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Biological & mathematical interpolation of receptors in tissue

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methods of receptor characterization, theories of receptors occupancy, scatchard's plot, schild's plot

methods of receptor characterization, theories of receptors occupancy, scatchard's plot, schild's plot

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  • 1. Dr AMIT RELHAN
  • 2.   A MACROMOLECULAR PROTEIN WHICH BINDS TO SPECIFIC FUNCTIONAL GROUPS OF ENDOGENOUS SUBSTANCE OR A CHEMICAL SUBSTANCE. DEFINITION OF RECEPTOR
  • 3.  Dose response curve  Maximal ceiling effect  Position of curve (EC50 & pD2)  Slope of curve
  • 4.
  • 5.   Claude Bernard- curare- injected frog skin – progressive dimunition of motor reflex ( electric stimulus to muscle – contraction)  Curare acting on neither muscle nor nerve- NMJ  Paul Ehrlich –preferrential accumulation of Pb in CNS  Differential staining of tissue using dyes  Arsenicals for T. pallidum- some sort of selectivity for parasite (magic bullet) Concept of receptor
  • 6.   Failure of arsenical in trypanosomes – lack of binding  Agents cannot act unless they are bound  Selectivity of binding
  • 7.   J.N.Langley –autonomic transmission & NM communiation  Frog gastrocnemius- nicotine- contraction  Blocked by curare ( even after degeneration of nerve)  Direct stimulation – contrction  Both curare & nicotine act on same substance  Receptive substance  ∞ conc. of drug (potency) & its affinity to receptive substance
  • 8.  Theories of drug receptor interactions
  • 9.   Studied antagonism b/w Ach & atropine in various muscle preparation  Effect of drug is proportional to the fraction of the receptor occupied by the drug  Maximum effects are produced when all receptors are occupied  Drug receptor complex breakdown at rate proportional to rate of complex formed  Saturability, Reversbility ,Dynamic equilibrium Classical receptor theory by Clark(1937)
  • 10.   Based on law of mass action(isotherm equation - langumuir)  k1  [L] + [R] < ===[LR]  k2  rate of association = K1[L][R]  rate of dissociation = K2 [LR]  At equilibrium, rate of association=rate of dissociation
  • 11.   K1[L][R]= K2[LR]  K1 /K2=[LR]/[L][R]=Ka –equilibrium association constant  1/Ka= Kd=[L][R]/[LR]  Kd –equilibrium dissociation constant
  • 12.  Equilibrium Dissociation constant (Kd). However, it is not possible to measure [R], So, Rtot = [R] + [LR] and [R] = Rtot -[LR] Scatchard equation
  • 13.  Scatchard plot
  • 14.   The Kd is the same for a given receptor and drug combination in any tissue, in any species (as long as the receptor is the same)  The Kd can therefore be used to identify an unknown receptor  The Kd can be used to quantitatively compare the affinity of different drugs on the same receptor
  • 15.   Studied-Ach induced contraction of frog rectus Ms.  Ach induced inhibition of electrically stimulated frog ventricle  Slope of log concentration- response curve  Linear correlation b/w occupancy &response
  • 16.
  • 17.
  • 18.   Slope of log conc.- response curve--- steeper than predicted from mass action equation.  Sometime even supramaximal conc.—not able to elicit maximal contractile response.  Dualism of homologus series of quaternary ammonium salt in muscle preparation • butyl-/lower member of series-full contraction • Hexyl/heptyl-higher member-weak contraction • Applied simultaneosly with butyl - antagonism Shortcomings of Clark theory
  • 19.  log
  • 20.
  • 21.   Webb (1950)-  when the cholinesterase of isolated rabbit auricles is blocked with physostigmine, the slope of the acetylcholine log-concentration-effect curve is about 10 times steeper than on normal auricles
  • 22.   Studied Dual behavior of phenylethylamine –in cat BP experiments  Agonistic and antagonistic effect –single recptor  Introduced intrinsic activity(IA)- ability of a drug to elicit effect  Effect ,E=α [LR]  For max response –maximal occupancy not required Ariens theory
  • 23.   Explained concept of partial agonism  Not able to explain steeper log dose response relationship than expected from equation
  • 24.   Alquist (1948)  Concentration- response curve of tissues or organs of different receptor systems obtained  rank order of potency was "adrenaline > noradrenaline > α-methyl noradrenaline > isoprenaline" in promoting contraction of blood vessel- α-adrenoceptors  the rank order was "isoprenaline > adrenaline > α- methyl noradrenaline > norepinephrine" in the heart-β-adrenoceptors
  • 25.   Clark equation– conc. Of drug & conc of drug receptor complex formed  Tabulated slopes of log conc response curve in literature- steeper than predicted  Ach & histamine on guinea pig ileum- greater response than predicted from receptor occupancy  Response not linearly ∞ to fractional receptor occupancy- only small fraction- max effect –receptor reserve Stephenson theory
  • 26.
