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!)
How is docking carried out? (3)
The most simple approach is to treat the ligand and the macromolecular receptor as rigid bodies
Or, the receptor is considered as a rigid body but the ligand is allowed to be flexible and to adopt different conformations (most common approach)
The most complex situation is where both receptor and ligand are considered to beflexible (computationally intensive).
How is scoring done? (3)
Takes in account the shape fit (between ligand and receptor), the chemical fit (H-bonding, electrostatic & hydrophobic interactions).
Good interactions are scored positively and bad ones negatively – based on potential energy grids to calculate interaction energies
Equation for free enthalpy? (1)
Check notes week 4
Molecular docking is generally used to answer which two different types of questions? (2)
Which compounds in my (virtual) compound collection could be active against protein A? (virtual screening)
How does the ligand-protein complex look that is formed by protein B and compound C? (single molecule docking)
What is virtual high throughput screening? (2)
Compounds are individually docked (in silico) and selected based on predict binding affinity
Prioritized list of compounds for purchase or individual synthesis
What are the advantages of VS? (3)
vHTS is much cheaper and is able to
process more compounds in less time
Over 50 million commercially
available compounds…(ZINC database)
Ultra-large screening capabilities are
available (100s millions virtual compounds)
What are the disadvantages of VS? (4)
Protein structure errors
Ligand conformation errors
Scoring errors
No de-solvation, flexibility
What is fragment screening? (3)
Aid discovery of new small-molecule therapeutics
It can also be used to establish the druggability of biological targets and discover alternative inhibition sites on already established ones.
What is a fragment? (2)
A fragment is a small, typically aromatic, organic molecule of molecular weight <250 Da, which is very soluble and chemically stable.
What is the benefit of using fragments in fragment screening? (2)
Allows an effective exploration of chemical space and a more rational design of the final molecule through iterations guided by structural knowledge of the site of binding
What are fragment libraries composed of? (2)
Mainly composed of flat aromatic or heteroaromatics
What is the Rule of 3? (4)
Molecular weight <300 Da
LogP <3
Hydrogen bond donors <3
Hydrogen bond acceptors <3
What is expected hit-rate? (1)
A consideration in deciding how many fragments to screen is the expected hit rate
What can be used to detect binding of fragments to biological targets? (4)
NMR (ligand- and protein-based)
X-ray crystallography
SPR
Isothermal titration calorimetry (ITC) and thermal shift
What are the advantages of SPR (4)
Ease of setting up the assay
Relatively high throughput
Requires low amounts of protein compared to other techniques used- it can also calculate binding affinity.
What is the advantage of Ligand-based NMR methods (1)
Can give details of multiple fragments binding simultaneously to proteins
Disadvantage of X-ray crystallography (2)
Cost of equipment
Specialised skills required to crystallise new proteins
What should good fragment offer? (1)
Offer immediate sites for elaboration
What is the aim of structure-activity relationships? (1)
To identify those parts of the molecule that are important to biological activity and those that are not
What is synthesizing analogues and why is it carried out? (2)
One functional group or substituent of the molecule is removed or altered
It is possible to find out which groups are essential, and which are not
How is ligand biding optimised? (3)
If alkyl groups are interacting with a hydrophobic pocket in the binding site, then varying the length and bulk of the alkyl group allows one to probe the depth and width of the pocket
If a drug contains an aromatic or heteroaromatic ring, the position of substituents can be varied to find better binding interactions
Addition of another functional group or substituent to the lead compound to probe for extra binding interactions with the target
What are Isosteres ? (2)
Atoms or groups of atoms which have chemical or physical similarities
Often been used in drug design to vary the character of the molecule in a rational way with respect to features such as size, polarity, electronic distribution, and bonding
What can isosteres be used for? (2)
To determine the importance of size towards activity, whereas others can be used to determine the importance of electronic factors
What is a bio-isostere? (2)
A group that can be used to replace another group while retaining the desired biological activity
What are bio-isosteres used for? (2)
Bio-isosteres are often used to replace a functional group that is important for target binding but is problematic in one way or another.
What is simplification? (1)
A strategy which is commonly used on the often-complex lead compounds arising from natural sources
Also used to remove chiral centres e.g. by replacing the carbon with a Nitrogen atom.
What is rigidification used for? (1)
Used to increase the activity of a drug or to reduce its side effects
To achieve good selectivity what is needed? (1)
To achieve good selectivity, it is necessary to check the drug’s potency against many other kinase enzymes
What is the general aim for designing drugs? (4)
To design drugs that will be absorbed into the blood supply, will reach their target efficiently, be stable enough to survive the journey, and will be eliminated in a reasonable period of time.
What are the relative hydrophilic/hydrophobic properties of a drug? (2)
The relative hydrophilic/hydrophobic properties of a drug are crucial in influencing its solubility, absorption, distribution, metabolism, and excretion (ADME)
The hydrophilic/hydrophobic balance of a drug can be altered by what? (1)
the hydrophilic/hydrophobic balance of a drug can be altered by changing easily accessible substituents.
How can side effects be reduced or eliminated? (1)
By varying seemingly harmless substituents
What are prodrugs? (2)
Prodrugs are themselves inactive, but they are converted in the body to the active drug
This can tackle problems with membrane permeability, bad taste or short duration of action
