cell and molecular pharmacology

lOMoARcPSD|6683673




• Positive allosteric modifiers (PAMs) which increase the

Cell and Molecular Pharmacology negative allosteric modifiers (NAMs) show promise as

CNS disorders
Lecture 1- Receptors and ion channels as targets for drug action
• PAMs and NAMs can only change the activity of the re

• Key proteins that act as targets for drug action: receptors, ion channels, enzymes,
activated so in the presence of the agonist.


transporters
• Non biased PAMs/ NAMs don't modulate a specific pa

• Receptor- a protein upon which agonist drugs act to initiate a cellular response. The
modulate a specific pathway from the receptor


receptor comprises a ligand binding site plus response elements Enzyme linked receptors

• A single protein complex can act as a receptor for more than one class of agonist such as
• Includes receptor tyrosine kinases and guanylyl cyclas

the GABA A receptor which has binding sites for GABA and benzodiazepines.  Receptors
• Binding of an agonist to an extracellular binding site le

are still main target of drugs intracellular, enzymatic 'response unit'

• Response element- whatever changes in the receptor to trigger a response
• Contain single transmembrane domain, though the activ

dimer. Some dimers are preformed such as the insulin
G-protein coupled receptors

• G-protein coupled receptors are the largest superfamily, they are also the most common
• Acts as receptors for a variety of polypeptide ligands

growth factors
type of receptors in which drugs bind

• All have 7 transmembrane spanning domains
• Chain of events to activate tyrosine- kinase receptors


• The response element activates a GTP binding protein on agonist binding
dimerization or a change in conformation of pre-forme


• There are 4 classes (A-D)
trans-phosphorylation of tyrosine residues on C-termin

enzyme activity but on dimerization bind tyrosine kinas
• Class D are vomeronasal pheromone receptors so are not considered here as they are
phosphorylate each other, the phosphorylated recepto
mostly only found in animals
sites for intracellular effector proteins that contain p
• Class A (Rhodopsin like) have a ligand binding pocket which is amongst the extracellular
such as SH2 domains, effector proteins with SH2 dom
ends of the transmembrane helices. There is a short extracellular N-terminal. Examples
• DAG and activates a MAP kinase pathway
include muscarinic receptors, adrenoceptors, dopamine receptors
• Receptor guanylyl cyclase such as for ANP act in a bro
• Protease activated receptors (PARs) are a type of Class A but have a unique activation
dimerization activates guanylyl cyclase activity with th
mechanism. Thrombin irreversibly cleaves the extracellular N-terminal tail to expose a
Nuclear receptors
new n-terminus that acts as a tethered ligand that activates the receptor.
• Regulate DNA transcription
• There are currently 4 types of PARs (1-4) which have slightly different cleavage sites
• Receptor usually found in nucleus though oestrogen rec
and tethered ligands
activation is chaperoned to the nucleus by HSP90
• All PARs are activated by thrombin except PAR-2 which is activated by trypsin
• Ligands include steroid hormones, thyroid hormones an
• Generally synthetic peptides that mimic the first six amino acids of the new tail act as
soluble to get into nucleus
agonists
• Ligand binding often induces dimerisation and activatio
• PAR-1 acts when thrombin removes a 41aa peptide creating the active tethered ligand
• Activated receptor binds selectively to hormone respo
(SFLLRN). The receptor couples to a variety of G-proteins with evidence of agonists
(act as DNA binding proteins)
demonstrating functional selectivity. Signalling is terminated by B-arrestin mediated
• More than 150 nuclear receptors have been identified
receptor desensitisation and receptor internalisation

• Vorapaxar is a PAR-1 reversible competitive antagonist approved for prevention of
Receptor operated (ligand gated) ion channels

• The response element incorporates an ion channel, ope
thrombotic cardiovascular events- it is antagonising a tethered ligand.

