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Question 1

what is disease target bashing?

Answer 1

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

Question 2

how were many medically used drugs first discovered

Answer 2

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

Question 3

describe an example of a drug being accidentally found?

Answer 3

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.

Question 4

what are phenotype based chemical screens?

Answer 4

using a drug and looking for a phenotype

Question 5

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

Answer 5

• disease target bashing- serendipitously - phenotype based screens

Question 6

give an example of a phenotype based screen for drugs

Answer 6

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

Question 7

what is the general principle behind chemical genetics screen?

Answer 7

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

Question 8

what is a forward chemical genetics screen?

Answer 8

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

Question 9

what is reverse chemical genetics?

Answer 9

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

Question 10

what are the major disadvantages of chemical genetics?

Answer 10

• 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!

Question 11

what are the advantages of chemical genetics?

Answer 11

• 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

Question 12

what are the downfall of using these large chemical sets?

Answer 12

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

Question 13

how do chemical sets compare to genome libraries?

Answer 13

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

Question 14

how many existing DFS as approved drugs are there?

Answer 14

around 1400

Question 15

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

Answer 15

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

Question 16

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

Answer 16

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

Question 17

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

Answer 17

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.

Question 18

when can looking at the dose response curve be important?

Answer 18

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

Question 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?

Answer 19

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

Question 20

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

Answer 20

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

Question 21

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

Answer 21

the drug you are using a partial agonist

Question 22

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

Answer 22

the drug is only a partial agonist

Question 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?

Answer 23

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.

Question 24

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

Answer 24

identified what the target of the drug is

Question 25

is finding a target of a drug easy?

Answer 25

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

Question 26

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

Answer 26

look at global expression profiling or use direct biochemical methods.

Question 27

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

Answer 27

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.

Question 28

what technique was used to identify the target of thalidomide?

Answer 28

affinity purification

Question 29

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

Answer 29

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

Question 30

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

Answer 30

SILAC: stable isotope labelling by amino acid culture.

Question 31

what does SILAC stand for?

Answer 31

stable isotope labelling by amino acid culture

Question 32

what is the SILAC process?

Answer 32

you have a cell ine that you label with a light isotope so that all of the proteins get labelled with a light isotope version and another cell line is labelled with a heavy isotope and then you take another small molecule (purple) and you have an immobilised version sitting on a bead and the soluble competitor that aren’t stuck on the bead and you ask who binds – there will be some non specific stuff which binds to the bead and you get very few things binding to your beads and it leaves thesebasically immoliblised not bound to anything. Then you do another condition where you just have the immobolised small molecule and none of the background competitor and this allows these to bind both the non specific and the specific stuff and then you use mass spec to see the difference between them by comparing which of the light and heavy isotopes are present for each protein. For each given protein, how much of the light isotope is present in the first experiment and then how much of the heavy stuff is there. So you compare what is bound to the bead in the first one (shouldn’t have actually bound to its target because immobilised) then you see what has bound to the beads in the second one (should be everything that has bound to the bead in the first as well as the actual target specific protein) and mass spec will reveal these differences. The proteins present in the heavy but not light will be the specific binding partners.

Question 33

list three ways that you can use yeast cells to identify the target protein of an implicated molecule

Answer 33

- synthetic lethality screen: you have a panel of single gene deletions in each yeast cell in a plate with wells. Then you use these deletions to look for cells that are made sensitive to the drug by these gene deletions and are killed when the drug is added. Those that give a phenotype with the drug added are sensitised. and the gene deleted is involved in the same pathway as the drug. You can then look at the ppathways that this gene is normally involved in and look at the gene expression profile of a knockout of the gene and this will give you an idea of the proteins that may be involved - recombinatnt loci sensitive mapping: you can also use the inbred recombinant strain idea where you cross two strains and you recombined strains and then map which loci confer sensitivity to this compound (by increasing dosage and seeing which one dies or give phenotype first) - then you can use an barcoded ORF library. in which you may for example has a mutant cell that is resistant to the phenotype of the small molecule. but when you add the gene by which it is getting resistance to it, so that it is no longer mutated, it is then killed by the small molecule. You then know by what mechanism it is that the small molecule is working. For example a cell may lack an apoptosis receptor which is activated by the small molecule, when you add this receptor back in, the cell dies. Or you have a small molecule that causes a disease phenotype but a mutant cell doesn't. you can use this fin what protein is conferring resistance to the disease phenotype

Question 34

what kind of proteins are good targets to the small molecules?

Answer 34

proteins such as kinases that have binding pockets that molecules can easily bind to and inhibit.

Question 35

what does synthetic lethality mean?

Answer 35

Synthetic lethality arises when a combination of mutations in two or more genes leads to cell death, whereas a mutation in only one of these genes does not, and by itself is said to be viable.[1] In a synthetic lethal genetic screen, it is necessary to begin with a mutation that does not kill the cell, although may confer a phenotype (for example, slow growth), and then systematically test other mutations at additional loci to determine which confer lethality. this infers a functional relationship

Question 36

how can you use a small molecule screen to find proteins involved in a disease phenotype.

