Section 3

Question 1

What are the industry standard drug discovery phases?
How long do they take?

Answer 1

Phase 0 - Target selection (3 months to 2 years)
Phase 1 - Screening (3-4 months) and Hit identification (3 months)
Phase 2 - Lead identification (6-9 months) and Lead optimisation (2 years)
Phase 3 - Candidate Drug Nomination
Phase 4 - Pre-clinical development

Question 2

What is the difference between actives, hits and leads?

Answer 2

Actives - the raw output from a HTS. Defined as IC50 values from a dose-response HTS assay.

Hits - any compound of defined structure which has biological activity at some defined threshold.

Leads - compounds resulting from structural optimisation of the biological, physical and pharmaceutical properties of a hit, which have the potential to become a drug candidate.

Question 3

Name 4 methods of hit discovery.

Answer 3

High throughput screening

Fragment based screening

Virtual screening

Fast follower/me too method

Question 4

How does high throughput screening discover hits?

Answer 4

Millions of compounds from a library are screened against a known target. Actives which appear active are investigated to confirm activity - these are hits.

Question 5

How does fragment-based screening discover hits?

Answer 5

Low molecular weight molecules are screened for low affinity hits. There are 2 methods:

1. Linking fragments - discover multiple fragments which bind to the active site separately, then link them to form one molecule.
2. Growing fragments - discover a fragment which binds to some of the active site but not all of it, then growth the molecule so it forms new interactions elsewhere in the active site.

Uses NMR, X-ray, and SPA screening due to poor differentiation between low MW molecules.

Optimisation relies on X-ray crystallography.

Question 6

How does virtual screening discover hits?

Answer 6

This computer-based method uses known actives to produce a 3-point pharmacophore model which new drugs can be based around. Has 2 methods:

Knowledge-based drug design: search in compound libraries for new compounds which match the pharmacophore pattern.

Structure based-drug design: generate a virtual set of compounds based on the pharmacophore, then build a receptor model to dock the new compounds in to look for hits.

Question 7

How does the fast follower/me too method discover hits?

Answer 7

A way of designing a new drug which is active at a clinically validated receptor, using existing chemical equity. Basically just replacing certain chemical groups in a known drug with chemically equitable/similar groups to make a new structure/drug.

Question 8

What are the pros and cons of high throughput screening?

Answer 8

Pros:
Uses various assays and can detect activity at a very low concentration range.
Doesn’t require an isolated protein target.
Large numbers of high molecular weight compounds are screened.

Cons:
The specific mechanism of action and binding site may not be known.
Often results in many false positives which must be identified and eliminated using a second assay (either with just the hits or with the whole library).

Question 9

What are the pros and cons of fragment-based screening?

Answer 9

Pros:
Few false positives as direct binding is detected.
Lower MWt/Log D hit starting points
Ligand efficiency is generally higher than conventional screening hits

Cons:
Relies on an assay where direct interactions between a small molecule and isolated target can be detected.
Fewer compounds are screened compared to HTS.
Evolving low-potency FBS hits to high-potency leads takes significant effort.

Question 10

What are the 4 categories of biological assays?

Answer 10

Enzymatic/biochemical assays

Binding assays

Functional assays

Phenotypical assays

Question 11

What do biochemical/enzymatic assays tell us?

Answer 11

Monitors formation of the product of an enzymatic reaction using an isolated and purified enzyme. This allows identification of enzyme inhibitors and activators, depending on the amount of product formed.

Since enzymes used are isolated, hits may not have appropriate pharmaceutical properties.

Question 12

What do phenotypic assays measure?

Answer 12

Changes in complex systems, regardless the specific molecular target.

Question 13

Describe the role of the catalytic triad in the cleavage of the amide bond during the mechanism of a serine protease.

Answer 13

1. Catalytic triad interact to deprotonate the serine through intramolecular H-bonding, forming a nucleophile.
2. The nucleophilic serine attacks the amide bond at the electrophilic centre of the amide carbonyl to produce an sp3 tetrahedral intermediate.
3. This intermediate breaks down as an amine is lost, leaving the acylated serine separate to the the main molecule.
4. The nitrogen of the histidine attacks the hydrogen on a water molecule. This remaining OH attacks the acylated serine.
5. A transfer of electrons results in regeneration of the catalytic traid and loss of a carboxylic acid.

Question 14

What is the catalytic triad in a serine protease?

Answer 14

Aspartic acid
Histidine
Serine

Question 15

How can a biochemical/enzymatic assay measure the inhibitory effects of different molecules on a serine protease enzyme?

Answer 15

FRET

Design a fluorogenic substrate with an attached amino acid sequence which is cleavable by the protease of interest. Under normal conditions, when the protease cuts the substrate, the fluorescent dye is released and emits a signal.

