Section 4

Question 1

What are the 4 types of screenware required for high throughput screening?

Answer 1

Chemware - the large compound libraries

Bioware - an assay to determine if a compound is active for the drug target.

Hardware - Plate readers (e.g., FRET), imagers, and automation of liquid handling apparatus (e.g., FLIPR).

Software - computers used for compound management and assay data acquisition.

Question 2

What types of cells can be used in a cell-based HTS assay?

Answer 2

Native cell line (already expresses the drug target)

Engineered cell line (engineered to express drug target).

Primary cells - native cells which can be obtained e.g., red blood cells.

Tissue - rarely used in HTS

Question 3

What is the difference between homogenous and non-homogenous assays?

Answer 3

Homogenous - “mix and read”. No need to separate, just add and measure.

Non-homogenous - need to separate e.g., filtration of bound and free label in receptor binding assay.

Question 4

Name 5 homogenous assays.

Answer 4

Scintillation proximity
FRET
Time resolved FRET
Fluorescence polarisation
Alpha screen

Question 5

Name 4 non-homogenous assays.

Answer 5

Membrane binding assays
Protein participiation assays
ELISA
Sandwich ELISA

Question 6

Describe the cathepsin K inhibition biochemical assay.

Answer 6

A pure protein (cell free) homogenous assay using fluorescence to monitor ligand binding to cathepsin K, a protease enzyme.

Upon binding of the non-fluorescent substrate, the protease cleaves it releasing the fluorophore aminomethylcoumarin (AMC). When excited by a certain wavelength, it will emit a fluorescent signal.

Presence of inhibitor will prevent binding of the substrate and release of AMC, so the fluorscent emission will be reduced.

Question 7

Describe the cathepsin S inhibition biochemical assay.

Answer 7

A pure protein (cell free) homogenous assay using fluorescence to monitor ligand binding to cathepsin S, a protease enzyme.

A substrate is flanked by 2 fluorophores. When they are in close proximity and one (the donor) is excited, it results in emission from the acceptor at a different wavelength.
Upon binding to cathepsin S, the substrate is cleaved in two, causing separation of the 2 fluorophores and reduced emission.

In the presence of an inhibitor, the substrate is not cleaved and the fluorescence isn’t diminished.

Question 8

How was the anticancer drug Vemurafenib developed?

Answer 8

During a fragment screening, a 7-azaindole fragment was found to bind to a section of a mutated B-Raf binding site. It was evolved to produce a 3-aminophenyl analogue which bound to the whole binding site, then optimised to produce vemurafenib.

Question 9

How was the matrix metalloproteinase inhibitor ABT-518 developed?

Answer 9

During a fragment screening, 2 fragments, acetohydroxamate and biphenyl, were found to bind weakly to 2 sections of the metalloproteinase binding site, using NMR. These fragments were combined and then optimised to produced ABT-518.

Question 10

How can fragments be combined?

Answer 10

Manually.

Self-assembly - if the 2 fragments are attached to reactive groups which are within conformation reach of each other, the lead molecule can spontaneously self-assemble in the active site.

Question 11

What must be done to hits from a virtual (in silico) screen?

Answer 11

Confirmation of activity using lab screening methods. Either:

Ligand-guided screening: screens for compounds similar to a known active ligand.

Receptor-guided screening: library database compounds are docked into a receptor binding site and ranked using a scoring function.

Question 12

What is virtual (in silico) screening?

Answer 12

Computerised molecular models of targets and ligands are aligned to determine potential complementary intermolecular interactions. Molecules with high levels of complementarity are flagged for synthesis and testing.

Question 13

What is scaffold-hopping?

Answer 13

A method of ligand-guided screening.

It involves the structure of an active ligand template being described computationally in terms of its size, polarity, H-bond donor and acceptor capabilities, and hydrophobicity etc.
This model is then compared to a library of compounds in terms of shape matching, pharmacophore searching, fragment replacement, and similarity searching.

Question 14

What are positive and negative controls in a high throughout screen?

Answer 14

Positive - highest possible measurement.

Negative - lowest possible measurement.

For example, in a FRET assay, the positive control would be the both the donor and acceptor fluorophore, while the negative control would be just one or neither.

Question 15

Name 5 methods to measure the quality of an assay.

Answer 15

Signal to background

Signal window

Coefficient of variation of signal and background

Signal to noise

Z’ factor

Question 16

How does the “signal to background” method assess the quality of an assay?

Answer 16

Signal to background = mean positive control signal/mean background

It indicates the separation of signal and background. This measure relates to the assay itself, not so much the performance of the equipment.

A minimum value of 3 is required.

