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TOPIC 2: PHARMACODYNAMICS Seminar 1 Drug-receptor interactions Molecule is fit in the active site.

Chemical interaction between ligand and binding site: Covalent binding Electrostatic interactions Hydrogen bond Hydrofobic interactions Van der Waals interactions What to know: the strength of the binding Measurement of ligand receptor binding How drugs-binding is measured: Measure receptor from tissue organ Homogenization: all cell will be disrupted you get all fractions of the cell membrane (receptors are inbedded in cellmembrane) Centrifuge: get rid of all cellular contents and all the enzymes clean membrane suspension Take increasing concentrations of the drug that you want to measure Radioactivity = indication of the drug binding to receptors (or fluorescently) Filter celmembrane suspension: only measure drug bound to receptor on filter (unbound drug will pass the filter) C=1 small amout of bindingsites are occupied by this drug Increase conc. drug more occupancy binding sites by drug Add a lot of radio-active drug small amounts bind + many will be in the solutin Conc=40 80% occupancy receptors

Experiment for measuring drug-membrane interaction Different concentrations (x-axis) measure % bound (y-axis) graph

MODEL FOR LIGAND-RECEPTOR BINDING (One arrow: covalent binding, usually reversible equilibrium)

Formation of [RL]: = association rate constant (more ligand or ligand there are the higher the chances they will meet eachother and bind) (speed of formation (amount RL formed in time) is dependent on ligand molecules that are swimming around) Dissociation of binding[RL]: = dissociation rate constant (decrease RL in time) (the more complexes the more likely that some of them will dissociate) (fewer chemical binding the higher At (dynamic) equilibrium: Direction of equilibrium: (big K) = association equilibrium constant = dissociation equilibrium constant affinity (=strength of binding) You want high affinity drugs, because you need only a low amount of drugs to have an effect, with lower changes of side effect. => Langmuir: [L] => ( ligand conc high approach RL will approach Rtot ) [L50]: concentration L when fractional receptor occupation is (so 50% of the receptors is occupied) You can determine Kdiss by RL of 50%: => [L50] = (moles/liter) You want high affinity for your drugs: Low high affinity High high affinity ( is inversion of also al concentration in M

Which drug is best to go with? (look for the highest affinity) A B 10-10 M 10-8 M 10 -1 10 M 108 M-1 1. Low means high affinity answer A 10-10 M 2. is inversion of (e.g. =10-8 =108) So high means high affinity 8 answer B 10 M => is direction of equilibrium and concentration at receptor occupancy

LOG-transformation Red curve: affinity = 3 nM 3nM = 3 10-9 M Log (3 10-9) = -8,5 When you look at 50% occupied (y-axis at 35) you see indeed -8,5 at the x-axis so its right: aff=3nM!! ( * = radio labeled) Problems drug receptor: Many drugs are antagonists: receptor is over reactive and you want to use drugs to block this, but both the normal ligand + drug want to interact with the receptor All drug must be (radio)labeled too expensive and not technically possible L (ligand) is interfering with the receptor, and the inhibitor also (I=inhibitor) Ki inhibitor interfering with the receptor (lower the Ki more active the drug will be) REVERSIBLY BINDING INHIBITOR

(with inhibitor) Two experiments: 1. Binding of radioactive ligand L* to the receptor yields 2. Binding of L* + in presence of an inhibitor also yields When you add an inhibitor apparent dissociation constant = *= * (High low affinity )

So *> => higher * apparent affinity * of ligand for receptor is lower!! Dependent on: [I] : the more [I] more will deviate from the real Kdiss Ki : lower Ki the better the antagonist will bind to the receptor the more the apparent affinity of the ligand will change from the real affinity Ligand affinity appears to be lower because it has to compete with the inhibitor: in presence of inhibitor a higher concentration needed (of ligand) to reach 50% R occupation COMPETIVE ANTAGONISM: reversibly inhibitor binding Drug binds reversibly inhibitor can dissociate always competition between agonist and drug

Normal (left) : [ = 100 With inhibitor (right) :


