Focus – Drug Discovery
Figure 1: Fragment-based drug discovery is
undertaken at Selcia’s biology facility in the
UK (shown) and the US
Bob Carling at
Selcia Limited
A variety of different biophysical techniques can be used in fragment-based
drug discovery, but none is a panacea for all biological targets, and it may often
be necessary to use several different technologies in combination to identify
suitable hit compounds.
Historically, chemical leads for
drug discovery programmes have
come from company compound
collection screens, natural product
screens and the elaboration of
natural ligands. High throughput
screening (HTS), where typically
100,000 to more than a million
compounds are tested against a
biological target, came into vogue
in the 1990s and is still prevalent
in the majority of
larger pharmaceutical
Keywords
companies today.
Beginning in the late
High-throughput
1990s, the concept of
screening (HTS)
fragment-based drug
Fragment-based drug
discovery (FBDD) was
discovery (FBDD)
reduced to practice
Surface plasmon
with the design of
resonance (SPR)
fragment libraries and
Biolayer interferometry
the birth of the first
(BLI)
FBDD biotechnology
Capillary electrophoresis
companies. FBDD
(CE)
is now widespread
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throughout the whole of the
pharmaceutical industry.
in order to identify higher affinity
(typically approximately 1 µM)
lead compounds.
Fragments are low molecular
weight (typically 150-250 Da)
ligands that are relatively weakly
binding (yet efficiently binding
for their size) and have affinities
typically in the region of 50-500 µM,
which can be structurally modified
to give new lead compounds
suitable for elaboration into
drug candidates. The process of
identifying low molecular weight
ligands for biological targets is
described as fragment screening
and this is almost invariably carried
out by testing a relatively small but
diverse fragment library (typically
500-2,000 fragments). This approach
is in direct contrast to traditional
HTS which involves testing vast
numbers (typically 100,000 to a
million) of higher molecular weight
(typically 300-500 Da) compounds
Higher rates of ligand identification
(typically three to 10 per cent) are
invariably observed with fragment
screening than with HTS (typically
less than one per cent), because
small fragments can sample
recognition space within biological
targets more efficiently than larger
and more sterically encumbered
molecules (1). Because the required
compound library size for efficient
fragment-screening is minimal
in comparison with HTS, smaller
pharmaceutical organisations or
academic groups are able to rapidly
identify good starting points for
drug discovery programmes and
so compete effectively within
the industry. The first medicine to
be discovered directly as a result
of fragment screening, the anti-
Innovations in Pharmaceutical Technology Issue 41
Images: Selcia Limited
Screening Techniques
for Fragment-Based
Drug Discovery
iptonline.com
melanoma drug Zelboraf, was
approved in 2011.
Fragment Screening
Techniques
The implementation of FBDD
in drug discovery has created a
demand for the development of
specialised and efficient biophysical
techniques that are capable of
detecting weak affinity interactions.
Examples of such techniques are
surface plasmon resonance (SPR),
nuclear magnetic resonance (NMR),
x-ray crystallography, capillary
electrophoresis (CE) and other
techniques as described below. The
relatively high number of possible
hit compounds identified through
fragment screening underlines the
need to use orthogonal screens
to confirm which hits should be
progressed further. For example,
if a fragment screening campaign
using CE as the primary screening
technique gives a four per cent
hit rate from a compound library
of 1,500 compounds, the 60
hits should be validated using
another screening technique
such as SPR before using the
more expensive and resourceintensive methodologies of
x-ray crystallography or NMR.
Techniques which monitor direct
binding to the protein target are
often subject to higher false positive
hit rates; therefore it is useful to use
a competitive technique, where
a known ligand is displaced by a
fragment, to confirm binding to a
specific site. Fragments which show
up as positive with two compatible
screening techniques are more
likely to be genuinely interacting
with the biological target and
therefore represent hits for further
optimisation. The subsequent
optimisation may be facilitated by
identifying and screening other
close analogues, modifying the hit
fragment or through structurebased design, which relies on x-ray
information from co-crystallisation
of the ligand with the biological
target. Many of the different
biophysical technologies that have
been used to measure the affinities
of weakly binding fragments to
biological targets are described
below with a concise summary
of their attributes.
High Concentration Bioassays
Bioassays are only reliable for
fragment screening if the assay
is robust in the presence of high
concentrations of ligand. Often
bioassays are unsuitable, with many
giving false positives as a result
of compound aggregation at the
concentration of the ligands used.
Throughput can be quite high, but
relatively poor sensitivity may not
allow the detection of weaker affinity
ligands (more than 10µM). However,
when bioassays work well, they
provide a rapid and quantitative
method for detection and protein
requirements are minimal.
Biolayer Interferometry (BLI)
BLI is a technique that measures
changes in an interference pattern
generated from visible light reflected
from a biolayer, which contains the
protein of interest, and an optical
layer (2). Fragments binding to the
biolayer shift the interference pattern
and generate a response profile.
As in the case of surface plasmon
resonance (SPR), BLI requires the
protein to be immobilised on a solid
support and for some proteins this
can be problematic, slowing down
feasibility studies and leading to
false positives.
