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pharmacology > basics + receptor theory > Flashcards

basics + receptor theory Flashcards

1
Q

define drug

A

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

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2
Q

define medicine

A

containing one or more, drugs, provides a therapeutic effect

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3
Q

what are excipients?

A

ingredients that stabilise the active drug in a medicine

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4
Q

what are biopharmaceuticals?

first gen vs second?

A

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

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5
Q

what is humira?

A

a monoclonal antibody used to treat rheumatoid arthritis

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6
Q

what is a potential draw back of biopharmaceuticals?

A

can be very difficult to get them into the CNS

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7
Q

what are the most common protein targets of drugs?

A

receptors
enzymes
transporters
ion channels

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8
Q

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

A

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

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9
Q

inverse agonist vs antagonist?

A

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

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10
Q

how do drugs typically affect ion channels?

give a simple example

A

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)

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11
Q

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

A

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

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12
Q

how do drugs usually target transporters?

give an example

A

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

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13
Q

most common/easy drug targets?

A

receptors and enzymes

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14
Q

what are the main synthetic and endogenous opiates?

A

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

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15
Q

how can opioids cause side effects?

A

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

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16
Q

how is heroin/fentanyl overdose treated?

A

naloxone - an antagonist for opioid receptors

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17
Q

ligand gated ion channels - describe the structure

A

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

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18
Q

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

e.g.curare?

A

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

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19
Q

what kind of LGIC are nAChRs?

A

pentameric cys-loop ion channels

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20
Q

briefly explain the structure of a GPCR

A

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

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21
Q

how many GPCRs are there?

A

more than 800

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22
Q

what is the general mechanism of a GPCR?

A

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

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23
Q

which G proteins effect which pathways?

A

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…

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24
Q

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

A

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

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25
Q

nuclear receptors - structure? what are the two classes?

A

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

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26
Q

what is FLIPR?

A

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

27
Q

kinase-linked receptors (specifically RTKs) typical structure?

A

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

28
Q

general mechanism of a kinase linked receptor?

A

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)

29
Q

cytokine receptors - how do they work?

A

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

30
Q

give two examples of disease caused by issues with receptors

A

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)

31
Q

what is KD?

A

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?

32
Q

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

A

biding to a receptor - this is occupation, which is governed by affinity

activating the receptor - this is activation and is governed by efficacy

33
Q

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

A

small KD = high affinity

34
Q

units of KD?

A

regular concentration units

35
Q

what orders are the forward and backwards reactions of agonist binding

A

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

36
Q

define occupation

A

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)

37
Q

define affinity
define efficacy
define specificity

A

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

38
Q

what is the equation for occupancy?

A

no. of receptors occupied / total no. of receptors

39
Q

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

A

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

40
Q

outline the steps in a radioligand binding assay

A
  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)
41
Q

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

A
  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

42
Q

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

A
  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

43
Q

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?

A

rectangular hyperbola. plateau = saturation

when you plot the radioligand concentration on a log scale, you get a sigmoid curve

44
Q

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

A

where the graph plateaus is 100% occupancy

the concentration at 50% occupancy is equal to KD

45
Q

what is the langmuir equation?

A

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

46
Q

what is the scatchard equation?

A

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)

47
Q

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

A

the curve furthest to the left, as it reaches 50% occupancy quicker than the rest

it will therefore have the lowest KD

48
Q

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?

A

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

49
Q

when making your radioligand what must be considered?

A

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

50
Q

what are the options for radioactively labelling your ligand?

A

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

51
Q

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

A

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

52
Q

why are things like albumin used in a binding assay?

A

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
53
Q

efficacy - agonist vs antagonist?

A

efficacy = ability of a drug to cause formation of an active receptor

agonist = has some level of efficacy

agonist by definition has 0 efficacy

54
Q

define EC50

A

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

55
Q

are % occupancy and % response equal?

A

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

56
Q

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

A

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

57
Q

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

A

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

58
Q

define a partial agonist

A

ellicits a response, but cannot cause max response
i.e. efficacy between 0 and 1

not to do with affinity

59
Q

when is EC50 = KD, and why?

A

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

60
Q

why are partial agonists desirable for treatments?

A

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

61
Q

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

A

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)

62
Q

what is potency and how is it determined?

A

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

63
Q

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

A

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

pharmacology (9 decks)

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