  • 27.   Concept of efficacy- capacity to start response  Response=f. (stimulus)=f.(e.y)  e=efficacy , y = fractional receptor occupancy  Explained dual behavior of homologus series  Lower /butyl-high efficacy- agonist  Higher/ hexyl-low efficacy- partial agonist  Affinity but no efficacy- antagonist
  • 28.   Nickerson 1956- 1% histamine receptor occupancy – max response in guiena pig ileum  Furchgott 1955- studied antagonism by β haloalkylamine on effect of adenaline - shift of only half log unit.  Goldstein 1974-studies on receptor antagonism  β haloalkylamine- irrevesible antagonism of histamine & catecholamine recpeptor  Low dose- only rightward shift of drc(spare receptor)  High dose- both rightward shift & ↓max effect Spare receptor
  • 29. Receptors are said to be ‘spare’ for a given pharmacological response when the maximal response can be elicited by an agonist at a concentration that not result in occupancy of the full complement of available receptors Spare receptor Emax Log Concentration Respones(%) Agonist alone Agonist with noncompetitive antagonist in presence of spare receptor Agonist with noncompetitiv e antagonist in absence of spare receptor
  • 30.   Studied competititive antagonism of adrenaline by ergotamine on rabbit uterus.  concepts of ‘dose-ratio’  Schild regresssion analysis –pA2 value & Kb  DR-1=[B]/Kb Gaddum equation  Log(DR-1)=log[B]-logKb Schild equation Schild & Gaddum
  • 31.
  • 32.
  • 33.
  • 34.
  • 35.
  • 36.
  • 37.
  • 38.   Excitation by agonist (eg.nicotine)—block function  Effect of agonist –fade with time  Excitation ∞ rate of drug receptor interaction than no. of receptor occupied  Agonists dissociates rapidly ,Kd-high  Antagonists slowly, Kd-low  Explained Persistent effect of an antagonist on a tissue  Explained tachypylaxis Paton theory(1961)
  • 39.   Receptors exist in discrete conformational states  Hill –O2 binding to Hb- steeper curve  MWC model-1965  2 conformational state of receptor- equilibrium in absence of ligand  Ligand binding –displacement of equilibrium to state having higher affinity  The extent to which the equilibrium is shifted toward the active state is determined by the relative affinity of the drug for the two conformations  Concept of coopertivity Allosteric theory
  • 40.   The binding of a ligand to a macromolecule is often enhanced if there are already other ligands present on the same macromolecule (this is known as cooperative binding). The Hill coefficient  Log(θ/1-θ)=nlog[L] - log Kd  θ – fraction of occupied site where ligand can bind  n >1 - Positively cooperative binding  n<1- Negatively cooperative binding  n=1- Non cooperative binding Hill langmuier equation
  • 41.   No explanation about constitutive active receptor  Not able to explain about GPCR effect coupling
  • 42.   Black & Leff et al- mathematical model  Diff. in relative potency order of ligands in tissues with different recp. Reserve  Receptor in diff. conformational state due to allostery  [LR]=[Rtot][L]/KA+[L]  Rectanguler hyperbola equation  Concept of transducer ratio,  τ =[Rtot]/Ke  τ- efficiency by which occupancy transduced to response Operational model of agonism(1983)
  • 43.   Effect, E=Em[LR]n/Ke+[LR]n  n>1 steep curve  n<1 shallow curve  n=1 linear relation  No insight into [LR] to E –linking event ,but provide τ- quantaive measurement of effect.
  • 44.   Leff & Hall et al. 2000  Difference in potency order for single receptor interacting with different G protein  Difference signal trasduction output from same receptor  To isolate pathway-pertusis toxin sensitive Gi/Go coupled signal or G protein selective disrupting peptide 3 state model /ternary complex
  • 45.
  • 46.
  • 47.   Seifert 2002- inverse agonist concept  Inverse agonist stabilize receptor in inactive conformation  IA-0 to -1 Agonist independent /constitutive activity
  • 48.
  • 49.  Methods of Characterization of Receptors 1. On basis of Pharmacological Responses 2. Radioligand binding studies 3. Molecular Cloning techniques 4. Analysis of biochemical pathway linked to receptor activation
  • 50.  On Basis of Pharmacological Responses a) Relative potency (Affinity) measurements of a series of Agonists b) Determination of Affinity or Dissociation constant of Antagonists c) Isomeric activity ratio of agonists
  • 51.  Relative potency(affinity) measurements of a series of agonists.  Alquist (1948)  Furchgott(1967)observed similar potency series adrenaline > nor-adrenaline > phenylephrine> isoprenaline  By calculating correlation coefficients of two systems e.g.sympathomimetics- bronchodilatation/vasodepression-0.96 (similar) Cardiac stimulation-bronchodilation-0.31(different)
  • 52.  Acetylcholine (ACh): One drug with different affinities for two different receptors (adapted from Clark, 1933) Muscarinic receptors EC50 = apparent Kd ~ 3 x 10-8 M, pD2 ~7.5 Nicotinic receptor EC50 = apparent Kd ~ 3 x 10-6 M, pD2 ~5.5
  • 53.  Different affinities of related agonist drugs for the same receptor: Different potencies (adapted from Ariëns et al., 1964)
  • 54.  Determination of affinity or dissociation constant of antagonist- p(A2) or p(KB)  Schild plot-Different tissue with similar receptors- same value e.g. acetylcholine –atropine in frog heart ,chick amnion ,mammalian intestine
  • 55.  A Schild plot -compares the reciprocal of the dose ratio versus the log of the antagonist concentration  Intercept on absicca- pA2 = log Kd, which represents the affinity of the competitive antagonist  pA2 (log molar concentration of antagonist producing a 2fold shift of the concentration response curve.
