Drug Discovery - Week 4 Screening

Question 1

What is high-throughput screening? (4)

Answer 1

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

Question 2

What are PAINS? (2)

Answer 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.

Question 3

What are the different ways PAINS give out false readouts? (3)

Answer 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

Question 4

What are the sources of compound libraries for HTS? (5)

Answer 4

Plant kingdom
Microorganisms
Marine Sources
Venoms and toxins
Animal sources

Question 5

What are the characteristics of a test and why is it important? (4)

Answer 5

The test should be simple, quick, and relevant, as there are many compounds to be analysed

Question 6

How is the test done and what does it involve? (2)

Answer 6

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

Question 7

What is cell-based screening? (1)

Answer 7

Examining the interaction between your target and compounds in a cellular context

Question 8

Advantages of cell-based screening (4)

Answer 8

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

Question 9

What are biochemical assays? (3)

Answer 9

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

Question 10

What is NMR? (3)

Answer 10

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

Question 11

Advantages of NMR screening (4)

Answer 11

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

Question 12

Disadvantage of NMR screening (1)

Answer 12

Disadvantages include the need to purify the protein and to obtain it in a significant quantity

Question 13

What is surface plasma resonance? (1)

Answer 13

An optical method of detecting when a ligand binds to its target

Question 14

What is needed for virtual screening? (5)

Answer 14

Protein Structure
Homology Model of Protein
Compound Library
Computer Software
Computer Power

Question 15

What are electronic libraries? (1)

Answer 15

File containing 3D atom coordinates for each entry or molecule

Question 16

What is X-ray Crystallography? (3)

Answer 16

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)

Question 17

What is Electron Microscopy? (3)

Answer 17

Determine large macromolecular
structures/assemblies

Beam of electrons & lenses are
used to image the biomolecule directly

Medium resolution (2-20 Å)

Question 18

What is Protein NMR? (3)

Answer 18

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!

Question 19

In molecular recognition what are the details that matter? (3)

Answer 19

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

Question 20

What is docking? (2)

Answer 20

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

Question 21

What is scoring? (2)

Answer 21

Evaluate (score) the different binding modes to identify the best ones and estimate the binding affinity (very hard!)

Question 22

How is docking carried out? (3)

Answer 22

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).

Question 23

How is scoring done? (3)

Answer 23

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

Question 24

Equation for free enthalpy? (1)

Answer 24

Check notes week 4

Question 25

Molecular docking is generally used to answer which two different types of questions? (2)

Answer 25

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)

Question 26

What is virtual high throughput screening? (2)

Answer 26

Compounds are individually docked (in silico) and selected based on predict binding affinity

Prioritized list of compounds for purchase or individual synthesis

Question 27

What are the advantages of VS? (3)

Answer 27

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)

Question 28

What are the disadvantages of VS? (4)

Answer 28

Protein structure errors
Ligand conformation errors
Scoring errors
No de-solvation, flexibility

Question 29

What is fragment screening? (3)

Answer 29

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.

Question 30

What is a fragment? (2)

Answer 30

A fragment is a small, typically aromatic, organic molecule of molecular weight <250 Da, which is very soluble and chemically stable.

Question 31

What is the benefit of using fragments in fragment screening? (2)

Answer 31

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

Question 32

What are fragment libraries composed of? (2)

Answer 32

Mainly composed of flat aromatic or heteroaromatics

Question 33

What is the Rule of 3? (4)

Answer 33

Molecular weight <300 Da
LogP <3
Hydrogen bond donors <3
Hydrogen bond acceptors <3

Question 34

What is expected hit-rate? (1)

Answer 34

A consideration in deciding how many fragments to screen is the expected hit rate

Question 35

What can be used to detect binding of fragments to biological targets? (4)

Answer 35

NMR (ligand- and protein-based)
X-ray crystallography
SPR
Isothermal titration calorimetry (ITC) and thermal shift

Question 36

What are the advantages of SPR (4)

Answer 36

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.

Question 37

What is the advantage of Ligand-based NMR methods (1)

Answer 37

Can give details of multiple fragments binding simultaneously to proteins

Question 38

Disadvantage of X-ray crystallography (2)

Answer 38

Cost of equipment
Specialised skills required to crystallise new proteins

Question 39

What should good fragment offer? (1)

Answer 39

Offer immediate sites for elaboration

Question 40

What is the aim of structure-activity relationships? (1)

Answer 40

To identify those parts of the molecule that are important to biological activity and those that are not

Question 41

What is synthesizing analogues and why is it carried out? (2)

Answer 41

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

Question 42

How is ligand biding optimised? (3)

Answer 42

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

Question 43

What are Isosteres ? (2)

Answer 43

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

Question 44

What can isosteres be used for? (2)

Answer 44

To determine the importance of size towards activity, whereas others can be used to determine the importance of electronic factors

Question 45

What is a bio-isostere? (2)

Answer 45

A group that can be used to replace another group while retaining the desired biological activity

Question 46

What are bio-isosteres used for? (2)

Answer 46

Bio-isosteres are often used to replace a functional group that is important for target binding but is problematic in one way or another.

Question 47

What is simplification? (1)

Answer 47

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.

Question 48

What is rigidification used for? (1)

Answer 48

Used to increase the activity of a drug or to reduce its side effects

Question 49

To achieve good selectivity what is needed? (1)

Answer 49

To achieve good selectivity, it is necessary to check the drug’s potency against many other kinase enzymes

Question 50

What is the general aim for designing drugs? (4)

Answer 50

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.

Question 51

What are the relative hydrophilic/hydrophobic properties of a drug? (2)

Answer 51

The relative hydrophilic/hydrophobic properties of a drug are crucial in influencing its solubility, absorption, distribution, metabolism, and excretion (ADME)

Question 52

The hydrophilic/hydrophobic balance of a drug can be altered by what? (1)

Answer 52

the hydrophilic/hydrophobic balance of a drug can be altered by changing easily accessible substituents.

Question 53

How can side effects be reduced or eliminated? (1)

Answer 53

By varying seemingly harmless substituents

Question 54

What are prodrugs? (2)

Answer 54

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