08/20/2015 SIM on Mechanisms of Drug Action

7:30 – 11:30 Principles and Perspectives

YL6: 01.24.01 Cecilia A. Jimeno, MD, FPCP, FPSEM

2
OUTLINE In all cells, two constituents exist :
I. Introduction IV. Enzyme-Associated • Effector or Target Tissues/Organs: substances
A. Concepts Related to Receptors concerned with carrying out chief functions of the cells
Receptor-Ligand A. Six Types • Receptors: liable to change and capable of setting the
Interactions B. Overview of the chief substance in action
B. Properties of Mechanics → Drugs combine with receptors and not on axon-endings
Receptors C. Insulin Receptors nor direct action on the chief substance
C. Cell-Surface V. Transcriptional
Receptors Regulator Proteins B. PROPERTIES OF RECEPTORS
II. Ion Channel Receptors A. Estrogen • Saturability – receptors are present in finite numbers
A. Nicotinic- VI. Regulator of Receptor • Reversibility – the binding of a drug to its receptor is a
Acetylcholine Function noncovalent interaction due to weak intermolecular forces
Receptor A. Process of
• Stereoselectivity – many naturally occurring agonists
B. GABA Receptor Regulation
exist as optical isomers (+ or -, 1 or d, S or R)
III. Seven-Transmembrane VII. Non-Receptor Mediated
→ The receptor should show a preference for the naturally
Domain Receptors Drug Actions
occurring stereoisomer (e.g. l- vs d- norepinephrine)
A. G protein-linked A. Metal Chelators
• Agonist Specificity – receptors show a preference for
Pathway B. Alkalizing Drugs
specific or closely-related agonists and not recognize
B. Adrenergic C. Acidifying Drugs
chemically dissimilar endogenous agents
Transmission Review Questions
C. Cholinergic Freedom Space • Tissue Specificity – binding should be present in tissues
Transmission References biologically sensitive to hormone or drug
Appendix → Binding should take place at concentrations consistent
with the physiological concentrations of the
endogenous agents
I. INTRODUCTION
2
Langley Experiment (1905)
A. CONCEPTS RELATED TO RECEPTOR- • Curare and Nicotine were given through systemic
LIGAND INTERACTIONS injections to experimental animals (chicks)
→ When injected, both drugs produce paralysis
• Observations made by the investigator:
→ Additional Nicotine after paralytic injection: produced
Generation( Change(in( more contraction of certain muscles
Agonist( Receptor( of(2nd( cellular(
messenger( activity(
→ Co-administration of Curare: potently reduced nicotine
response
→ After isolating the muscles by severing the nerves, the
Figure 1. Receptor-drug interactions leading to changes in cells. same results were found
The binding of agonists (endogenous substances or drugs) with their • Conclusions:
receptors elicits generation of second messengers. These signaling → Direct electrical stimulation of a muscle would cause
mechanisms lead to changes in cellular activity.
contraction despite the pharmacological paralysis
→ Contractible substance is different from drug-
• Agonist – initiates a change in cellular activity or elicits a
sensitive substance of the protoplasm
biological effect once complexed with a receptor
nd → Protoplasmic receptive substance must exist which
→ Binding usually triggers release of 2 messengers
the drugs compete for directly
which lead to change in cellular function
• Antagonist or blockers – bind to receptors and inhibits B. CELL SURFACE RECEPTORS
the biological effect of an agonist but do not initiate a
change in cellular function and devoid of any biological
activity
• Two factors which determine whether a chemical agent
will have effects as a drug:
→ Affinity – measure of the tightness with which a drug
binds to a receptor
→ Intrinsic Activity – measure of the ability of a drug to
produce physiologic effects once bound to a receptor
• Agonists have both affinity and intrinsic activity while
antagonists usually only has affinity for the receptor.
• Ligands – agents that bind to receptors and include
drugs, hormones, autacoids, growth factors,
neurotransmitter, etc.
• Receptor – any cellular molecule that a drug binds to Figure 2. Signaling mechanisms involving cell-surface receptors. A.
initiate its effects Ligand binds to an ion channel receptor B. Ligand binds to a domain
of a transmembrane receptor, which is coupled to a G protein. C.
Ligand binds to the extracellular domain of a receptor that activates a
kinase enzyme. D. Lipid-soluble ligand diffuses across the membrane
to interact with its intracellular receptor. (Lippincott’s Illustrated
Reviews Pharmacology, 5e)

