HTS

Question 1

Why do we need screening? ***

Answer 1

1. Rational drug design takes time & money
2. it is based on previous experience
3. Natural products often contain potential drug molecules
4. Screening can identify agents that act on a new target
5. screening my find a whole new drug structure class
6. screening could find new drug for pharma

Question 2

what are the Screening targets ***

Answer 2

receptors, enzymes, hormones, factors, ion channels (for pain), nucleic acids (HIV drugs), nuclear receptors

Question 3

What is screened?

Answer 3

Combinatorial chemistry, privileged motifs (core chemical structure), literature and patents, ligand design, endogenous ligands, chemogenomics, natural products (most common), random screening

Question 4

What is combinatorial chemistry?

Answer 4

Synthesis technologies to make compound libraries rather than single products
HT purification equipment, solid-and solution-phase chemistry

Question 5

What is chemogenomic?

Answer 5

use genetic similarities in target protein families for drug development (change in structure) - (chemistry + genomics)

Question 6

what is the Success rate of general screening

Answer 6

is very low,
one in a million
we do not want high HITS because it means there is something wrong

Question 7

what is Diversity based screening

Answer 7

Brute-force and luck

you screen everything! (big pharma)

Question 8

what is Focused based screening

Answer 8

Eliminate some chemicals before screening, increase success by 100x (in small pharma)
e.g. eliminate those ones with CYP DI,
focus on whichever library that is already generated hits
virtual/in silica screening - sindie the computer

Question 9

What is In Silica Screening??

Answer 9

Virtual screening

• Eliminate chemical structures with unfit molecular size/lipophilicity
• 3D pharmacophore models (comparison with existing drugs - when company wants to copy)
• docking algorithms base don crystal structure of the receptor (X-ray crystallography - you might know what the receptor looks like)

Question 10

What is Cellular signalling??

Answer 10

The way cells communicate and function (eg. measuring increase in Ca2+ when GPCR is activated)
in most drug targets,
they are becoming more complex,

Question 11

What are the measurements of cellular signals

Answer 11

measurement is like drug analysis but harder
Ca2+ - (essential,
no chromaphore, you can measure this inside a living cell by fluoresecnce Ca2+ censor)

Question 12

What is fluorescence Ca2+ sensor?

Answer 12

Fluorescence sensors for Ca2+

they get brighter when they bind to calcium

Question 13

functional screening in GPCR

Answer 13

Overexpress new GPCR
screen a library of compounds to find an activator of the receptor, by measuring Ca2+ concentration inside the cells
By using fluorescence microplate readers as an equipment

Question 14

New sensors for other cellular signals

Answer 14

cAMP, NO, IP3

but these need more advanced techniques

Question 15

What is high content screening?

Answer 15

it’s like ‘taking pictures of cells’

it defines spatially and temporarily in the context of structural/functional integrity

Question 16

What is assessed in High Content Screening??

Answer 16

nuclear morphology/size
receptor internalisation
cell morphology/motility
membrane permeability
mitochondrial membrane potential
neurite outgrowth
DNA condensation
apoptosis/necrosis markers
calcium homeostasis

Question 17

Example of HTS assay - stopping DI before drug is developed

Answer 17

PXR is a transcription factor that regulates the expression of CYP3A4 drugs
So, we develop a HTS assay to detect drugs which activate PXR so that we can stop this.

Question 18

METHOD of PXR activating drug for HTS assay

Answer 18

1. We want to make the plasmid to make it express PXR
2. cell transfected with reporter
3. The reporter has luciferase so it makes light instead of cyp3a4
4. When the test drug comes in activates PXR, it lights up
5. The robot detects the light
6. Any drug with the light is thrown away because it means it is an activator of PXR (DI will be present)