In the presence of an inhibitor, the protease will be unable to cleave the substrate and the fluorescent dye will not be released. Therefore, the degree of inhibition relates to level of fluorescent emission.

Question 16

What do binding assays measure?

Answer 16

Ligand binding assays provide a measure of the interactions that occur between two molecules via radiolabels, fluorescent labels, or MW changes.

Don’t measure the effect of binding, but rely on the idea that binding of a drug will displace the natural ligand.

Question 17

What do functional assays measure?

Answer 17

The downstream effects of a binding event.

For example, calcium mobilisation assays using a calcium sensitive dye to measure the calcium flux associated with Gq-protein couple receptor activation or inhibition.

Question 18

When and what was the first use of powdered foxglove plant?

Answer 18

1775 - congestive heart failure.

Question 19

What are some symptoms of poisoning due to ingestion of Foxglove plant?

Answer 19

Nausea
Diarrhoea
Headache
Skin irritation
Visual disturbances

Question 20

When was Digoxin first isolated from Foxglove plant?

Answer 20

1875.

Question 21

Which medications are based on ephedrine, the active substance of the Ephedra sinica plant?

Answer 21

Amphetamine and methylphenidate used for ADHD.

Question 22

Which medications are based on paclitaxel, the active substance of the Pacific yew tree?

Answer 22

Docetaxel and cabazitaxel which are anticancer agents.

Question 23

What are the key structural features of morphine and related drugs, meperidine and fentanyl?

Answer 23

A tertiary alkyl amine that’s at least 4 atoms away from an aromatic ring.

Question 24

Name 3 drugs derived from microbes.

Answer 24

Penicillin G (antibiotic from mould)

Cephalosporin C (antibiotic from fungus)
Cefotaxime (structure derived from cephalosporin C)

Doxorubicin (anticancer from soil fungus)

Epothilones (anticancer from soil fungus)

Question 25

What side effect of doxorubicin can be reduced when the structure is altered to make a new drug?

Answer 25

Cardiotoxicity

Mitoxantrone has simplified chemical structure and reduced cardiotoxicity.

Question 26

What change was made in the structure of anticancer agent epothilone B to Ixabepilone?

Answer 26

Ester (lactone) to amide (lactam) switch to improve metabolic stability.

Question 27

What are the pros and cons of using endogenous ligands as a starting point?

Answer 27

Advantages:
Likely to be active.
Likely have prior knowledge regarding natural ligand binding and the effect on the protein target.

Disadvantages:
Will act similarly to endogenous ligand so may affect all receptor subtypes, resulting in low specificity.
Strategy not useful if the target substrate or ligand is not known.

Question 28

Name 4 examples of drugs whose structure is based on the endogenous ligand.

Answer 28

Cimetidine (histamine)
Propanolol (noradrenaline)
Methotrexate
Sumatriptan (5-HT)

Question 29

How do HIV protease inhibitors work?

Answer 29

During HIV replication, HIV protease recognises the Phe-Pro segment in the S1 pocket of the Gag-Pol precursor protein and cleaves the amide bond.

HIV protease inhibitors are designed to mimic the tetrahedral intermediate of the cleavage, due to their secondary alcohol. They contain the recognition segment so they bind with high affinity, but have no cleavable amide bond.

Question 30

What 2 types of activity has Azidothymidine been reported to have and when were they discovered?

Answer 30

1960s - anticancer

1974 - anti retrovirus
1980s- anti-HIV (HIV is a retrovirus)

Question 31

What are the 2 types of synthetic libraries?

Answer 31

Diversity library - a random set of compounds that have been purchased or synthesised for previous drug discovery programmes. Will have drug-like properties but no basis for activity at the current target.

Targeted library - a set of compounds which have been designed (or purchased) that possess structural features that suggest they may have activity at the target.

Question 32

What does it mean if a compound has drug-like properties?

Answer 32

Has acceptable ADME properties to serve through the completion of human phase 1 trials.

Question 33

What Is the difference between physicochemical and biochemical properties?

Answer 33

Physicochemical properties are influenced by interaction between structural properties of the compound and the physical environment.

Biochemical properties are influenced by interactions between the structural properties of the compound and proteins.

Question 34

What is Lipinski’s rule of 5?

Answer 34

Dictate whether a drug will have good oral absorption based on its physicochemical properties.

1. <5 H bond donors (sum of all OH and NH groups)
2. <10 H bond acceptors (sum of all Ns and Os). (doesn’t apply to biological transporters)
3. MW<500
4. LogP<5

Violation of 1 rule doesn’t result in poor absorption, but after this, absorption worsens as more rules are broken.

Question 35

What is the affect of adding non-polar and polar groups to a chemical scaffold?

Answer 35

Non-polar groups are added to increase binding to lipophilic pockets in the active site.

Polar groups are added to increase hydrogen bonding to the active site.