YL6: 01.24.01 Group 12: Buenaseda, Cabaña, Fernandez, Hernandez, Jimenez, Martinez, Medina, Neric, Perez, Quiwa, Viloria 1 of 13
1. Ion-channel receptors aka ligand-gated ion channels
2. Seven-transmembrane Domain (G-protein coupled)
3. Enzyme-associated receptors
4. Transcriptional regulator proteins aka Intracellular
receptors
II. ION CHANNEL RECEPTORS
• Also known as ligand-gated channel, channel-linked
receptors or ionotropic receptors
• Receptor and ion channels are closely associated in the
membrane
• Binding of the agonist opens the channel causing net
movement of ions into or out of the cell
• The membrane events are triggered by a change in
membrane potential as a result of the change in
intracellular ionic concentration
A. NICOTINIC ACETYLCHOLINE RECEPTOR
Figure 3. Nicotinic acetylcholine (Ach) receptor, with extracellular
fluid above and cytoplasm below. Composed of five subunits, the
receptor opens a central transmembrane ion channel when Ach
binds to sites on the extracellular domain of its α (alpha) subunits
(Katzung’s Basic and Clinical Pharmacology, 13e)
Site and Mechanism of Action
• 5 sub-units: 2 α (alpha), 1 β (beta), 1 γ (gamma), and 1 δ
(delta)
• Acetylcholine binds to the α-subunits of the receptor
• This causes a conformational change that allows bulk
+
influx of Na
2+ +
• Some Ca also flows in, and some K flows out
• 2 ACh molecules are required for channel to open,
one for each α-subunit
Physiological Effect: Muscular Contraction at
the Neuromuscular Junction
Figure 4. Nicotinic transmission at the skeletal neuromuscular
junction (Google Images)
YL6: 01.24.01 SIM on Mechanisms of Drug Action: Principles and Perspectives
1. Acetylcholine (ACh) from choline and acetate is
synthesized
2. ACh is released from the nerve terminal
3. It then crosses the synapse and binds to acetylcholine
receptors in muscles
4. Acetylcholine binds to the 2 alpha subunits of the
pentameric acetylcholine receptor
• This step can be inhibited by Tubocurarine
5. Conformational change of the receptor happens allowing
+
sodium ions (Na ) to enter
+
6. Na influx produces an excitatory postsynaptic potential
(EPSP)
7. The EPSP depolarizes the muscle membrane, generating
an action potential
• Resting membrane potential of Na-K pump: -90
millivolts (mV)
+ +
• Depolarization: Na channels open leading to Na influx
(-90mV → +)
+ +
• Repolarization: Na channels close, K late opening, K+
efflux repolarizing membrane potential (+ → -90mV)
+ +
• Hyperpolarization: K channels still open, K efflux
hyperpolarizing membrane potential
+
• K late closing followed after by the returning to resting
membrane potential
8. Depolarization triggers contraction.
9. Acetylcholine is broken down by acetylcholinesterase
(found in the membrane) into choline and acetate
10. The cycle repeats
Clinical Uses: Tobocurarine
• A competitive neuromuscular blocker
• Binds to and antagonises nicotinic ACh receptors on
postsynaptic nerve terminal
→ Without the binding of ACh to the receptor, there would
be no influx of sodium ions
→ As a result, depolarization does not occur leading to
inhibition of skeletal muscle excitation and contraction
• They can bind to receptors but are not flexible enough to
allow channel opening
• Administered intravenously; used in surgery for
prolonged relaxation and to stop reflex actions
→ Onset is 2-3 minutes and effects last from 40-60
minutes
→ NOT metabolized
→ Excreted via the bile (30%) and urine (70%)
• Causes flaccid paralysis of:
→ Extrinsic eye muscles (double vision)
→ Small muscles of face, limbs, pharynx
→ Respiratory muscles (hence, patients should be on
ventilator)
• Does not:
→ Affect consciousness
→ Affect pain sensation
→ Cross the blood brain barrier
• Side effects include:
→ Histamine release due to ganglion block which can lead
to bronchospasm
→ Hypotension and tachycardia
→ Excessive brochial and salivary excretions.
• NOT being used any more because the new
alternative drugs are more interesting
→ The effect of new drugs appears more quickly after
their administration and ceases more frankly after their
discontinuation.
→ New drugs are less ganglionic blocking agents and less
histamine liberators.
→ The drugs used currently are pancuronium,
vecuronium, tracrium, rocuronium, mivacurium and
cisatracurium
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 B. GABA RECEPTOR
Figure 5. GABA Receptor (google images)
• Gamma Aminobutyric Acid (GABA) receptor
• Major inhibitory neurotransmitter in the CNS
• Macromolecular protein composed of a chloride ion-
selective channel
• Binding sites for GABA, barbiturates, and
benzodiazepines
→ Barbiturates and Benzodiazepines – enhance
GABAA receptor function through different allosteric
regulatory sites
• Composed of a combination of different isoforms of α, β,
γ, δ, polypeptide subunits
Site and Mechanism of Action
• Released from GABA-containing neurons
• Binds to GABAA and GABAB receptors
→ Interaction with GABAA receptors
! Produces a flow of negatively charged chloride
ions into neurons, thus producing membrane
hyperpolarization
→ Interaction with GABAB receptors
! Produces a flow of positively charged potassium
ions out of neurons, also producing membrane
hyperpolarization
Physiological Effect: Hyperpolarization
Figure 6. Synapses on postsynaptic membrane. Image C exhibits
hyperpolarization. Note that the charge is lower than the resting
neuron’s. (Google Images)
• A spinal motor neuron can transmit both an excitatory or
inhibitory signal
• Resting: normal intra-neuronal potential
YL6: 01.24.01 SIM on Mechanisms of Drug Action: Principles and Perspectives
→ Resting Membrane Potential: -65 mV
• Excitation: decrease the voltage to a less negative
value (more excitable)
→ Excitatory Postsynaptic Potential (EPSP)
! A positive increase in voltage above the normal
resting neuronal potential
! When it rises high enough in the positive direction, it
can initiate an action potential in the neuron
! Threshold for Excitation: -45 mV
• Inhibition: increase the voltage to a more negative value
(neuron is less excitable)
→ Primary mechanism of GABA receptor
→ Inhibitory Postsynaptic Potential
! Inhibitory synapses open mainly chloride channels
! Opening the chloride channels will allow negatively
charged chloride ions to move from the extracellular
fluid to the interior
− Makes the interior membrane potential more
negative than normal (-70 millivolts)
! As such, chloride influx increases the degree of
intracellular negativity, which is called
hyperpolarization
− This inhibits the neuron because the membrane
potential is even more negative than the normal
intracellular potential
Clinical Uses: Diazepam
Figure 7. Mechanism of benzodiazepine (google images)
• A type of benzodiazepine
! CNS depressant which slow down the nervous
system
• Main use: treating seizures, relieve anxiety, muscle
spasms, control agitation from alcohol withdrawal,
anticonvulsant, sedative
• Diazepam binds to a specific subunit on the GABA
receptor
! Binding of GABA increases the total conduction of
chloride ions across the neuronal cell membrane
! As a result, the difference between resting potential
and threshold potential is increased and firing is less
likely
! This increases the inhibitory processes in the
cerebral cortex.
• Because of the role of diazepam as a positive allosteric
modulator of GABA, binding to benzodiazepine
receptors causes an inhibitory effect
• With long-term high-dose use of benzodiazepines (or
ethanol), there is an apparent decrease in the efficacy of
GABA-A receptors (tolerance).
• When high-dose benzodiazepines is abruptly
discontinued, this “down-regulated” state of inhibitory
transmission is unmasked, leading to characteristic
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 withdrawal symptoms such as anxiety, insomnia,
autonomic hyperactivity and, possibly, seizures.
• Common side effects of diazepam include muscle
weakness, drowsiness, dizziness, incoordination and
slurred speech.
• Other examples: Alprazolam (Xanax), Clonazepam
(Klonopin), Diazepam (Valium), Lorazepam (Ativan),
Triazolam (Halcion)
→ Predominant for both hormonal and non-hormonal first
messengers (ACTH, epinephrine, LH, FSH, TSH,
prostaglandin)
• cGMP, NO, and Co: contraction and relaxation of smooth
muscle cells and sensory cells
• Lipid second messengers: regulatory functions and
immune response mediation
• Calcium: keeps resting intracellular calcium levels low

