Pharmacology - Lecture 2 PDF

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Pharmacology - Lecture 2 The Receptor concept 

PHARMACOLOGY is... What drugs do and how they do it

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We now recognise that the vast majority of drugs exert their effects through specific interaction with cellular macromolecules (receptors).

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The ‘receptor’ concept was first proposed in the early 1900s by J.N. LANGLEY (‘receptive substances’) and Paul EHRLICH (‘chemoreceptors’)

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However, for almost 30 years such views were challenged and disparaged. Wider acceptance only came through the application of quantitative analysis to drug interactions with cells and tissues.

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“A drug will not work unless it is bound” – Paul Ehrlich

The Occupation Theory 

In the Occupation theory, A.J. Clark assumed that the intensity of drug effect was directly proportional to the number of receptors occupied – i.e. The higher the concentration of the drug, the more the response was seen from a system, until it is was saturated (He believed that the occupation of 50% of available receptors results in 50% of the maximal response)

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Mathematically he made the equation; (Dose-Response Where E is the functional effect, Emax is the maximal functional effect, [A] is the drug (agonist) concentration, and KD is the dissociation constant.

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By plotting functional effect (“response”) E on the y-axis and log10 [A] on the x- axis a sigmoidal curve is generated which describes the relationship between drug concentration and tissue response.

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Plotting a response (E, here shown as the E/Emax ratio on the ordinate axis) against the concentration of agonist ([agonist]) produces a rectangular hyperbola. LINEAR LOGGED

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Re-plotting the data for log10 [agonist] produces a sigmoidal curve often referred to as a concentration-response (or dose-response) curve -

“Normally we see the data in a linear representation but we should turn it into a logged value. You need to be comfortable for logs”

The Schild Plot 

Clark, Gaddum and subsequently Schild, showed that agonist concentration-response curves are rightward-shifted in the presence of a competitive antagonist for the receptor

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e.g. atropine right-shifts the concentration-response curve for acetylcholine in an ileum smooth muscle preparation

This effect can be exploited to determine the binding affinity of the competitive antagonist at the receptor – the Schild plot

Experimental method: 

Schild developed a method of getting quantitative information about antagonists – The Schild plot - “All pharmacologists should know what a schild plot is”

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He took ileum from a guinea pig and put it in an organ bath. He added increasing concentration of Ach (agonist) to the tissue and received a dose-response curve and stopped when he no longer saw an increase in the response of the tissue

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Then he washed some antagonist in the tissue. He then repeated the dose response curve, which shifted it to the right. So we need to add more agonist to produce the same response

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Gaddum actually logged the data – He plotted the data of the log of the antagonist concentration, against the log of the Dose Ratio – 1 (The DR shows how far the curve shifts in the presence of that concentration of antagonist)

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We can then extrapolate the line back to find the pA2 value, to give the affinity for the antagonist to the receptor

Receptor-ligand affinity and cellular change 

People quantified what antagonism was – Agents that pocess affinity for a receptor but do not cause the receptor to generate a cellular response

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Agonism have affinity for receptors and cause an effect

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E.J. ARIDNS proposed that drug-receptor interactions can be defined by two factors: AFFINITY (a measure of the ‘attachment’ of the drug to the receptor), and INTRINSIC ACTIVITY (a measure of the ability of the bound drug to evoke a cellular effect).

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AriGns (1954) designated the intrinsic activity of full agonists to be 1, antagonists to be 0 and partial agonists to possess intrinsic activities between 0 and 1. But it was later realised that this was not exactly true and that you do not need all the receptors to be occupied to get a maximal response

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Quantification of both receptor occupancy and cellular response led to the realization that full agonists can cause similar (maximal) functional responses while occupying different fractions of

the total number of receptors (i.e. there are often ‘spare’ receptors that do not require to be occupied for a maximal response to be evoked by some agonists.

Method to prove this: 

Take a pieces of guinea pig ileum and add drugs to inactivate some of the receptors. We can see that with a full agonist and no inactivation, we get a full dose-response curve result. If we inactivate some of the receptors, we still achieve an optimal response (Disproves the Occupation Theory), we just see a rightward shift of the curve. If we inactivate more receptors again, then there is a reduced overall response. Therefore, around 90% of the receptors are called ‘spare’ receptors as we can activate 10% of the receptors and still produce a maximal response

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If we use a partial agonist, we never achieve a maximal response compared to the full agonist. I f we inactivate receptors, we see a proportionate decrease in the response we receive. So not all the receptors in the tissue do not have to be occupied to achieve a response

The efficacy concept 

R.P. STEPHENSON (1956) – defined EFFICACY as a property of the agonist that would permit two drugs to occupy different proportions of receptors yet produce equal responses.

