Fragment Based Drug Delivery

Question 1

What is fragment based screening? (3)

Answer 1

• Concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance.
• These compounds act as starting points for the development of drugs
• Within the hit identification and hit-to-lead discovery phases

Question 2

Steps in FBDD (4)

Answer 2

• Fragment library (commercial libraries, customized libraries, virtual libraries)
• Screening methods (X-ray crystallography, DSF/SPR, NMR spectroscopy, Mass spectrometry, Docking, Others)
• Confirmation methods (X-ray crystallography, NMR spectroscopy, SPR/ITC/DSF, Biochemical assay, Docking, Cell-based assay)
• Fragment growth (Fragment linking, Fragment growth, Merging/scaffold hopping, AI-guided design, Linking with a compound)

Question 3

What is a fragment hit? (2)

Answer 3

• a molecule of low molecular weight that has been validated to bind to a target protein, can be an effective chemical starting point for a drug discovery project
• Ability to find and progress fragment hits could potentially be improved by enhancing understanding of their binding properties

Question 4

What strategies are used to improve fragment hits? (2)

Answer 4

• NMR spectroscopy, SPR (surface plasmon resonance), X-ray crystallography, and thermal shift assays
• This is due the fragment hits having weaker affinities, so biochemical assays that HTS uses can’t be used as an accurate measure of fragment binding

Question 5

Advantages of FBDD over HTS (2)

Answer 5

• HTS involves identifying large, complex molecules that display moderate affinity for binding to the target as starting points for optimisation to develop suitable drug candidates - tuning already complex compounds.
• FBDD involves identifying small-molecules that display weak affinity for binding to, but high-quality interactions with, the target and then combining these to develop suitable drug candidates - assembling high-quality simple compounds

Question 6

What makes a good fragment? (8)

Answer 6

• Molecular shape complementarity to match the biological target binding pocket
• Electrostatic distribution to complement the biological target binding pocket
• Enthalpy-driven binding interactions via directed hydrogen bonds and polar interactions with biological target binding pocket to increase specificity
• Favourable binding entropy via desolvation of apolar groups
• Dispersion forces which drive non-polar/hydrophobic interactions
• Less complexity to drive interactions, and avoid functionalities that interfere with binding
• Avoid high flexibility, which leads to lower affinity due to entropic costs
• Inclusion of polarizable groups that enable protein binding site adaptations

Question 7

Differential Scanning Fluorimetry (DSF) (3)

Answer 7

• When heated proteins unfold at a temperature (Tm) determined by buffer composition and sequence
• Ligands that bind the protein stabilise the structure and increase Tm
• Dyes bind hydrophobic portions of the protein and fluorescence increases, allowing us to measure Tm

Question 8

Surface Plasmon Resonance (SPR) (5)

Answer 8

• Measures binding of ligand to a protein immobilised onto the surface of a chip
• Changes in the intensity of light reflected from a gold surface allow us to monitor binding
• Provides KD values
• Less high throughput than DSF
• Does not prove fragments inhibit activity

Question 9

Rule of 3 (4)

Answer 9

• MW/Da ≤300
• Hydrogen bond donors ≤3
• Hydrogen bond acceptors ≤3
• cLogP ≤3

Question 10

Rule of 5 (4)

Answer 10

• MW/Da ≤500
• Hydrogen bond donors ≤5
• Hydrogen bond acceptors ≤10
• cLogP ≤5

Question 11

Applications of X-ray crystallography in FBDD (6)

Answer 11

• Often soak a mixture of >10 fragments into a crystal and see what sticks
• Provides binding location
• Also provides information on key interactions
• Provides information on how to grow and improve the hit
• Can provide information on how to combine fragments
• It is used as a screening method and as a confirmation method