Lecture 2

Question 1

What are the main different types of bioassays

Answer 1

Whole organism (measure heart rate, blood pressure, etc).

Organ

Cell culture

Organ slice

Question 2

What is an advantage to using tissue bath physiometry?

Answer 2

advantages: you have the whole organ rather than just individual cells, so you can study this under conditions that mimic physiological conditions. There may be various things interferingwith the drug reaching the target organ, so you can get around this by having the whole target organ
present.

Question 3

What is sulforhodamine B?

Answer 3

Dye for SRB assay. Taken up by living cells and can be washedaway from cells that dont take it up. Can do this for adherent cells or cells in suspension. Can then take the dye up and then resuspend the extracted dye and you get a colored product you can measure.There is a direct correlation between the extracted dye and the number of living cells present. Darker dye indicates more cells. Used to measure cell proliferation in response to a drug. If the cells aggregate (dark spots in the well) it can lead to inaccurate results by affecting the due uptake.

Question 4

What are some measurable cell behaviors/ effects for assays?

Answer 4

Cell proliferation, cytotoxicity, cell cycle arrest, cell survival.

Question 5

How do you test for cell proliferation?

Answer 5

Use sulforhodamine B and then do a cell count with a hemocytometer.

Question 6

How do you test for cell cytotoxicity?

Answer 6

Dye exclusion (use trypan blue which is excluded from living cells). You then count the number of living cells (blue cells are dead).

Could also do an apoptosis assay using Annexin V binding and mitochondrial membrane potential. Apoptosis leads to a loss of mitochondrial membrane potential due to holes in the mitochondria.

Question 7

How do you test for cell cycle arrest?

Answer 7

Flow cytometry using a DNA binding dye. Or use fluorescence microscopy: mitotic index (DAPI) BrdU (analog of thymidine) incorporation: S phase arrest. DAPI intercalates in the DNA.

Question 8

How to test for cell survival?

Answer 8

Colony Forming Assay: measure of cell survival. Can treat cells with a drug, plate them out so you have relatively few cells per well, let them grow into visible colonies. Used because you may get cells proliferating in response to a drug, but over time they may die. so it can test for toxic effects that take a while to develop (long term effect of drugs).

Question 9

What is organ slice assay?

Answer 9

Let’s you carry out the experiment on a portion of the organ over a substantially longer period of time compared to what you’d be able to do with the whole organ.

Question 10

Why is it important to use more than one animal model? Why can’t you often use a whole organ in these assays?

Answer 10

Cats taking the anti-thyroid drug showed hemolytic effects. Hemolysis was found to occur in the
liver, so they took slices of liver from human and animal livers to look at hemolysis effect. Combined blood from animal with slice of liver and compared to a control for hemolysis. With a whole liver, you wouldnt get good perfusion of the blood and it wouldn’t last very long. Better than using individual cells becasue in this case you have all cell types of the liver present. The same effects weren’t seen in different animals.

Question 11

Can flow cytometry be used to look at the number of cells in different phases of the cell cycle? Explain the peaks shown in the notes.

Answer 11

Yes. Why large peak for G1? Cells are in G1 for longer than any other part of the cell cycle. There is a troph for
S phase because it represents all of DNA division. This allows for a gradient of DNA content, starting at
diploid and eventually reaching tetraploid by G2. In S phase, some are partially replicated and so they have
a little but more than diploid while others are almost completely replicated. Youd cant distinguish between G2 and M phase in this
assay. If you did want to, you could use cell cycle specificmarkers (proteins only present/ active in M phase) to distinguish.

Question 12

What is a problem with some of the methods for receptor isolation and how would this affect your decision of which to use?

Answer 12

One problem during receptor isolation is heat generation because it denatures proteins and makes them lose their activity. Sonication generates the most heat of the 3 listed, followed
by homogenization. Detergent lysis causes very minimal heat generation so doesn’t cause damage. Sonication is quick, so thats one reason it may be used instead of homogenization.Tumor
tissue is hard to homogenize, so sonnication may be used for that. work with small samples and do on ice. Detergents often interfere with protein
function (break down membranes and release proteins, so if membrane is important for function it is lost). So wouldnt use detergent for transmembrane protein isolation. Detergents can also interefere with
drug binding to receptor.

Question 13

How can receptors be isolated?

Answer 13

Generally need to break up the cell.

• tissue/ cell disruption: can do homogenization, sonication (short bursts of sound energy), and detergent lysis.
• sub-cellular fractionation: differential centrifugation.
• further purification with physical/ biochemical/ immunological methods.

Question 14

What are some important assumptions made when determining the Kd?

Answer 14

• the association of drug and receptor occurs as a result of a simple bimolecular interaction. 1 mole of drug bound to 1 mole of receptor.
• the reaction is at equilibrium.
• the receptor concentration is much lower than the total drug concentration.