III. SEVEN TRANSMEMBRANE Table 1. Examples of G-Protein-coupled Receptors

PROTEIN RECEPTORS

• Also known as G-protein-coupled receptors and

metabotropic receptors
• Activated receptor couples to a GTP-binding protein and
dissociates while the activated alpha-subunit may then
directly activate or inhibit an enzyme system to produce a
diffusible intracellular second messenger
• Two common enzyme systems are adenylate cyclase
and phospholipase C

A. G-PROTEIN LINKED PATHWAY

B. ADRENERGIC TRANSMISSION

• Adrenergic Agonists
→ Group of agonists that produce their effects by
activating adrenergic receptors
→ Also called sympathomimetics because their
response is very similar to that of the sympathetic
nervous system
! Bronchodilation
! Increase cardiac muscle contractility
! Pupillary dilatation
→ Follows receptor specificity
Figure 8. G-protein linked pathway (google images)
! Epinephrine – α1, α2, β1, β2
! Norepinephrine - α1, α2, β1
Mechanism of Action
! Dopamine - α1, β1, dopamine
1. Ligand binds with G protein-coupled receptor (GPCR) ! Phenylephrine - α1
2. G-protein is activated via conformation change ! Albuterol - β2
3. Heterotrimeric G-protein releases alpha subunit (GTP-
bound) Mechanisms of Adrenergic Receptor Activation
4. Activated alpha subunit stimulates adenylate cyclase
5. Activated adenylate cyclase converts ATP to cAMP
(cyclic adenosine monophosphate) which leads to
activation of:
• cAMP-dependent protein kinase (protein kinase A or
PKA)
→ Four cAMP molecules are required to activate a
single PKA
• Cyclic nucleotide-gated ion channels
• Exchange proteins
6. Protein kinase A phosphorylates specific proteins and a
cascade of enzymes
7. Cellular response induced

Physiological Effect: Second Messenger

• Three types of second messengers
2+
→ Hydrophilic messengers: IP3, cAMP, cGMP, Ca
→ Hydrophobic lipid messengers: DAG and PIP3
→ Gases: NO, CO, ROS

Clinical Uses
• cAMP:
→ Mobilizes stored energy Figure 9. Synthesis and release of norepinephrine from cholinergic
neuron (Lippincott’s Illustrated Reviews of Pharmacology, 5e)
→ Conserves water by the kidneys
→ Regulates the production of adrenal and sex steroids

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• Direct Receptor Binding • Mechanism of action:
→ Most common mechanism by which drugs activate → Salbutamol binds to β2 receptors in the airways of the
peripheral adrenergic receptors lungs, causing conformational change in the receptor
→ Produces its effect by binding to adrenergic receptors and activating the alpha subunit of G protein
and mimicking the actions of natural transmitters → GTP is then used to activate Adenylate Cyclase
• Promotion of Norepinephrine (NE) Release → Adenylate Cyclase converts ATP to cAMP
→ Acts by affecting the terminals of the sympathetic → cAMP activates a cAMP-dependent protein kinase
nerves to cause NE release (pKA)
→ e.g. Amphetamine → Activation of pKA would decrease intracellular
• Inhibition of NE Reuptake calcium concentration through:
→ It acts by blocking NE reuptake so NE accumulate ! Calcium influx into the sarcoplasmic reticulum
within the synaptic gap and increase receptor activation ! Calcium efflux in the sarcolemma
→ e.g. tricyclic antidepressants like cocaine ! Inhibition of the actin-myosin interaction
• Inhibition of NE Inactivation → This would lead to muscle relaxation of the airway or
→ MAO is inhibited which inhibits NE in terminals of bronchodilation
adrenergic neurons leading to increase in the amount • Salbutamol is in inhaler form because you would rather
of NE available for release and receptor activation give the drug locally.
→ If taken systemically, you will experience the adverse
Table 2. Classification of Adrenergic Agonists
Catecholamines Noncatecholamines
side reactions, as an extension of its pharmacologic
Can be given NO YES
effects:
orally ! Tremors or shakiness
Duration of Action Brief Longer half-life ! Increased blood sugar
Can cross the NO YES (considered ! Increased wakefulness; may lead to insomnia
Blood-brain less polar) ! Increased heart rate; palpitation
barrier
C. CHOLINERGIC TRANSMISSION
Examples Epinephrine, Albuterol and
norepinephrine, phenylephrine • Involves the release of a neurotransmitter acetylcholine
dopamine and its application to a postsynaptic neuron
Clinical Use: Norepinephrine Acetylcholine in the Synaptic Gap
• It has almost the same function as epinephrine except that
it is only specific to α1, α2, and β1 receptors
→ It does not have an effect on the lungs
→ It acts mainly on blood vessels and the cardiac muscle
• Therapeutic use
→ Myrdiasis during ophthalmologic procedures
→ Overcome AV heart block
→ Restore cardiac function in cases of cardiac arrest
• Adverse effects
→ Hypertensive crisis
→ Dysrhythmias
→ Angina pectoralis
Clinical Use: Salbutamol
• Synonymous to Albuterol or Levalbuterol
• It is an adrenergic drug usually taken by inhalation which
has effects similar to epinephrine Figure 11. Synthesis and release of acetylcholine from cholinergic
→ Stimulating drug that has a bronchodilator effect neuron (Lippincott’s Illustrated Reviews of Pharmacology, 5e)
• Moderately to highly selective to β2-adrenergic
receptors • Ach is released from a pre-synaptic neuron into the
• Indication: To relieve bronchospasm in bronchial asthma, synaptic cleft
chronic bronchitis, emphysema and other chronic • Ach binds to pre-synaptic receptors
obstructed bronchospasm or airway resistance diseases → Auto-modulation of the presynaptic cholinergic neuron
• Although it is only sensitive to β2-adrenergic receptors, in • Ach is degraded by acetylcholinesterase into
high concentrations, it could activate β1-adrenergic → Choline
receptors causing tremors and tachycardia → Acetyl-CoA
• Ach binds to postsynaptic receptors
! Cholinergic Response
Nicotinic Cholinergic Transmission

