Bioassay development part 2

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
Enzymes as pharmaceutical targets
• Enzymes catalyse the conversion of a substrate in to a
product.
• This is essential for almost all physiological processes and
may be the causative agent of a pathological state.
• Enzymes are also excellent targets for treatment of
infections.
– The enzymes present in the pathogen may not be present in the host.
– If present their amino acid sequence may have diverged sufficiently during
evolution to provide a therapeutic window.

Enzyme catalysis - Michaelis-Menten
• For reactions which can be described by the simple steps above, the
rate is given by the Michaelis-Menten equation.
• Under steady state conditions the rate of formation and breakdown of
the enzyme-substrate complex is equal.
• The efficiency of enzymes can vary from very low to limited only by
diffusion (catalytically perfect).

Enzyme catalysis – Measurement of the initial rate
• To measure enzymatic rates requires the accurate measurement of an
enzyme catalysed reaction under conditions where [ES] is constant.
• In a time course of reaction this occurs early on, before substrate is
consumed sufficiently to alter the rate of ES formation.
• Depending on the catalytic rate of the enzyme this can be very fast.
• To measure this value accurately requires rapid reagent mixing and
detection.

• Suitable detection of reaction products.
– Simultaneous recoding of reaction products during experiment.
– Ability to stop reaction and measure products offline.
• Rapid initiation of the reaction.
– Rapid mixing of enzyme and reagents.
– Ability to pre-mix enzyme and reagents before triggering start of reaction.

Methods for detection of reaction products -
Spectrophotometry
• Many substances absorb light in
the ultraviolet or visible regions
of the spectrum.
• By shining a light of known
wavelength through a solution
the concentration can be
measured.
• This is calculated using the
Beer-Lambert law:

Spectrophotometry
• This can be measured in a
cuvette-based or plate-based
spectrophotometer.
• Choice depends on the method
used and the volume of
information required.

Spectrophotometry
• Almost all spectrophotometers
work using a monochromator.
• This splits white light in to a
spatially separated spectrum of
UV or visible light.
• By moving the slit position and
width a small range of
wavelengths can be sampled.

Spectrophotometry
• This approach can be used
wherever the substrate and
product differ substantially in
their absorption at a specific
wavelength.
• An example of this is alcohol
dehydrogenase.
• The NADH product absorbs light
at 340 nm in comparison to
NAD+.
• The sensitivity is limited by the
path length (normally 1cm or
less for a cuvette) and the
extinction coefficient.

Spectrophotometry
• The advantage of spectrophotometry is it can be monitored
over time without altering the reaction conditions.
• It can also be adapted to high throughput screening.
• However, a small extinction coefficient will severely limit the
sensitivity of this approach.
• A disadvantage is that other components of the reaction
may also have significant absorbance at the required
wavelength.
– This decreases the overall sensitivity of the assay.

Spectrofluorimetry

Spectrofluorimetry
• Fluorescence is defined as the “Emission of radiation, generally
light, from a material during illumination by radiation of a higher
frequency”.
• The difference in wavelength between the absorbed and emitted
photon is referred to as the Stokes shift.

Spectrofluorimetry
• This shift in wavelength is utilised
in a spectrofluorimeter.
• By blocking the excitation
wavelength with a filter the
emission is detected against a
very low level of background
signal.
– c.f. spectrophotometry where the aim
is to detect a decrease in intensity of a
bright light.
• This makes spectrofluorimetry a
highly sensitive technique
(theoretically ~100 times more
sensitive than spectrophotometry).

Spectrofluorimetry
• Can be used whenever the
substrate or product differ in their
fluorescent properties.
• An example of a highly fluorescent
drug is quinine used to treat
malaria.
• Quinine blocks the
biocrystallisation of heme in to
hemozoin inside the parasite.
• This results in build-up of toxic
heme leading to their death.
• It is also the flavouring using in
tonic water.
Quinine

Spectrofluorimetry

Spectrofluorimetry
• The advantage of spectrofluorimetry is it can be monitored
over time without altering the reaction conditions.
• It can also be adapted to high throughput screening.
• It is also theoretically more sensitive than
spectrophotometry.
• However, it is only useful if either the substrate or product
are fluorescent
– Although it may be possible to engineer them to be.

