Bioassay development part 1

Principles of Drug Discovery &
Development
Bioassay development
B19FE – Semester 2
8 Lectures
Dr Colin Rickman
(c.rickman@hw.ac.uk)

B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 2
The process of drug development
• A multi-step process initiated by advances made in basic biomedical
research.
• At any stage retrograde steps may be made in the process as a result
of failure.
• Throughout this process the cost incurred increases steeply.

Bioassays
• Any assay involving a biological sample.
• They typically are either quantal or graded.
– Quantal assays ideally provide a yes or no answer.
– Graded assays measures a property under multiple conditions.
• They can be used to test:
– Drug/Target interactions – either presence or a measure of a kinetic
property.
– Influence of a drug on target functions – such as disruption of protein-
protein interactions or enzyme kinetics.
– Physiological outcome of drug on disease state.
• Graded assays can be adapted to give a quantal result
through the use of defined thresholds.

Bioassays
• Bioassays fall in to two broad categories.
• High throughput bioassays are used in high throughput screens (HTS).
– They provide small amounts of information from millions of small scale bioassays.
– Ideally provide a QUANTAL “yes” or “no” answer which is 100% accurate.
• High content bioassays are used in lead identification and optimisation.
– They provide very large amounts of information from a small number of bioassays.
– Need to provide accurate GRADED information with a high degree of sensitivity.

Bioassays – High Throughput Screening

Bioassays – High Content Screening

Bioassay considerations
• What property is to be measured?
– Binding of compound to target.
– Influence of compound on target interactions with other molecules.
– Effect of compound on an enzymatic process.
– Result of compound on a downstream cellular process.
• How is this performed and what is the readout?
– High throughput versus high content.
– Monitored over time or an endpoint.
– Measured using light, heat, radiation.
• What biological system is going to be used?
– Using purified targets in solution (in vitro).
– Cell based assays (in vivo).
– Animal based assays (in vivo).

Bioassay development lecture plan
• Measurement of binding.
– Recap of mathematical principles (KD, KON, KOFF).
– Radio-ligand binding.
– Isothermal titration calorimetry.
– Surface plasmon resonance.
• Measurement of kinetics.
– Recap of mathematics on enzyme kinetics (KM, VMAX).
– Rapid mixing and sampling techniques.
– Flash photolysis.
• Cell-based assays.
– Gene expression reporter constructs.
– Förster resonance energy transfer

Measurement of binding
• Binding is the process of a
ligand (A) binding to a target (B)
resulting in a complex.
• The target can be DNA, protein
or lipids and the ligand can be
endogenous or exogenous (a
drug).
• Most drugs in development
today target proteins due to their
wide variation in structure and
properties (allowing specificity)
and their key role in cell
physiology and pathology.
Example: Influenza neuraminidase
bound to oseltamivir (Tamiflu).

• Measurement of the affinity of a ligand for its target is important to
distinguish between similar compounds in the lead optimisation stage.
• Optimisation of affinity allows lower concentrations of a drug to be used
therapeutically.
– This minimises side effects which typically have lower affinities for the drug than the
target.
– Also minimises costs if the drug is expensive to synthesise.
• So how is the affinity of a ligand for a target measured?

• To calculate the affinity we need to make some assumptions:
• All receptors are equally accessible to ligands.
• All receptors are either free or bound to ligand. The model ignores any
states of partial binding.
• Neither ligand nor receptor are altered by binding.
• Binding is reversible.
• For binding analysis theory and the influence of inhibitors see lectures
given in B19FD (on Vision with notes for these lectures).

Measurement of binding – Radioligand binding
assays
• This is the general process to
perform a radioligand binding
assay.

assays
• The target can be either a purified
protein or cells expressing surface
receptors.
• Purified proteins can be fused to an
affinity tag (e.g.. GST, myc, biotin)
to allow immobilisation on beads.
• The radiolabelled ligand can either
be synthesised de novo
incorporating 3H.
– Expensive for each molecule but highly
specific and no change to properties.
• Or labelled post synthesis on an
aromatic hydroxyl group (tyrosine)
by 125I.
– Can degrade and may change properties
of compound.

assays
• It is important that the reaction
reaches equilibrium for all of the
different concentrations of ligand
used in the assay.
• The lower the concentration the
longer it takes to achieve
equilibrium.
• This is achieved more quickly at
higher temperatures.
• However, purified proteins may be
unstable above 4oC and cell
viability will be hindered
above/below 37oC.

assays
• Unbound ligand is typically removed
by filtration or centrifugation of the
sample.
• The sample is normally washed to
remove as far as possible unbound
ligand.
• Calculation of affinity is performed
by non-linear regression.
• The ionic composition of solutions
and the temperature the experiment
is performed at all influence the
measured affinity and must be
standardised.
• By setting a threshold of detected
radiation above which binding is
deemed to have occurred this can
be applied to HTS for a small
number of concentration points.