Question 36

What is the ideal cLogP for oral drug-like compounds?

Answer 36

Between 1 and 4

Question 37

What is the ideal molecular weight for oral drug-like compounds?

Answer 37

between 250 and 400

Question 38

How were targeted libraries used to develop anti-HIV drugs?

Answer 38

1. Recognised that HIV protease is an aspartic acid protease.
2. Screened compounds for a similar acid protease, renin, against HIV protease and found that some synthetic peptides known as dog renin inhibitors inhibited HIV protease.
3. Studied the interactions and screened other targeted libraries for drugs with the necessary structure.

Question 39

What 3 types of activity has Topiramate been reported to have and when were they discovered?

Answer 39

1990s - anti-epileptic

2004 - migraines

2012(with phentermine) - chronic weight management

Question 40

What is a combinational library?

Answer 40

A large library of structurally related compounds. Used to complement the results of diversity library screening.

Question 41

What is the process of parallel synthesis?

Answer 41

1. An automated synthesiser performs the synthesis of multiple products at once.
2. The intermediate is purified using a direct purification system.
3. Following sample handling, the sample is evaporated.

Question 42

What is combinational chemistry?

Answer 42

The systematic, repetitive, covalent connection of a set of different building blocks of different structures to each other to yield a large array of diverse substituents around a common scaffold.
Compounds are produced using solid-phase synthesis or using split-pool synthesis.

Question 43

What is resin-based synthesis (aka solid-phase synthesis)?

Answer 43

Compounds are assembled on beads of resin by a repetitive sequence of combine, divide, and couple.

Question 44

What is the process of resin-based synthesis (aka solid-phase synthesis)?

Answer 44

1. Attach resin bead to a linker
2. Attach compound to other end of linker.
3. Deprotection (removal of Boc protecting groups) unmasks primary amine on compound.
4. Compound is coupled with another compound to produce a dipeptide.
5. Deprotection (removal of Boc protecting groups) unmasks primary amine on the second compound.
6. This continues until a peptide chain is produced.
7. A strong acid (e.g., hydrofluoric acid) is used to release the peptide from the solid support.

Question 45

What are Boc protecting groups>

Answer 45

A tert-butyloxycarbonyl (Boc) group which binds to amines to protect them from hydrolysis or nucleophilic attack during organic synthesis mechanisms such as solid-phase synthesis.

Question 46

What is split-pool synthesis?

Answer 46

Used to make combinational compound libraries.

1. 3 pots containing resin are coupled to building blocks A, B and C to give resin-A, resin-B and resin-C as starting points
2. These are then mixed together (pooled) to give a mixture of the 3 monomers
3. The mixture is then divided (split) into 3 equal portions
4. A coupling reaction is performed on each pot with a unique monomer
5. The process is repeated.
6. The products are cleaved from resin and assayed. Results must be deconvoluted to find the active components.

Question 47

What is parallel synthesis?

Answer 47

Used to make combinational compound libraries.

Each type of monomers is combined with each type of another monomer to produce dimers.

So for example, monomers A-D are combined with resin (A-resin, B-resin) etc. These monomers are then combined with monomers E-H, so resin-A-E, resin-A-F etc.

Question 48

What are DNA-encoded libraries?

Answer 48

Used to make combinational compound libraries.

1. Attach each building block to a linker which terminates with a short strand of DNA. The strand is specific sequence which is a tag for that building block.
2. React a second building block to the linker and extend the DNA strand with another sequence which is a tag for the second building block.
3. Continue adding new building blocks and unique DNA tags to produce a library.

The library then undergoes an affinity-based assay (filter wash):

1. Incubate target protein with DNA-encoded library to allow binding of those with strong affinity.
2. Weak and none binders are eluted.
3. Heat the complexes to denature the proteins and elute the selected binders.
4. Elucidate the structure of binders using PCR amplification or DNA sequencing to identify the compounds with binding affinity.
5. Re-synthesise off-DNA for conformation of activity.
6. Optimise chemical structure to generate lead compounds.

Question 49

What are 3 ways to achieve diversity in a collection of molecules for diversity-orientated synthesis?

Answer 49

Skeletal diversity - Generate different scaffold which provide more attachment points.

Stereochemical diversity - generate different stereoisomers to access different binding patterns with protein targets.

Functional group diversity - insert different functional groups around the core structure.

Question 50

What is the Ugi reaction?

Answer 50

A method which produces single compounds with relative stereochemistry associated with it.

4 components (benzylamine, an aldehyde, a phenylacetonitrile, an acrylate/malonic acid amide) are combined to produce a much larger structure. In this structure is a diene and a dieneophile which, if they are close together, can undergo a 2 plus 4 cycloaddition reaction to generate a fused system. The 2 nitrogens in the amides can be alkylated. Also, a cross metathesis reaction can occur to form more fused rings.