• • Mediate rapid electrophysical responses
Class B (Glucagon like) receptors have an intermediate length N terminal tail which

incorporates the ligand binding site and contains 6, well conserved cysteine residues
• Includes NAChR, ionotropic glutamate receptors, vanil


• Examples of class B- CGRP receptor
• All are multimers comprised of several (4/5) subunits

• The functional CGRP receptor is formed from two proteins, the 7TM component (CRLR)
• Also includes IP3 and ryanodine receptor on endoplasm

and the receptor activity modifying protein (RAMP1)

• RAMPs have now been shown to be able to interact with a range of class B GPCRs. The

protein cannot function without the RAMP which is a single TM domain protein which

subtly monitors their activity

• RAMP can change the same receptor to have a different function depending on the RAMP

• Class C (metabotropic glutamate like) receptors have a long N-terminal tail that

incorporates the ligand binding site and acts like a venus fly trap to close in on the ligand

• Examples of class C- GABA B receptor

• Functional GABA B receptors are formed as dimers of a GABA B1 and B2, these both

, lOMoARcPSD|6683673




Lecture 2- Radioligand binding less than the actual number of disinteg

dpm can be calculated by dividing the c

• These assays investigate the interaction between a drug and a binding site. 7. The dpm is directly proportional to the

However, they do not provide any information as to whether the drug is an agonist sample. The specific activity of the rad

or antagonist etc. convert the dpm from a sample into the

• Bmax- number of receptors present • Scintillation proximity assay (SPA) is a bead based a

• KA- affininty constant scintillant so there is no need for filtration. It emits l

• KD- dissociation constant particle

• K+1- forward rate constant
• Receptor density is often expressed as fmol/mg prote

• KA is also equal to the forward rate constant divided by the backward rate
makes an allowance for differences in the number of c


constant
• Bmax maximal binding capacity is carried out by differ

• KD is 1/KA so can also we determined by the backward rate constant divided by
lignad are incubated with a aliquots of homogenate con

protein or number of cells. As the ligand concentration
the forward rate constant

• At high concentrations of ligand then all receptors will be bound= Bmax
increases


• • A bmax experiment can be used to detect changes in r
KD is defined as the concentration of ligand which occupies 50% of the receptors
regions or tissues of the body including brain regions,
ie occupies 50% Bmax

• The units of KD are concentration
species, in the same specified at different stages of d


• A Scatchard plot linearly shows the saturation curve. It can be used to work out
pharmacological or other interventions

• KD is constant under specified conditions and is known
KD
constant for the reaction. It is a measure of the chem

• The numerical size of KD varies inversely with the ene

for a particular drug recepror interaction

• Low KD- high affinity

• High Kd- low affinity

• Reasons for determining Kd- to identify receptor subt

affinity following pharmacological or other interventio

• IC50- the amount of unlabelled drug required to redu

to 50% of its value in the absence of the displacer

• The Cheng and Prusoff equation is used to find the rel

displacing drug and its binding affinity for the recepto

• Ki = IC50/(1+(L/Kd)) where L is the concentration o

dissociation constant of the radiolabelled ligand and KI

the binding of the unlabelled displacer for the recepto

• In displacement experiments, a high concentration of

• Radioactive particles spontaneously breakdown over time and give off energy- able to prevent all binding of the radioligand to the re

disintegration • This residual radioactivity is not regarded as represen

• Radioactivity is quantified by the number of disintegrations in unit time receptor and is termed non- specific binding (NSB)

• 1Bq- 1dps 1TBq-6x1013dpm
• NSB may represent drug absorbed by membrane fragm

• 1Ci- 2.22x1012dpm
released by washing or material bound to non-receptor


• • NSB is said to represent very low affinity binding to h
Specific activity is amount of radioactivity associated with an amount of
linerarly with ligand concentration
compound

• • NSB is easy to control for
Units of specific activity- TBq/mmol or Ci/mmol
• Specific binding= total binding- NSB
• Labelled ligands are normally either tritium (H3) or iodine 125

• • Saturation curves are rectangular hyperbolae but can
Tritium gives off a beta particle which is low energy, it is stable and does not
generating a Hill plot which generates hill coefficients
alter the properties of drug

• • If the interaction between the drug and the receptor
Iodine 125 is high energy, relatively unstable, can alter properties of drug, easy
then nH should be unity
to detect and there is a need to protect workers

• • If the nH is significantly between 1 and 0.9 this can in
The ideal radioligand for a binding assay is a compound, has high affinity for the
the binding of the drug decreases the likelihood of fur
receptor, high selectivity for the receptor site, low binding to other non-receptor
• If the nh is between 1 and 1.1 this can indicate positive
binding sites and is high stability

• There are three types of assay- kinetic experiments, saturation experiments and
drug molecule increases the likelihood of further bindi

• Another interpretation of a low nH is that this is evide
displacement assays

, lOMoARcPSD|6683673




Quantitative PCR • Can make a 3D model of a receptor based on informati

• Standard PCR tells you if the gene is there but not how much of it is there find the binding site of a ligand based on the structure

• qPCR monitors the reaction in real time • Allows you to screen compounds on the computer

