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http://www.mc.uky.edu/pharmacology/instruction/pha824mp/PHA824mp.html

Macromedia Shockwa e isnecessary to iew theanimations on this page

PHA 824

RECEPTORS

DR. MICHAEL T. PIASCIK

This material is available at http://www.mc.uky.edu/pharmacology/mtp_research.aspBackground information can be found in chapters 1 and 2 of the olan te!t and inchapter 1 of the "agiela te!t.

#earning $b%ectives

The student should know:

The definition of a drug The concept of affinity and those factors that lead a drug binding to a receptor

the different types of receptors

the concept of intrinsic activity

the difference between a full and partial agonist

the properties of a competitive antagonist and how it differs from an irreversible receptoragonist

the concept of a therapeutic inde! and how it is calculated

the concept of spare receptors and signal amplification

Below is a hypotheti al patie!t sit"atio! whi h ill"st#ates how $asipha#%a olo&i p#i! iples ai' i! the "!'e#sta!'i!& o( )a#io"s li!i al sit"atio!s.The i!(o#%atio! !ee'e' to "!'e#sta!' this ase will $e p#ese!te' i! the le t"#eso! Receptor Theory, Adrenergic Receptors and Cardiovascular Pharmacology.

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&hristopher Thomas is a retired musician. $n several visits over a period of a year younote his blood pressure is 1'2/()* 1+,/(,* 1')/((* and 1+-/(-. e has a history ofsmoking and has chronic bronchitis. e also has elevated lipid and cholesterol levels."ou wish to begin therapy with an antihypertensive medication. There are severalchoices of drugs which can be prescribed in this situation. Two of the potential choices

are pra osin* which is a competitive alpha0receptor blocker* and propranolol* which is acompetitive beta0receptor antagonist. "ou choose to prescribe pra osin 1 mg b.i.d. Thene!t day r. Thomas calls and complains that he took his medication and shortlythereafter became di y and passed out.

&onsider the following uestions which you will be able to answer after completion ofthe lecture se uences listed above:

*"estio!s+

3hat are the various drug classes which can be used to treat hypertension4

3hat are the pharmacologic and nonpharmacologic approaches to the therapy ofhypertension4

3hat is the agonist blocked by pra osin4

3here are the receptors pra osin blocks located and why does this cause hypotension4

3hat factors in this case made pra osin a better choice than propranolol4

3hy did r. Thomas faint and should this be a cause for concern4

Re epto#+ 5ny cellular macromolecule that a drug binds to initiate its effects.

D#"&+ 5 chemical substance that interacts with a biological system to produce aphysiologic effect .

5ll drugs are chemicals but not all chemicals are drugs. The ability to bind to a receptoris mediated by the chemical structure of the drug that allows it to interact withcomplementary surfaces on the receptor. 6rugs that interact with receptors can beclassified as being either a&o!ists or a!ta&o!ists . $nce bound to the receptor anagonist activates or enhances cellular activity. 7!amples of agonist action are drugs thatbind to beta receptors in the heart and increase the force of myocardial contraction ordrugs that bind to alpha receptors on blood vessels to increase blood pressure. The

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binding of the agonist often triggers a series of biochemical events that ultimately leadsto the alteration in function. The biochemicals that initiate these changes are referred toas se o!' %esse!&e#s . A!ta&o!ists have the ability to bind to the receptor but do notinitiate a change in cellular function. Because they occupy the receptor* they canprevent the binding and the action of agonists. ence the term antagonist. 5ntagonists

are also referred to as $lo ,e#s.

-a to#s o)e#!i!& D#"& A tio!

Two factors that determine the effect of a drug on physiologic processes are a((i!ity and i!t#i!si a ti)ity .

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A((i!ity is a measure of the tightness that a drug binds to the receptor.

I!t#i!si a ti)ity is a measure of the ability of a drug once bound to the receptor togenerate an effect activating stimulus and producing a change in cellular activity.