Figure 10. Cascade of salbutamol similar to epinephrine.

(oregonstate.edu)
Figure 12. Nicotinic Cholinergic Mechanism (CNSforum.com)

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• Binding of 2 molecules of ACh to nicotinic acetylcholine
receptor (nAChR) results to conformational change
→ This forms a monovalent cation-selective pore
through the postsynaptic membrane
→ Net inflow of ionic current occurs
• Once the nAChR is activated, depolarization takes place
→ Sympathetic synapse is stimulated
→ Triggers the release of norepinephrine
• Very brief: <10 ms
• Rapidly hydrolyzed by acetylcholinesterase in the
synaptic cleft
• Receptor bound Ach dissociates quickly from the “closed”
receptor
Muscarinic Cholinergic Transmission
Figure 13. Muscarinic Cholinergic Transmission (CNSforum.com)
• Produces parasympathetic response
→ Occurs at the end organs innervated by the
parasympathetic component of the autonomic nervous
system (ANS) and in the central nervous system (CNS)
• All muscarinic receptors are G-protein couple receptors
→ Subclasses:
! M1, M4, M5 – CNS: memory, arousal, attention,
analgesia
! M2 – Heart: lower heart rate
! M3 – Smooth muscle: bronchial tissue, bladder,
exocrine glands
• Binding of Ach to Muscarinic Cholinergic Receptor
(mAChR) produces indirect ligand-gated effects
• Variety of second messengers mediate the effects of
muscarinic transmission:
→ Inhibition of adenylate cyclase ! reduction of cAMP
→ Stimulation of phospholipase C ! release DAG and IP3
→ G-protein activation
• Tissue responses from cascading series of catalytic
events
→ Slower (ex. Kinase activation leading to protein
phosphorylation)
• Compared to those from direct action of G-proteins
+
→ Faster (ex. Opening of cardiac K channel)
Phospholipase C and Muscarinic effect
Figure 14. Phospholipase C mechanism (Google Images)
YL6: 01.24.01 SIM on Mechanisms of Drug Action: Principles and Perspectives
• Phospholipase C is an effector molecule/enzyme found
in the cell membrane
• Interaction between the muscarinic receptor and GTP-
binding protein C ! leads to G-protein activation !
activation of phospholipase C
• Activated phospholipase C then splits phosphatidyl-4,5-
bisphosphate (PIP2) into secondary messengers:
→ Diacylglycerol (DAG) activates protein kinase C which
phosphorylates other target proteins leading to cellular
responses
→ Inositol-1,4,5- triphosphate (IP3) goes to the ER
2+
where it causes an increase in the release of Ca from
intracellular storage sites (mitochondria and ER)
• Increase in cytosolic calcium causes smooth muscle
contraction
Clinical Use: Respiratory Tract (PNS)
• Control of airway caliber
→ Pulmonary reflexes ! afferent nerve pathways
→ Interactions of efferent vagus and mediators or
modulating transmitter substances ! efferent nerve
pathways
→ Cholinergic muscarinic receptors and post-receptor
mechanisms in target organ (Ach and GPCR mediated)
• Bronchodilatation vs. Bronchoconstriction
→ Sympathetic (adrenergic) tone ! bronchodilation
→ Parasympathetic (cholinergic) tone !
bronchoconstriction
→ Non-adrenergic, non-cholinergic (NANC) fibers !
innervations of the respiratory tree
• Bronchoconstriction
→ When protein kinase C is activated, bronchoconstriction
is promoted
→ Rapidly adapting (“irritant”) receptors
! C-fiber endings
− Increases during ozone-induced hyper reactivity
− Increased bronchial reactivity
! Vagus nerve – parasympathetic
− Airway smooth muscle cells: muscarinic receptors
− Parasympathetic postganglionic neurons:
acetylcholine
! Anti-cholinergic agents ! bronchorelaxation
− Chronic obstructive pulmonary disease
− Acute asthma exacerbations
− β-adrenergic agonists contraindicated
Clinical Use: Atropine
Figure 15. Atropine (Google Images)
• Anticholinergic
• Used in COPD and asthma
→ However, stronger bronchodilation effects can be
achieved by the beta adrenergic drugs like Salbutamol.
• Atropine and smooth muscles of the airway
→ Inhibits the muscarinic actions of acetylcholine on
structures innervated by postganglionic cholinergic
nerves as well as on smooth muscles
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 → Major action: competitive antagonism
! Overcome by increasing the concentration of
acetylcholine at receptor sites of the effector organ
! Atropine competes with acetylcholine for muscarinic
receptors
! Atropine does not prevent the release of Ach or
block its synthesis
• Atropine and respiration
→ Both smooth muscle and secretory glands of the airway
receive vagal innervations and contain muscarinic
receptors
→ Even in normal individuals, administration of atropine
can cause some bronchodilation and reduce secretion
→ The effect is more significant in patients with airway
disease
• Atropine is not solely used for bronchodilation, because
the M3 receptor is all over the body and will have systemic
effects. Other uses include:
→ Pre-medication for surgery
→ Decrease in secretions
→ Increase in bladder contraction
→ Increases heart rate
IV. ENZYME-ASSOCIATED RECEPTORS
• Also known as kinase-linked receptors
• These are membrane receptors which incorporate an
intracellular protein kinase
• Signal transduction generally involves dimerization of
receptors, followed by autophosphorylation of tyrosine
residues
• They are involved mainly in events controlling cell growth
and differentiation and act indirectly by regulating gene
transcription
A. SIX TYPES
1. Tyrosine Kinase Receptor – phosphorylate specific
tyrosines on a small set of intracellular signaling proteins
2. Tyrosine-Kinase-associated Receptor – associate with
intracellular proteins that have tyrosine kinase activity
3. Receptor-like Tyrosine Phosphatases – remove
phosphate groups from tyrosines of specific intracellular
signaling proteins
4. Receptor Serine/ Threonine Kinases – phosphorylate
specific serines or throenines on associated latent gene
regulatory proteins
5. Receptor Guanylyl Cyclases – directly catalyze the
production of cyclic GMP in the cytosol
6. Histidine-Kinase-Associated Receptors – reactivate a
“two-component” signaling pathway in which the kinase
phosphorylates itself on histidine and then immediately
transfers the phosphate to a second intracellular signaling
protein
B. OVERVIEW OF THE MECHANICS
1. Ligand binding to tyrosine kinase receptor
" Cross-phosphorylation of receptor domains on multiple
tyrosines
2. Kinase activation + phosphotyrosine production
" Phosphotyrosine as docking sites for a set of
intracellular signaling proteins (binding via their SH2
domains)
3. Receptor coupling to GTPase Ras ! activation of
cascade of serine/threonine phosphorylations
(converging on a MAP-kinase) OR activation of another
protein that docks on activated receptor tyrosine
kinases (PI 3 kinase) which generates specific inositol
phospholipids
YL6: 01.24.01 SIM on Mechanisms of Drug Action: Principles and Perspectives
" Inositol phospholipids as docking sites in the plasma
membrane for signaling proteins with PH domains (e.g.
PKB)
Remember: LUBRICATING
• Ligands are released into the extracellular space
• Upon
• Binding to their
• Receptor
• Specific Interactions cause a
• Conformational change within the catalytic domain
• This Activates the connected enzyme
• Phosphorylation generates Tethering sites where
• Intracellular effector proteins bind
• These further relay the signal to the Nucleus resulting in
changes in
• Gene expression
C. INSULIN RECEPTORS
Figure 16. Mechanism of action of insulin receptor (google images)
Mechanism of Action in Terms of Glucose
Uptake
1. Insulin binds to α sub-unit of insulin receptor, inducing
the dimerization of the β sub-units
2. Tyrosine kinase receptor phosphorylates IRS-1
3. IRS-1 recruits GRB2 (which activates the Ras pathway)
and activates PI3 Kinase
4. PI3 Kinase phosphorylates the active site of PIP2,
resulting into PIP3
5. PIP3 activates Akt
6. Activation of Akt leads to a series of activation of key
proteins, resulting to the recruitment of GLUT 4 to the
membrane and the increased glycogen synthesis by
glycogen synthase
V. TRANSCRIPTIONAL REGULATOR
HORMONES
• Receptors are intracellular proteins hence ligands must
first enter cells
• Mediated via intracellular receptors
• Steroids bind to cytosolic and nuclear receptors.
• In the nucleus, steroid receptor complex binds to specific
segments of DNA, initiating mRNA synthesis and protein
synthesis. The outcome can be: tissue hypertrophy, or its
reverse, depending on the type of cell. New proteins may
also be secreted.
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 A. ESTROGEN

