Lectures 1-4 - Radioligand binding + quantitative Flashcards
Give three examples of drugs that show how molecular pharmacology is important in clinically relevent drugs
Atorvastatin
Ramiprill
Sertraline (an SSRI)
all widely used, all require knowledge of the protein-ligand interactions to function
Explain examples of pharmacological drugs created through targeting endogenous ligands
Humria - an Ab for TNF-a (an inflammatory cytokine)- a biologic that binds to TNF-a and inhibits inflammation
Keytruda Ab against PD-1 (PD-1 involved in signaling tumours)
Eylea another Ab that targets VEGF (a growth factor)
Reasons why molecular pharmacology studies are increasing
Identify endogenous and exogenous ligands to study function and for pharmacological purposes
Isolate, sequence, clone and express receptors and enzymes (compare WT and mutatnt)
Explain how Isolating, sequencing, cloning and expressing receptors and enzymes (compare WT and mutatnt) can be done in molecular pharmacology
Introduce mutations to predict what would change interaction between ligand and receptor, done through:
- Functional assays (in vitro screening) e.g. introduce receptor into transgenic system, take a tissue slice ad look at response e.g. look for receptor activation
- Model systems (e.g., cell lines for in vitro screening) - transfect cell line with receptor and measure the interaction of different drugs and molecules with receptor
- Structures: ligand-binding and function (rational drug design in silico modelling – make predictions of interactions if you change binding pocket)
- Knowledge of in vivo system (including ‘orphan’ receptors ( receptor where we don’t know what the ligand is especially G protein coupled receptors common in vivo can be used to find out about orphan receptors by e.g. by knocking out receptor))
Explain the molecular methods for receptor characterisation
- tissue with rich source of receptor protein and a selective high affinity ligand (fishing rod) (used in 80s for GABA-A, tissue used was bovine cerebral cortex, ligand used was 3H-flunitrazepam)
- Ligand has now labeled receptor in tissue (Autoradiography uses brain slice instead of homogenate and sees disruption of receptors by imaging)
- Characterise binding proerties of the receptor (its pharmacilogical profile, drug interactions)
- To isolate receptor - treat with detergent to libetare the receptpr protein from membrane and then isolate and purify by e.g. affinity chromatography
- Purified receptor, next:
- Further analysis: biochemical/biophysical/structural
- could raise antibodies for it (immunocytochemistry) to find out where localised
- Reconstitution (put into artificial membrane) and therefore discovery of function
- Partial sequence of the receptor protein, first 30/40 as from N terminus, can then predict the gene sequence that encodes protein sequence and then screen cDNA libraries to identify whole receptor, primary structure if subunit can be predicted
- Once cDNA identified: expression studies, transfection for drug screening; allowing a receptor profile to be made
BUT this is longer way, now shortcuts using bioinformatics and genetics
Explain the uses of bioinformatics as a shortcut once a protein/gene of interest has been identified
- Clone cDNA or synthesis gene de novo, allowing for expression studies
This leads to 2 things:
- solubilisation/purification - then biochemical/biophysical/structural analysis
- stable transfection and characterisation/screening
- Study of endogenous receptor (CRISPR, siRNA etc.)
