Special Technical Feature
Fragment-based Drug Discovery,
an Accessible Approach to New Therapeutics
Martin Scanlon* and Raymond Norton*
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052
*Corresponding authors: [email protected] and [email protected]
The past decade has witnessed a drop in the number of
new FDA-approved drugs reaching the clinic, although
that decline appears to have halted temporarily (1). In
certain key areas, antibiotics being a prime example, the
output of new therapeutics from big pharma has fallen
alarmingly (2). New approaches to the development of
therapeutic leads are needed. In this article we describe
fragment-based drug discovery (FBDD), which offers one
particularly promising way forward. FBDD, moreover, is
readily accessible to the academic community, and in this
article we outline some of the requirements for a successful
FBDD campaign.
Introduction
The initial phase of FBDD employs one or more of a
number of sensitive biophysical techniques to detect
the binding of small molecular entities (fragments) to a
target protein. Indeed, the development of biophysical
techniques with sufficient sensitivity and throughput to
enable fragment screening has been a key innovation that
has enabled FBDD to flourish. Many of the key concepts
of FBDD had in fact been discussed in the literature long
before they were demonstrated experimentally. The
notion that binding free energies could be additive was
proposed by Jencks in 1981 (3). This idea was developed
by Andrews et al. (4), who reported the contribution of
individual functional groups to binding energies and may
have also been responsible for one of the first of an evergrowing number of measures to probe the efficiency with
which a ligand binds to its receptor. Peter Goodford’s
GRID provided an early computational tool to map the sites
where particular functional groups could bind to proteins
(5). However, it was not until 1996, when researchers
from Abbott Laboratories described their approach of
SAR-by-NMR (structure activity relationships by nuclear
magnetic resonance), that the first practical demonstration
of FBDD emerged (6). The field has developed rapidly
since then, and there is now an almost mind-boggling
array of techniques for finding hits, metrics to select the
most promising candidates, and strategies for enhancing
their potency and selectivity at the desired target.
FBDD involves identifying small (typically <300 Da)
ligands (‘fragments’), which, because of their small
size, tend to bind with relatively low affinity, and then
developing these to produce larger, higher-affinity ligands
(7,8). The major advantage of FBDD over more traditional
high-throughput screening methods is that FBDD provides
a more rapid and effective means of identifying ligands for
a protein target. Because there are fewer possible fragmentsized molecules than lead- or drug-sized molecules, FBDD
Vol 44 No 3 December 2013
samples chemical space far more efficiently than traditional
approaches and therefore requires far fewer compounds
to be tested to identify suitable hits as starting points
for development. Furthermore, there is some evidence
to suggest that the lead compounds that emerge from
FBDD have better physicochemical properties (described
by Lipinski’s ‘rule of five’ (9)) than those from traditional
drug discovery approaches, and as such are more likely to
result in orally bioavailable compounds (10).
A recent analysis of compounds that have emerged
from FBDD programs on the Practical Fragments blog
(http://practicalfragments.blogspot.com.au/2013/01/
fragments-in-clinic-2013-edition.html), hosted by Dan
Erlanson, lists 26 fragment-derived compounds in various
stages of clinical evaluation, and notes that this number is
likely to be an underestimate. Indeed, the recent approval
of vemurafenib, a B-Raf(V600E) inhibitor developed by
Plexxikon for late-stage melanoma, validates FBDD as an
approach to support the development of clinically useful
drugs (11). Moreover, FBDD also has the capability to
develop inhibitors of protein-protein interactions (PPIs),
about which the pharmaceutical industry has had major
reservations in the past as drug targets. That skepticism,
however, is gradually being eroded as blockers of such
interactions progress to the clinic. Several companies have
employed FBDD to develop inhibitors of the β-secretase
enzyme (BACE1) that have been taken into clinical trials
for the treatment of Alzheimer’s disease. Similarly,
inhibitors of the protein folding chaperone Hsp90 and
inhibitors of proteins involved in apoptosis (Bcl-2 and
Bcl-xL) have progressed to clinical trials for the treatment
of cancer. This demonstrates that FBDD has the capacity
to generate lead inhibitors of PPI targets.
The ingredients of a successful fragment-based drug
discovery program are a stable biomolecular target
that can be produced in milligram quantities, a wellconstructed fragment library, one or more biophysical
screening methods, and access to medicinal chemistry
expertise to develop promising hits. It helps to have a
high-resolution structure of the target, determined by
either X-ray crystallography or NMR spectroscopy (Fig.
1). As all of these are accessible in an academic research
environment and do not require access to sophisticated
robotic platforms or dedicated assay development,
there is growing interest in FBDD within the academic
community in Australasia.
Choosing a Fragment Librar y
Fragment screens can be undertaken with commercially
available libraries, although most practitioners prefer to
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Special Technical Feature
Fig. 1. Overview of fragment-based drug screening workflow.
The fragment library is screened using one or more biophysical methods (in this example STD NMR), then
hits are validated by orthogonal biophysical methods (here SPR and two-dimensional protein-detected NMR),
ranked according to chemical class and ligand efficiency, then elaborated synthetically. High-resolution
structural feedback is invaluable for efficient elaboration of fragments. 1D, one-dimensional NMR spectrum;
STD, saturation transfer difference; SPR, surface plasmon resonance.
create their own. Analysis of commercially available
libraries (http://www.cambridgemedchemconsulting.
com/resources/hit_identification/FragmentLibrary
Profiles.html) indicates that there is remarkably little
overlap between the compound collections offered by
different commercial vendors, suggesting that purchasing
fragments from multiple vendors may generate a
more diverse library. Most reported libraries consist of
fragments with an average molecular mass of ~200 Da
(12). As smaller compounds are better able to sample
chemical space (13,14), diverse screening libraries can
be constructed with relatively few compounds. Most
reported libraries contain fewer than 3,000 fragments.