• A family of molecules that stimulate the development and

maintenance of female characteristics and sexual
reproduction
• Most common human forms are estradiol and estrone
• These are steroid molecules produced and secreted by
ovaries
• They act on tissues with estrogen receptors
• Target tissues include: breast, uterus, brain, bone, liver
and heart

Figure 19. Effect of estrogen on cell proliferation (2018 Trans)

• Steps in gene activation:

→ Step 1: Binding of Estrogen to Receptor
! Estrogen crosses the nuclear membrane and binds
to the estrogen receptor α with AF1 and AF2
domains
→ Step 2: Nucleus
! Estrogen receptor acquires a changed shape and
attaches to co-activators
→ Step 3: Gene Transcription and Protein Synthesis
! Activation of gene transcription and protein synthesis
through DNA direct binding
! Growth factor formation which leads to proliferation
# Breast Cancer
Figure 17. Chemical structure of estrogen (2018 Trans)

• Effects of estrogen
→ Beneficial
! Programs the breast and uterus for sexual
reproduction (cell proliferation)
! Controls cholesterol production in ways that limit the
buildup of plaque in the coronary arteries
! Preserves bone strength by helping to maintain the
proper balance between bone buildup and
breakdown
→ Harmful
! Promotes cell proliferation in breast and uterus !
increases risk of developing cancer
Figure 20. Mechanism of estrogen receptor activation (2018
Trans)

Selective Estrogen Receptor Modulator

(SERM)
• Blocks estrogen receptors only in certain tissues but
mimic the action of estrogen in other tissues
• Tamoxifen selectively blocks estrogen in breast
tissues but activates estrogen receptors in uterine
endometrial cells

Figure 18. Estrogen inducing proliferation of existing mutant cells

may lead to spontaneous new mutations (2018 Trans)