- In silico modelling/screening
Explain what types of binding sites drugs bind to in tissue
Some are high affinity specific receptor sites for the drug
Some are low affinity nonspecific binding sites for the drug e.g., a lipophilic drug may simply partition into lipid membranes
Disadvantage as drugs will partition into the membrane, that’s not receptor binding
Explain what radioligand binding is and what it helps to do
Take tissue e.g. brain and something that will bind to receptor of interest that is radiolabeled
Radioligand binding techniques allow us to resolve the specific receptor binding, and to characterize this
Receptors are typically:
Glycoprotein
Multi-subunit
Expressed at low density – effective at transducing responses so don’t need high density
Reversible binding of drugs
High affinity
What is the rationale used when resolving specific receptor binding
Receptor binding is finite and saturable
Non-specific binding is non-saturable
By what logic/process do we determine specific receptor binding
Ligand will bind to specific receptors, non-specific receptors and it will partition through membrane
We resolve this by knowing that receptor binding is finite and saturable
Add an excess of unlabeled old ligands – displace drug that’s bound to receptor, now the specific will be labeled with unlabeled ligands
BUT wont displace binding to non-specific binding sites, but may partition into membrane
Total – non-specific = specific binding
Describe the process of designing a radioligand binding assay
- Decide on the source of the receptor
- Identify appropriate radioactive ligand(s)
- Identify a means to separate bound ligand from unbound ligand
- Identify a means for distinguishing specific from non-specific binding
-> Estimate the amount of ligand bound at different concentrations of ligand
Explain the different sources of receptor used in radioligand binding
Solubilised receptor – take tissue and treat with receptor to solubolise receptor
Homogenised membranes (e.g. synaptosomal preperation) - take brain tissue and homogenise
Freshly isolated or cultured cells
Cells transfected with cloned receptor (often used in pharmaceutical field)
Tissue homogenates or fresh slices of tissue (5-30 µm thick)
Animal models (sometimes human e.g. post mortem brain to measure receptor density in psychiatric conditions)
Explain how an appropriate radioactive ligand is identified for radioligand binding
If choosing a ligand (imaging, quantification) usually we want:
- High affinity for receptor (KD < 1 nM)
- Slow dissociation kinetics – goes hand in hand with high affinity
- Antagonists > Agonists – reason: agomnist bind – induces confomrational change that brings about signalling transduction cascade, affinity may change. Antagonist meand you can study structure characteristics, also antagonists have a higher affinity than agonists
- High selectivity with minimal non-specific binding (never avoid non-specific completely but want to reduce)
Be compatible with labelling and stable under experimental conditions (label should not influence binding, cause radiolysis, etc.)
Explain the advantages of using a radioactive label in receptor binding experiments
V high sensitivity
Easy incorporation
Usually no biological interference
Simple detection
Low expense
Explain the disadvantages of using a radioactive label in receptor binding experiments
Detection not usually multiplex
Not often real time measurements
Needs care in terms of safety (radiation)
Explain when fluorecent labels are used
when you want to multiplex a receptor, can look at binding simultaneous of different ligands by labelling with different tags
Measurements can also be done in real time
What usual isotopes are used in radioligand binding
tritium (3H), carbon-14 (14C), phosphorous-32 (32P), sulphur-35 (35S), iodine-131 (131I)
How do we measure radioactivity?
q = number of disintegrations per second. Usually use disintegration per minute (d.p.m) - very sensetive measure (si unit) so not often used, Usually use:
Curie- Ci = 2.22×1012 d.p.m (a measure of radioactivity)
What is the specific activity of a radiolabelled drug
Specific activity (SA) = activity/mole e.g., 20 Ci/mmol
Explain properties of [3H]
β emitter
SA ~ 30 Ci/mmol
half-life ~12 years (from 30 to 15)
Explain properties of [32P]
β emitter
SA ~ 9000 Ci/mmol
half-life ~14 days (have to correct for half-life in analysis)
Explain properties of [131I]
γ emitter (more of a biohazard)
SA ~ 2000 Ci/mmol
half-life ~60 days
How is radioactivity measured in solution?
Scintillation Counter
Explain cpm vs dpm
counts per minute is data straight from Scintillation counter, needs to be corrected into dpm as not 100% accurate
What are ways to separate bound ligands from unbound ligands
Filtration – collects bound ligand, can wash away free ligand RW for details
Centrifugation – particulate tissue is sedimented at bottom of tube, cut bottom of tube of then put into the machine
Sedimentation – tissue/cell culture, apply suction and select cells at bottom of well, again cut and put into machine for counting
equilibrium dialysis - semi-permeable membrane in chamber, only soluble (unbound) ligands can pass through
size exclusion chromatography - Receptor and ligand bind to form complex, column loaded with porous beads, free ligand is a small molecule, everything else big, only small molecules can get into beads, delaying their passage through the column
Explain ways in which to distinguish between specific and non-specific binding
Homologous competition:
- Radiologand, and cold ligand, same drug, one labelled, one not
- [Radiologand] constant
- [Cold ligand] variable
Heterologous competition:
- Radiolabeled drug, another drug, that binds to the same receptor, use this cold analogue to displace (doesn’t have to be cold, just has to displace)
- End up with a dataset showing amount of radioactivity plotted against log of [cold competitor]
When you get to point where cant displace binding anymore, that is the non-specific binding
Total – non-specific = specific binding
What info can be derived from calculating specific receptor binding
The affinity of the receptor for a specific ligand Kd (equilibrium dissociation constant)
The pharmacological profile of a receptor
The number of receptor sites in a tissue Bmax (maximal binding affinity)
Characteristics of the drug receptor interaction e.g., cooperativity
Structure-function relationships
What are the disadvantages of radioligand binding?
