basics + receptor theory

Question 1

define drug

Answer 1

chemical of known structure causing a biological effect, can be synthetic or plant derived small molecules, or can be biopharmaceutical

Question 2

define medicine

Answer 2

containing one or more, drugs, provides a therapeutic effect

Question 3

what are excipients?

Answer 3

ingredients that stabilise the active drug in a medicine

Question 4

what are biopharmaceuticals?

first gen vs second?

Answer 4

can be oligonucleotides -
DNA or RNA used in gene therapy, regenerative medicine with engineered stem cells etc…

proteins -
e.g. insulin, antibodies
First generation = copy of endogenous proteins
Second generation = engineered proteins, slightly altered to improve performance

Question 5

what is humira?

Answer 5

a monoclonal antibody used to treat rheumatoid arthritis

Question 6

what is a potential draw back of biopharmaceuticals?

Answer 6

can be very difficult to get them into the CNS

Question 7

what are the most common protein targets of drugs?

Answer 7

receptors
enzymes
transporters
ion channels

Question 8

what are some key properties of receptors as drug targets to note?

Answer 8

they are accessible as often are on the cell surface

they are relatively specific, but at very high doses a drug can affect other targets causing unwanted side effects (concentration is critical)

the same receptor may be present in an area you do not want to target, e.g. if your receptor is also found in the lungs you do not want to cause something like asphyxiation

Question 9

inverse agonist vs antagonist?

Answer 9

an inverse agonist produces an effect opposite to that of an agonist, and inverse agonists can also be “full” or “partial” the way agonists can. An antagonist produces no effect on its own but blocks the effects of both agonists and inverse agonists

Question 10

how do drugs typically affect ion channels?

give a simple example

Answer 10

as blockers or modulators - causing prolonged opening OR shutting of the channels

lidocaine/lignocaine is a small molecule that acts to block vol-gated Na+ channels as a local anesthetic for a sore throat (strepsils)

Question 11

what are the three main ways in which a drug can act on enzymes?

Answer 11

Inhibitors

False substrates - a molecule that mimics the structure of a substrate for a particular enzyme but is not actually metabolized by that enzyme - I think its a from of competitive inhibitor. produce abnormal metabolites???

Prodrugs bind to endogenous enzymes, resulting in production of an active drug

Question 12

how do drugs usually target transporters?

give an example

Answer 12

Drugs can target transporters by inhibiting them (at AS or away from it)
Drugs can act as false substrates - competitive inhibition
prozac/fluoxetine = antidepressant binding to serotonin transporters

Question 13

most common/easy drug targets?

Answer 13

receptors and enzymes

Question 14

what are the main synthetic and endogenous opiates?

Answer 14

morphine and heroine (which is just diacetylmorphine)
fentanyl - 100x more potent than morphine

endogenous opiates are endorphins like met-enkephalin, which are peptide hormones involved in reward pathways

Question 15

how can opioids cause side effects?

Answer 15

Mu opioid receptors are also found in the neurons controlling gut motility, so a common side effect is constipation

Question 16

how is heroin/fentanyl overdose treated?

Answer 16

naloxone - an antagonist for opioid receptors

Question 17

ligand gated ion channels - describe the structure

Answer 17

ligand binding site on apical side, several transmembrane domains

usually several protein sub units coming together

amino acids lining the pore are important for specificity toward certain ions

Question 18

LGICs - explain why different subunits within the same receptor family can be used?

e.g.curare?

Answer 18

nicotinic ACh receptors are all over the body, so if we only wanted to target the ones in the CNS for example, we could try and find a drug that is selective for these based on the fact that different subunits are present

e.g. curare only works as as an antagonist for nAChRs in skeletal muscle

Question 19

what kind of LGIC are nAChRs?

Answer 19

pentameric cys-loop ion channels

Question 20

briefly explain the structure of a GPCR

Answer 20

7 transmembrane domains, apical ligand binding site, an associated trimeric G protein with a, b and y subunits

coded for as one protein/by one gene

very fast (not as fast as LGIC)

multiple subfamilies based on their endogenous agonists

Question 21

how many GPCRs are there?

Answer 21

more than 800

Question 22

what is the general mechanism of a GPCR?

Answer 22

Upon ligand binding, GDP is swapped for GTP, the alpha subunit of the G-protein dissociates from the b/y subunit to both mediate different downstream paths. very fast
Alpha subunit has GTPase activity, eventually hydrolyses its GTP to GDP and system resets.
When things separate/reunite as a result of GTP-GDP swap or vice versa - just mention that its a change in affinity

Question 23

which G proteins effect which pathways?

Answer 23

Gs (inc) and Gi (dec) work on adenylate cyclase which makes cAMP which activates PKA
Gq activates phospholipase C to turn PIP2 into DAG (stays in the membrane, can activate PKC) and IP3 (works on channel in ER to cause Ca2+ influx) etc…

Question 24

what molecule is useful when a stimulus for a GPCR is prolonged?

