Lecture 3 Flashcards
What is the goal of High thru put screening?
Identify hits (active compounds in comparison to controls)
That could be leads (active compound that could be a therapeutic)
To compress timelines from target ID to drug discovery
What are the parts of HPS? part of what part of discovery?
early discovery
Screening automation
Assay techniques
Liquid handling
Informatics
Compound storage and handling
What are 3 drugs that are FDA approved that where HPS hits?
Ibrutnib— biochemical assay
Trametinib— cell based assay
Dasatinib—-HER2 targeting HTS
What well sized were used n 80s, 90s, and now?
HTS is feasible due to advancements in ?
50-200, 20-50, 2-10
Increased thruput, cost reduction, beyond human capacity
automaton and liquid handling
What are considerations for time in HTS?
Relevant for large screens >100k compounds—> ultra HTS is 100k
Need a miniaturized assay, amenable to robotic liquid handling and automation
Need a simplified assay—> mix and measure, homogenous are ideal
What are assay considerations for cost?
Reagent costs—> biochemical assay ( purified target, buffers, detection reagent) or cellular assay (cell lines, cell culture media, detection reagents)
Consumable costs— plates, pipet tips
Assays that needs expensive stuff are cost prohibitive
What is the average HTS assay cost?
0.1 and 1 dollar per well—> does not include assay development or hit follow up!
What has the high cost of HTS led to?
Screen focused libraries rather than full deck screens
What are assay considerations for quality ?
Few false positives and negatives, validated binders, S/N, S/B and Z factor
What is S/B and S/N and Z factor?
S/N— signal to noise ratio—> mean sig—mean background/SD of background
S/B signal to background ratio —-> Mean high signal /compared to mean low signal
SN is always higher than S/B*
- –>consider both signal dynamic range and data variation to determine robustness
What can false positives be due to?
What are sources of PAINS?
Physiochemical properties
Aggregators, insoluble compound
How do we , avoid false positives?
Re test— does it hit again?
Concentration response curve— what is potency?
Null assay— does it have the same effect in cells not expressing the target?
Confirmation of chemical material
Orthogonal—- confirm compound activity of target
Similarity searching!— test analogs
What is the timelines and output for HIT assessment?
2 weeks, conformed and primary hits
2-3 months—— 10-20 on target hits
What are the two main strategies of HTS?
Phenotypic and target based screening
Difference between phenotypic and target based screening?
Pheno: identify compound that can cause phenotypic change in invivo model
unknown target
Screen in cells of animals
May be more physiologically relevant
__»>Thruput is low
Deconvolution
Target based screening:
Screening against a known and validated target to identify compounds based on hypothesis for that target
Know target
Screening against purified protein or cell lines over expressing target
–»>High thru put
What are biochemical assays, biophysical, and cellular?
-Monitor in test tube via enzyme assay, protein protein interaction
Can use abs, fluorescence or chemilinensense
High thruput
- monitor direct binding of compound to the target
SPR, thermal shift, NMR,
Lower thruput
Asssays that monitor activity in cells
-reporter assay, receptor signaling, protein protein interaction
Use fluorescence or chemluminescence
High thru put
What is absorbable, fluorescence and chemiluminse ?
(least sensitive–most inferens)
Measures light absorbed—DHFR assay (most interference)
Measures light emitted as a result of light absorbed (FRET)
(most sensitive–least inferens)
Measures light emitted as result chemical reaction (ELISA)—least interferents
what is a robust assay parameter relating to S/B and Z?
=10 and z=1
why is z important? what is the equation?
measures separation between pos and negative controls
—1-3(sd signal+SD background)/mean sig-mean background
