists can act as functional
antagonists when in competition with higher
efficacy agonists.

• Methadone for heroin abuse treatment.
• Used to wean off abused drugs.
• Basically competition between the full and
  partial agonist.

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Receptor antagonists.

• Prevent agonist-mediated responses by preventing
  a drug from binding and eliciting its normal
  response.
• Intrinsic activity 0.
• No sensitivity to Na or GTP.
• Antagonists are measured by the selectivity,
  affinity for their receptor, and potency.

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Receptor antagonists

• Competitive antagonist.
• Reversible or irreversible.
• Bind to the same site as the endogenous ligand or
  agonist.
• Can be over come!
• Their presence produces a right-ward shift in
  both the binding and dose-response curves.
• No change in Emax or Bmax.
• Similar dose-response curve shapes indicates the
  presence of a competitive agonist (competing for
  the same binding sites).

A agonist alone B antagonist (one
concentration) AB agonist antagonist
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Non-competitive antagonist

• Does not prevent formation of the DR complex, but
  impairs the conformation change which triggers a
  response.
• Bind to a site different than the agonist binding
  site at an allosteric site (use a hemoglobin
  example.).
• Cannot be overcome by adding more agonist
• Emax and Bmax are reduced but EC50 remains the
  same for the unaffected receptors.
• Dose-response curves will have different shapes
  indicating different binding sites.

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Irreversible antagonists.

• Binds in an irreversible manner, usually by
  covalent modification of the receptor.
• EEDQ (non-selective)
• N-ethylmalemide (NEM) or other sulfhydryl or
  alkylating agents (non-selective).
• Antibodies
• Molecular control (mutation) EXAMPLE
• Prevents binding at the atomic level.
• Effectively and practically lowers the number of
  receptors capable of binding an agonist.
• Adding more agonist is useless
• Only cure Make New Receptors by Protein
  Synthesis.

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Receptor subtypes

• First learned for the histamine receptor.
• histamine activation by agonist produces smooth
  muscle contraction.
• The residual activity in gastric secretion, even
  in the absence of muscle contraction, indicated
  the presence of histamine-sensitive receptors.
• Conclusion Receptor Subtypes.
• Receptor subtypes are characterized by
• Binding differences (selective ligands)
• Function
• Molecular cloning analysis revealing amino acid
  differences.

Contraction antagonist
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Opioid receptor subtypes

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Stopping the GPCR signal

• Endogenous GTPase within Ga subunit
• Proteolysis of receptor-rare
• NT re-uptake or enzymolysis
• RGS proteins-regulators of GTPase
• Receptor internalization/down-regulation.

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Receptor desensitization

• A loss of agonist affinity, but not receptor
  number after chronic agonist stimulation.
• Best example is b2-AR.
• Activation of PKA/GRKs
• Phosphorylation
• - b-arrestin
• uncoupling of receptor and G-protein
• results in a rightward shift of the binding
  curve DESENSITIZATION.
• KD of isoproterenol (1 ? 100 nM) goes up
• affinity goes down
• number of receptors does not change (Bmax does
  not change).
• b-arrestin binds with clathrin AP-2 binding site.
• Complex internalizes into membrane-bound
  endosomes.
• Endosomes internalizes
• transient decrease in surface receptor number.

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Receptor desensitization
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100 50 0
untreated
response
Chronic treatment
0 10 9 8 7 6
5 4 3
-log agonist M
Receptor desensitization
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Adapted from Lefkowitz, 1998 (JBC, vol., 273)
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Receptor down-regulation

• Proteolytic degradation of receptor
• producing a net loss in total cell receptor
  number.
• PKC involvement during endocytosis
• Bmax can decreases (60) KD remains the same
• Use of endosomes and lysosomes.

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Receptor down-regulation
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100 50 0
untreated
response
Chronic treatment
0 10 9 8 7 6
5 4 3
-log agonist M
Receptor down-regulation
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