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Efficacy – How a system generates a response, once a ligand is bound Where the stimulus (S) to a receptor by an agonist is defined as the product of the efficacy of the agonist (ε) and the fraction of receptors occupied by the agonist (Rfract).

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In the 1960s FURCHGOTT investigated the relationship between receptor number and receptor occupation further. These experiments provided clear evidence that Stephenson’s efficacy is a

tissue-dependent phenomenon. Furchgott re-defined this parameter as ‘INTRINSIC EFFICACY’ where Where e is efficacy, ε is intrinsic efficacy and [Rtot] is the number of receptors.

Reflections 

The vast majority of these developments in quantitative pharmacology were made using classical pharmacological preparations

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Radio-ligand binding methods (first introduced in 1970s) allowed ligand-receptor interactions to be quantified for the first time

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N.B. All of these developments occurred before any receptor had been purified/cloned (only occurring for GPCRs in 1980s)

Landmarks in Cell Signalling 

Agonist  Receptor  Response

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Sutherland & Rall (1953-1958) - Discovered cyclic AMP (adenosine cyclic 3’,5’-monophosphate) and the hormone-sensitive enzyme which makes it – adenylate (or adenylyl) cyclase. Provided a mechanistic outline of the pathway linking hormone (adrenaline) receptor binding in liver cells to the breakdown of glycogen to glucose.

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Birnbaumer & Rodbell (1966-1969) - Proposed that the receptor protein that senses the hormone is separate from adenylate cyclase

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To receive an extracellular signal, the ‘receptor’ is likely to be in the plasma membrane – is the enzyme activated on agonist binding part of the receptor?

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The collision coupling (mobile receptor) model (Jacobs & Cuatrecasas, 1976) proposed that receptors and effectors (e.g. adenylyl cyclase) can be separate entities that can interact (‘collide’) within the fluid-mosaic membrane.

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Orly & Schramm (1976) - Demonstrated that receptor and effector (adenylate cyclase) are separate proteins using hybrid/fusion approach.

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Their experiemnt is as follows;

Evidence for guanine nucleotide-binding proteins: 

Rodbell et al. – Experiments attempting to ‘reconstitute’ the signal transduction pathway leading from hormone binding to cyclic AMP generation o

Thoroughly washed membranes isolated from adipocytes

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Add: hormone (e.g. adrenaline), ATP, Mg2+ [adenylyl cyclase known to require Mg2+ for catalysis]

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Highly variable results

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Analysis of the ATP used in experiments showed varying batch-to-batch contamination of ATP by GTP (guanosine 5’-triphosphate)

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Pure ATP – little cAMP; GTP-contaminated ATP – high cAMP generation

Therefore, GTP is also necessary for efficient transduction of receptor- agonist binding into a second messenger response

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Bourne (1975) - Isolation of the mutant S49 lymphoma cell-line which did not make cyclic AMP when stimulated by hormone – hypothesized that cell-line was deficient in adenylate cyclase (hence cell-line called cyc-cells).

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Pfeuffer (1975) - Chromatographic purification of a GTP-binding protein

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Ross & Gilman (1977) - Demonstrated (using components purified from S49 lymphoma and cyc-cells) that adding-back GTP and a 40 kDa GTP-binding protein could reconstitute hormone-stimulated adenylate cyclase activity. Thus, cyc cells possess adenylate cyclase, but lack the GTP-binding protein necessary for allowing the receptor to stimulate (or inhibit) adenylate cyclase.

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Cassel & Selinger (1978) - Reported a cholera toxin-sensitive GTPase activity – proposed a sequence of events whereby receptor causes GTP-for-GDP exchange on the GTP-binding protein (G-protein) which in turn activates the effector (adenylate cyclase) – activation is terminated by GTP hydrolysis.

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Lefkowitz et al. (1981/1986) - Purification of G protein-coupled receptors (GPCRs) – β1 and β2adrenoceptors. Cloning of the β2-adrenoceptor (currently >300 GPCRs cloned and partially characterized).

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Simon et al. (1987-1992) - Cloning of several G-protein subunits – GPCR-linked G-proteins are heterotrimeric consisting of 3 separate protein components α (alpha), β (beta) and γ (gamma) subunits.

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Gilman and others (1989-1992) - Cloning of the mammalian adenylate cyclase enzyme family (AC1-AC9).

Establishment of the central dogma of GPCR signalling, whereby receptor activates G-protein, and Gα (and/or Gβγ) subunits activate or inhibit an effector protein (which can be a second messenger generating enzyme (e.g. adenylate cyclase) or an ion channel (e.g. G-protein-regulated, inwardly rectifying K+ (GIRK) channels))....