5ffinity and intrinsic activity areindependent properties of drugs. 5gonists have $oth affinity* that is*the ability to bind to the receptor*as well as intrinsic activity* theability to produce a measurableeffect. 5ntagonists* on the otherhand* o!ly ha)e a((i!ity for thereceptor. This property allowsantagonists to bind to the receptor.

owever* because antagonists donot have intrinsic activity at thereceptor no effect is produced.Because they are bound to the

receptor* they can prevent binding of agonists. This is a diagram of a 0protein coupled receptor.8otice how the amino acids that make up the receptor protein can contribute functional groups toallow a drug to bind to this receptor.

The binding of a drug to a receptor is determined by the following forces:

1. ydrogen bonds

2. 9onic bonds

+. an der 3aals forces

'. &ovalent bonds

/!'e#sta!'i!& A((i!ity

To bind to a receptor the functional group on a drug must interact with complementarysurfaces on the receptor. The binding of a drug* illustrated here as 6* to the receptor*illustrated as ;* can be described by this e!pression.

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This is a reversible reaction and when at e uilibrium* the rate of drug0receptor comple! formation 0, or 1> nanomolar. There is an i!)e#se#elatio!ship between the ?d and affinity. The smaller the ?d* the greater the affinity. 5drug that has a dissociation constant of 1 nanomolar is said to have higher affinity than adrug that has a dissociation constant of 1 micromolar. This is because 1 nanomolar ismuch smaller than 1 micromolar.

By appropriate substitution of the e uations above we can write:

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This e0"atio! 'es #i$es the $i!'i!& o( '#"&s to #e epto#s a!' states that thea%o"!t o( '#"& $o"!' to the #e epto# is 'epe!'e!t o! the drug concentrationand Kd.

Question: WHAT percentage o the total receptor population !ill "e occupied

!hen the concentration o the drug is e#ual to the dissociationconstant$ Help %olution

This points out that when a drug is given at a concentration e ual to its dissociationconstant* ->@ of the receptors will be occupied. The greater the affinity* the less drugwill be re uired to occupy ->@ of the receptors.

/!'e#sta!'i!& the Co!se0"e! es o( Re epto# O "pa! y

9t is apparent that for a drug to produce an effect it must first bind to a receptor. To

understand the relationship between receptor occupancy and the generation ofmeasurable physiologic effect* we make the assumption that magnitude of thephysiologic response A E1 is proportional to the amount of drug bound to the receptorA

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activity describes the ability of a drug induce changes in receptor structure leading toalterations in cellular activity. 3e can now write:

Therefore* the ability of a drug to produce a physiologic effect is dependent on receptoroccupancy Awhich is in turn governed by D3 a!' K' and the propensity of the drug toactivate the receptor A e . 3hile similar* you should understand that e uations C1 and C2calculate different parameters. 7 uation C1 determines the degree of #e epto#o "pa! y . 7 uation C2 Awith the presence of e calculates the effect of a '#"& o! a("! tio!al #espo!se .

Question: AT WHAT concentration o agonist !ill the e ect &'('ma)* "e e#ual to

+ - o the ma)imal response$ .Help Sol"tio! +This concentration is also referred to as the Effective Dose-50 or ED 50 .

-"ll a!' Pa#tial A&o!ists

3hile the precise mechanism is not known* agonists have the ability to impart a

stimulus to the receptor such that cellular signaling is activated. 5gonists differ in theirpropensity to deliver an activating stimulus to receptors. 5s a result* agonists can befurther divided into ("ll and pa#tial agonists:

-"ll A&o!ists : &ompounds that are able to elicit a ma!imal response following receptor occupation and activation.

Pa#tial A&o!ists : &ompounds that can activate receptors but are unable to elicit thema!imal response of the receptor system.

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6rugs which are full agonists are arbitrarily assigned an intrinsic activity value of 1.Dartial agonists* which cannot produce the same ma!imal effect as full agonists willhave intrinsic activity values less than 1. The effect of partial and full agonists one uation C 2 is apparent. Because partial agonists have e values less than 1* the valueof 7/7ma! will be some fraction of the value obtained with a full agonist.

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Dose Respo!se C"#)es

6ose0response relationships are a common way to portray data in both basic andclinical science. Eor e!ample* a clinical study may e!amine the effect of increasingamounts of an analgesic on pain threshold. To present the data* the concentration of the

drug would be plotted on the !0a!is and the effect on pain threshold would be presentedon the y0a!is. 5 plot of drug concentration A> to 1>>>0fold. This necessitatesa long F0a!is. To overcome this problem* the log of the drug concentration is plottedversus the effect. 5 plot of the log of

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&lonidine and etho!amine arepartial agonists. &lonidine has ahigher affinity but a lower intrinsicactivity than does etho!amine.9ntrinsic activity affects the

magnitude of the response.

