Pharma 1A: Cell Biology, Drug Targets (G Proteins) and Drug-Receptor Interactions

Which G protein targets adenylyl cyclase (AC)?

Gs

What is the signaling cascade for Gs?

Activation of AC, leading to increased cAMP production and PKA activation

Example receptor for Gs?

Beta 1 adrenoceptor

Example cellular effect of Gs?

Increase in force of heart contraction

Which G protein targets phospholipase C (PLC)?

Gq

What is the signaling cascade for Gq?

Activation of PLC, leading to production of IP3 and DAG. DAG activates protein kinase C. IP3 causes calcium release from intracellular stores

Example receptor for Gq?

Alpha 1 adrenergic receptors

Example cellular effect of Gq?

Contraction of vascular smooth muscle

Which G protein (alpha subunit) inhibits adenylyl cyclase?

Gi

What is the signaling cascade for Gi (alpha subunit)?

Inhibition of AC, decrease in cAMP production, and PKA activity.

Example receptor for Gi (alpha subunit)?

Muscarinic acetylcholine receptors

Example cellular effect of Gi (alpha subunit)?

Oppose actions of beta 1 adrenoceptor, decrease in force of heart muscle contraction

Which G protein (beta gamma subunit) targets adenylyl cyclase?

Gi (beta gamma subunit)

What is the signaling cascade for Gi (beta gamma subunit)?

Targets potassium channels, causing opening and resulting in muscle relaxation

Example receptor for Gi (beta gamma subunit)?

Acetylcholine receptor

Example cellular effect of Gi (beta gamma subunit)?

Slow down heart rate

Describe the drug target and mechanism of action of digoxin

Digoxin treats heart failure. it targets the K/Na pump. Digoxin binds to the extracellular part of the pump, inhibiting it. This means Na cannot leave thus Ca2+ levels rise in the cell, causing heart contraction to happen with more force.

Draw a diagram to illustrate the signal transduction pathway for the beta1- adrenoceptor in the heart. Include the receptor, G protein subunits, transduction mechanism and intracellular cascade that leads to a change in cardiac muscle contraction.

Adrenaline activates the beta 1 GPCR. This causes a conformational change in the receptor. GTP replaces GDP bound to the G protein. This causes the alpha subunit of the Gs G protein to split from the beta-gamma subunit of the G protein. The alpha subunit activates adenylyl cyclase, which converts ATP to cAMP. cAMP activates protein kinase A, which phosphorylates voltage-gated calcium channels in the cell membrane. The influx of calcium is increased, which leads to stronger muscle contraction

name the 4 main types of receptors

1. ligand gated ion channels

2. g protein coupled receptors

3. kinase linked receptors

4. nuclear receptors

name and briefly describe the 3 methods of chemical signalling

1. paracrine

   1. Signaling cells release molecules affecting nearby target cells in the same tissue

2. endocrine

   1. Endocrine glands release hormones into the bloodstream.

3. synaptic

   1. Between neurons or between a neuron and a target cel

in the signal transduction pathway activated by Gq-protein coupled receptors, what are the names of the first and second effectors and first and second messengers?

First Effector: Phospholipase C (PLC)

Second Effector: Protein Kinase C (PKC)

First Messenger: Ligand binding to Gq-coupled receptor

Second Messengers:

• Inositol Trisphosphate (IP3)

• Diacylglycerol (DAG)

Some clinically approved anti-cancer agents target kinases. Explain what kinases are and why they are targeted in certain cancers.

kinases - enzymes that catalyse the transfer of phosphate groups from ATP to specific target molecules.

Kinases are targeted in certain cancers because

• Mutated or overactive kinases can contribute to uncontrolled cell growth and survival

• Some kinases function as oncogenes when mutated or overexpressed

Aspirin can be used as an anti-thrombotic drug without having a systemic effect on other tissues. Explain the underlying mechanism

Aspirin binds irreversibly and thus inhibits COX. COX converts acid to prostaglandins and thromboxanes. Prostaglandins - inflammation. Thromboxanes - blood clotting. Specific activtiy because thromboxanes only involved in platelets and more prostaglandins can be produced by body if needed.

Explain the 8 steps of how glucose stimulates insulin secretion from β cells in the pancreas. Which ion channel is targeting by sulfonylurea’s? How does this help patients with diabetes?

• Step 1: Glucose enters β cells via GLUT2 transporters.

• Step 2: Glucose undergoes glycolysis, producing ATP.

• Step 3: Elevated ATP levels lead to the closure of KATP channels.

• Step 4: Membrane depolarization occurs.

• Step 5: VGCCs open due to membrane depolarization.

• Step 6: Calcium influx into the β cell.

• Step 7: Increased intracellular calcium stimulates insulin exocytosis.

• Step 8: Insulin is released into the bloodstream.

  Sulfonylureas target ATP-sensitive potassium (KATP) channels on β cell membranes. By targeting KATP channels, sulfonylureas enhance insulin release from β cells, aiding in glucose regulation.

What is the primary function of enzymes?

