Day 1 Flashcards
Goal of secondary screens
Prevent late-stage attrition from bottleneck shifting downstream.
Hit
A molecule capable of interaction with a pharmacologically relevant protein with robust dose-response activity in a primary screening. Known structure
Lead
A compound with the desired pharmacological effect
Forward Screen
First perform phenotype assay.
Then, Id target.
Reverse Screen
Perform in vitro say on known target, then perform secondary screens of efficacy, specificity and phenotype in vivo.
RNAi mechanism
> 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.
Paramters for siRNA knockdowns, especially when using multiple siRNAs
- Knock-down efficacy
- off-target effects
- Transfection toxicity
Pooled screening
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)
Array library screening
sgRNAs delivery to discreet populations. Identification of populations of interest instead of selection.
Chemical libraries
Pharma: 1 million compounds, EPFL: 100,000
+ custom libraries.
Molecule storage: 10 mM in DMSO
Molecular shape for selecting screening drugs
In addition to the Lipinksi rule of 5 molecular shape is used at the BSF as a criterion for selecting chemically diverse compounds.
Lipinski’s Rule of 5
Rules for orally available molecules.
- Molecular weight below 500.
- Lipophilicity less than 5 (log partition water, octanol)
- > 5 H bond donors.
- > 10 H bond acceptors.
Instrumentation at the BSF
- Robotic arms
- liquid dispensers and handlers
- Centrifuge/peeler/sealer/washer/shaker
- hotels and cell incubators.
Target families for assays
GPCR, Transporters, Nuclear receptors, Ion channels, Kinases/Phosphatases, Proteases, Epigenetic, ubiquitination, Metabolic (amino acids, nitrous oxide,…)
Diversity of Assay:
- Cell based vs. not
- Binding vs. functionality
- mono vs. coculture
- High vs. low throughput.
- 2D vs. 3D.
Single strategy Assay Development for Screening
Ask:
- Which parameters impact assay?
- How do I control for these parameters to ensure high quality?
HTS compatibility
- Homogenous assays preferred (mix & read)
- Limited number of steps -> simplification of protocol leads to a lot of time being saved.
- Incubation time and temperature (RT preferred, since the robots are at room temperature.)
- Reproducibility
Z’
= 1 - 3*Σ SD/ abs(Δ μ)
Cannot be higher than 1. Lower than 0 means you have a terrible screen (no conclusions possible)
Looking for ATP binding site competitor
Start with a library of ATP derivatives.
Cyclo-oxygenase
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.
IC50 value
y = min + (Max-min)/1+(x/IC50)^nH
Diclofenac
Potent NSAID