Spa#e Re epto#s

Thus far we have made the assumption that the relationship between receptoroccupancy

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Because of this hyperbolicrelationship betweenoccupancy and response*ma!imal responses are elicitedat less than ma!imal receptor

occupancy. 5 certain number of receptors are Gspare.G Hparereceptors are receptors whiche!ist in e!cess of thosere uired to produce a fulleffect. There is nothingdifferent about spare receptors.They are not hidden or in anyway different from otherreceptors.

5ssume an agonist with a ? 6 I -> n and an eI1.

!n a linear occupancy response system !n a non linear occupancy"responsesystem with #$ %.& and #$2

'ccupancy (esponse

%) nM $ %*2) nM $ 284) nM $ 44&) nM $ &)

%)) nM $ **2)) nM $ 8)

1J2,''->JJ,>

'ccupancy

(esponse#$%.&

(esponse#$2.)

1> n I 1J2> n I 2,'> n I ''-> n I ->1>> n I

JJ

2>> n I,>

2&42**+&,,

%))

-2&*88

%))%))

%))

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5I igh ;eceptor ;eserve

BI edium ;eceptorreserve

&I8o ;eceptor ;eserve

A!ta&o!ists

A!ta&o!ists e5hi$it a((i!ity for the receptor$"t 'o !ot ha)e i!t#i!si a ti)ity at thereceptor. 5n antagonist that binds to thereceptor in a reversible mass0action manneris referred to as a o%petiti)e a!ta&o!ist .Because the antagonist does not haveintrinsic activity* once it binds to the receptor*it blocks binding of agonists to the receptor. 5

key point about competitive antagonists is that like agonists* they bind in a #e)e#si$le%a!!e# . This has important implications regarding the effect competitive antagonistshave on the configuration of the dose0response curve of agonists. Because competitiveantagonists bind in a reversible manner* agonists* if given in high concentrations* candisplace the antagonist from the receptor and the agonist can then produce its effect.The antagonist action can* in effect* be s"#%o"!te' . Because the antagonist can becompletely displaced* the agonist is still able to produce the same ma!imal effectobserved prior to antagonist treatment. owever* because higher agonistconcentrations were necessary to displace the antagonist* the agonist dose0responsecurve is shifted to the right in the presence of a competitive antagonist. This can beillustrated with two e uilibrium e uations:

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The antagonist B3 and agonist D3 are competing for the same limited number ofreceptors R3. The drug that binds to the receptor in the highest concentration will bedetermined by two factors.

These factors are the a((i!ities o( the a&o!ist a!' a!ta&o!ist (o# the #e epto# a!'thei# #elati)e o! e!t#atio!s . 9n the presence of a competitive antagonist e uation C2is modified as follows:

3here B3 is the concentration of antagonist and K$ is the affinity e!hibited by theantagonist for the receptor. 9nspection of this e uation will reveal that the affinity of theagonist* ?d* is modified by the term 67 B39K$1. 9f the concentration of antagonist

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'. The agonist dose0response curve in the presence of a competitive antagonist isdisplaced to the right parallel to the curve in the absence of agonist.

I##e)e#si$le Re epto# A!ta&o!ists

5nother type of antagonist is referred to as an i##e)e#si$le #e epto# a!ta&o!ist . Theproperties of irreversible antagonists are markedly different from competitiveantagonists. 9rreversible receptor antagonists are chemically reactive compounds.These ligands first bind to the receptor. Eollowing this binding step* the ligand thenreacts with the functional groups of the receptor. The conse uence of this chemicalreaction is that the ligand becomes covalently bound to the receptor. Because achemical bond is formed* an irreversible ligand does not freely dissociate from thereceptor. 9t remains attached to the receptor for a long period of time. The synthesis of

new receptor protein may be re uired to generate a receptor free of an irreversibleblocker. Because the ligand is covalently bound to the receptor* the binding of agonists*and hence their pharmacologic activity* are blocked. /!li,e o%petiti)e a!ta&o!ists;the $lo ,i!& a ti)ity o( i##e)e#si$le #e epto# a!ta&o!ists a! !ot $e o)e# o%e $yi! #easi!& the a&o!ist o! e!t#atio! . The antagonism therefore cannot be overcomeby increasing the agonist concentration. ;ecall* that the effect of an agonist isproportional to the active drug0receptor comple!es formed. Because an irreversiblereceptor antagonist reduces the total number of active receptors*

