EXAM 2 - Principles of Biological Assays & Lead Compound Identification Flashcards
Describe the difference between in vitro and in vivo.
in vitro - ‘within the glass’
* test tube, plate, culture dish
in vivo - ‘within the living’
* whole animal models (in mice)
When measuring how active a compound is against our drug target, would you use in vivo or in vitro assays? Why?
In vitro assays
* identify a lead compound - first initial small molecule that has activity against the target (assays measure the activity)
* develop the lead into a Drug Candidate (SAR) - makes the drug more potent, selective, etc.
What output does a colorimetric/absorbance assay give?
Change in color as a function of concentration
* amount of light absorbed is directly related to the concentration of solute present in a solution
What output does a luminescence assay give?
Visible light emission based on a chemical or enzymatic reaction
What output does a fluorescence assay give?
Visible light emission based on the absorption of and emission of light at differing wavelengths
What is a biochemical assay?
Identifies how compounds interact with an isolated target in an artificial environment.
* receptor-binding, enzymatic activity, protein-protein interactions
Wha is a cell-based assay?
Analyzes a measurable effect of a given target within a cell: its natural environment
* output must correlate to target - you don’t want to ask “was it bc my target binded to something else?”
Biochemical assays: Explain what information an analytical assay gives.
A biochemical analytical assay tells us if the molecule/compound of interest binds to the target or not.
* does the molecule bind to the target?
Biochemical assays: Explain what information a functional assay gives.
A biochemical functional assay tells us if the compound of interest actualy inhibits the function of the target
Biochemical assays: Explain the difference between direct and coupled assays.
Direct - product of a single enzymatic reaction produces a readable output (uncommon)
Coupled - product from primary enzymatic reaction is used as a substrate in a seperate reaction that has a readily detectable output
Describe how to read a colorimetric assay in regard to cytotoxicity/anti-cancer activity.
Quantifies the ability of a potential drug to prevent cell growth
* living cells –> more enzymatic activity –> more Formazan (purple color)
* non-living cells –> less enzymatic activity –> less Formazan (yellow - MTS)
How can you use luciferase assay to determine kinase activity?
Luciferase assays are coupled with other reactions
* kinases phosphorylate proteins
* if a kinase is active –> converts ATP to ADP –> produces less light
* if kinase is inactive –> won’t convert ATP to ADP –> produces MORE light
What is a fluorescence assay? What does it tell you?
A fluorescence assay can measure apoptosis through emission of visible light.
What is caspase? What is its significance?
Caspase is a protease that regulates apoptosis
* it cleaves proteins to release fluorophores –> emit light
* the fluorophore must be unbound for there to be fluorescence
- inhibitor caspase drug –> no cleavage by caspase –> bounded fluorophore –> no light (low fluorescence)
- bound = no phosphorylation = high polarization = no light
What does fluorescence polarization assay tell you?
How much of the light coming out is still polarized?