Kdiss= 10-7 M

Max occupancy can be reached at high [L]: [ = 100 same maximum can be reached Curve shifts to the right Kdiss*= 10-6 M higher Kdiss means lower affinity* (affinity = 10x lower) Curve with reversibly binding: Curve shift to the right (at the time of inhibitor binding) lower apparent affinity Same maximum can be reached: use very high concentration of ligand ligand wins the battle: able to fully occupy the receptors (not enough inhibitor to occupy receptor)


[ ] with inhibitor (=[ ]*) is always decreased because of irreversibly inhibitor binding Two experiments: 1. Binding of radioactive ligand L* to the receptor yields [ ] (maximal respons/binding) 2. Binding of L* + in presence of an inhibitor also yields When you add an inhibitor apparent maximal binding = [ *=[ [ ]* < [ ] => apparent maximal binding of ligand to receptor is lower (dependent on [I] en Ki) ]*

NON-COMPETIVE ANTAGONISM: irreversibly inhibitor binding

Normal (left) : [ = 100 & affinity = 10 With inhibitor (right):


M (bij Y=100/2=50)

[ *= 60 at high [L] decreased maximal binding maximum binding NOT reached! Affinity = 10-7 M (bij Y=60/2=3) affinity doesnt change There can be an antagonist that will bind on the same binding site of the receptor where the agonist binds ( ) two situations of antagonism: Reversible binding competitive (when agonist can dissociate from binding site of receptor an antagonist can bind receptor => competition) Irreversible binding non-competitive (covalent binding is irreversible) There can be an antagonist that will bind on another binding site (not the same as the ligand) it can induce conformational changes both reversible- and irreversible binding of the antagonist non-competitive antagonism => allosteric modulations: antagonist binding to receptor not the active site but elsewhere change in receptor structure (conformation) antagonist can bind receptor anymore Linear transformations: Scatchard [RL]=

Straight line: y = b a x y= b= a= =intersect y-axis =slope

x = [RL] = intersect X-axis

Alternative approach of binding: Instead of increasing concentrations of L* => one fixed concentration L* + increasing concentrations of inhibitor Low concentration inhibitor [I] huge binding: increase L* Increase [I] decrease radioactive ligand L* 500 drugs are too many to label each so here only make one drug radioactive (the ligand) instead of all drugs, that you would like to investigate: L* IC50 = conc of non-labeled drug leading to 50% of binding measure of affinity of inhibitor receptor but IC50 Ki Low IC50 high inhibitor binding High IC50 bad inhibitor binding: need more to displace this drug from the receptor Fractional receptor occupation = r= Without competitive inhibitor: r = With competitive inhibitor: r = * [I] = 0 maximal binding r= * [I] = IC50 half-maximal binding r=0,5 Cheng-Prusoff equation in presence of inhibitor

Seminar 2 Drug - receptor interaction There are different kinds of receptor that are target for drugs: Ligand-gated ion channels (ionotrope receptoren) GPCR (metabotrope receptoren) Kinase-linked receptors Nuclear receptors Measurement of receptor action/activation Defect in smooth muscle from GIT take a gut segment put the piece of muscle tissue in a organ bath add drug on the top of tube drug will go through will reach tissue: occupy receptors Transducer can measure the strength or magnitude of contraction (so longer or stronger contraction) Start with concentration 1x10-9 M nothing really happens (conc is so low that not many receptors get occupied) Then increase the concentration tissue starts to contract Increase of contraction is smaller at the start At the top: increases is getting smaller receptor occupancy is getting almost satisfied (At high concentration an extra dose of the drug has less effect than a raise of dose at lower concentrations) S-shape curve: very similar as binding-curve NE = norephrine; PE = phenylephrine Best werking drug( NE): at low [NE] it has already a larger effect than PE

AGONIST-INTERACTION WITH RECEPTORS Drug = agonist of receptor Many [AR] low Kdiss high affinity: best drug Many [A][R] high Kdiss low affinity: bad drug [AR]=

If effect directionally proportional to concentration of occupied receptors (=cell of 100 receptor all 100 receptors are occupied full respons): E= = intrinsic activity 01 = 1 drug = full agonist

no other drug can get a higher respons than this drug structure change (by binding receptor) is optimal = 0 drug = antagonist can occupy receptor, but dont lead to a effect: resulting effect = 0 binding will change structure, but now it will have a completely n wrong structure 0< <1 drug = partial agonist partial respons, even though all receptors are occupied it can bind and change the structure receptor, but not in the most optimal way When all receptor are occupied: E= E= becomes E becomes Emax = = efficacy (maximal respons)