• The reaction is monitored by fluorescent markers which only fluorescence once bound to • This can then be checked with traditional binding expe

the product therefore the fluorescence increases after each cycle

• For good quantitation a standard curve or a stable reference gene is required Lecture 4- Pharmacological Analysis of Receptors

• A threshold is used on the standard curve to see how many cycles it takes each sample to

reach the threshold, the less cycles it takes the more of the gene of interest there is in
• Scatchard plot is based on the equation: B/Xd=Bmax/K

the sample
• The slope of the Scatchard plot is KD

DNA microarrays
• Where it interceptors the x axis is the bmax

• Uses DNA hybridisation where one stand is anchored to a substrate sample labelled with
• Kd is a physiological constant like Avagadro’s constant

fluorophore, when there is that gene it fluoresces a colour
• The kd is the same for a given receptor and drug comb

• Can do a 2 channel or normal cells vs tumour cells species, anywhere in the universe

• Label each set with a different colour probe
• The Kd can therefore be used to identify an unknown r

• Gives a large amount of raw data
• The Hill Langmuir equation Pa= (Xa/Ka)/(Xa/Ka + 1)


X-ray crystallography
• Ka is a measure of agonist affinity

• A traditional method
• Ka is the concentration of agonist required to occupy 5

• A large crystal of the protein is made and placed in a powerful x-ray beam which gives
• Ka is a function of agonist and receptor combination

off an image which you can predict the structure from
• An agonist binds to a receptor and elicits a response

• It is very difficult to crystallise lipophilic proteins such as membrane proteins, so these
• The EC50 is the effective concentration which produc

cannot be analysed by this technique a functional measure and a product of both affinity an

• The X-ray beam and/or -196 degrees temperature can destroy the protein so giving you
• IC50 is the concentration needed to produce 50% inhi

no results
• The hill slope is described by the slope of the sigmoid

Serial Femtosecond crystallography describes the numerical relationship between ligand an

• The new way of doing X-ray crystallography
• A hill slope of 1 could mean a one to one relationships b

• Uses micro crystals and shines free electrons as a laser, they are pulsated at
of more than 1 could mean more than 1 ligand per rece

couple mean less than one ligand per receptor
femtosecond duration and the results are analysed as before

• Does not require large crystals
• EC50 does not give you the KA because most receptor

• Less energy applied to protein so less damage
give the response e.g. more than one ion channel openin

• Can be done at room tmep so the structure is more like normal
• Partial agonists- has affinity for the receptor but low

• Can be used on X-ray sensitive proteins
maximum response


Transgenic animals
• A partial agonist is so because there is a flip state bet


• Usually mice are used as they are cheap
and having efficacy


• • Inverse agonist- reduce the number of activated rec
Can be made knock out or knock in

• • Bias receptors- activate more than one messenger sy
Norally done in the germline phase however this can be complicated as the gene may be
than another
used in development of the animal so the animal might be changed in other ways

• • Competitive reversible agonists- have affinity but ze
Temporal transgenic can be used so that the gene isn’t knocked out until later in life so
receptor but induces no response. It binds in such a wa
that the animal grows up as the normal

• • KB is the KD for antagonists
Spatial transgenic can be done to target the gene in only a specific area such as muscle
• pA2- the negative log of the concentration of antagon
so it is much more specific and can see the effect of a receptor in just one location if it
concentration response curve 2-fold to the right
is normally in many

RNA interference
• pA2= KD, this means we do not have to do binding expe


• Uses a natural antiviral defence mechanism in cells
of the KB


• • pA2 is independent of the agonist used
Long double stranded RNAs specific for a target gene can be used to silence the
• The KB is better at defining receptor affinity than th
expression of the target gene in a variety of cell types via RNA interference pathway

• dsRNAs get processed by RNAase III enzyme and endoribonuclease DICER into small
binding data (KB) with functional data (pA2), competit

and the same receptor coupled to different G proteins
interfering RNAs

• • Competitive irreversible antagonists- has zero effica
siRNAs assemble into RNA induced silencing complexes

• activated RISCs bind to complementary transcripts by base pairing interactions between
induces no response however it binds in suc a way to pr

permanently bound to the receptor
the siRNA anti-sense strand and complementary mRNA then bound mRNA is degraded
• Irreversible competitive antagonists are time depende
resulting in gene silencing

• • The Furchgott analysis uses an irreversible competitiv
can target genes to find out what is switched off and also to find out how second