Answer 24

arrestin - back up system to turn off an activated GPCR when stimulus is prolonged

Question 25

nuclear receptors - structure? what are the two classes?

Answer 25

cytosolic - will always have a ligand binding domain and a DNA binding site Ligands must be able to cross plasma membrane (lipid soluble) like steroid hormones Class 1 - steroid receptors, homodimers come together when agonist binds Class 2 - non-steroid hormone receptors - heterodimers that bind different ligands - different subunits come together when agonists bind

Question 26

what is FLIPR?

Answer 26

Fluorescence imaging plate reader Put cells expressing GPCR in your wells, screen 100,000s of chemicals per day to identify potential agonists/antagonists

Question 27

kinase-linked receptors (specifically RTKs) typical structure?

Answer 27

Enzymatic (intracellular) domain that does the phosphorylation is built into the receptor or is an accessory protein Has a single transmembrane domain to anchor Extracellular ligand binding domain

Question 28

general mechanism of a kinase linked receptor?

Answer 28

Two receptors typically come together as a dimer upon activation, and one of the dimers can phosphorylate the other (trans phosphorylation). Often the receptor when activated will phosphorylate another molecule that activates a monomeric G protein (like Ras)

Question 29

cytokine receptors - how do they work?

Answer 29

Cytokine receptors (tyrosine kinase linked receptors again) - These are an example of a RTK that have an accessory protein providing the enzymatic domain to phosphorylate tyrosine residues they dimerise when activated, and also cross phosphorylate each other, and downstream targets that enter the nucleus to control transcription

Question 30

give two examples of disease caused by issues with receptors

Answer 30

Myasthenia gravis (muscle wastage, autoimmune condition where antibodies destroy NAChRs at NM junction) Thyroid hypersecretion (the body makes an antibody that chronically activates the receptor)

Question 31

what is KD?

Answer 31

KD is the rate of the reverse reaction (ligand-receptor complex unbinding back to ligand and receptor) divided by the rate of the forward reaction (binding) KD is a measure of affinity it can be defined as the concentration of a drug at which 50% occupancy is acheived?

Question 32

for an agonist, there are two distinct steps. what are they?

Answer 32

biding to a receptor - this is occupation, which is governed by affinity activating the receptor - this is activation and is governed by efficacy

Question 33

what does a small KD tell us about a drug's affinity for a certain receptor?

Answer 33

small KD = high affinity

Question 34

units of KD?

Answer 34

regular concentration units

Question 35

what orders are the forward and backwards reactions of agonist binding

Answer 35

Forward reaction is 2nd order as it depends on concentration of both A and R Reverse reaction is 1st order as it only depends on concentration of AR

Question 36

define occupation

Answer 36

Occupation = proportion of receptors with bound drug, relative to total number of present receptors. Can only be between 0 (no drug) and 1 (all receptors occupied)

Question 37

define affinity define efficacy define specificity

Answer 37

Affinity = how well a drug binds to a receptor, so applies to both agonists and antagonists (KD) Efficacy = a drug’s ability to activate a receptor, efficacy only applies to agonists specificity = how a drug interacts structurally with a receptor

Question 38

what is the equation for occupancy?

Answer 38

no. of receptors occupied / total no. of receptors

Question 39

when trying to determine a drug's affinity for a receptor, why cant we look at/measure the response it evokes?

Answer 39

the size of response is not linearly related to affinity. this can be due to signal cascades measuring the size of a response is also not possible if your drug is an antagonist

Question 40

outline the steps in a radioligand binding assay

Answer 40

1. radioactively label your ligand of interest 2. prepare your tissue sample/cell line with desired receptor 3. measure radioactivity from total binding (specific + non-specific) 4. measure radioactivity from non-specific binding 5. calculate specific binding (take non away form total)

Question 41

in a radioligand binding assay how do you measure total binding?

Answer 41

1. get an array of test tubes with your cell line/sample 2. to these test tubes add different concentrations of your radioligand (0.1, 0.3, 1, 3 etc... for example) 3. wash away unbound ligand in these tubes, the radioligand will bind to the target receptor, as well as other sites in your sample you are not interested in the radioactivity from these tubes is therefore a measure of specific and non-specific binding i.e. total binding

Question 42

in a radioligand binding assay, how and why do you measure non-specific binding?

Answer 42

1. to another lot of test tubes with your sample, add your radioligand (in same concentrations as you did to measure total binding) 2. ALSO add a large excess of 'cold ligand' (not labelled) 3. the cold ligand outcompetes the radioactive one for the target receptor binding sites, leaving the radioactively labelled ligand to mostly be involved in non-specific binding so the radioactivity measured here is taken as a measurement of non-specific binding now that you have calculated total binding AND non-specific binding, just minus the later from the former and you get specific binding

Question 43

when you plot specific binding (radioactivity counts/receptor against concentration of radioligand added), what shape is the graph? what shape is it when made semi-logarithmic?