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Appli atio!s to The#ape"ti s

Eew drugs interact with one and only one receptor. Huch a drug would be said to be spe i(i * that is producing effects by specifically interacting with a single receptor. ostdrugs interact with several receptors and thus have the capability to produce distinctlydifferent pharmacologic effects. Home of these effects could be beneficial* some couldbe to!ic. Huch a drug would be said to be a sele ti)e. The factors that determine whichparticular effect of a drug will be observed are the a((i!ity and i!t#i!si a ti)ity o( a'#"& .

To illustrate this point consider the following e!ample. 5 drug is capable of producingactions at 2 distinct receptors. 5t each of these receptors* the ligand has a differentaffinity as well as pharmacologic effect.

Re epto# Syste% < 7+

? 6 I '>.>* intrinsic activity 1.>*effect0 lowering of systemic arterial blood pressure.

Re epto# Syste% < 2+

? 6 I '>.>* intrinsic activity 1.>*effect0 lethal ventricular arrhythmias.

Thus* this drug could either be a highly beneficial therapeutic agent or a lethal poison. 5n overwhelming ma%ority of drugs used in clinical practice produce their therapeuticeffects due

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to interactions at multiple pharmacologic receptors. This also illustrates that whether thedrug will be beneficial or poisonous depends on the skill and knowledge of the individualprescribing the agent.

The The#ape"ti I!'e5

The therapeutic inde! is the ratio of the 76-> of a drug to produce a to!ic effect to the76-> to produce a therapeutic effect. Eor the drug e!ample above* the 76-> for thebeneficial effect of blood pressure lowering is >.' n while the 76-> for to!icity is '>n . Therefore* the therapeutic inde! will beL

T9I

76 ->Ato!icity

76 ->Atherapeutic

I '>.>n>.'n

I 1>>

A')a! e' Co! epts Re&a#'i!& Pa#tial A&o!ists

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Dartial agonists have lower intrinsic activities than full agonists but values greater thancompetitive antagonists. 5t certain concentrations partial agonists actually can beantagonists.

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Reaso!s (o# the =o!li!ea# Relatio!ship Betwee! Re epto# O "pa! y a!'Physiolo&i Respo!se

To understand how the relationship between occupancy and response can be non linear let us analy e the components which contribute to the response.

P#otei! Co"ple' Re epto#s

0protein coupled receptors are a large family of receptors that serve as the site ofaction for many drugs. The name reflects the fact that the activity of these receptors isregulated by interaction with &"a!i!e !" leoti'e #e&"lato#y p#otei!s Ahence 0proteins . 6espite ma%or differences in the physiologic responses they activate and thevariety of second messengers involved* the structure of all 0protein coupled receptorsis similar. 0protein coupled receptors have a single polypeptide chain which passesthrough the cell membrane seven times. This arrangement results in the formation ofloops on both the e!tracellular and intracellular sides of the membrane. Heven clustersof hydrophobic amino acids make up the membrane spanning domains of the receptor.The membrane spanning regions also form a binding pocket with which agonists andantagonists interact. The intracellular loops are thought to be necessary for theinteraction with 0proteins and second messenger systems.

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The p#otei! Re&"lato#y Cy le

9n cellular signaling pathways involving 0proteins* thereceptor/agonist comple! does not interact directly withthe en yme which generates the second messenger.