• Estrogen receptors
→ Trigger gene activation
→ Normally reside in the nucleus, along with DNA
molecules
• Estrogen and breast cancer
→ High estrogen levels may trigger breast cell proliferation
and increases the risk of developing mutations
! This leads to an increased risk of developing cancer
Figure 21. Mechanism of SERMs (2018 Trans)

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Clinical Use: Tamoxifen
• Tamoxifen binds to estrogen receptors. Thus, estrogen
can no longer bind to these receptors. This blocks
activation of growth promoting proteins.
• Mechanism
→ Step 1: Binding of Tamoxifen to Receptor
! Tamoxifen crosses the nuclear membrane and
competitively binds to estrogen receptor α at the AF2
domain
→ Step 2: Nucleus
! Unlike estrogen, when Tamoxifen binds to its
receptor, it does not change the receptor’s shape.
! The complex recruits co-repressors (NCoR, SMRT)
and stops genes being switched on by estrogen
! This would block the action of estrogen, stopping the
proliferation of breast cancer cells.
3. Downregulation
" Process of reducing a response to a stimulus
→ Example: cellular decrease in receptors to a
molecule (hormone or neurotransmitter # reduction
in cell’s sensitivity to the molecule
→ Form of negative feedback
" Accounts for drug tolerance
4. Supersensitivity
" Extreme upregulation as a result of decreased
activation (exposure to an antagonist or inhibition of
transmitter synthesis or release)
" Upon withdrawal of the antagonist, there would be an
overshoot phenomenon because of the increased
number of receptors (spare receptor effect)
" One example would be the upregulation of adenosine
receptors upon increased intake of caffeine (antagonist)
→ When caffeine stops being taken, the increased
number of adenosine receptors leads to the
increased effect of adenosine since caffeine no
longer competes for the receptors

Table 3. Comparison between desensitization, tachyphylaxis, down

regulation, and supersensitivity
Duration of Temporality Reversibility
exposure of change
Desensitization Short Gradual Reversible
Tachyphylaxis Short Sudden Reversible
Downregulation Long Gradual Irreversible
Supersensitivity Long Gradual Irreversible
Figure 22. Mechanism of Tamoxifen (2018 Trans)
Categories of Desensitization
• On uterine cancer • Homologous Desensitization
→ While Tamoxifen acts as an anti-estrogen in breast → Loss of responsiveness exclusively of the receptors
cells, it mimics the action of estrogen in other tissues that have been exposed to repeated or sustained
such as the uterus activation by an agonist
! Estrogen-like effects stimulate proliferation of uterine • Heterologous Desensitization
endometrium → Desensitization of one receptor by its agonists also
! Increased risk for uterine cancer results in desensitization of another receptor that has
• On Bone not been directly activated by the agonist
→ Will stimulate bone formation or deposition
Mechanisms of Desensitization
VI. REGULATION OF RECEPTOR FUNCTION 1. Intracellular proteins may block access of a G-protein to
the activated receptor molecule
• Receptors are regulated in: " Continuous activation of the receptor causes β-arrestin
→ Number to bind to an intracellular loop of its β-adrenoceptor,
→ Location inhibiting access to the G-coupling protein
→ Sensitivity " Reaction may be reversed quickly by removing the β-
• Changes can occur over a short time (minutes) and longer agonists
periods (days) 2. Agonist-bound receptors may be internalized by
endocytosis, removing it from further exposure to
A. PROCESS OF REGULATION extracellular molecules
" Example of receptor molecules that can be reinserted:
1. Desensitization morphine receptors
" Caused by continuous exposure to a constant " Following receptors are degraded: β adrenoceptors and
concentration of an agonist over a short amount of epidermal growth factor receptors
time
" The same concentration of agonist becomes much less
effective at producing the same level of effect
" Clinical significance: may limit therapeutic response to
drugs
2. Tachyphylaxis (rapid + protection)
" Rapid progressive decrease in response to a dose
after repetitive administration
→ Due to either depletion of either neurotransmitters
that facilitate the drug’s effect, or receptors to which
the drug or neurotransmitter binds
! Example: relapse of an episode of depression
after full recovery despite continued drug
treatment
Figure 23. Mechanism of desensitization (1-5) and
downregulation (1-3 & 6). (Katzung 13th ed.)

YL6: 01.24.01 SIM on Mechanisms of Drug Action: Principles and Perspectives 9 of 13