  • 56.
  • 57.
  • 58.  Different pA2 values (affinities) for different receptors of some clinically useful drugs: The basis of therapeutic selectivity
  • 59.
  • 60.
  • 61.  ISOMETRIC ACTIVITY RATIO  IAR=Antilog (negative molar EC50 of L-isomer minusEC5O of D-isomer)  High ratio – specific interaction  eg-Isoprenaline (L) -35 times potent than (D)  Similarity of ratio to Enantiomorphs- similar receptors
  • 62.  Experimental Condition for characterization RESPONSE-Should be 1.Solely due to action on one type of receptors 2.Not be due to release of other active substance 3.Concentration of free drug –at steady level 4.Proper control –to any change in sensitivity of agonist 5.Sufficient time-for antagonist to act in equilibrium.
  • 63.  USE OF RADIOLABELLED LIGANDS
  • 64.  RADIOLABELLED LIGANDS a) Direct binding studies  Eg labelled bungarotoxin binds specifically and irreversibly to cholinergic receptors  Phenoxybenzamine –an irreversible alpha blocker .  Relative proportion of B1 and B2 receptor  4:1-Heart  2:1cerebral cortex  1:3 lungs
  • 65.
  • 66.  Scatchard Plots  Kd and R tot cannot be measured directly.  plot the ratio of bound/unbound drug,[LR]/[L] versus bound drug[LR].  intercept of the line with the abscissa -total number of receptors available(Bmax)or R tot.  Kd (the dissociation constant) from the negative reciprocal of the slope of the line
  • 67.  b) Indirect radioligand binding/Competition binding  The affinity of un-labeled compounds –by competition binding. - - displacement from the binding site.  The concentration of un-labeled ligand which displaces half of the tagged is interpolated from the curve and refered to as the IC50 value.
  • 68.   The IC50 value -used to estimate the affinity of the unlabeled ligand -Ki values.  Rank order Ki values are a type of fingerprint for a receptor subtype.  Comparison between EC50 values (rank order potency) and Ki/KD values (rank order affinity)
  • 69.  If Bmax remains unchanged and the slope of the lines decreases with increasing concentrations of the compound, the displacement is competitive. Unchanged slope and decreased Bmax indicate that the displacement is noncompetitive The lower the IC50 or Kd, the higher the affinity
  • 70.  Ki value  Ki, the inhibitory (or affinity) constant of the displacer compound  when the displacement is noncompetitive (Ki = IC50)  for a competitive displacement -Cheng-Prusoff equation  Ki = IC50/(1 + [L]/Kd)  [L]= concentration of the radioactive ligand.
  • 71.
  • 72.   Peroutka & Snyder (1979)  5HT1 – high affinity for [3H]5HT  5HT2 – low affinity for [3H]5HT, but high affinity for [3H] spiperone
  • 73.
  • 74.  Allosteric interaction  The affinity shift, -the ratio of radioligand affinity in the presence (KApp) to that obtained in the absence (KA) of each concentration of antagonist.  A plot of log (affinity shift1) versus log [antagonist] should yield a straight line  slope of 1 for a competitive interaction,  curvilinear plot for an allosteric interaction.
  • 75.
  • 76.
  • 77.  Protean Agonism  After Proteus, the Greek god  Ligands act as partial agonists in quiescent silent systems  As inverse agonists in systems that show a high level of constitutive activity.  Agonist produces an active conformation of lower efficacy than a totally active conformation
  • 78.  Molecular Cloning  Heterogeneity of receptor, distinct sequences and tissue distribution  Receptor-labelling tech. made it possible to extract and purify receptor material  Firstly this approach used on Nicotinic ACh recp. in 1970  Transgenic & receptor knockout mice- subtypes of receptor
  • 79.   Sequence homology of receptor  Alpha1a,1b,1d – 70%  Alpha 1 & 2 – only30%
  • 80. Images of cAMP Transients in Cultured Aplysia Sensory Neurons. The cell was loaded with a fluorophore that would allow for the quantification cAMP concentrations within the cell. A: Free cAMP in the resting cell is < 5 X 10-8 M. B: Stimulation with serotonin, activates adenylate cyclase increasing cytoplas cAMP to ~ 1 X 10-6 M (red), especially within fine processes with a high surface to volume ratio. Thurs, within 20 sec of stimulation, the intracellula [cAMP] increased ~ 20-fold.
  • 81.   Greengard et al. – nigrostriatal cAMP for dopamine receptor identification  D1 - increase cAMP  D2 – decrease cAMP
  • 82.