Answer 36

You can have a drug which induces a disease phenotype but you find a mutant that does not express this phenotype when exposed to the small molecule. You then use the ORF library barcode principle to find what gene it is that is giving resistance. this would likley involve gain of function mutations maybe?

Question 37

How can you use RNAi to ID a pathway being effected by a small molecule (3)?

Answer 37

- first you can carry out a screen using RNAi on the same animals to see if any RNAi give the same phenotype as the small molecule. This will suggest that the gene/protein being effected is the same in both. - Also you can use RNAi to find out what protein is being targeted in small molecules that cause cell or embryo death. This can be done by scaling back ht dose until the small molecule is not killing anymore. You can then add RNAi library to each well and which ever probe causes death-- you know that this is working in the same pathway or using the same gene/protein - another way you can use RNAI is to work out whether a small molecule is activating to inhibiting a pathway. so first you identify a molecule that causes a phenotype. When you also suspect that a certain pathway is involved from looking at the phenotype, you can then use RNAi probes that you know enhance or suppress a pathway. This will help you to determine what the small molecule is doing to the pathway. When you use a suppressor RNAi with a drug that is inhibiting the pathway, it should make the phenotype worse. But if the pathway is enhancing the pathway then it should counteract the suppressing RNAI. By looking at hither the change in dose response. you can work this out.

Question 38

what is the guilt by association mechanism of finding out what the mechanism by which a small molecule acts to produce a phenotype?

Answer 38

you can look at the expression profile of a cell or tissue when the drug is applied. You can then compare this to expression profile libraries of other drugs or gene pertubartaions and look for similar profiles. The gene or process involved in the known similar profile can provide you with clues for what the unknown drug is doing

Question 39

what is polypharmacology?

Answer 39

describes drugs that have many downstream targets and effects

Question 40

when can polypharmacology be good, give an example?

Answer 40

magic "shotgun" targets many different implicated pathways- the antidepressant drugs were seen as being dirty because they had lots of targets but more specific drugs did not work at all- it is better to target lots

Question 41

what is small molecule epistasis?

Answer 41

when you plot a three dimension graph showing he response of the phenotype to the addition to two drugs you can work out whether the drugs are acting synergistically in parallel with alternative or required targets or with serial targets.

Question 42

describe the different forms of small molecule epistasis

Answer 42

...

Question 43

what are the best model organisms for chemical genetics and why?

Answer 43

zebrafish, because they are cost effective, good genetic took available, high fecundiity, good for the amount of the chemical required (not a lot), amenable to a multi-well format, good chemical absorption, good for controlled behaviour and good for controls.

Question 44

other than the best, which is worms, which other two organisms are good for chemical experiments

Answer 44

worms but their chemical absorption is fairly poor so you need a lot of chemicals. but they are amenable to multi well plate, good to observe, good genetic tools and good for controls. Flies are then the next best but even worse for chemical absorption and need a lot of chemical and they are not amenable to multi well plate! their chorion is permeable to small molecules., you can view embryo development, good for automation

Question 45

should you perform chemical genetics on mice?

Answer 45

no- very poor chemical absorption, not good for high throughput because can't be put in plates etc, and very hard to monitor embryonic behaviour and phenotype

Question 46

what is the main thing you need to consider for high throughput forward screens and chemical genetics?

Answer 46

need to be able to put in wells mostly and easily observed and screened.

Question 47

what does it say about controls?

Answer 47

..

Question 48

give the example of gridlock to explain how chemical genetics can be used to find drugs for diseases and/or for identifying the pathways involved in developmental processes

Answer 48

the gridlock mutation causes vasculature to be lost in development. The scientist then looked for chemicals which could rescue this phenotype. He then found that the target of drugs that did this were PI3K and found that this was involved in arterial specification pathway. The drugs only partially inhibited the PI3K which is important because a knock out would have just given a lethal probably

Question 49

give an example of when using a drug to find out a development process has been successful where a genetic know out approach would not have been

Answer 49

gridlock, this was a mutant with perturbed vasculature development. a molecule rescued it by partially inhibiting a PI3K pathway. the partiality of this was significant as a full inhibition would have simply caused a lethal probably which is not helpful at all.

Question 50

describe an example of when a protein that was implicated to bind to a molecule in vitro was shown to be replicatable in vivo

Answer 50

The example was the target of thalidomide. there are different steps to this. 1. they showed that the small molecule had the same effect on the fish as it did on humans- short limbs- this suggests the same functions and therefore likely same target 2. They also showed that a morpholino against cerebral (the suggested implicated gene) caused the same phenotype. So this suggested that thal acts by inhibiting cerebron functioning 3. they then did somehting very sophisticated. They injected thal into the fish and therefore got no limbs. They injected cerebron into the fish during development with thal and still got no fins. They then worked out the binding pocket that thal was binding to in cerebron and mutated it so that it could no longer bind, produced the mRNA for this and then injected it during development- the fish grew fins.