Question 15

Why is it important to have much less receptor present than total drug concentration?

Answer 15

This is important because if you have a receptor concentration that is
pretty close to the range of total drug that your’e adding, you may
end up saturating the amount of receptors (super saturation), so you
don’t have enough receptor to take up all of the drug. Also, the receptor
total is going to be about equal to the free receptor.

Question 16

Why is studying drug binding important?

Answer 16

The only way to determine KD unambiguously. KD apparent can be wrong if occupancy assumptions are not met. Can also identify/ confirm identity of drug receptor. Doing a drug binding assay satisfies these.

Question 17

What’s the relationship between Kd and time needed to reach equilibrium? Do you always have to wait for the reaction to reach equilibrium before measuring?

Answer 17

Lets say there are two drugs, one with a very large Kd and one with a very small Kd, which would take longer to reach equilibrium? The one with the smaller numerical Kd would
take longer to reach equilibrium .There is an inverse relationship between Kd and time required to reach equilibrium. May not need to exactly reach equilibrium for a drug you want to
test, if you can figure out the relationship between how far away from equilibrium you are at a given time; its possible to estimate with some mathematical manipulation. Typically working
with a situation in which the receptor concentration is much less than the drug concentration.

Question 18

Do you always need a purified receptor to study it?

Answer 18

Does the drug in question bind to more than one receptor? If there is any reason to suspect that it binds to more than one, then you really do need a purified receptor. Seen with glucocorticoid receptor where it had nonspecific binding. Unless you can use the SAR studies to see if you can displace the labeled drug with nonlabeled conjimer, ideally you just want to isolate the receptor.

Question 19

What are some approaches to drug binding assays in pharmacology?

Answer 19

Equilibrium Approaches:

• equilibrium dialysis
• equilibrium gel filtration (Hummel dryer method)
• affinity chromatography.

Nonequilibrium Approches

• nonequilibrium gel filtration.
• batch (or other selective) adsorption methods.

Question 20

What are some advantages to using equilibrium approaches?

Answer 20

• measure the amount of drug-receptor complex in the presence of free drug so there are no problems with drug-receptor complexes that dissociate rapidly.
• excellent with low affinity drug-receptor complexes, if one can obtain a sufficiently high concentration of the receptor.

Question 21

Will equilibrium approaches work with high affinity complexes?

Answer 21

It will still work with high affinity. But when you have a low affinity (large Kd), in which the drug
receptor complex can fall apart fairly readily and so if you remove all of the drug in the process
of doing the assay (seen with the nonequilibrium methods), then the complex may fall
apart before you can do the measurement. So this method works as long as you have enough of
the receptor. So limitation: situations where obtaining the pure receptor is hard, you need enough
receptor close to the Kd value. If you havnt done the assay then you dont know exactly what
the Kd value is, but based on the bio response assays youll have a guess. If the Kd app is really high,
then you dont want to try a nonequilibrium approach.

Question 22

What are some limitations to equilibrium approaches?

Answer 22

• requires a significant amount of receptor.
• the receptor concentration must be in a concentration range that is close to the binding constant for the complex. If the bound drug receptor complex concentration is substantially below the free drug concentration, it will be impossible to make the measurement.
• time intensive.
• possible problems of drug or receptor sticking to the wall of the chamber. Plastic can cause drugs or receptors to stick to it. Nonequilibrium uses glass.

Question 23

Equilibrium dialysis

Answer 23

Apparatus consists of two chambers separated by a semi-permeable membrane. The drug can pass across the membrane, the receptor cannot. Must bring the system to equilibrium and keep at equilibrium. The drug concentration on the side containing the receptor is free drug plus drug-receptor complex. So one determines the amount of drug bound by determining the difference in drug concentration between the two sides. So drug receptor complex concentration: DR= Dtotal on receptor side - Dtotal on free drug side. By comparing the DR concentration with D concentration, you can get a log dose curve or scatchard plot.

Question 24

Non equilibrium approaches

Answer 24

The drug receptor complex is separated from the free drug to make the measurement.
Advantages:
-total separation of the drug receptor complex from the free drug so there is no free drug present to confound measurement.
-excellent with high affinity complexes. (Small Kd values wanted)
- fast

Disadvantage: works poorly with low affinity receptors because with no drug in equilibrium with receptor, there is a tendency for some of the drug to dissociate from the receptor in the process of running the assays (more of an issue with traditional assays that use long glass columns). So if it dissociates before you can quantitate, its bad. Faster spin columns can sometimes work with moderate Kd values.

Question 25

Affinity chromatography

Answer 25

Drug molecule connected to solid support resin by a linker. Receptor added to column containing affinity resin. Gradient if free drug is run through column. Record free drug concentration required to elute receptor.