Question 17

How does the “signal window” method assess the quality of an assay?

Answer 17

Signal window = mean positive control signal - mean background

Indicates the magnitude of a response value. A minimum signal window (1000pcm) is required to avoid reproducibility issues. This measure relates to the assay itself, not so much the performance of the equipment.

Question 18

How does the “coefficient of variation of signal and background” method assess the quality of an assay?

Answer 18

%CV = 100 x (SD/Mean)
Done for both positive control signal and background to get 2 values which can be compared.

Provides a measure of the variability of the signal. Indicates the assay stability, liquid handling, and instrumentation.

Question 19

How does the “signal to noise” method assess the quality of an assay?

Answer 19

(calculation too complicated to write)

In screening, this provides a complete picture of the performance of a HTS assay.

Typically a value of 10 is acceptable.

Question 20

How does the “Z’ factor” method assess the quality of an assay?

Answer 20

(calculation too complicated to write)

Most widely used parameter to evaluate performance of a HTS assay.
Above 0.4 is acceptable.

Question 21

What are some common sources of errors in high throughput screens?

Answer 21

Liquid handling

Robotic failures

Plate geometry (positioning of plate in machine)

Unintended changes in compound concentration due to evaporation during assay or storage.

Question 22

How do false positives occur in high throughput screens?

Answer 22

Pan assay interference compounds with reactive functional groups which form aggregates that act non-specifically with the target. These compounds can be identified and and excluded, although may be desirable and be left in.

Compounds which interfere with the assay signal. For example, fluorescent ligands or luminescence inhibitors.

Question 23

What are some common criteria of excluding less promising hits?

Answer 23

Top percentage (e.g., 1%, 2% of compounds).

Percentage inhibition (e.g., if results in less than 40% of control activity, exclude).

Difference of a compound from control e.g., must affect the signal beyond 3 standard deviations from the control.

Question 24

How do beta-secretase inhibitors work in Alzheimer’s disease?

Answer 24

Beta-site APP cleaving enzyme 1 (BACE-1 aka beta-secretase) is a transmembrane aspartyl protease which cleaves amyloid precursor protein (APP) to produce beta-amyloid peptides (A-beta). This results in production of A-beta peptides in the brain, neuronal cell death, and the subsequent symptoms associated with Alzheimer’s disease.

Beta-secretase inhibitors therefore prevention beta-amyeloid peptide production, reducing Alzheimer’s symptoms.

Question 25

What are the 4 key residues in the beta-secretase/BACE-1 binding site?

Answer 25

Catalytic site - Asp32 and Asp228
S3 pocket - Gly74 and Gly13

Question 26

Outline the development of the Alzheimer’s drug, Verubecestat.

Answer 26

1. Fragment screen done on NMR fragment library.
2. Isothioureas found to bind with low affinity to catalytic region and S3 pocket of beta-secretase (BACE-1).
3. Isothiourea hit 2 was chosen and optimised to produce isothiourea hit 3.
4. Isothiourea hit 3 was analysed using x-ray crystallography to determine its position in the active site. It was found that a central structure in the isothiourea was suscptible to hydrolysis so needed to be modified.
5. Based on the hit 3 structure, a 2-aminopyridine series and an iminohydantoin series were developed. From the iminihydantoin series, compound 4 was developed.
6. Compound 4 bound to the BACE-1 active site but weakly. The ring system was modified to better fit the binding pocket which increased potency.
7. Eventually verubecestat was developed, which possessed a pyridyl ring which occupied the binding pocket.

Question 27

What is the effect of cannabinoids on human glycine receptor alpha-1 (hGlyR-alpha1)?

Answer 27

Human glycine receptor alpha-1 is an ligand-gated chlorine channel with inhibitory transmission. When chloride ions pass through, there is slight hyperpolarisation of the membrane which dampens neuronal transmission.

Cannabinoids such as THC bind to the residue S229 of the channel and potentiate the inhibitory effect, leading to reduced pain transmission.

Question 28

Describe the virtual screening method used to find approved drugs with Glycine receptor potentiation activity.

Answer 28

1. 1549 FDA-approved drugs were screened against 180 conformers (with and without various membrane lipids) of the hGlyR-alpha-1 at the S229 binding site.
2. The top 25 hits were selected based on predicted binding energies. These were tested on models with and without lipids.
3. 16 hits which bound to both lipid and non-lipid target structures were filtered to ensure they weren’t pan-assay interference compounds and were not structurally similar to THC
4. A cell-based assay using xenopus oocytes was done to confirm activity. The channel was activated with glycine before addition of one of the 16 compounds or THC (control). Electrophysiology was used to measure the activity of the channel. 15 compounds were successful.