Cannot distinguish between agonists, partial agonists, antagonists
Non-physiological environment
Long incubation times may lead to receptor desensitisation
What are the advantages of radioligand binding?
No pharmacokinetic confounds
Provides a direct measure of ligand affinity for a specific receptor, Kd
Provides a direct measure of receptor density, Bmax
Excellent approach for defining the molecular characteristics of drug-receptor interactions
Suitable for high throughput drug screening
Excellent approach for defining pharmacological profile of a receptor
What information can be obtained about receptors?
the affinity of drug-receptor interactions
the number of drug binding sites
their pharmacological properties
structure-function relationships
What are the definitions of ligand and bound?
‘ligand’ is a molecule that binds to a receptor
‘bound’ is the amount of ligand bound to a receptor
Explain how quantitative studies are carried out in radiologand binding
- Incubate the tissue/cells etc. with radio-labelled ligand
- Separate the free drug from the bound drug by centrifugation or filtration
- estimate the amount bound at different concentrations of ligand
- Use other drugs to displace the ligand to charaterise the pharmacology of the receptor binding site
- Introduce mutations into the receptor protein to investigate structure-function
- Bound ligand plotted against the ligand concentration
Explain hoe specific binding is determined from a graph showing ligand conc. against bound ligand, what other things can be determined from this
Total binding = specific + non-specific
Determine non-specific binding in the presence of an excess of unlabelled ligand to saturate the specific sites
Specific binding = total - non-specific
2 key measures from this: affinity (Kd ) and receptor density (Bmax)
The lower the Kd, the higher the affinity
Why bother with quantitative studies
Pharmacological profiling (ligands for receptor subtypes)
Identification and isolation of receptors
Quantifying receptor number
What equation can you derive if you assume law of mass action, a neglagible amount of drug is bound, and there is equilibrium, from (L) + (R) <-> L-R complex (B)
k1[L][R] = k-1[B]
(where K1, k-1 = rate constants)
What is the equation of Kd (equilibrium dissociation rate constant)
k-1/k1
Why do we use Kd over Ka in determining Bmax
The units are easier to work with:
Ka has units of litres/mole
Kd has units of mol/L I.e. molar
What is the equilibrium equation?
B/Bmax = [L]/[L] + Kd
Bmax is the total number of receptors
L is the total drug concentration
B is the concentration of the bound drug
KD is the equilibrium dissociation constant of the drug
When half the receptors are occupied B=
Bmax/2
Kd = L
What does Bmax measure?
Total number of receptors (so receptor density)
Whats a typical range of values for Bmax?
10-12 to 10-15 moles/mg protein
steps to calculate amount of protein
- Convery c.p.m to d.p.m (will be given a counting efficiency)
- convert Specific activity to Ci/mol (e.g. if in ci/mmol, x by 10*3)
- Work out d.p.m per mole by multiplying SA by 2.2 x1012 (1 Ci = 2.2 x 1012 dpm)
- Work out how many mol of ligand bound by doing dpm (step 1)/dpm per mol (Step 3)
- Account for amount of protein - (will be given amount of protein in each assay tube - divide by this (e.g. 0.13mg) to get value in mol/mg
- repeat for all rows/repeats
How do you work out Bmax and Kd?
Once the mol/mg have been discovered (could be a different unit e.g. fmol)
can plot this against concentration of radiolabelled ligand used (x - axis) and the draw curved line
Bmax, is maximum value of bound ligand (top y-axis value)
Kd, take half the Bmax, and draw line down to y-axis from. interception point on curve, this will tel, you Kd in whatever unit conc. was in e.g. nM