Methods for detection of reaction products – Coupled
assays - Chemiluminescence
• If the reaction substrate or
products are not naturally
chromophores/fluorophores (and
then can’t be engineered to be
so) a coupled reaction can be
used.
• This feeds the reaction products
from the test enzyme in to a
subsequent reaction generating
a detectable output.
• A simple example of this is the
use of the luciferase enzyme
from fire flies.

Methods for detection of reaction products – Coupled
assays - Chemiluminescence
• Luciferase catalyses the
reaction of luciferin with oxygen
to form oxyluciferin and light.
• This process requires ATP.
• If the enzyme reaction to be
assayed produces ATP then it
can be coupled to the luciferase
reaction and light output
measured.
• Alternative coupling assays can
make use of NADH production
and assayed using
spectrophotometry.

Radioactivity
• Radioactive detection of products is the most sensitive
approach available (~106 times more sensitive than
spectrophotometry).
• Radioactive 3H or 14C can be incorporated in to the
substrate during synthesis.
• However it cannot be detected simultaneously during the
reaction.
– The reaction must be stopped for detection.
• The product and substrate must be separated to allow
specific detection.
– Normally both product and substrate are radioactive.

Radioactivity
• Separation can be achieved by
thin layer chromatography or
electrophoresis followed by
scintillation counting.
• Alternatively, separation by
HPLC can be combined with
simultaneous detection of
radioactive components.
• If the product and substrate are
sufficiently different in their
chemical properties they may be
simply separated using a filter
pad.
Isotope Half-life Max energy of
emission (MeV)
14C 5730 yr 0.156
3H 12.35 yr 0.0186
32P 14.3 days 1.709

• Suitable detection of reaction products.
– Simultaneous recoding of reaction products during experiment.
– Ability to stop reaction and measure products offline.
• Rapid initiation of the reaction.
– Rapid mixing of enzyme and reagents.
– Ability to pre-mix enzyme and reagents before triggering start of reaction.

Rapid initiation of the reaction – Continuous flow
• Two syringes (containing enzyme and substrate) are compressed at a
constant rate.
• They mix thoroughly and pass down the flow tube.
• The flow rate must be sufficiently high to ensure a turbulent flow.
– For a 1 mm diameter tube a flow rate in excess of 2 m s-1 is required.
• The advantage is a dead time as low as 10 µs, however, large amounts
of substrate and enzyme are consumed.

Rapid initiation of the reaction – Stopped flow

Rapid initiation of the reaction – Stopped flow
• The reaction mix in stopped flow fills the stopping syringe which once
filled to the required level sends a trigger to the detector and stops the
flow.
• This allows recording with a dead time of 0.5 ms and observations over
several minutes.
• In comparison to the continuous flow approach, stopped flow requires
only 100 – 400 µl.
• Both continuous flow and stopped flow are ideal for spectrometric
recording.

Rapid initiation of the reaction – Quenched flow
• The quenched flow technique is an adpatation of the continuous flow
approach which does not require simultaneous detection during the
experiment.
• The quencher can contain an acid or denaturant to stop the reaction
after a period of time determined by the flow rate and l.
• The minimum dead time is approximately 5 ms with a maximum
recording time of around 150 ms.
• The quenched reaction can then be analysed (often radioactivity).

Rapid initiation of the reaction – Flash photolysis
• Flash photolysis requires the use of caged compounds which may be
the main substrate or a required co-factor (more common).
• A pulsed laser source (340 nm) provides the energy to break the caged
compound releasing the active molecule (in this case ATP).
• This allows pre-mixing of enzyme and substrate minimising dead time.
• However, this approach is limited by the availability of caged
compounds.

Enzymes as pharmaceutical targets - Summary
• Enzymes are a common therapeutic target permitting the
regulation of a biochemical process.
• Michaelis-Menten kinetics provides a standardised means
of measuring enzyme kinetics and the influence of targets
on this reaction.
• Spectrophotometry, spectrofluorimetry, radioactivity and
coupled reactions are the principle means of detection.
• Stopped flow is the most popular means for the rapid
mixing and measurement of initial rates and velocities.
• Every assay needs to be specifically designed for the
enzyme reaction to be assayed.

Bioassay development part 2

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