Enzymes catalyze biochemical reactions. Examples of enzyme drug targets include proteases, kinases, and polymerases.

What is the primary function of enzyme-linked receptors?

Enzyme-linked receptors transduce extracellular signals by activating intracellular enzymatic activity. Examples include receptor tyrosine kinases (RTKs)

Where are nuclear receptors primarily located within the cell?

Nuclear receptors are located in the cell nucleus. They bind to ligands, such as hormones, and regulate gene expression, influencing various cellular processes.

What is the role of voltage-sensitive ion channels in action potential propagation?

Voltage-sensitive ion channels open or close in response to changes in membrane potential, allowing the rapid flow of ions, contributing to the propagation of the action potential.

What is the general role of Ca2+ in signal transduction?

Ca2+ serves as a second messenger, regulating diverse cellular processes by modulating enzyme activity, protein conformation, and vesicle fusion.

What is the primary function of ligand-gated ion channels?

Ligand-gated ion channels open or close in response to ligand binding, regulating ion flow. An example of a drug target in this group is the nicotinic acetylcholine receptor

define “competitive” drug antagonism

binds at the agonist recognition site preventing access of normal ligand

define “non-competitive” drug antagonism

does not bind at the agonist site but inhibits agonist binding in

another way

define “uncompetitive” drug antagonism

this is when binding occurs to an activated form of the receptor (i.e. “use-dependent”).

define “physiological” drug antagonism

when the effect of a neurotransmitter or hormone is countered by the action of another neurotransmitter or hormone

true or false: antagonism can be reversible OR irreversible

true

What are the two effects an antagonist can have on the concentration-response curve to an agonist?

depression of the maximal response

RIGHTWARD displacement of the curve.

What is depression of maximal response?

when an antagonist partially or fully prevents the agonist from eliciting its full response.

when does depression of maximal effect occur?

when the antagonist binds irreversibly or competes with the agonist for binding

what does rightward displacement of the curve suggest (in a conc-response curve of an agonist where an antagonist is present)?

a decrease in the potency of the agonist in the presence of the antagonist

how does the antagonist affect the agonist's binding?

makes it more difficult for the agonist to bind or activate the receptor

what happens to the concentration response curve in rightward displacement?

it requires higher concentrations of the agonist for the same effect

What is competitive irreversible antagonism?

Antagonist competes with the agonist for the same site but binds irreversibly (covalently).

How does irreversible binding affect antagonism?

Irreversible binding means that antagonism is not overcome by increasing agonist concentration.

What does the graph look like in competitive irreversible antagonism?

A decreased maximal response with a shorter curve, as antagonism is not overcome by increasing agonist concentration.

What causes the decrease in maximum response in this antagonism?

The irreversible binding of the antagonist reduces receptor number until there are not sufficient numbers to maintain the maximum response.

What is non-competitive antagonism?

Antagonist binds at a site distinct from that used by the agonist.

What is the first type of noncompetitive antagonist?

Allosteric modulator - Binds and changes the binding of agonist to its site.

What is the second type of noncompetitive antagonist?

Binds and antagonizes response by blocking later in the pathway from receptor activation to response.

Give an example of the second type of noncompetitive antagonist.

e.g., enzyme inhibitor or calcium channel blocker.

What is physiological antagonism?

When one neurotransmitter/hormone antagonizes another via an action on an independent molecular target.

Give an example of physiological antagonism involving histamine and omeprazole.

Histamine stimulates acid secretion, omeprazole blocks this effect.

Provide an example of physiological antagonism related to heart rate.

Noradrenaline increases heart rate, Acetylcholine decreases heart rate.

Describe a physiological antagonistic action involving the pupils.

Noradrenaline dilates the pupil, acetylcholine constricts pupils.

Illustrate physiological antagonism with an example related to blood pressure.

Endothelin increases blood pressure, nitrates decrease.

What is the therapeutic index of a drug? Define the numerator and denominator. What is it a measure of?

The therapeutic index: the ratio of the dose that produces therapeutic effects to the dose that produces toxic effects.

LD50/ED50

• LD50 - the median lethal dose

• ED50 - median effective dose

What information does the KD provide?

KD is a measure of the affinity of binding of the drug to the receptor

KD = [drug] that binds to 50% of the available receptor

In terms of ligand-receptor binding, what is the significance of a low Kd?

A low Kd signifies that the ligand binds tightly to the receptor, requiring lower concentrations for effective binding.

identify which curve is for the i) agonist only ii) agonist + [B] iii) agonist + [2B] iv) agonist + [3B] where B is antagonist

What does a Schild plot for KB look like (ie what are the x and y axes) and how can we i) identify if the antagonist is competitive ii) calculate KB?

i)If the slope is equal to 1 then the antagonist is considered competitive. ii) If the slope is equal to 1, the intercept with the x-axis gives the –log KB

What is Kd? Give the formula

KD is the concentration of drug that occupies 50% of the binding sites: Kd = [D] [R] / [DR]

The receptor occupancy formula is strictly valid under what 5 circumstances?