Advantage: no need to label drug.
Disadvantage: requires DR complex with long half life i.e. Kd less than or equal to 100nM. Half life= time to reach equilibrium. If your Kd is larger than 100nM, then this method doesn’t work well.

Question 26

Classical gel filtration

Answer 26

Makes use of cross-linked polysaccharide beads that allow small molecules to penetrate but excludes large molecules. Beads of many sizes and degrees of exclusion are available commercially. Usually run in glass columns that filter by gravity, but there are many kinda of spin columns that are run in a centrifuge to speed passage through the columns.

Question 27

Affinity chromatography (steps)

Answer 27

1. Load receptor onto column containing resin-bound ligand.
2. Run gradient of free ligand into column to compete with bound ligand for receptor.
3. Record free ligand concentration at which receptor is eluted from the resin. Since free drug is eluted, it doesn’t need to be labeled.

Question 28

How does gel filtration work

Answer 28

There are pores in the beads. The chosen size allows for small molecules such as the free drug to travel through the pores, as well as around the beads. The drug receptor complex and free receptor are too big so they have to travel around the beads only. The volume outside if the gel beads is small than the volume within, so the DR and R elute first. Use radio labeling.

Question 29

How can you get better separation with gel filtration?

Answer 29

Choose the right pore size. Can also use FPLC forced pressure chromatography. Basically working to pump the solution through the column rather than waiting for gravity flow.

Question 30

Gel filtration parameters

Answer 30

• bead volume: volume of the column taken up by the beads and surrounding buffer.
• void volume: Bed volume - bead volume (aka excluded volume).
  Can determine void volume by putting something known to be too large to flow through the beads, and is something that you can readily detect. For instance, a dye molecule conjugated to a large protein. Look at what fractions of the dye show up at certain times. Gel filtration can be used to find Kd or to determine if two drugs bind to different sites.

Question 31

Gel filtration problem example: a column has a bed volume of 10 ml and a bead volume of 7ml. You load 100 microliters of a drug + receptor mixture on the column and collect 1 ml fractions. Assuming that the free drug can completely enter the beads and the DR complex is completely excluded, in which fractions do you expect to first detect free drug and drug-receptor complex?

Answer 31

DR will come out first and will come out in excluded volume (10-7)= 3ml. So it will start to come out in tube number 4. First 3 mls that come out will represent excluded volume that was already in the column, and by
the fourth tube youll start to see DR complex coming through. Then finaly you should see free drug coming through with the void volume.

Question 32

What are the keys to successful separation of DR from free drug using gel filtration?

Answer 32

• choose a bead size that excludes all R.
• volume sample (D+R) should be less than 5% of the column volume (100ml column = less than 5ml of solution loaded).
• large length to diameter ratio for the column.
• run separation slowly (can run faster if using FPLC system). If you try to speed it up using gravity it can mess up your results. Typically want a tall thin column as opposed to a short wide column.

Question 33

Hummel dryer method

Answer 33

Pre-equilibrate column with non-radiolabeled drug. Bump in drug concentration is due to more of the label coming off in fractions
with the receptor also present. If you have a lower number of
drug relative to receptor, you may actually get a dip instead.
Height of peak is proportional to amount of DR complex.

Question 34

What dictates of you do equilibrium gel filtration?

Answer 34

So if we have a high Kd and low affintiy, we can do equilibrium gel
filtration. Done the same way, but the column is pre-equilibrated with
free drug at the concentration that we are measuring. So in addition to
having a fixed concentration of drug added to a fixed concentration of
receptor in a tube, and then adding it to the top, we also have a buffer
in which the gel is suspended pre-equilibrated by running an excess of
the drug concentration. This can be time intensive unless you use multiple columns.

Question 35

Non equilibrium gel filtration

Answer 35

2 peaks seen. First is receptor bound to drug and the second is the free drug.

Question 36

Do you want to do nonequilibrium gel filtration if you have a small Kd?

Answer 36

If you have a numerically small Kd, generally a Kd less than micromolar concentration is considered
high affinity. Want a high affinity situation for gel filtration because then the drug and receptor would
stay together well enough to keep the DR in tact as it goes through the volume. Something with a
micromolar or above Kd would probably tend to fall apart once free drug is removed. So you would
get a falsely low DR complex measurement at a given drug concentration. So we ant a small Kd, and high affinity.

Question 37

Properties of colchicine binding protein and interactions with vinca alkaloids and podophyllotoxin (competitor).

Answer 37

showed that tubulin was associated with a number of drugs known to depolymerize microtubules. Can use these peaks to analyze if there is competition binding. If a competitor is added, then the first peak will disappear. So this would be like in the presence of Podophyllotoxin. If Vinca
Alkaloids were added, then you would get the same peaks as seen above since it doesnt compete with binding of colchicine to microtubules (there are multiple drug binding sites).