;ather* an intermediate or transducing protein couplesthe receptor to the second messenger generating system.This is the role of the 0protein . There is not a single 0protein* but a family of 0proteins which functions toregulate second messenger systems. p#otei!s o!sisto( th#ee s"$"!its+ alpha; $eta a!' &a%%a. 9n theresting state the receptor is not occupied by an agonistand the 0protein e!ists as trimer of the alpha* beta andgamma subunits with 6D bound to the alpha subunit. 9nthis state* 0proteins are poor activators of intracellularsignaling systems. 5gonist binding to the receptor

promotes the dissociation of the 6D and binding of TD.TD binding promotes the dissociation of the alphasubunit from the beta and gamma subunits. 9t is the TDbound alpha subunit that activates effector en ymesystems. The alpha subunit is also a TDase and is thusable to hydroly e the TD. The hydrolysis of TD to 6Ddeactivates the alpha subunit and terminates theactivation effector systems. The alpha subunit/ 6D

comple! is then re0associated with the beta and gamma subunits to complete theregulatory cycle. The 0protein heterotrimer is again available for interaction with areceptor and activation of second messenger generating systems. Therefore* the rate at

which the TD is hydroly ed regulates the time the 0protein is active. The longer the0protein is active* the more second messenger can be generated

9n a responding system which has a linear relationship between occupancy andphysiologic response* there is a direct proportionality between the degree of receptor

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activation and the generation of second messengers. 3hile this is difficult toconceptuali e* it can be thought of as a small amount of receptor occupancy producinga small increase in the level of the second messenger. This small amount of secondmessenger activates a small increase in physiologic response. 9n the more realisticnonlinear occupancy versus response system* a small degree of receptor occupancy

generates a large increase in second messenger levels which in turn generate an evenlarger physiologic response. The signal is amplified at every step of the signaltransduction process. 9n this fashion* then* a small degree of receptor occupancy leadsto a large physiologic response. &onsider the following e!ample. 9n a given beta0receptor system* ->*>>> c5 D molecules are needed to yield a full response. 9n a linear response relationship* ->*>>> receptors would have to be occupied to give a fullresponse. owever* in a nonlinear system* only 1>> would be re uired to achieve a fullresponse.

Re&"latio! o( Re epto# -"! tio!

&ontinuous e!posure of an agonist results in a phenomenon referred to asdesensiti ation. The same concentration of agonist becomes less and less effective atproducing the same level of effect. 3hen this desensiti ation occurs very rapidly* it isreferred to as tachyphyla!is. ;ecent evidence has suggested potential mechanisms bywhich the process of tachyphyla!is and desensiti ation occur. The receptor becomes

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phosphorylated in the third cytoplasmic loop and c0terminal tail. The phosphorylatedreceptor is less efficient at activating 0protein and also e!hibits lower affinity foragonists. The receptors can also be removed from and se uestered away from the cellsurface. These events indicate that second messengers not only regulate intracellularprocesses but are also capable of regulating the receptor systems which generate

them.

I!)e#se A&o!ists

Traditionally* 0protein coupled receptors were thought to be inactive and that agonistoccupation was re uired to allow the receptor to assume an active conformation.;ecently* though* it has been suggested that the receptor can be active without thepresence of agonist. The term for this is constitutive activity. &onstitutively activereceptors are thought to be coupled to second messenger pathways in the absence ofagonists.

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This has led to the postulate that in addition to traditional agonists* drugs can functionas inverse agonists. 9nverse agonists bind to constitutively active receptors and shift thee uilibrium to the formation of the inactive conformer. 9n this system an inverse agonistwould actually reverse receptor activity. The concept of inverse agonism has added alevel of comple!ity to our thinking of drug action. 5s the diagram below illustrates* thespectrum of drug activity can range from a full agonist to a full inverse agonist.

The relevance of constitutively active receptors and inverse agonists to normalphysiology and pathophysiology has not been established. That being stated* theconcept of a constitutively active receptor does offer insights which could help to e!plain

pathophysiologic conditions. 9f the process of disease induced the e!pression of aconstitutively active receptor* the receptor would no longer be under the influence of thesympathetic nervous system. This could occur in hypertension with a constitutivelyactive D&; being e!pressed in any number of areas including the brain* kidneys orperipheral blood vessels. 9n this scenario* drugs with inverse agonist properties couldprove to be safe* rational therapeutics.

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&omments to Htephanie 7delmann * #ast odified:&opyright M 2>>>* Nniversity of ?entucky &handler edical &enter Terms* &onditions O Drivacy Htatement

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