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PHAR 503 > 3. Drug Screening and Discovery > Flashcards

3. Drug Screening and Discovery Flashcards

1
Q

describe historical drug discovery. is there an advantage or disadvantage?

A

anecdotal evidence of natural agent allows us to find drugs

ADVANTAGE: drug has already been biologically activated so there is a high success rate

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2
Q

describe target-based screens. is there an advantage or disadvantage?

A

make drugs based on specific genes

DISADVANTAGE: drug has not been biologically activated so there is a high failure rate

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3
Q

describe phenotype-based screens. is there an advantage or disadvantage?

A

screen drugs more broadly based on phenotype, not one enzyme

ADVANTAGE: high success rate

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4
Q

how would you conduct a phenotype-based screen for cancer drug?

A

slow down cancer cell proliferation, rather can curing cancer

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5
Q

how would you conduct a phenotype-based screen for a diabetes drug?

A

get rid of glucose in bloodstream, rather than fixing the insulin, etc.

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6
Q

what would the read-out for a target-based assay be?

A

specific activity –> enzymatic activity, receptor activation etc

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7
Q

what is alphafold?

A

AI-based drug discovery

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8
Q

what are 3 types of cells used for cell-based assays?

A
  1. immortalized cell lines
  2. primary cell cultures from tissue
  3. induced pluripotent stem cells
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9
Q

how are cells immortalized?

A

either cell lines come from cancer cells or cell is immortalized by a virus

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10
Q

why is media red?

A

stained with phenol red as a pH indicator –> cells take up glucose and release acidic lactose, turning media yellow

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11
Q

why do we do cell-based assays in a biosafety cabinet?

A

bacteria can contaminate and use up nutrients bc they grow very quickly –> need clean system with enough nutrients

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12
Q

what type of cells are better than immortalized cells? why?

A

induced pluripotent stem cells –> can differentiate into any cell line, perfect model for human disease

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13
Q

where do induced pluripotent stem cells come from (3)?

A
  1. healthy subjects
  2. diseased patient
  3. diseased patient genetically reversed to healthy (isogenic)
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14
Q

what type of read-out do we see with cell-based assays?

A

increase/decrease of phenotype

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15
Q

how can we screen using in vivo assays?

A

usually too hard to do drug screens in animals but can use genetically engineered ZEBRAFISH in dish

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16
Q

what are the 8 criteria for a screening assay?

A
  1. quick, simple, robust, relevant
  2. positive read-out
  3. direct read-out (little handling)
  4. good dynamic range, z’ factor
  5. in vitro for enzymes
  6. ex vivo w cells, tissues, zebrafish
  7. cost effective
  8. end up with a lead compound
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17
Q

what does it mean for a screening assay to be robust?

A

gives good measurement with solid result

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18
Q

describe quenching experiment with positive vs negative read-out
- downsides of negative read-out?

A

POSITIVE: drug increases protease activity = screen for wells that have more fluorescence
NEGATIVE: drug reduces caspase activity = screen for wells that have less fluorescence (darker)

hard to interpret negative read-out –> how do you know cells aren’t dead or protocol wasn’t followed?

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19
Q

what does a good dynamic range mean?

A

big difference btwn good and GREAT results

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20
Q

describe this phenotype-based screen in zika virus paper where green = zika virus and red = caspase 3 (indicates apoptosis)

A

drug emricasan allows virus into the cell (lots of green) but theres no red so there’s no caspase-3 activity or apoptosis

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21
Q

describe this phenotype-based screen in alzheimer’s disease

A

more lipid droplets in ApoE4 than ApoE3

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22
Q

what is Z factor? is 10% good or bad?

A

measure of how different the signal is from the baseline

10% increase in activity is not good bc of biological variation

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23
Q

what are the 3 considerations before a target-based screen?

A
  1. what is the evidence that acting on the target will cure the disease?
  2. what is the evidence that the target is drugable?
  3. what is the evidence of selectivity
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24
Q

what are the 3 considerations before a phenotype-based screen?

A
  1. what is the evidence that interfering with the phenotype will cure the disease?
  2. how relevant is the phenotype for the disease
  3. is the cell type and stimulus relevant for the disease
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25
Q

how do we know that inhibiting an enzyme etc. will cure the disease?

A

previous evidence from KO mice, human mutations, etc. will indicate

26
Q

how do you acquire evidence for selectivity?

A

must check how many substrates an enzyme has for ex –> even if not published, doesn’t mean they don’t exist

27
Q

how many drugs can you target with HTS?

A

1000s in academia or 100,000s in industry

28
Q

what is the motto for HTS? what does it mean?

A

“fail fast, fail cheap”
if you find 50 leads, don’t have capacity to test them all so want to find reasons to fail the drugs as fast as possible

29
Q

what affinity do we want for positive hits in HTS?

A

between 30uM - 1nM

30
Q

what is a FIRST IN CLASS drug? what type of screening is most effective?