At EC50 or [A50] [R]= [AR] = [Rt] (half of the receptor are occupied) Kdiss = EC 50 -Log = - Log EC50 = pD2 = potency Potency has 2 measure dimensions: Kdiss = EC 50 in concentration (M) pD2 = - Log EC50 dimensionless (EC 50 in M so maybe you have to convert it into M) E.g. Kdiss = EC 50 = 3,2 x 10-8M pD2 = 7,49 Potency vs. Efficacy

*dotted line indicates the same dose for both drugs Drug A lower EC 50 than drug B higher potency than drug B So low EC 50 high pD2 good agonist Drug A higher efficacy than drug B So low EC 50 high pA2 good antagonist (inhibitor)

ANTAGONIST-INTERACTION WITH RECEPTORS An agonist and a antagonist want to bind to one receptor at the same time competitive-antagonism Antagonist will inhibit the effect of the agonist. EA = Increase inhibitor [I] => than EA (effect will decrease) Increase extra agonist [A] => than EA (effect will increase) = competitive antagonism COMPETITIVE ANTAGONISM: inhibition can be overcome by increasing [A] Normal curve (left): without inhibitor [B]=0 Second curve: presence of 1M inhibitor * shift of curve to right means that it is competitive antagonism EC50 = measurable concentration Kdiss = affinity

Determination of the Ki value of an antagonist Method 1: EC50(B) = Method 2 : DR = dose ratio Log transformation: pA2 = -Log Ki = -Log [I] + Log (DR-1) = measure of affinity of antagonist (pD2 = measure of potency of an agonist with EC50) A B 10,2 pA2 10 M 109,6 M High pA2 (low EC50) = good antagonist here drug A High pD2 (low EC50) = good antagonist Linear transformation: Arunlakshana-Schild ( only by competitive antagonism) pA2 = -Log Ki = -Log [I] + Log (DR-1) X = Log[I] vs. Y-axis= Log(DR-1) (Y-axis approaches 0, but not real intercept) X-axis intercept = pA2 Make four dose respons curves!! *= (only holds when my assumption is true) measure [I], EC50(B) or EC50

NON-COMPETITIVE ANTAGONISM By adding a non-competitive inhibitor: only efficacy (maximal respons) decreases, potency stays the same (for both reversible and irreversible antagonism) 2 options non-competitive antagonism: via allosteric regulation on receptor (extracellular) (not binding on the binding-site of the agonist) via intracellular signal transduction route Irreversible binding is always non-competitive!! In nature most kinds of antagonist are non-competitive because they bind on several places. In reality there are more competitive antagonist, because we made them up

NON-COMPETITIVE ANTAGONISM (IRREVERSIBLE) At higher concentrations: non-competitive antagonism (1, 10) At lower concentrations: competitive antagonism (100, 100) But where a drug binds and how determines whether is competitive or non-competitive so a drug cant be both!! Explanation for these curves: We used two drugs: drug A and drug B We did two experiments: 1. Drug binding fractional occupancy of receptors (=effect) 2. Respons (e.g. contraction in muscle) Curves: Occupancy (both drugs) => both drugs bind in a similar way to the receptor cant discriminate between the two drugs (drug A and B: same maximal binding=Rtot & the same affinity of 1M) Response (full agonist) A => at high concentration reaches the maximum of binding Response (partial agonist) B => even at high concentration not able to get the full response *vertical arrow on x: concentration where the maximum of 100% is reached) *horizontal arrow on y: at the same concentration where maximum of A is reached 20% receptor occupancy => so 20% of receptor occupation yields 100% effect => so EC50 Kdiss => here full agonist with receptor reserve EFFECT vs. RECEPTOR OCCUPATION (=maximal binding) Full agonist: occupy all receptors full response Partial agonist: occupy all receptors no full response Antagonist (see X-axis): occupy all receptors no response Full agonist + receptor reserve: occupy less than 100% receptors 100 % effect (=full response) Full agonist + threshold (partly X-axis): first receptors get occupied leading to no effect, but after an certain moment (threshold) occupation of receptors will lead to 100% effect (full response) Q How will be the curve of full agonist in presence of an competitive antagonist? A Like a partial agonist?? Tissue properties (physiologic relevance/adaptation) Receptor reserve: 100% reached if not all receptors are occupied, so there are spare receptors (spare = dont need them) 1) effect on direction of equilibrium ( [A] and [R] determine the direction) so if you have high [R], you need less [A] for same effect (to get same amount [AR]) 2) if receptors disappear: with receptor reserve just increase [A] still maximal effect possible Receptor threshold: before threshold no effect, but after 100% receptor occupation full response 1) Discrimination between organs if you want that only a specific organ reacts to a rise of concentration [A], theres an threshold for certain level [A] in one organ (will react at high [A]) and theres no threshold in another organ for example (and will react at low [A]) 2) no effect through basal hormone levels (e.g. always adrenaline present) with threshold you will not react to basal level [A], but when the [A] is increased above the threshold it will lead to an effect. Drug properties: potency, intrinsic activity, efficacy