Measurement of binding – Isothermal titration
calorimetry
• Isothermal titration calorimetry
(ITC) measures the enthalpy
change which occurs upon
binding.
• Ligand is added stepwise to a
chamber containing the binding
target.
• With each injection the
concentration of ligand
increases in the chamber
allowing a binding curve to be
built up.

calorimetry
• In general two cells are used –
one containing the sample
reaction and another a reference
cell.
• The heat energy injected by the
machine in to the sample cell or
reference cell to maintain the
temperature between the two
cells is used to measure the
enthalpy change of each ligand
injection.

calorimetry

calorimetry
• The top graph shows the energy
input to counteract the binding of
ligand to target.
• This can be converted in to a
binding curve from which the
enthalpy change of binding and
the stoichiometry can be
calculated.
• The gradient of the slope around
the point of inflexion is the KA
which is 1/KD.
• Disadvantage of this approach
is it needs large amounts of
target and ligand.

Measurement of binding – Surface plasmon
resonance (Biacore)
• Surface plasmon resonance
(SPR) has been adapted to
measure binding kinetics and
affinities in solution.
• In comparison to ITC which
measures binding as a change
in energy, biacore measures
interactions using a special
property of light.

resonance (Biacore) – Total Internal Reflection (TIR)

• When light hits an interface between two materials with different
refractive indices the light bends.
• If the light hits this interface with a sufficiently shallow angle the light
will reflect off the interface in a process called total internal reflection.

• When light hits an interface between two materials with different
refractive indices the light bends.
• If the light hits this interface with a sufficiently shallow angle the light
will reflect off the interface in a process called total internal reflection.
• Perpendicular to this surface an evanescent electromagnetic wave is
set up which can interact with a gold layer and generate a SPR.

resonance (Biacore)
• The SPR is an electronic oscillation at the boundary between the gold
layer and the medium.
• As such is it is very sensitive to local changes to the surface of the gold
(such as a target binding a ligand).
• This results in a slight change in the angle of the reflected light which is
detected by the biacore machine and used to report interaction.

resonance (Biacore)
• Target is immobilised on to the gold surface.
• Light undergoes total internal reflection through a prism generating SPR.
• As ligand is added to the sample through a microfluidic channel binding occurs.
• This alters the SPR causing a slight deflection in the angle of the reflected light.
• This shift in angle is recorded in real time as the binding occurs.

resonance (Biacore)

resonance (Biacore)
From this you can measure:
• The concentration of immobilised target (excess ligand in solution) or
ligand concentration (if immobilised target is in excess).
• The rate of association and dissociation – KON/KOFF.
• The affinity – KD.
• The target surface can also be regenerated for the next experiment.

resonance (Biacore)
Edin Univ. Centre for translational and chemical biology

Measurement of binding – Microscale
thermophoresis

thermophoresis

thermophoresis
• By examining the flow of molecules through the temperature gradient at
different ligand concentrations you can determine binding affinities.
• This requires the constant molecule to be fluorescently labelled (or
using intrinsic tryptophan fluorescence.
• This process uses very small amounts of material.
• Also compatible with crude lysates.

Measurement of binding - Summary
• Detection of binding can be important both in hit
identification (although a physiological outcome of the
ligand is probably more important to establish) and lead
identification/optimisation.
• Radioligand and microscale thermophoresis binding is
applicable to both in vitro and in vivo analysis of
interactions but does require labelling.
• ITC and Biacore both allow label free detection of binding
and calculation of binding/kinetic rate constants.
• The measurement of binding affinities is an essential step
for the selection and optimisation of leads, iterative
increase in specificity and ultimately the decrease of
dosage and off-target effects.

Bioassay development part 1

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