Day 1

Question 1

Goal of secondary screens

Answer 1

Prevent late-stage attrition from bottleneck shifting downstream.

Question 2

Hit

Answer 2

A molecule capable of interaction with a pharmacologically relevant protein with robust dose-response activity in a primary screening. Known structure

Question 3

Lead

Answer 3

A compound with the desired pharmacological effect

Question 4

Forward Screen

Answer 4

First perform phenotype assay.

Then, Id target.

Question 5

Reverse Screen

Answer 5

Perform in vitro say on known target, then perform secondary screens of efficacy, specificity and phenotype in vivo.

Question 6

RNAi mechanism

Answer 6

> 200 nt dsRNA is transfected in cells, which is then processed into 20-25 nt siRNAs. These are unwound in the formation of RNA-induced silencing complexes (RISCs).
These cleave cognate RNA.

Question 7

Paramters for siRNA knockdowns, especially when using multiple siRNAs

Answer 7

• Knock-down efficacy
• off-target effects
• Transfection toxicity

Question 8

Pooled screening

Answer 8

All the sgRNAs are delivered to a single vessel of cells at low multiplicity of infection. Selection for transducer cells and specific phenotypes are carried out. (Positive or negative selection of target population)

Question 9

Array library screening

Answer 9

sgRNAs delivery to discreet populations. Identification of populations of interest instead of selection.

Question 10

Chemical libraries

Answer 10

Pharma: 1 million compounds, EPFL: 100,000
+ custom libraries.
Molecule storage: 10 mM in DMSO

Question 11

Molecular shape for selecting screening drugs

Answer 11

In addition to the Lipinksi rule of 5 molecular shape is used at the BSF as a criterion for selecting chemically diverse compounds.

Question 12

Lipinski’s Rule of 5

Answer 12

Rules for orally available molecules.

1. Molecular weight below 500.
2. Lipophilicity less than 5 (log partition water, octanol)
3. > 5 H bond donors.
4. > 10 H bond acceptors.

Question 13

Instrumentation at the BSF

Answer 13

• Robotic arms
• liquid dispensers and handlers
• Centrifuge/peeler/sealer/washer/shaker
• hotels and cell incubators.

Question 14

Target families for assays

Answer 14

GPCR, Transporters, Nuclear receptors, Ion channels, Kinases/Phosphatases, Proteases, Epigenetic, ubiquitination, Metabolic (amino acids, nitrous oxide,…)

Question 15

Diversity of Assay:

Answer 15

• Cell based vs. not
• Binding vs. functionality
• mono vs. coculture
• High vs. low throughput.
• 2D vs. 3D.

Question 16

Single strategy Assay Development for Screening

Answer 16

Ask:

1. Which parameters impact assay?
2. How do I control for these parameters to ensure high quality?

Question 17

HTS compatibility

Answer 17

1. Homogenous assays preferred (mix & read)
2. Limited number of steps -> simplification of protocol leads to a lot of time being saved.
3. Incubation time and temperature (RT preferred, since the robots are at room temperature.)
4. Reproducibility

Question 18

Z’

Answer 18

= 1 - 3*Σ SD/ abs(Δ μ)

Cannot be higher than 1. Lower than 0 means you have a terrible screen (no conclusions possible)

Question 19

Looking for ATP binding site competitor

Answer 19

Start with a library of ATP derivatives.

Question 20

Cyclo-oxygenase

Answer 20

COX is important in the inflammatory response. COX-1 has a constitutive role, whereas COX-2 is inducible with cytokines.
Cox 1 has an important physiological role in maintaining tissue homeostasis.

Converts Arachidonic acid to PGG2.

Question 21

IC50 value

Answer 21

y = min + (Max-min)/1+(x/IC50)^nH

Question 22

Diclofenac

Answer 22

Potent NSAID