Capillary Electrophoresis (CE)
CE is a high-resolution technique
that can detect both high- and
low-affinity molecular interactions
(3). The main advantage of CE is
that it is solution-based (under
near physiological conditions) and
unmodified protein is generally
used in the assay. As a result,
feasibility studies can be carried out
very rapidly. Other advantages are
that CE uses very little protein, there
Innovations in Pharmaceutical Technology Issue 41
is no need for purified protein, there
is a low false hit rate and no limit on
protein size. CE is also an excellent
technique for the detection of weak
protein-protein interactions using a
fluorescently labelled target protein.
CE does not yet give any insight into
the nature of the docked ligand nor
generate any information on the
kinetics of binding which surface
plasmon resonance may provide.
Differential Scanning
Fluorimetry (DSF)
DSF is a thermal shift method
that relies on the measurement of
protein unfolding by the increase in
fluorescence of a specific dye that has
increased affinity for the hydrophobic
parts of the protein exposed during
the unfolding process (4). Fitting of a
thermogram provides the transition
midpoint (Tm), while the difference in
the temperature of this midpoint in
the presence and absence of ligand
is related to its binding affinity. This
technique may not identify small
efficient binders and is not commonly
used for fragment screening.
Isothermal Titration
Calorimetry (ITC)
The use of ITC for fragment
screening is a relatively new
practice; two samples are mixed
together, and the change in
temperature is measured precisely
(5). If one solution contains a protein
and the other contains a small
molecule, the enthalpy (ΔH) as well
as the overall free energy (ΔG) of
binding can be determined (and
hence the affinity). In practice, weak
fragment interactions are often not
detectable by ITC but the technique
has value as an orthogonal screen.
Mass Spectroscopy (MS)
MS has also been used as a
fragment screening technique.
It has the advantages of low
protein consumption, provides
information on the stoichiometry
of binding and is automated. It
does have drawbacks, including an
upper protein size limit of 50kDa,
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interactions in a sensitive and labelfree detection format, but it requires
significant technical expertise
(8). It is possible to evaluate large
libraries of compounds but, as in
the case of ITC, the protein must
be immobilised which can lead
to slower assay development and
high false positive rates. It can be
used as a primary or an orthogonal
screen offering the advantages of
low protein consumption and the
possibility of obtaining kinetic
and thermodynamic validation of
hits – but this, and the detection
of protein-protein interactions,
are often challenging.
Weak Affinity
Chromatography (WAC)
Figure 2: Selcia’s
CEfragTM Screen is
based on capillary
electrophoresis
high buffer sensitivity and high
sensitivity to non-specific effects.
Microscale
Thermophoresis (MST)
MST is a relatively new, solutionbased technique in the fragment
screening arena. It is based
on the directed movement of
molecules along temperature
gradients, an effect which is termed
thermophoresis. The thermophoresis
of a fluorescently labelled molecule
(A) typically differs significantly
from the thermophoresis of a
molecule-target complex (AT) due
to size, charge and solvation entropy
differences. This difference in the
molecule’s thermophoresis is used
to quantify the binding in titration
experiments under constant buffer
conditions. However, the rate of
screening of fragments is relatively
slow as there is presently inadequate
automation available. It may become
a useful orthogonal screen in the
short term.
Nuclear Magnetic
Resonance (NMR)
Ligand based NMR fragment
screening is most commonly used
(6,7). Saturation-transfer difference
(STD) NMR detects magnetisation
that is transferred from a receptor
protein to a bound ligand. STD
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has a lower limit on protein size
of 20-30kDa and, while screening
of mixtures is efficient, protein
consumption is high and it relies
on expensive instrumentation and
needs expert analysis. Another
ligand-based adaptation of NMR is
target immobilised NMR screening
(TINS) which requires significantly
reduced amounts of protein (7).
Binding is detected by comparing
the 1D NMR spectrum of a fragment
obtained in the presence of an
immobilised biological target
relative to the 1D NMR spectra
obtained with a control protein.
Target-based NMR screening
works by measuring changes in
proton-carbon or proton-nitrogen
correlation signals for a cold
labelled protein in the presence
of an active ligand. NMR can
detect nanomolar to millimolar
interactions and provides structural
information on the binding site,
but it requires large quantities of
isotopically labelled protein and it
is not effective for target proteins
with a molecular weight of more
than 70kDa.
Surface Plasmon
Resonance (SPR)
SPR has emerged as a powerful
tool for studying biomolecular
WAC has recently been utilised for
fragment screening (9). The protein
of interest is covalently immobilised
on a high-performance liquid
chromatography (HPLC) column
packed with modified silica gel.
Fragments are injected into the
column, and those with affinity
for the immobilised protein elute
more slowly than fragments with
no affinity for the biological target.
The fragments can be detected
with either UV spectrometry or
mass spectrometry.