Question 38

Selective adsorption methods

Answer 38

These are non-equilibrium methods that are rapid and inexpensive.
Examples are the use of activated charcoal, that adsorbs aromatic compounds and can quickly deplete the free drug concentration by centrifugation. Or adsorption of the target (receptor) onto charged surfaces.
These approaches often have important limitations due to the possible rapid dissociation of the drug receptor complex, poor adsorption efficacy, dissociation of the complex from the absorptive surface, etc.
- wouldn’t work well if the drug bound to charcoal can still bind to the receptor, resulting in the receptor being peletted out as well and not in the supernatant.

Question 39

Batch method of adsorption

Answer 39

Can do batch methods. Have an antibody to tubulin that is bound to
beads that you pour into a column.
As you pour the DR complex over,
it gets trapped. Often times when dealing with a crude extract, even
though you centrifuge, you may get particulate matter which causes
the column to clog. So using a batch method would help with this.Batch method is simply an alternative to the column method.

Question 40

Can filter paper be used to separate receptors?

Answer 40

If a receptor has a net negative charge, you can use a filter paper that has the opposite charge and use it to pull out the receptor: has been used for isolating tubulin-colchicine complexes.

Question 41

Other DR binding methods

Answer 41

HPLC : high pressure liquid chromatography. Excellent for small molecule separation/ identification (measures free drug). If the drug is too big, it may not separate enough.

Fluorescence spectroscopy: increase or decrease in fluorescence in response to drug binding. Most receptors have aromatic amino acids close to the surface, which can undergo fluorescence.

Circular Dichroism (CD) spectra: change in absorbance difference between left and right rotated polarized light upon drug binding.

Surface Plasmon Resonance

Question 42

How can fluorescence be used to study DR binding?

Answer 42

In some cases, after binding you have an aromatic amino acid becoming exposed, thereby increasing
fluorescence, in other cases you have the aromatic amino acid being pulled in deeper into the receptor,
resulting in fluorescence quenching. The latter is a more accurate way to measure drug binding to
receptor. Can also be looking at drug fluorescence. Advantages of fluorescence spectroscopy: 1.) no
radioactivity involved, 2.) you dont have to separate the drug from DR complex. So you can work with
a fairly low affinity interaction and dont have to worry about dissociation because the drug is always present in
solution.

Question 43

What would you expect to see using a spectroscopic method?

Answer 43

Two peaks. Shift in fluorescence in response to drug binding to receptor.
Advantage: can make 2 measurements, both of
which are proportional to amount of drug bound. So if something goes wrong with one measurement
you can tell based on the other measurement.

Question 44

What’s the difference when measuring fluorescence of drug binding and looking at quenching vs increased fluorescence.

Answer 44

In drug quenching, the higher the concentration of drug the lower amount of fluorescence. The opposite is true if dealing with increased fluorescence in response to drug binding.

Question 45

Surface Plasmon Resonance (SPR)

Answer 45

Receptor is immobilized on a dextran matrix attached to a thin layer of nanogold particles

Drug at various concentrations flows over the dextran-gold surface

Polarized light is reflected off of the gold layer

The angle of reflection is altered by binding of the drug in a manner proportional to the amount of drug bound

Real time measurements allows determination of off and on rate constants

Kd can be calculated from koff/kon ratio

Question 46

Why is gold used in SPR?

Answer 46

thin layer of gold provides a good mirror for polarized light. As you bind drug you alter the ability of gold layer to reflect the polarized light (changes the angle), which is proportional to the amount of drug bound. So the more drug bound
to the receptor, the greater perturbation of reflected polarized light. So by measuring the angle of reflection you can get the amount of binding and can directly measure the rate constant from which you can derive the Kd.

Question 47

What may factor into the decision to use SPR?

Answer 47

very expensive mainly due to instruments. Since the free drug is present, this is basically an
equilibrium-type method. So you can use this with low-affinity binding situations.

Question 48

What is shown in the SPR method that’s unique?

Answer 48

What they are actually showing is binding of an antibody to some tethered antigen. Typically
we’re looking for drug receptor binding, with the receptor linked to the dextran surface and the drug is
free in solution. Whats shown is somewhat reversing this idea, so you can do it in both ways. The key feature is
that whatever is bound, needs to have its surface that is going to interact with its free agent not hindered by the
attachment. So lets say we have the receptor attached to the surface, but the attachment blocks the binding site to the drug.
Obviously this wouldn’t work well. Its generally easier to tether the receptor without interfering with drug binding,
so typically the receptor is covalently attached to the dextran.

Question 49

What are some things to consider when choosing a method to measure drug binding?

Answer 49

• need for a labeled drug. SPR doesn’t.
• sensitivity (how much receptor do you need?) SPR is the most sensitive.
• stability of DR complex.
• susceptibility of receptor to denaturation (important for assays that take a long time).