basics + receptor theory Flashcards
define drug
chemical of known structure causing a biological effect, can be synthetic or plant derived small molecules, or can be biopharmaceutical
define medicine
containing one or more, drugs, provides a therapeutic effect
what are excipients?
ingredients that stabilise the active drug in a medicine
what are biopharmaceuticals?
first gen vs second?
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
what is humira?
a monoclonal antibody used to treat rheumatoid arthritis
what is a potential draw back of biopharmaceuticals?
can be very difficult to get them into the CNS
what are the most common protein targets of drugs?
receptors
enzymes
transporters
ion channels
what are some key properties of receptors as drug targets to note?
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
inverse agonist vs antagonist?
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
how do drugs typically affect ion channels?
give a simple example
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)
what are the three main ways in which a drug can act on enzymes?
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
how do drugs usually target transporters?
give an example
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
most common/easy drug targets?
receptors and enzymes
what are the main synthetic and endogenous opiates?
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
how can opioids cause side effects?
Mu opioid receptors are also found in the neurons controlling gut motility, so a common side effect is constipation
how is heroin/fentanyl overdose treated?
naloxone - an antagonist for opioid receptors
ligand gated ion channels - describe the structure
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
LGICs - explain why different subunits within the same receptor family can be used?
e.g.curare?
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
what kind of LGIC are nAChRs?
pentameric cys-loop ion channels
briefly explain the structure of a GPCR
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
how many GPCRs are there?
more than 800
what is the general mechanism of a GPCR?
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
which G proteins effect which pathways?
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…
what molecule is useful when a stimulus for a GPCR is prolonged?
arrestin - back up system to turn off an activated GPCR when stimulus is prolonged
nuclear receptors - structure? what are the two classes?
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
what is FLIPR?
Fluorescence imaging plate reader
Put cells expressing GPCR in your wells, screen 100,000s of chemicals per day to identify potential agonists/antagonists
kinase-linked receptors (specifically RTKs) typical structure?
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
general mechanism of a kinase linked receptor?
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)
cytokine receptors - how do they work?
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
give two examples of disease caused by issues with receptors
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)
what is KD?
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?
for an agonist, there are two distinct steps. what are they?
biding to a receptor - this is occupation, which is governed by affinity
activating the receptor - this is activation and is governed by efficacy
what does a small KD tell us about a drug’s affinity for a certain receptor?
small KD = high affinity
units of KD?
regular concentration units
what orders are the forward and backwards reactions of agonist binding
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
define occupation
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)
define affinity
define efficacy
define specificity
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
what is the equation for occupancy?
no. of receptors occupied / total no. of receptors
when trying to determine a drug’s affinity for a receptor, why cant we look at/measure the response it evokes?
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
outline the steps in a radioligand binding assay
- radioactively label your ligand of interest
- prepare your tissue sample/cell line with desired receptor
- measure radioactivity from total binding (specific + non-specific)
- measure radioactivity from non-specific binding
- calculate specific binding (take non away form total)
in a radioligand binding assay how do you measure total binding?
- get an array of test tubes with your cell line/sample
- to these test tubes add different concentrations of your radioligand (0.1, 0.3, 1, 3 etc… for example)
- 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
in a radioligand binding assay, how and why do you measure non-specific binding?
- to another lot of test tubes with your sample, add your radioligand (in same concentrations as you did to measure total binding)
- ALSO add a large excess of ‘cold ligand’ (not labelled)
- 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
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?
rectangular hyperbola. plateau = saturation
when you plot the radioligand concentration on a log scale, you get a sigmoid curve
from a binding curve, achieved from a radioligand binding assay, what can you learn
where the graph plateaus is 100% occupancy
the concentration at 50% occupancy is equal to KD
what is the langmuir equation?
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
what is the scatchard equation?
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)
if you compare binding curves of different drugs with the same receptor (or vice versa), how do you know which has highest affinity?
the curve furthest to the left, as it reaches 50% occupancy quicker than the rest
it will therefore have the lowest KD
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?
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
when making your radioligand what must be considered?
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
what are the options for radioactively labelling your ligand?
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
how is ligand and receptor integrity protected in a binding assay?
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
why are things like albumin used in a binding assay?
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
efficacy - agonist vs antagonist?
efficacy = ability of a drug to cause formation of an active receptor
agonist = has some level of efficacy
agonist by definition has 0 efficacy
define EC50
the effective conc. that gives 50% of maximal response in THAT TISSUE
are % occupancy and % response equal?
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
100 % occupancy is not required for maximum response - what is this called and why is it the case?
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
what is the hill equation for calculating what size response a certain agonist concentration will cause?
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
define a partial agonist
ellicits a response, but cannot cause max response
i.e. efficacy between 0 and 1
not to do with affinity
when is EC50 = KD, and why?
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
why are partial agonists desirable for treatments?
Partial agonists are very desirable as treatments because they cannot ‘max out’ a system
what happens when you apply a partial agonist with a full agonist?
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)
what is potency and how is it determined?
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
how do agonists vary for receptors in different tissues and why?
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