3. Continuous activation of the receptor-effector system may
lead to depletion of some essential substrate required for
downstream effects
" Example: nitroglycerin tolerance - occurs due to the
depletion of thiol cofactors
" Reaction may be reversed by replicating the needed
substrate
VII. NON-RECEPTOR MEDIATED DRUG
ACTIONS
A. METAL CHELATORS
Chelating Agents
• Drugs used to prevent or reverse the toxic effect of a
heavy metal on any cellular target, or to accelerate the
elimination of the metal
• Contain one or more coordinating atoms to form
coordinate covalent bonds with the heavy metal ion.
→ Hence, they do not act on a receptor
• Are considered nephrotoxic since some redistribute the
metals to the kidney.
• Chelators play an important role in oxygen transport and
photosynthesis
→ Some are biological catalysts
• Chelate = metal ion + chelating agent
EDTA
• Used as treatment for heavy metal poisoning (i.e. lead)
→ Administered intravenously
2+ 2+
• Forms complexes with Ca and Mg
→ Frequently used in soaps and detergents
→ As a chelator, it is administered with calcium disodium
to prevent life-threatening calcium depletion
• Used as a stabilizing agent in food industry
→ Deactivates the enzymes
→ Promotes color retention
→ Improves flavor retention
→ Inhibits rancidity
• Mechanism
→ Has 4 carboxyl groups and 2 amine groups
→ A hexadentate ligand, forming up to 6 coordinate
covalent bonds with an ion or metal
→ High thermodynamic stability depending on properties
of the metal and the chelating agent
→ Higher K constant being more stable
Indications
• Use for lead poisoning, lead nephropathy
• Symptoms of lead encephalopathy and/or blood lead level
> 70 mcg/dL (lead encephalopathy must be treated with
combination dimercaprol (BAL) for 5 days preceding
C10H12CaN2Na2O8)
• May be useful for zinc and manganese poisoning and
certain heavy radioisotopes
• Lead encephalopathy must be treated with combination
BAL preceding Edetate Calcium Disodium
Figure 24. EDTA chemical structure (above) and EDTA: metal
th
complex stability constants (below)(Katzung 13 ed.)
YL6: 01.24.01 SIM on Mechanisms of Drug Action: Principles and Perspectives
Contraindications
• Mostly no absolute contraindication to EDTA
• Exceptions: severe allergy to EDTA
• Acute Lead Encephalopathy
• Relative contraindication: renal dialysis
• Discontinue if oliguria (due to renal failure) occurs or pain
occurs at injured site
Side Effects
• Fatigue, nasal congestion, abdominal cramps, nausea,
vomiting, diarrhea, headache, low blood pressure, skin
problems, fever, lacrimation, mucocutaneous lesions,
glycosuria, myalgia, hepatotoxicity, increased urinary
frequency, abnormal changes in ECG
• Unsafe: > 3 g EDTA/day, or taking it > 5-7 days
• Too much can cause:
→ Kidney damage
→ Dangerously low calcium levels
→ Death
Examples
• Biologically significant chelating agents
→ Heme – major component of hemoglobin
! Contains a porphyrin that binds to iron (II) ion
→ B12 – the only metal-containing essential vitamin
! A chelate agent that binds to cobalt (II) ion
→ Dimercaprol – first medical chelating agent; used as
antidote for the Lewisite gas (arsenic) during WWII
B. ALKALIZING DRUGS
• Alkalinizing drugs confer alkalinity, and are used to
increase blood pH level and treat metabolic acidosis.
• Sodium bicarbonate is used in cooking as a leavening
agent. Because baking soda is an alkaline, it reacts with
acidic ingredients in foods, such as citrus, yogurt, lemon
juice, and honey, producing bubbles of carbon dioxide as
a byproduct, making dough rise.
Features
• Increase blood pH and possibly urine pH
→ Are used to increase blood pH and treat metabolic
acidosis
→ Are also used to raise urine pH level to help remove
certain substances, such as phenobarbital, after an
overdose
• Is also used in raising the stomach pH level in cases of
heartburn
• Pharmacokinetics
→ Sodium bicarbonate isn’t metabolized. It’s filtered and
reabsorbed by the kidneys.
→ Less than 1% of the filtered drug is excreted.
• Pharmacodynamics
→ In the blood, sodium bicarbonate separates to provide
bicarbonate ions that are used in the blood buffer
system to decrease the hydrogen ion level and raise
the blood pH level. (Buffers prevent extreme changes
in the pH level by taking or giving up hydrogen ions to
neutralize acids or bases.)
→ As the bicarbonate ions are excreted in urine, the urine
pH level rises.
→ After conversion to bicarbonate, sodium citrate and
lactate alkalinize the blood and urine in the same way.
10 of 13
 Acetazolamide
• Acetazolamide inhibits carbonic anhydrase located
intracellularly (cytoplasm) and on the apical membrane of
the proximal tubular epithelium
• Carbonic anhydrase catalyzes the reaction of CO2 and
+
H2O, leading to H2CO3, which spontaneously ionizes to H
and HCO3- (bicarbonate)].
+ +
• The decreased ability to exchange Na for H in the
presence of acetazolamide results in a mild diuresis
• Additionally, HCO3- is retained in the lumen, with marked
elevation in urinary pH

Therapeutic Uses
• Glaucoma
→ Decreases the production of aqueous humor by
blocking carbonic anhydrase in the ciliary body of the
Figure 25. Role of bicarbonate in raising blood pH level (google eye.
images)
→ Useful in the chronic treatment of glaucoma but should
Indications not be used for an acute attack
• Reduces stomach acid • Urinary alkalization
• Used as an antacid to treat heartburn → Helps in the excretion of certain compounds like uric
• Other uses acid, cystine and other weak acids
→ Can be used to make urine less acidic • Metabolic alkalosis
• Helps kidneys get rid of uric acid (helping to prevent gout → Used when alkalosis is due to excessive use of
and kidney stones) diuretics in patients with severe heart failure
• Treat metabolic problems (acidosis) caused by kidney • Acute mountain sickness
disease → Decreases pH of CSF and brain, increasing ventilation
and lessening symptoms
Contraindications
• Contraindicated for patients with metabolic alkalosis Pharmacokinetics
• Also contraindicated for patients with hypocalcemia • Increase in urine pH within 30 minutes, maximal at 2
→ Alkalosis may produce tetany hours, persists for 12 hours after a single dose
Adverse Reactions • Secreted by proximal tubule S2 segment
• IV-administered sodium bicarbonate
Adverse Effects
→ Overdose
! Leads to metabolic alkalosis (evidenced by • Hyperchloremic metabolic acidosis
hyperirritability, tetany, or both) • Renal stones
→ Water retention and edema in patients with renal • Renal potassium wasting
impairment, or heart failure • Drowsiness, Nausea, Vomiting
! Due to high sodium content • Paresthesia
→ Oral sodium bicarbonate • Hearing dysfunction
! Gastric distention and flatulence • Altered taste
! Nausea, bloating
Contraindications
Examples
• Decreased excretion of NH4+ which is harmful for patients
• Intravenous sodium bicarbonate with hepatic cirrhosis
→ Baros • Urine alkalinity
• Oral sodium bicarbonate → Decreased calcium salt solubility
→ Tums → Increased renal calculi formation
C. ACIDIFYING DRUGS • Kidney or Liver disease
→ Depressed serum sodium/potassium levels
• Acts to correct acid-base imbalances in the blood → Risk of developing hepatic encephalopathy
• Decreases the pH of the blood and increase the • Chronic noncongestive single-closure glaucoma
concentration of hydrogen ions. → May permit organic closure of the angle to occur while
worsening glaucoma
→ Masked by lowered intraocular presure

