Drug Discovery - Week 4 Screening Flashcards
What is high-throughput screening? (4)
Particularly effective in identifying new lead compounds
This involves the automated testing of large numbers of compounds typically, several million compounds
It is important that the in vitro test should produce an easily measurable effect that can be detected and measured automatically
This effect could be cell growth, an enzyme-catalysed reaction that produces a colour change, or displacement of radioactively labelled ligands from receptors
What are PAINS? (2)
Pan-assay interference compounds (PAINS) arechemical compounds that can cause false positive results in high-throughput drug screening assays
Most PAINS function as reactive chemicals rather than discriminating drugs.
What are the different ways PAINS give out false readouts? (3)
Some are fluorescent or strongly coloured, which in certain assays, give a positive signal even when no protein is present
Other compounds can trap the toxic or reactive metals used to synthesize molecules in a screening library or used as reagents in assays. These metals then give rise to signals that have nothing to do with a compound’s interaction with a protein
Other PAINS coat a protein or sequester metal ions that are essential to a protein’s function, or they may alter proteins chemically without fitting specifically into a binding site
What are the sources of compound libraries for HTS? (5)
Plant kingdom
Microorganisms
Marine Sources
Venoms and toxins
Animal sources
What are the characteristics of a test and why is it important? (4)
The test should be simple, quick, and relevant, as there are many compounds to be analysed
How is the test done and what does it involve? (2)
The test is done in vitro- i.e. on isolated cells, tissues, enzymes, or receptors.
In vitro tests do not involve live animals; instead, they use specific tissues, cells, or enzymes
What is cell-based screening? (1)
Examining the interaction between your target and compounds in a cellular context
Advantages of cell-based screening (4)
Mechanism of action studies
Identifying the potential for cytotoxicity
Defining the agonist or antagonist properties of your compounds
Distinguishing compounds that can cross the membrane of cells for intercellular targets
What are biochemical assays? (3)
Measure the modulation of isolated disease targets and key off-targets by test compounds
They are routinely used to guide the optimisation process
Biochemical assays often use plate readers that quantify a variety of read-outs
What is NMR? (3)
Determine the molecular structure of compounds
A compound is radiated with a short pulse of energy that excites the nuclei of specific atoms such as hydrogen, carbon, or nitrogen
Once the pulse of radiation has stopped, the excited nuclei slowly relax back to the ground state, giving off energy as they do so
Advantages of NMR screening (4)
It is possible to screen 1000 small-molecular-weight compounds a day with one machine- more if cocktails of multiple molecules can be screened together
The method can detect weak binding, which would be missed by conventional screening
It can identify the binding of small molecules to different regions of the binding site
Screening can be done on a new protein without needing to know its function
Disadvantage of NMR screening (1)
Disadvantages include the need to purify the protein and to obtain it in a significant quantity
What is surface plasma resonance? (1)
An optical method of detecting when a ligand binds to its target
What is needed for virtual screening? (5)
Protein Structure
Homology Model of Protein
Compound Library
Computer Software
Computer Power
What are electronic libraries? (1)
File containing 3D atom coordinates for each entry or molecule
What is X-ray Crystallography? (3)
Most common method.
Protein needs to be crystallized!
Crystals diffract the X-ray beam to give diffraction pattern & determine the distribution of electrons.
High resolution (<2 Å)
What is Electron Microscopy? (3)
Determine large macromolecular
structures/assemblies
Beam of electrons & lenses are
used to image the biomolecule directly
Medium resolution (2-20 Å)
What is Protein NMR? (3)
Proteins in solution, study flexible
proteins, ensemble of protein structures
Protein subjected to strong magnetic
field, observed resonances of atomic
nuclei that are close to each other
Labour intensive!
In molecular recognition what are the details that matter? (3)
Interatomic distances and interaction angles should be measured and assessed against databases (CSD or PDB)
Potent ligands should bind in a strain-free manner
Physicochemical properties of molecules should be considered: solubility, lipophilicity and pKa values
What is docking? (2)
Place the ligand within the defined binding site in different orientations or ‘binding modes’
This docking problem involves many degrees of freedom: translational, rotational, configurational
What is scoring? (2)
Evaluate (score) the different binding modes to identify the best ones and estimate the binding affinity (very hard!)