1. Equilibrium 2. Drug concentration at receptors same as that applied to system 3. 1 drug molecule combines with one receptor molecule 4. A negligible amount of the drug added is bound 5. Binding of one drug molecule does not influence the binding of another

What does the Kd tell you about the biological response?

NOTHING

In a drug-receptor binding semi-logged curve (with P on y axis), the higher the affinity, the ____the value of KD and the more left or right? ward the binding curve will be along the concentration axis

The higher the affinity, the lower the value of KD and the more leftward the binding curve will be along the concentration axis

In a drug-receptor binding curve (with P on y axis), the position of the binding curve along the concentration axis is governed by _______

the position of the binding curve along the concentration axis is governed by affinity

"The ability of an agonist to produce a pharmacological effect in a cell/tissue depends on which two parameters?

"

1. Binding of drug to receptor (determined by its affinity) 2. Following binding, activation of the receptors and production of response (determined by efficacy)

define Pharmacodynamics

What the drug does to the body

define Pharmacokinetics:

What the body does to the drug

Give 6 reasons why drugs show side effects and toxicity.

1. Drugs are insufficiently selective. 2. Drugs are very selective but target sites can affect multiple body processes, causing toxicity/side effects. 3. The prolonged use of the drug may lead to long-term structural/functional change. 4. Lack of knowledge of disease process 5. Patient variability 6. Drug interactions

Define agonist

A drug that evokes a response

define antagonist

A drug that prevents the action of another drug, naturally occurring hormone/transmitter

define efficacy

efficacy is the ability to activate a receptor and evoke a biological response

Which one out of agonist and antagonists have efficacy?

agnost only has efficacy

what are the different effects of histamine on i) smooth muscle ii) gastric muscle

"i) contracts smooth muscle in e.g. bronchi. ii) stimulates gastric secretion

"

How can histamine analogues be tailored? Give examples of H1 and and H2.

H1 antagonists: designed to counteract allergies. H2 antagonists: designed to address ulcers

receptor theory: ____ interact with ______ in a ______ manner to produce a change in the ______ of the receptor

Drugs interact with receptors in a reversible manner to produce a change in the state of the receptor

in receptor theory, in short, what determines the selectivity of drug effects?

Mutual affinity of drugs and receptors determines the selectivity of drug effects

in receptor theory, in short, what can explain the activity of agonists, partial agonists and antagonist drug activity?

Competition of mutually exclusive molecules for the same receptors explains agonist, partial agonist and antagonist drug activity

what is a partial agonist?

partial agonists occupy all receptors to evoke their maximum response

how did Langley know both nicotine and curare were acting on the same or closely related receptors?

Langley observed that when nicotine was applied to certain tissues, it produced physiological responses, suggesting that nicotine was interacting with specific receptors on the cells. When curare was applied, the effects of nerve stimulation by nicotene were blocked – L found that curare competitively inhibit/block nicotine actions. when curare was present, the effects of nicotine were diminished or prevented. The fact that curare could compete with and inhibit the actions of nicotine implied that both substances were interacting with the same/closely related receptors.

The higher affinity the _____ the binding

The higher affinity the stronger the binding

"Lower the KD the ______ the affinity

"

"Lower the KD the higher the affinity

"

The _______ the EC50, the more potent the substance

The lower the EC50, the more potent the substance

receptor occupancy reflects the proportion of _______ ________ _____ _______.

receptor occupancy reflects the proportion of receptors bound by ligands.

how affinity relates to receptor occupancy: at lower concentrations, high-affinity ligands can effectively occupy a _____ percentage of receptors because they bind tightly.

how affinity relates to receptor occupancy: at lower concentrations, high-affinity ligands can effectively occupy a high percentage of receptors because they bind tightly.

define the "maximum tissue response"

a response that is the maximum a portion of biological tissue can achieve.

partial agonists reside in which DR state most of the time, inactive DR or active DR*?

inactive DR

the concentration-response and occupancy curves are similar. What is one KEY difference (apart from their properties)?

The CRC is NON super-imposable

define EC50. what kind of graph can you derive this from?

The concentration of drug that gives 50% of the maximum response. The CRC will give you this.

Efficacy can be considered as time DR complex spends in ________ state

Efficacy can be considered as time DR complex spends in DR* (active) state

The higher the efficacy, the ____ the separation between binding and functional response curves. Why?

The higher the efficacy, the greater the separation between binding and functional response curves. Tells us there is a higher proportion of spare receptors

The greater the efficacy, the ______ the separation between KD and EC50

The greater the efficacy, the bigger the separation between KD and EC50

You have spare receptors/receptor reserve when...

you don’t need to occupy and activate all receptors to get the tissue’s maximum response

true or false - Magnitude of response IS NOT proportional to receptor occupancy

TRUE

______ agonists (low efficacy) have an EC50 close to KD

“weak” agonists (low efficacy) have an EC50 close to KD

______ agonists have high efficacy (i.e. EC50 <<< KD)

“strong” agonists have high efficacy (i.e. EC50 <<< KD)