Question 51

describe a different in vivo experiment, not involving thalidomide, that proved a protein binding partner for a small molecule.

Answer 51

a group found a drug that caused pigment dispersal in the zebrafish. They used affinity purification and found it found to alcohol dehydrogenase, they then thought that the drug acted by activating alcohol dehydrogenase (maybe by doing MOs?). - in vivo they added the drug and showed that it caused pigment dispersal. Then they added a drug which inhibited alcohol dehydrogenase alone and showed that it did nothing. Then they added the drug and inhibited ADH and showed that the drugs ability to cause pigment dispersal was inhibited. This showed that it acted in the predicated pathway

Question 52

how was chemical genetics used t identify pathways involved in axis development

Answer 52

zebrafish were screened with drugs that cause shortened fish and therefore caused DV patterning perturbations- they identified dorsopmorphin

Question 53

how can drug screens be helpful for seeing if a drug is safe for commercial use?

Answer 53

many drugs interrupt ERG changes which cause heart problems- and often when they effect fish the also effect humans so you can screen fish for this

Question 54

describe a chemical genetic screen that focussed on hasematopoietic stem cells. (2) one being very interesting!

Answer 54

- they found markers for harm stem cells in zebrafish and then used in situ to view the HSC count in fish. - they then applied drugs and looked for those which increased stem cell proliferation. - this was needed to expand HSC counts from umbilical cord transplants. - they then sought to address their problem directly by actively seeing if the drug could perform in a transplant situation in zebrafish transplanatation. They took two strains of fish. One which expressed a GFP marker universally and the other which expressed a RFP reporter univserally. you then harvest the HSC from the green fish and incubate them with different chemical for 4 hours. Then you take the HSC from a normal fish and keep them on ice. Then you mix each chemical green set with a normal red set and then put them into a irradiated fish that has no stem cells. You then leave the stem cells to grow and look for any chemical which either prevent growth and more importantly increase it. - They found prostaglandin helped increase HSC proliferation

Question 55

explain how people have used chemical genetics to look at drugs that can improve heart regeneration

Answer 55

zebrafish hearts can regenerate. So if you freeze or cut a third of the zebrafish heart it can regrow. Because it is hard to dissect many fish hearts, instead you can look at drugs which perturb heart development and then once you have found candidate s you can then try these select molecules on heart region

Question 56

describe how jasons lab looked at drugs that affected sleep and why the zebrafish is particularly amenable to these studies.

Answer 56

zebrafish can be plated and visualised very easily. drugs can be added to each plate in an automated way and they can be observed. Jason added drugs and measure sleep pattern and activity. By using programmes and set parameters he was able to look at different aspects of behaviours under different drugs, such as total sleep time, rest or sleep activity drugs the night or during the day. He then clustered unknown drugs with drugs that were known that had similar affects in an attempt to find out what they were.

Question 57

why are zebrafish a good target for sleep studies?

Answer 57

drugs tend to affect humans and fish in the same way- not always though

Question 58

what are the three summary points from masons work?

Answer 58

. Drug Discovery/Prediction— Clustering drugs by behavior links drugs with unknown targets to compounds with known targets2. Broad Conservation -- Drugs of the major neurotransmitter pathways have conserved effects on sleep in zebrafish and mammals3. New Sleep/Wake Regulators-- Small molecule screen implicates several molecular pathways in sleep/wake behavior.

Question 59

what is an annoying feature of genetics in fish?

Answer 59

they can often contain 2 copes

Question 60

if you have a phenotype and you want to know the pathways involved in it, how can you use drugs to this, briefly give an example.

Answer 60

you can have a phenotype and you can look for a drug which ameliorates or gives an opposite phenotype. You can then select these drugs and apply them to your phenotype fish and see if they rescue it. Jason did this with hyperactivity during the sleep phase- treated with a drug with ameliorated but importantly only in sleep- wasn't just a general sedative.

Question 61

explain step by step how the gridlock experiment was carried out

Answer 61

- they had the gridlock mutant, they then exposed it to different drugs and found which ones rescued it- they then tried the drug at different times in development to see when it was needed to have an effect - they then use real time PCR to quantify the expression of several genes they believed to play roles in angioblast cell fate determination or migration - the expression of ENGF was upregulated in the mutants after exposure to the drug - when they saw this they then injected the mutant with VEGF and saw that it could do the same job- they then looked to see if the molecule could have the same effect on human umbilical vein endothelial cells and saw that it did - they saw that other molecules which were rescuing the phenotype had PI3K as their targets (they inhibited its action)- they knew that PI3K acted in the VEGF pathway - then look at the activation of downstream effectors of this pathway when apply the small molecule - they needed to perturb some actors that gave lethal phenotypes when perturbed so the created a mosaic by injecting into the two cell stage - they did this by injected a labelled plasmid to identify mutant cells - they found that the mutants cells had the fate expected if the perturbation in the gene resulted in a different fate