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PHAR 503 > lecture III: drug screening & discovery > Flashcards

lecture III: drug screening & discovery Flashcards

1
Q

Historical drug discovery

A

Natural medicines, previous drugs were known, then refined.
→biological effect was discovered before target validation

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2
Q

Historical drug discovery steps

A
  1. Biologically (clinically) observed agent
  2. Bioassay
  3. Biological hit
  4. Lead optimization
  5. Preclinical & clinical studies
  6. Drug
  7. Medically validated target
  8. Better Drugs → high success rate!
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3
Q

Target-based drug discovery

A

“Genes-to-drugs”

  • genome sequencing would reveal new targets
  • main issue: many targets are not biologically validated
  • drugs can be developed, but they don’t work in vivo
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4
Q

Target-based drug discovery steps

A
  1. Molecular genetics (target selection)
  2. Target-based HTS assay
  3. Target hit
  4. Lead optimization
  5. Preclinical and clinical studies
  6. Drug → high failure rate :(
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5
Q

Phenotype-based drug discovery

A

Return to biology first!

  • one specific protein is not targeted, but rather a certain phenotype
  • not really interested on how the drug works, but as long as you get the desired phenotype, then you go with it
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6
Q

Phenotype-based drug discovery steps

A
  1. Human cell systems
  2. Biological hit
  3. Lead optimization
  4. Preclinical and clinical studies
  5. Drug
  6. Medically validated target
  7. Better drugs → high success rate!
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7
Q

Cell-based assays

A
  1. Immortalized cell lines
    →cancerous, human embryonic kidney, HEK293 cells, HeLa
  2. Primary cell culture from tissue
  3. Induced pluripotent stem cells (iPSC)
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8
Q

Biosafety cabinet

A

Allows work in a sterile environment

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9
Q

Human iPSC

A

iPSC are derived from skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state that enables the development of an unlimited source of any type of human cell needed for therapeutic purposes.

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10
Q

Human iPSC types

A
  1. from healthy subjects
  2. from disease patients
  3. from diseased patient genetically reversed to healthy (isogenic)
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11
Q

iPSC target-based screen read-out

A

target protein specific

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12
Q

iPSC phenotype-based screen read-out

A

increase/decrease of a phenotype

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13
Q

Zebrafish assay

A

A type of in vivo assay where Zebrafish can be used to assess the toxicity of drug candidates in early screening assays, sometimes in a high-throughput manner.

→can be target-based or phenotype-based
→advantage: whole system

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14
Q

High-throughput screening (HTS)

A

High-throughput screening (HTS) is a drug discovery process that allows automated testing of large numbers of chemical and/or biological compounds for a specific biological target, for example through binding assays.

→issue of false positive hits
→“fail fast, fail cheap”

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15
Q

Key elements for developing a HTS screening assay

A
  1. Quick, simple, robust, relevant
  2. Positive read-out
    →i.e. fluorescence increases upon drug binding
  3. Direct read-out
    →as little handling as possible
  4. Good dynamic range
    →good z factor
  5. Proper model: in vitro / in vivo
  6. Cost effective

→hopefully you can then identify a lead compound!

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16
Q

Examples of phenotype-based screens (lecture)

A
  1. ZIKA virus decreases CAS3 (responsible for apoptosis), but with addition of drug, CAS3 inhibition decreases.
    →read-out: CAS3 inhibition
    →not the best as it is NOT a positive read-out
  2. Lipid droplets in AD. ApoE4 expressing cells have more lipid droplets as compared to ApoE3.
    →read-out: number of lipid droplets per cells
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17
Q

Consideration before a target-based screen

A
  1. Evidence that modification of the POI will cure the disease
  2. Evidence that the target is drugable
  3. Evidence of selectivity
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18
Q

Consideration before a phenotype-based screen

A
  1. Evidence that the phenotype will cure the disease
  2. Relevance of the phenotype for the disease
  3. Relevance of cell type and stimulus for the disease
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19
Q

Considerations before a drug screen (!!)

A

Choose the disease and the clinical outcome you anticipate (cure or amelioration). Is there a target? Multiple? Which one do you select?

  1. Evidence that a modification will cure the disease
    →use KO mice, literature review, human mutations
  2. Evidence that the target is drugable
    →consider similar pharmacological compounds that may need optimization
  3. Evidence of selectivity
    →expression pattern of protein, unique phenotype/cellular pathway, localization
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20
Q

HTS: positive hit range

A

Positive hits are active in the range of 30 μM - 1 nM

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21
Q

NME

A

New Molecular Entity

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22
Q

NME success order: first-in-class drug

A
  1. Phenotype-based screening (37%)
  2. Biologics
  3. Target-based screening
  4. Modified natural substances
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23
Q

NME success order: follower drug

A

1.Target-based screening (51%)
2. Biologics
3. Phenotype-based screening
4. Modified natural substances

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24
Q

Best drug options: overall

A
  1. Synthetic small molecule
  2. Biologic
  3. Natural substance
  4. Natural product
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25
Q

Best drug options: phenotype-based screens

A
  1. Synthetic small molecule
  2. Natural product
  3. Natural substance
  4. Biologic
26
Q

Best drug options: target-based screens

A
  1. Biologic
  2. Synthetic small molecule
  3. Natural substance
  4. Natural product
27
Q

Library selection: Natural world

A

Natural sources have some form of biological activity
→serve as lead compound
→usually have complex structures
→EX: cocaine, digoxin, quinine, nicotine, muscarine, penicillin

  1. Plants
    →trees, flowers
  2. Microorganisms
    →fungi, bacteria
  3. Animals
    →frogs, snakes
  4. Biochemicals
    →neurotransmitters, hormones
  5. Marine chemistry
    →corals, bacteria, fish
28
Q

Library selection: Synthetic world

A

Synthetic libraries contain compounds synthesized by company where the starting point may have been a natural substance.

