Theme 4 Lecture 13

Question 1

What is the role of high throughput screening

Answer 1

High throughput screening is crucial in early stages of drug discovery to find out active molecules

Question 2

Describe the typical setup of HTS

Answer 2

Assaying many compounds in multi well plates.

Question 3

What are the three main types of HTS

Answer 3

hts, fragment and virtual screening

Question 4

What is the capacity of high throughput screening?

Answer 4

Over 100k a day

Question 5

What are the 4 essential components needed for HTS

Answer 5

Chemware-Library
Bioware-assays
Hardware-Automation
Software-Data

Question 6

What are the characteristics of an ideal high-throughput screening assay?

Answer 6

A: Reliability, reproducibility, robustness, simplicity, user-friendliness, rapid and sensitive

Question 7

What are the considerations for developing the HTS assay(tech,re,pharm,sm,auto)

Answer 7

Evaluating the technology for the assay, generating reagents, pharmacologically validating the assay, and miniaturization and automation of the assay.

Question 8

What is needed to be taken into account when designing and assay(compound characteristic)

Answer 8

Nature of the drug target, technical considerations and what the compound characteristic is

Question 9

What features categorize high-throughput screening assay types?(Bm,Ap,s,tech,tar)

Answer 9

Biological material used, overall assay approach, signal or readout detection, assay configuration, specific technology employed, and target class

Question 10

Which two main classes of biological material are used in HTS assays?

Answer 10

Pure systems and cell based systems

Question 11

Do you always need a drug target to do a high throughput screen?

Answer 11

No, you can do a phenotypic screen.

Question 12

How does the nature of biological material influence HTS (whole/pure)

Answer 12

Whole cell systems and cell free determines types of assays and the tech needed to be used

Question 13

What the the two main approaches to HTS

Answer 13

target based and phenotypic based(assaying without knowing the drug target)

Question 14

What are common readout signals

Answer 14

Absorption, fluorescence, luminescence (photoluminescence, chemiluminescence, radioluminescence).

Question 15

Homogeneous (no need to separate) and non-homogeneous(mem and elisa)

Answer 15

H-scintillation proximity, FRET, fluorescence polarisation and alphascreen
NH-Membrane binding assays, protein precipitation assays and ELISA.

Question 16

How HTS can be categorised through target class GPCR,kinase/proteins

Answer 16

Receptors, ENZYMES, ion channels and protein interactions

Question 17

what are the types of whole cell assays?R,Ae,cal,me

Answer 17

Whole cell binding assays, reporter gene assays (e.g., luciferase or GFP under control of a pathway sensitive promoter), Aequorin for Ca2+ reporting, Indicator assays with calcium sensitive dyes, membrane potential sensitive dyes, and high content assays for monitoring various cell properties.

Question 18

Pro crys, li,co,h3 and mass

Biophysical assay technologies

Answer 18

Protein crystallization and ligand-protein co-crystallization, 3H and 15N-NMR, Surface Plasmon Resonance, Isothermal titration calorimetry, Differential Scanning Fluorimetry, Mass spectrometry.

Question 19

Describe the biochemical assay for Cathepsin K. (Homogenous assay)

Answer 19

It’s a homogeneous assay using cell-free pure protein, with fluorescence intensity (Flint) as the readout, detecting increased fluorescence when Cathepsin K cleaves the substrate.

Question 20

Explain FRET for Cathepsin S

Answer 20

Cell free pure protein(biochemical assay)
Substrate has two fluorophores
Measures red fluorescence when S is cleaves the substrate

Question 21

What is the principle behind indicator assays for intracellular calcium?

Answer 21

Calcium sensitive dyes
Measures alterations in fluorescence and calcium levels

Question 22

think about specific response via signalling pathways

reporter assay screening (luciferase)

Answer 22

Luciferase genes controlled by specific response elements indicate specific activity.

Question 23

What are imaging assays?(cellular behaviour)

Answer 23

These monitor changes in cellular behaviour like NFAT translocation or cannabinoid receptor internalisation.

Question 24

What is the concept of fragment based screening? NMR

Answer 24

Offers smaller molecules for screening, but are less potent but offer more room for development.

Question 25

Virtual Screening? Ligand and receptor gudied

Answer 25

Computational models to match drug targets with chemical libraries

Question 26

fit,al

Receptor guided approach

Answer 26

Using fitting points and a genetic algorithm to map and score optimal ligand-receptor poses.

Question 27

Ligand guided approach (known ligand)

Answer 27

Known ligand based on properties like size, polarity, H-bond capabilities, and hydrophobicity, using a computational model of the ligand.