Theme 4 Lecture 13 Flashcards
What is the role of high throughput screening
High throughput screening is crucial in early stages of drug discovery to find out active molecules
Describe the typical setup of HTS
Assaying many compounds in multi well plates.
What are the three main types of HTS
hts, fragment and virtual screening
What is the capacity of high throughput screening?
Over 100k a day
What are the 4 essential components needed for HTS
Chemware-Library
Bioware-assays
Hardware-Automation
Software-Data
What are the characteristics of an ideal high-throughput screening assay?
A: Reliability, reproducibility, robustness, simplicity, user-friendliness, rapid and sensitive
What are the considerations for developing the HTS assay(tech,re,pharm,sm,auto)
Evaluating the technology for the assay, generating reagents, pharmacologically validating the assay, and miniaturization and automation of the assay.
What is needed to be taken into account when designing and assay(compound characteristic)
Nature of the drug target, technical considerations and what the compound characteristic is
What features categorize high-throughput screening assay types?(Bm,Ap,s,tech,tar)
Biological material used, overall assay approach, signal or readout detection, assay configuration, specific technology employed, and target class
Which two main classes of biological material are used in HTS assays?
Pure systems and cell based systems
Do you always need a drug target to do a high throughput screen?
No, you can do a phenotypic screen.
How does the nature of biological material influence HTS (whole/pure)
Whole cell systems and cell free determines types of assays and the tech needed to be used
What the the two main approaches to HTS
target based and phenotypic based(assaying without knowing the drug target)
What are common readout signals
Absorption, fluorescence, luminescence (photoluminescence, chemiluminescence, radioluminescence).
Homogeneous (no need to separate) and non-homogeneous(mem and elisa)
H-scintillation proximity, FRET, fluorescence polarisation and alphascreen
NH-Membrane binding assays, protein precipitation assays and ELISA.
How HTS can be categorised through target class GPCR,kinase/proteins
Receptors, ENZYMES, ion channels and protein interactions
what are the types of whole cell assays?R,Ae,cal,me
Whole cell binding assays, reporter gene assays (e.g., luciferase or GFP under control of a pathway sensitive promoter), Aequorin for Ca2+ reporting, Indicator assays with calcium sensitive dyes, membrane potential sensitive dyes, and high content assays for monitoring various cell properties.
Pro crys, li,co,h3 and mass
Biophysical assay technologies
Protein crystallization and ligand-protein co-crystallization, 3H and 15N-NMR, Surface Plasmon Resonance, Isothermal titration calorimetry, Differential Scanning Fluorimetry, Mass spectrometry.
Describe the biochemical assay for Cathepsin K. (Homogenous assay)
It’s a homogeneous assay using cell-free pure protein, with fluorescence intensity (Flint) as the readout, detecting increased fluorescence when Cathepsin K cleaves the substrate.
Explain FRET for Cathepsin S
Cell free pure protein(biochemical assay)
Substrate has two fluorophores
Measures red fluorescence when S is cleaves the substrate
What is the principle behind indicator assays for intracellular calcium?
Calcium sensitive dyes
Measures alterations in fluorescence and calcium levels
think about specific response via signalling pathways
reporter assay screening (luciferase)
Luciferase genes controlled by specific response elements indicate specific activity.
What are imaging assays?(cellular behaviour)
These monitor changes in cellular behaviour like NFAT translocation or cannabinoid receptor internalisation.
What is the concept of fragment based screening? NMR
Offers smaller molecules for screening, but are less potent but offer more room for development.
Virtual Screening? Ligand and receptor gudied
Computational models to match drug targets with chemical libraries
fit,al
Receptor guided approach
Using fitting points and a genetic algorithm to map and score optimal ligand-receptor poses.
Ligand guided approach (known ligand)
Known ligand based on properties like size, polarity, H-bond capabilities, and hydrophobicity, using a computational model of the ligand.