X-Ray (10)
X-ray crystallography can be
considered to be the ‘gold
standard’ of orthogonal fragment
screening techniques because it
can definitively confirm whether or
not a hit fragment is really binding
to a protein target (10). When
x-ray confirmation is achieved,
it may also be possible to assess
how to enhance binding affinity
by using modelling techniques
or by the purchase/synthesis of
related analogues. Different ligands
may occupy different volumes of
the binding site which may allow
‘fragment linking’ to enhance
binding affinity.
These techniques can be very
expensive in terms of time and
Innovations in Pharmaceutical Technology Issue 41
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resources as x-ray crystallography
typically requires 10-50mg of
target protein with a purity of more
than 95 per cent and throughput
is low. Fragment ligands being
evaluated need to be soluble in
the crystallisation medium and no
affinity information is obtained from
a crystallographic experiment. X-ray
crystallography also has a high false
negative rate and some targets are
not amenable to structural biology.
Because the costs associated with
establishing a fragment library and
a suitable biophysical technique
capable of detecting weak ligandprotein interactions are minimal
in comparison with the cost of
establishing, maintaining and
screening a standard-sized HTS
collection, it is possible for smaller
pharmaceutical organisations
to compete effectively in the
fragment-based drug discovery
(FBDD) arena.
Fragment Collections
The success of a fragment screening
campaign is highly dependent on
the quality and diversity of the
fragment collection (library) that
the biological target is screened
against, together with the sensitivity
and reliability of the biophysical
techniques used to identify hits (11).
Simply put, a fragment screening
campaign can only ever be as good
as the fragment library that the
biological target is screened against.
The ideal fragment library consists
of 500-2,000 low molecular weight
(150-250 Da), good water solubility
(more than 3mM), highly pure (more
than 95 per cent) fragments of low
lipophilicity (ClogP less than three),
with three or less rotatable bonds
containing less than three hydrogen
bond donors and six potential
hydrogen bond acceptors. More
recently, there has been increased
interest in molecules with a more
3D character, exploring different
vectors. The establishment of a
suitable fragment collection is a
relatively inexpensive undertaking
compared with the establishment
and administration of the vast highthroughput screening compound
collections historically accumulated
by large pharmaceutical companies.
Conclusion
FBDD has firmly established itself
in the pharmaceutical sector
as a viable approach towards
finding new starting points for
drug discovery programmes.
None of the different biophysical
techniques described above is a
panacea for all biological targets
and it may often be necessary to
use several different technologies
in combination with each other to
identify suitable hit compounds.
Perhaps the future of FBDD relies
on more collaboration between
various small FBDD providers, who
may have expertise in the use of just
one biophysical technique or just
one means of target modification.
A recent example of this strategy
is the collaboration between
Heptares and Selcia to discover
fragment ligands (using Selcia’s
CEfrag® platform technology) that
specifically bind to a stabilised
G-protein coupled receptor (StaR®)
drug target purified by Heptares.
References
1.Hann et al, Molecular
complexity and its impact
on the probability of finding
leads for drug discovery,
J Chem Inf Comput Sc 41(3):
pp856-864, 2001
2.Wartchow et al, Biosensorbased small molecule
fragment screening with
biolayer interferometry,
Journal of Computer-Aided
Molecular Design 25(7):
pp669-676, 2011
3.Austin et al, Fragment
screening using capillary
electrophoresis (CEfrag™) for
hit identification of heat shock
protein 90 ATPase inhibitors,
J Biomolec Screening, 2012
Innovations in Pharmaceutical Technology Issue 41
4.Niesen et al, The use
of differential scanning
fluorimetry to detect
ligand interactions that
promote protein stability,
Nature Protocols 2:
pp2,212-2,221, 2007
5.Ladbury et al, Adding
calorimetric data to decision
making in lead discovery: a
hot tip, Nature Reviews Drug
Discovery 9: pp23-27, 2010
6.Shuker et al, Discovering highaffinity ligands for proteins: SAR
by NMR, Science 274(5292),
pp1,531-1,534, 1996
7.Siegal et al, TINS, Target
immobilized NMR screening:
An efficient and sensitive
method for ligand discovery,
Chemistry & Biology 12(2):
pp207-216, 2005
8.Neumann et al, SPR-based
fragment screening: advantages
and applications, Curr Top Med
Chem 7(16): pp1,630-1,642,
2007
9.Duong-Thi et al, Weak
affinity chromatography as a
new approach for fragment
screening in drug discovery,
Anal Biochem 414(1):
pp138-146, 2011
10.Gill et al, Identification of
novel p38alpha MAP kinase
inhibitors using fragmentbased lead generation,
J Med Chem 48(2):
pp414-426, 2005
11.Visit www.cambridgemedchem
consulting.com/resources/
hit_identification/fragment_
collections.html
Bob Carling has been Head of Medicinal
Chemistry at Selcia Limited since 2006
and has worked in the drug discovery
industry for more than 30 years. Working
alongside colleagues, he has helped to
establish Selcia’s Fragment-Based Drug
Discovery platform. Prior to joining Selcia, Bob obtained
a BSc at the University of Liverpool and a PhD at the
University of Cambridge, and has previously worked at
Merck, ICI and Reckitt & Colman as a medicinal chemist.
Email: [email protected]
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