A

new drug completely –> new structure, new strategy

phenotype-based screen is most effective

31
Q

what is a FOLLOWER drug? what type of screening is most effective?

A

old drug has been optimized/modified but remains in same drug class

target-based screen is most effective

32
Q

what type of screening is used for biologicals?

A

target-based by definition!

33
Q

what type of screening is best for small molecules and natural compounds?

A

phenotypic

34
Q

what is the chemical space?
how many chemicals? how many are known?

A

all possible molecules
10^18-10^200 but only 10^7 are known

35
Q

describe the chemical space in the context of drugs

A

there is limited space in the chemical space available for each type of drug –> must maximize this space and create libraries

36
Q

what are 5 libraries in natural world?

A
  1. plants
  2. microorganisms
  3. animals
  4. biochemicals
  5. marine chemistry
37
Q

how are libraries selected in the synthetic world? (2)

A
  1. chemical synthesis
  2. combinatorial and parallel synthesis
38
Q

what are 4 advantages of natural compound libraries?

A
  1. evolved for biological advantage (not random)
  2. potent
  3. cover many chemical types
  4. serendipity –> wouldn’t have found otherwise
39
Q

what are 3 disadvantages of natural compound libraries?

A
  1. hard to synthesize
  2. hard to produce, make extracts, reproducibility
  3. may have poor bioavailability
40
Q

what is the Lipinski rule of 5?

A

to determine oral bioavailability:
1. <5000 Da
2. Lipophilicity –> logP
3. # of groups that can donate H atoms <5
4. # of groups that can accept H atoms <10

41
Q

how is the partition coefficient measured?

A
  1. add water and octanol in vial and add drug
  2. shake, then let 2 phases separate
  3. P = [drug in octanol]/[drug in water], take log
42
Q

how does P change if the drug is more lipophilic?

A

higher P if more lipophilic bc higher concentration in octanol

43
Q

how does P change if the drug is more hydrophilic?

A

lower P if more hydrophilic bc higher concentration in water

44
Q

what is an ideal logP for oral bioavailability

A

mostly hydrophilic but a little bit lipophilic –> <5

45
Q

why do you need a good balance of lipophilicity and hydrophilicity?

A

drug must be lipophilic enough to cross hydrophobic membranes but hydrophilic enough to go thru blood

46
Q

how do drugs achieve good oral bioavailability? explain!

A

AMINE GROUPS –> can be charged or uncharged based on environment

SI has pH 6 –> amine is not protonated/non-ionized and can easily cross membranes for absorption

blood has pH 7.35-7.45 –> amine is protonated/ionized and can easily travel thru blood

47
Q

what kinds of molecules are screened in synthetic libraries?

A

screen ones that follow Lipinski rule and often only screen ones with same core structures with small variations

48
Q

what are 3 methods to design novel compound library?

A
  1. Parallel synthesis
  2. Combinatorial synthesis
  3. Copper-catalyzed azide-alkyne cycloaddition (CuAAC)
49
Q

describe parallel synthesis (3 steps)

A
  1. take 3 vials w core structure and add different small additions
  2. repeat and add different small additions until all have the same parts in different orders
  3. gives 3 slightly different molecules to increase chemical space
50
Q

describe combinatorial synthesis (3 steps)

A
  1. all 1 vial, start with A1, A2, A3
  2. then add B1, B2, B3 to each –> 9 molecules
  3. add C1, C2, C3 to each –> 27 molecules
51
Q

what is a downside of combinatorial synthesis?

A

compounds are all mixed, not a clean compound –> but still possible to accurately screen

52
Q

what is another name for CuAAC?

what is the principle?

A

click chemistry

azide easily reacts with alkene when copper is present

53
Q

what is fragment-based lead discovery?

A

screen low-molecular weight compounds for specific binding, then repeat for a second specific fragment and link the 2 binding components via CuAAC/click chemistry

54
Q

why is fragment-based lead discovery helpful?

A

chances of finding a large molecule that fit into a binding site are lower than finding a few smaller molecules that fit into a part of the binding site separately then combine

55
Q

what are 6 advantages of fragment-based lead discovery?

A
  1. high level of diversity
  2. smaller libraries
  3. easier to synthesize
  4. smaller fragments
  5. adhere to Lipinski
  6. higher success probability
56
Q

what are 3 advantages for synthetic libraries?

A
  1. characterized so can be synthesized easily
  2. stratified for Lipinski
  3. stratified for toxic compounds
57
Q

what are 2 disadvantages for synthetic libraries?

A
  1. many compounds are variations on a common structure (limited chemical diversity)
  2. loss of serendipity
58
Q

how do we optimize a lead compound?
2 alternatives?

A

systematically modify functional groups to see their importance for biological effect and affinity
1. resolve structure of compound and target
2. model compound with Alphafold

59
Q

what does a structure-activity relationship tell us about the drug?

A

can determine pharmacophore

60
Q

what is a pharmacophore?

A

3D structure required to elicit biological effect with specific groups, conformations, angles, relative positions in space, etc.

PHAR 503 (10 decks)

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