D. OSMOTIC DIURETICS

• The PCT and descending limb of the Loop of Henle are

freely permeable to water. Any osmotically active agent,
that is filtered by the glomerulus but not reabsorbed
causes water to be retained in these segments and
promotes a water diuresis.
• As such, osmotic diuretics act on the PCT and Loop of
Henle: significantly increase the osmolarity of plasma and
tubular fluid, thus preventing water reabsorption and
extracting water from the intracellular compartment
Figure 24. Role of carbonic anhydrase in sodium retention by
epithelial cells of the renal tubule (2018 Trans)

YL6: 01.24.01 SIM on Mechanisms of Drug Action: Principles and Perspectives 11 of 13

Mannitol
Figure 25. Chemical structure of Mannitol (google images)
• The most frequently used osmotic diuretic with a 6-carbon
sugar structure that cannot be absorbed from the GI tract
• It is administered through IV injection
• It is freely filtered at the glomerulus but poorly reabsorbed
at the tubules; hence, it remains in the lumen and keeps
water in this compartment due to its osmotic effect
Mechanism of Action
• Mannitol increases the osmolarity of renal tubular fluid
and prevents reabsorption of water.
→ Remember: Water always follows sodium
→ Water staying in lumen → excretion → diuresis
• Sodium is diluted in this retained water in the renal
tubules, leading to less reabsorption of this ion.
→ This leads to an osmotic diuretic effect with urinary
excretion of water, sodium, chloride, and bicarbonate
ions. Urinary pH is not altered by mannitol-induced
osmotic diuresis.
→ Negates the effects of ADH (Anti-diuretic hormone)
• IV administration of mannitol also increases plasma
osmolarity, thus drawing fluid from intracellular to
extracellular spaces.
→ May result in an acute expansion of the intravascular
fluid volume.
→ Redistribution of fluid from intracellular sites decreases
brain bulk and may increase renal blood flow to the
medulla.
Therapeutic Uses
• Increase of urine volume
→ Increases water excretion in preference to sodium
excretion
• Reduction of Intracranial and intraocular pressure
→ Alter Starling forces: water leaves cells, reducing
intracellular volume
• Reduce cerebral edema (lower ICP), assuming the blood
brain barrier is intact
• Prophylaxis against acute renal failure
• Acute oliguria
Pharmacokinetics
• Poorly absorbed by GI Tract, thus, given parenterally
• Not metabolized, excreted by glomerular filtration within
30-60 minutes
Toxic and Adverse Effects
• Extracellular volume expansion and hyponatremia
• Dehydration
• Hyperkalemia
• Can complicate heart failure
• Pulmonary Edema
• Headache
• Nausea
• Vomiting
• These complications can be avoided by careful attention
to serum ion composition and fluid balance.
YL6: 01.24.01 SIM on Mechanisms of Drug Action: Principles and Perspectives
REVIEW QUESTIONS
1. Why is salbutamol usually given using an inhaler?
a. Because when given using an inhaler, it is exposed to
several tissues in the body, making it more effective
b. Because when given using an inhaler, it is filtered by
the liver first so as to prevent toxicity
c. Because when given orally, it skips past the portal
circulation and immediately acts on tissues of the
lungs.
a. Because when given intravenously, it may act on the
B2 receptors on the heart, causing chest pain
2. Benzodiazepines mimic the structure of GABA, hence
it acts as an agonist. How does it act upon the
skeletal muscles?
a. It closes the GABA-activated chloride channel, letting
fewer chloride ions in, causing excitation
b. It opens the GABA-activated chloride channel, letting
chloride ions in, preventing excitation.
c. It closes the GABA-activated sodium channel, letting
sodium ions in, preventing excitation.
b. It closes the GABA-activated sodium channel, letting
fewer sodium ions in, causing excitation.
3. What is the end effect of ACE?
a. Puts patient at risk for alkalosis because it promotes
the excretion of acidic urine
b. Lowering the pH of blood, since it promotes the
excretion of bicarbonate.
c. Excretion of mercury because it is a chelating agent
c. Excretion of lead because it is a chelating agent.
4. Even if acetylcholine acts on both the muscarinic and
nicotinic receptors, these receptors are very different.
Name one difference.
a. Muscarinic receptors activate the sympathetic
response while nicotinic receptors activate the
parasympathetic.
b. Muscarinic receptors activate the parasympathetic
response while nicotinic receptors activate the
sympathetic.
c. The effect of muscarinic receptors lasts only as long as
the receptor is bound to acetylcholine. The effect of
nicotinic receptors lasts longer because of the
secondary messengers.
5. How do chelating agents inactivate heavy metals?
a. They make the environment more acidic so the metal
cannot bind
b. Chelators call up macrophages to swallow the metals
c. Chelating agents facilitate metal to metal binding so
the metals do not interact with the body's cells.
d. They form coordinate covalent bonds with the metal,
rendering the metal unable to bond with proteins,
lipids, or carbohydrates.
Answers: d, b, b, b, d
REFERENCES
1. Katzung B, Trevor A. Basic and Clinical Pharmacology.
th
13 ed. New York, NY: McGraw-Hill Education;
2015.
2. Cruz N, Cagayan S, Jimeno, C. Self-Instructional Module
on Mechanisms of Drug Action.
3. Harvey R. Lippincott’s Illustrated Reviews: Pharmacology
th
Edition. 5 ed. Philadelphia, PA: Lippincott Williams
& Wilkins; 2012.
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 APPENDIX

Figure 1. Mechanisms of signal transduction, G protein-coupled receptor, Tyrosine Kinsase receptor, and Intracellular receptors (Lippincott’s
Illustrated Reviews Pharmacology, 5e)

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