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1

what is disease target bashing?

finding a gene which is involved in a disease, make a mutant of it and then carry out a drug screen to find out drugs that ameliorate the symptoms and then find out what pathways this targets and how the drug can be used to treat the disease

2

how were many medically used drugs first discovered

serendipitously, accidentally finding a phenotype in animals and then working out what the drug was doing from there

3

describe an example of a drug being accidentally found?

warfarin, was originally put on grass which was eaten by cows, When the cows ate this they would die from bleeding out. This was because warfarin is involved blood coagulation and prevents it so wounds can't heal.

4

what are phenotype based chemical screens?

using a drug and looking for a phenotype

5

what are three ways in which traditional drug discovery occurs within chemical genetics

- disease target bashing- serendipitously - phenotype based screens

6

give an example of a phenotype based screen for drugs

using an already known drug that has a known function and seeing if it has other functions

7

what is the general principle behind chemical genetics screen?

you have many wells with organisms of cells in. You then apply different drugs to each well and then screen for a phenotype. if there is a phenotype of interest you take the drug that you added to this plate and identify what its binding partner is. This protein is then implicated the phenotype an the process that was perturbed in order to form it

8

what is a forward chemical genetics screen?

it is phenotype based- you have many wells with organisms of cells in. You then apply different drugs to each well and then screen for a phenotype. if there is a phenotype of interest you take the drug that you added to this plate and identify what its binding partner is. This protein is then implicated the phenotype an the process that was perturbed in order to form it

9

what is reverse chemical genetics?

it is target based. You over express a protein of interest, you then apply drugs and see which one can bind to it or modulate the protein function. then you add this drug to a cell or organism and see if there is a phenotype

10

what are the major disadvantages of chemical genetics?

• Whole Organism vs. Single Target Screening (can't always use entire organisms- can for worms and zebrafish that can easily be put in plates)• Polypharmacology– Multiple Targets• Target Identification is Difficult• Dose Matters• Not all pathways are druggable!

11

what are the advantages of chemical genetics?

- High-Throughput – especially with automation• Target specific protein domains vs. whole gene knockouts (but you can do this point mutations)• Target with precise timing• Combinatorial Screen is feasible• Main Effects and Side Effects are screened simultaneously

12

what are the downfall of using these large chemical sets?

although there are many drugs, not all biological processes are targetable and there isn't a drug to target ever process. whereas with genome libraries, for most organisms, 80-90% of the genome has been sequenced and can therefore be potentially targeted in some way

13

how do chemical sets compare to genome libraries?

for most of the model organisms 80%-90% of the genome has been sequenced but even though there are around 1400 existing drugs, they can't target ever biological process

14

how many existing DFS as approved drugs are there?

around 1400

15

what are the benefits of using an DFA approved drug screen?

You know that these drugs have FDA approval and so dont cause any unexpected side effects

16

what are the pros of cons of carrying out a screen using registered chemicals?

you can find unexpected or uninvestigated drugs but you have no idea whether they will be at all safe to use humans

17

what is a dose response curve? what are the x,y axis?

the dose goes along the x and the individual response of severity of phenotype goes along the y. They are a way of plotting what dose is required for the maximal or minimal effect.

18

when can looking at the dose response curve be important?

when you know that your drug has a harmful side effect as some concentrations but beneficial at others. You can plot the dose response curve for both of these response and see it there is much overlap

19

what does it mean if there is a large overlap between the dose response curve for the benefit and the dose response curve for toxicity?

the drug can't be used- there is a too greater risk

20

why would you want to plot the dose against the % of maximal response?

because if a really high dose not give you maximal response, this would suggest that your drug is only a partial agonist

21

what does a depressed sigmoidl cruve within a graph demonstrating the dose compared o the %maximal response tell you?

the drug you are using a partial agonist

22

what does it mean if you have 100% receptor occupancy but not 100% of the maximal response of the protein?

the drug is only a partial agonist

23

when you are plotting % response of a process against the concentration of the drug, what does a shift to the left or a shift to the rightof th curve mean? what about a shift up?

if the curve shifts to the left it means it has increased in potency and the opposite to a shift to the right. if it shifts up it means it ha increased in efficacy.

24

so once you have identified a drug which causes a phenotype in a forward genetic screen, what is the next step?

identified what the target of the drug is

25

is finding a target of a drug easy?

no! the target of warfarin wasn't fun until around 50 years after it started being used commercially as a drug.

26

what are the two main methods used to identify the target of a drug?

look at global expression profiling or use direct biochemical methods.

27

what is the affinity purification method of finding a binding partner for a chemical?

you column that contains your molecule which has been tagged to the base of the columnal tray. Then you dump lots of proteins that are binding candidates onto the tray. and then you wash off everything until all proteins are gone apart from the on that is bound. You can wash off the protein itself and then use mass spectrmetry to identify this protein. the target of thalidomide was found using this method. You can then do other tests to show that this is intact what the binding partner is causing the phenotype. For example you can engineer a zebrafish fish express the protein but without the binding site of the drug. If the phenotype doesn't occur in this animal then you know it is caused by binding this protein.

28

what technique was used to identify the target of thalidomide?

affinity purification

29

what must you remember when using in vitro methods to identify the targets of chemicals

may not also be replaceable in vivo. you must always carry out in vivo expimernts to show the same is in vivo

30

as well as affinity purification, what is another method used to identify the target of a chemical?

SILAC: stable isotope labelling by amino acid culture.