Isoreceptors and selectivity

isoreceptor = GPCR (histaminereceptor)

Different subtypes isoreceptors: H1, H2, H3 and H4 AC = adenyl cyclase pKi = inhibition constant So H1 increase adenyl cyclase In common have these subtypes: histamine = endogenous agonist (for all these receptors) N.B. drug binding test whether drug is agonist or antagonist most is not relevant, only competition. So if you add two agonist that compete, one can be seen as an inhibitor of the other and therefore you van express affinity as Ki. (see extra assignment 1) For an certain drug (thioperamide): H3 receptor pKi = 8,8 H4 receptor pKi = 7,6 If pKi of two isotypes differ: pKi, average = 8.8-7.6=2,1 => their selectivity differs with a 16-fold = 101,2 (inverse log transformation) so H3 selective drug Selectivity = drug has preference for a binding receptor over another receptor (difference in affinity) * -fold selectivity for receptor with the biggest pKi Curve with one plateau Drug doesnt discriminate between the two receptor has equal affinity for both (no selectivity) S-shaped curve Curve with two plateaus: Drug must have selectivity for one of both subtypes of receptors!! also two EC50s Here: At low [A] this drug is activating this receptor and is occupied all of this receptor isotype At higher [A] starts to attack the other receptor isotype Two EC50s (for each subtype 1 EC50): * 1st subtype: 10-10 M (at 35%= half of first plateau) pD2 = -Log 10-10= 10 * 2nd subtype: 10-6 M (at 85%= half of second plateau) pD2 = -Log 10-6= 6 => pKi, av = 10-6=4 (inverse log:) 104=10.000-fold selectivity for 1st subtype Partial agonism Both drugs activate the receptors so are both agonists Hypothesis: both have the similar affinities for receptor Isoprenaline = full agonist; oxprenolol = partial agonist partial agonism is not involved by the binding: partial agonist binds with the same affinity to a receptor as full agonist Isoprenaline causes optimal change of receptor structure optimal binding to G-protein Oxprenonol causes less optimal binding to G-protein => Binding of an agonist causes change in affinity of G-protein binding to the receptor (by binding iso higher affinity G-protein (than by binding oxp)

Inverse agonism Histamine receptor (GPCR) Receptor is in activated (R*) state by agonist OR in non activated state (R) Agonist (e.g. histamine) = compound that converts the receptor in activated state that leads to signal transduction * add histamine increase in cAMP Antagonist (e.g. buramide) = inhibits the effect of the agonist on a receptor (fewer receptors are occupied by receptors), but itself cant cause an effect (effect=0) = opposite effect of agonist * add buramide decrease in cAMP Inverse agonist(e.g. cimetidine) = inhibits the effect of the agonist on a receptor, but can cause an effect * add cimetidine decrease in cAMP to real 0 level / base-level => constitutive receptor activation (receptors are always activated in some way: 20% of receptors is occupied) Inverse agonism: conformational selection Old model: agonist interferes with receptor get activated complex AR* New model: receptor can be in activated- or non-activated state (=property tissue with certain receptor) * 10% is constitutive activity Agonist: Kdiss > Kdiss* (drug has higher affinity for R*) more signal transduction(more active state) Inverse agonist: Kdiss < Kdiss* (drug has higher affinity for R) less signal transduction (more resting state) Antagonist: Kdiss = Kdiss* (equal affinity)