Although some of the larger companies have collections
with significantly more fragments, there is little literature
to suggest that this provides a significant advantage
for the implementation of FBDD. However, there is an
increased requirement to maintain and curate these
larger libraries, imposing significant additional costs.
Fragment screening is typically undertaken at relatively
high fragment concentrations (300–500 µM), as the small
size of the fragments dictates that they usually interact
weakly with their targets. Therefore, fragment solubility
is a key consideration in designing the library. For a
general screening library, diversity, purity and suitability
for the proposed screening technique(s) are also key
considerations (15-17). Some of the issues associated with
establishing a bespoke fragment library are described in
recent articles by Doak et al. (12) and Francis et al. (18).
Screening the Librar y
Screening is mostly undertaken using biophysical
techniques, as conventional biochemical methods are often
not sufficiently sensitive to identify the modest (often mM)
affinity of fragments for the target protein. Many such
techniques are used to identify fragment hits, each with
its own strengths and weaknesses, and it is advisable to
employ at least two orthogonal methods to eliminate false
positives (Table 1).
At the Monash Institute of Pharmaceutical Sciences, we
screen our in-house library in cocktails of six fragments by
Page 10
ligand-based NMR methods initially, and score as positive
those fragments that show clear responses in at least two
and preferably three ‘ligand-detected’ NMR methods
(saturation-transfer difference (STD), Carr-PurcellMeiboom-Gill (CPMG) and WATER-LOGSY), all of which
detect ligand binding to the target over the µM–mM
affinity range. If there is a peptide or similar ligand for the
target-binding site, we further filter those positive hits on
the basis of whether they compete with the known ligand.
Finally, we confirm that those fragment hits give a clear,
concentration-dependent response by SPR and/or HSQC
NMR. A critical evaluation of the strengths and weaknesses
of various methods for fragment screening and follow-up is
provided by Davis and Erlanson (19)
Transforming Fragment Hits into Potent Ligands
Fragments that are identified as hits in a primary screen
must subsequently be elaborated to increase their potency.
This can be achieved through a range of different strategies
exemplified in a number of recent reviews of successful
FBDD campaigns (20-22).
The relatively high hit rates that are observed in FBDD
dictate that it is not uncommon to have several hits
to choose from after the primary screen is completed.
Therefore, a key aspect of fragment elaboration is to select
from the primary screen the most promising candidates
for development. Ideally the screening library should be
free of problematic compounds such as those identified
as ‘Pan Assay Interfering compounds’ (PAINS (23)) and
those containing reactive functional groups (24). Fragment
hits are often ranked not on their affinity for the target, but
rather their ligand efficiency, which is commonly defined
as [ΔGbinding/(number of heavy atoms)] (25) and is a more
accurate descriptor of the quality of binding interactions
present in the complex. The aim of the elaboration process
is to maintain the ligand efficiency of the compound whilst
increasing its potency.
One of the design criteria in quality fragment libraries is
that there are numerous commercially available analogues
for each fragment, which allows the initial exploration of
structure-activity relationships (SAR) to commence without
AUSTRALIAN BIOCHEMIST
Vol 44 No 3 December 2013
Special Technical Feature
Table 1. Screening methods commonly employed for FBDD.
Screening technique Protein
requirement
Limitations
Strengths
Ligand-detected
NMR
Moderate
High false positive rate.
Protein-detected
NMR (e.g. twodimensional HSQC)
High
Surface plasmon
resonance (SPR)
Low
Labeled protein required. Generally
only suitable for proteins <35 kDa
produced in bacterial expression
systems.
Protein must be immobilised on a
surface in an active conformation.
Suitable for screening of mixtures.
Capable of detecting binding below
fragment KD. Provides valuable QC for
fragment in screening data.
Can identify binding site. Provides KD
estimate.
X-ray
crystallography
Moderate
Isothermal titration
calorimetry
Weak affinity
chromatography
Thermal shift assay
High
Low
Low
High false negative rate. Low
throughput. No information on
affinity.
Low throughput.
Protein must be immobilised on
column.
Not applicable to all targets. Requires
high concentration of fragments.
having to synthesise compounds. Once it is established
that elaborated fragments show promising SAR, it’s time
to engage medicinal chemists in the project. Their input is
essential for the efficient and effective transformation of
weakly binding fragments into sub-µM ligands that can
serve as valuable biological tools and eventually therapeutic
leads. Structural input is also important in guiding the
medicinal chemistry elaboration of promising fragments.
Ideally, compounds can be soaked into crystals or cocrystallised with the target protein. If a crystal structure is
not available, high-resolution NMR can provide structural
details of protein-fragment complexes.
An example of the value of structural data in designing
elaborated ligands is shown in Fig. 2 for fragments that were
found to bind to the integrase enzyme of HIV-1 (26).
High throughput. High sensitivity.
Capable of detecting binding below KD.
Provides KD estimate.
Provides detailed structural
information on the complex.
Provides thermodynamics (KD, ΔH, ΔS)
and stoichiometry of binding.
Capable of screening mixtures. High
throughput.
Relatively inexpensive and high
throughput.
Conclusions
This field offers many exciting opportunities for the
biomedical community. The tools of the trade required
for FBDD are readily available in an academic setting and
the small size of the screening library means the cost is
not prohibitive. We hope you share the enthusiasm of
the authors for the prospects it offers to produce new
biological tools and eventually new therapeutics.
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Fig. 2. Crystal structures of HIV-1 integrase-fragment complexes.
A and B. Two fragments were identified that bound in different orientations within the same pocket of the enzyme.
C. Superposition of the two fragments suggests a strategy for merging their structures. (Adapted from (26)).
Vol 44 No 3 December 2013
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Feature
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