  1. Chemical synthesis
  2. Combinatorial synthesis
  3. Parallel synthesis
29
Q

Library selection: Virtual world

A
  1. Computer-aided drug design
30
Q

Natural compound library advantages

A
  1. Probably pre-selected/evolved for biological activity
  2. Serendipity
  3. Potent
  4. Cover large chemical diversity
31
Q

Natural compound library disadvantages

A
  1. Difficult to synthesize
  2. Difficult to produce, do extracts, reproducibility
  3. Bioavailability may be suboptimal
32
Q

Chemical space

A

The chemical space, a set of all possible molecular structures.

Beilstein database → 10^7

33
Q

3 phases of drug action

A
  1. Pharmaceutical phase
  2. Pharmacokinetic effect
  3. Pharmacodynamic effect
34
Q

What is the ideal administration of a drug

A

Orally! (oral bioavailability)

35
Q

Drug action: pharmaceutical phase

A
  • Dissolution of drug from pill
  • Survive gastric juices, survive food-degrading enzymes
  • Pass through gut cells (sufficient lipophilicity)
36
Q

Drug action: pharmacokinetic phase

A
  • Pass into the blood stream (sufficient hydrophilicity)
  • Distribution to tissue (every cell max. 20-30 μm away from capillary)
  • Pass between cells, through cells, transporters

→essentially, oral bioavailability!

37
Q

Drug action: pharmacodynamic phase

A
  • Drug interacts with molecular target, pharmacological effect
38
Q

Bioavailability

A

Bioavailability is a subcategory of absorption and is the fraction of an administered drug that reaches the systemic circulation.

39
Q

The Lipinski rule of 5

A

An indicator for the potential ability of a drug to be orally absorbed (bioavailability).
An orally active drug has no more than one violation of the following criteria:
1. MW≤ 500 kDa
2. HBD ≤ 5
(expressed as the sum of OH & NH groups)
3. HBA ≤ 10
(expressed as the sum of O & N atoms)
4. logP ≤ 5

40
Q

Partition coefficient

A

Measure of how much of the drug is in a hydrophilic vs. hydrophobic phase.

→ a measure of the drug’s lipophilicity
→ P = [drug in octanol] / [drug in aq. solution]

41
Q

Octanol

A

Hydrophobic organic solvent

42
Q

Oral bioavailability

A

Ability to cross hydrophobic cell membranes and be water soluble for distribution

43
Q

Which functional group do many drugs contain? What’s the purpose?

A

Amines.

→pH in different parts of the body (small intestine, stomach, blood) differ, thus you need a drug that can easily cross all of these different parts.
→amines have a pKa ranging from pH 6-8
→amines can be p+ or not depending on the environment’s pH (all parts of your body have
a pH within the amine’s pKa range, so drugs with amines are always switched between their different forms)

44
Q

Synthetic library characteristics

A

Fun! go read slide 28 :)

45
Q

Synthetic library: Parallel synthesis

A

Method to design a novel compound library.

Many wells starting with core structure and then to each well, you add a new reactant. In the second reaction, you switch up the new reactant.

→small scale
→each vial has one compound
→preferred method!

46
Q

Synthetic library: Combinatorial synthesis

A

Method to design a novel compound library.

Everything mixed in and see how it reacts with the core structure.

→small scale
→mixture of compounds in one vial
→obtain way more products

47
Q

Fragment-based lead discovery (FBLD)

A
  1. Screen for 1st ligand: Screening of low-molecular weight compounds for specific binding, then optimize.
  2. Screen for 2nd ligand: Repeat screen for second specific fragment, then optimize.
  3. Link: The two binding compounds are linked.
48
Q

Advantages of FBLD

A
  1. High level of diversity is possible
  2. Easier to synthesize
  3. Higher success probability
49
Q

Click chemistry

A

A variation of FBLD consisting of a class of biocompatible small molecule reactions commonly used in bioconjugation, allowing the joining of substrates of choice with specific biomolecules.

50
Q

How can you screen FBLD?

A
  1. NMR
  2. Bioassays
51
Q

Synthetic library advantages

A
  1. Characterized, can be synthesized
  2. Stratified for Lipinski’s rules of 5
    →higher likelihood for oral bioavailability
  3. Stratified for toxic compounds
52
Q

Synthetic library disadvantages

A
  1. Low serendipity
    →many compounds are variations on a single structural theme
  2. Limited chemical diversity
53
Q

Computer-aided drug design characteristics

A

Fun! go read slide 33 :)

54
Q

Computer-aided drug design method

A

Alphafold!!!

55
Q

Specificity

A

To cause one particular action in all biological systems

56
Q

Selectivity

A

To bind to one target, not to others

→less side effects

57
Q

Sensitivity

A

The lower the dose needed to cause an effect, the more sensitive the system is

58
Q

Structure-activity relationship: morphine & naloxone

A

Naloxone is a morphine antagonist. It was synthesized to possess the same pharmacophore as morphine, but with higher affinity, thus it will knock morphine out when introduced in our system.

59
Q

Pharmacophore of morphine

A
  1. Phenol-OH
  2. Aromatic ring
  3. Tertiary amine
60
Q

How can you test structure-activity relationships?

A
  • by systematically modifying functional groups to see their importance for biological effect
  • by resolving the structure of compound and target
  • by modelling the compound on theoretically derived Alpha fold structures
61
Q

Pharmacophore: spacial views
(draw it)

A
  1. 2D → binding groups
  2. 3D → angle between binding groups
  3. 3D → bonding type

PHAR 503 (6 decks)

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