Answer 43

rectangular hyperbola. plateau = saturation when you plot the radioligand concentration on a log scale, you get a sigmoid curve

Question 44

from a binding curve, achieved from a radioligand binding assay, what can you learn

Answer 44

where the graph plateaus is 100% occupancy the concentration at 50% occupancy is equal to KD

Question 45

what is the langmuir equation?

Answer 45

conc of specifically bound = (Bmax i.e. the y value where graph plateaus X conc. of drug added) all over conc of drug added + KD

Question 46

what is the scatchard equation?

Answer 46

B/F = (Bmax - B) / KD B = conc. of bound F = conc of free radioligand added (same as Xa) Bmax = max number of receptors/binding sites where the graph plateaus (Y value)

Question 47

if you compare binding curves of different drugs with the same receptor (or vice versa), how do you know which has highest affinity?

Answer 47

the curve furthest to the left, as it reaches 50% occupancy quicker than the rest it will therefore have the lowest KD

Question 48

if you added a drug to two different tissues from different parts of the body BUT looking at the same target receptor, and the curves did not matchh, what could you conclude?

Answer 48

if the curves don't match, the affinity between drug and receptor do not match the receptors in the two different tissues must be different subtypes

Question 49

when making your radioligand what must be considered?

Answer 49

Purity - Must be SUPER pure Must also be the same enantiomer Degradation - Don't want it to degrade - solutions include Free-radical scavenger like ethanol, cold, avoid light

Question 50

what are the options for radioactively labelling your ligand?

Answer 50

Common radioactive tracers - swap a H for a radioactive H, hardly affecting structure and has super long shelf life However gives off a lot of radioactivity and has long half life - not safe, expensive to work with, hard to make More common is iodine 125, much easier to work with and is cheap, shorter half-life, however it may alter structure of ligand so can be a pain

Question 51

how is ligand and receptor integrity protected in a binding assay?

Answer 51

Incubation - ligand and receptor integrity must be protected from degradation, so often ice the experiment to stop enzyme activity, plus other additives to stop things like e.g. oxidation

Question 52

why are things like albumin used in a binding assay?

Answer 52

Ligands can often bind non-specifically to the tube they are in, the filter used, glass etc… So we use anti absorbents to reduce chances of non-specific binding, e.g. albumin to coat and bind to the plastic first etc… * to avoid confusion - when measuring non-specific binding, this is to molecules/other receptors in your tissue sample/cell line

Question 53

efficacy - agonist vs antagonist?

Answer 53

efficacy = ability of a drug to cause formation of an active receptor agonist = has some level of efficacy agonist by definition has 0 efficacy

Question 54

define EC50

Answer 54

the effective conc. that gives 50% of maximal response in THAT TISSUE

Question 55

are % occupancy and % response equal?

Answer 55

no they are not - e.g. a max response can be caused at a concentration that is less than what is required for maximum occupancy amplification in a signal cascade is also why they are not equal efficacy is independent of affinity

Question 56

100 % occupancy is not required for maximum response - what is this called and why is it the case?

Answer 56

this is receptor reserve in some cases as little as 5% occupancy is needed for a max response This excess of receptors is like a safety net to keep probability of an agonist finding it’s receptor very high, and conc. Of agonist needed very small (conservative), we can also afford to lose a lot of receptors while stilll getting a max response

Question 57

what is the hill equation for calculating what size response a certain agonist concentration will cause?

Answer 57

response = (conc. that gives max response)^n over [Xa]^n + EC50^n where Xa = conc. of agonist in question where n = slope factor or hill slope, a measure of the number of molecules that need to bind to a receptor to activate it

Question 58

define a partial agonist

Answer 58

ellicits a response, but cannot cause max response i.e. efficacy between 0 and 1 not to do with affinity

Question 59

when is EC50 = KD, and why?

Answer 59

for partial agonists this is because a partial agonist must reach 100% occupancy to produce the maximum response it is capable of in that tissue so conc for 50% response is equal to conc for 50% occupancy so KD = EC50

Question 60

why are partial agonists desirable for treatments?

Answer 60

Partial agonists are very desirable as treatments because they cannot ‘max out’ a system

Question 61

what happens when you apply a partial agonist with a full agonist?

Answer 61

shifts conc./response curve to the right, as the partial agonist competes and acts as an antagonist (this is resolved by raising full agonist conc. So it out-competes)

Question 62

what is potency and how is it determined?

Answer 62

potency is a relative term, used in comparison to a standard drug you must do a full conc/response comparison between drugs to see which is more 'potent' example - you measure the response cause by two drugs at 3.0 M, but they both have the same max. response, and have already reached it, so they appear to have the same potency, but one of them reaches this max response much quicker and you wouldn't know

Question 63

how do agonists vary for receptors in different tissues and why?

Answer 63

an agonist's EC50 WILL vary for the same receptor in different tissues and the presence of spare receptors an agonist's AFFINITY will not vary between (the same kind of) receptor