A comparative study of fragment screening methods on the p38α kinase: New methods, new insights

Article abstract of DOI:10.1007/s10822-011-9454-9

The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets.

Full text of DOI:10.1007/s10822-011-9454-9

J Comput Aided Mol Des (2011) 25:677–687
DOI 10.1007/s10822-011-9454-9
A comparative study of fragment screening methods
on the p38a kinase: new methods, new insights
•
•
•
•
Scott J. Pollack Kim S. Beyer Christopher Lock Ilka Mu¨ller
•
•
•
David Sheppard Mike Lipkin David Hardick Peter Blurton
•
•
Philip M. Leonard Paul A. Hubbard Daniel Todd Christine M. Richardson
•
•
•
•
•
•
•
Thomas Ahrens Manuel Baader Doris O. Hafenbradl Kate Hilyard
Roland W. Bu¨rli
Received: 15 April 2011 / Accepted: 24 June 2011 / Published online: 6 July 2011
Ó The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract The stress-activated kinase p38a was used to
evaluate a fragment-based drug discovery approach using
the BioFocus fragment library. Compounds were screened
by surface plasmon resonance (SPR) on a BiacoreTM T100
against p38a and two selectivity targets. A sub-set of our
library was the focus of detailed follow-up analyses that
included hit confirmation, affinity determination on 24
confirmed, selective hits and competition assays of these
hits with respect to a known ATP binding site inhibitor. In
addition, functional activity against p38a was assessed in a
biochemical assay using a mobility shift platform (LC3000,
Caliper LifeSciences). A selection of fragments was also
evaluated using fluorescence lifetime (FLEXYTETM) and
microscale thermophoresis (Nanotemper) technologies. A
good correlation between the data for the different assays
was found. Crystal structures were solved for four of the
small molecules complexed to p38a. Interestingly, as
determined both by X-ray analysis and SPR competition
experiments, three of the complexes involved the fragment
at the ATP binding site, while the fourth compound bound
in a distal site that may offer potential as a novel drug
target site. A first round of optimization around the
remotely bound fragment has led to the identification of a
series of triazole-containing compounds. This approach
could form the basis for developing novel and active p38a
inhibitors. More broadly, it illustrates the power of com-
bining a range of biophysical and biochemical techniques
to the discovery of fragments that facilitate the develop-
ment of novel modulators of kinase and other drug targets.
Keywords p38a Á Surface-plasmon resonance Á
Fragment screening Á X-ray crystallography
Abbreviations
FLT
SPR
Fluorescence lifetime
Surface plasmon resonance
MSA Mobility shift assay
MST Microscale thermophoresis
PIN
CA
Percent inhibition
Carbonic anhydrase
Introduction
In the context of preclinical drug discovery, fragments can
be defined as partial structures of lead- or drug-like mol-
ecules with molecular weights typically ranging from 150
to 300 Da. The quality of a fragment can further be defined
by its lipophilicity, conformational flexibility, hydrogen
bonding capacity and other parameters. Screening of
fragment libraries with subsequent hit optimization has
become a validated approach for hit identification and an
attractive complementary method to high-throughput
screening [1, 2]. It offers the potential for identifying
chemical starting points with high ligand efficiency [3]
whereby most atoms of the fragment are engaged in target
Electronic supplementary material The online version of this
article (doi:10.1007/s10822-011-9454-9) contains supplementary
material, which is available to authorized users.
S. J. Pollack Á C. Lock Á I. Mu¨ller Á D. Sheppard Á M. Lipkin Á
D. Hardick Á P. Blurton Á P. M. Leonard Á P. A. Hubbard Á
D. Todd Á C. M. Richardson Á K. Hilyard Á R. W. Bu¨rli (&)
BioFocus, Chesterford Research Park, Saffron Walden, Essex
CB10 1XL, United Kingdom
e-mail: Roland.Burli@glpg.com
K. S. Beyer Á T. Ahrens Á M. Baader Á D. O. Hafenbradl
BioFocus, Gewerbestrasse 16, 4123 Allschwil, Switzerland
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J Comput Aided Mol Des (2011) 25:677–687
interaction. Due to their size, fragments are inherently
more prone to fit in many receptor pockets and therefore,
fragment libraries typically yield higher hit rates than
collections of more elaborated lead-like molecules. Many
screening techniques have been used and extensively
described, each displaying potential advantages and
disadvantages.
whereby roughly 80 percent are commercial. It has been
generated to be of high quality in terms of purity, solu-
bility, and diversity. To ensure the best possible starting
points for medicinal chemistry projects, the following cri-
teria were applied for fragment selection: the library was
generated for broad applicability with no bias applied
towards any particular protein class or route of adminis-
tration. The Rule-of-Three [10] was used as guidance and
fragment candidates were further filtered in silico to
remove molecules with unwanted reactive functionalities.
All fragments were assessed for chemical purity with a
minimal requirement of 90% as determined by standard
analytical methods (HPLC). The aqueous solubility of
these fragments was also tested, with a minimum solubility
of either 750 lM (LC/MS kinetic solubility determination)
or 1 mM (turbidity analysis). This filter process ensured
that high quality, soluble fragments would form the basis
for binding experiments and minimize false positive out-
comes or interference in SPR screens and allow a high
success rate in crystallisation experiments. An overview of
the StarDropTM [11] calculated physicochemical properties
of this library is illustrated in Fig. 1.
As part of a case study, we herein compare several
screening technologies: a high-concentration biochemical
method using the Caliper LC3000 mobility shift, a high-
concentration biochemical fluorescence lifetime platform
and a biophysical binding assay applying surface plasmon
resonance. Microscale thermophoresis was used to study a
few selected hit compounds.
For this study we selected p38a, a well-characterized
protein target, to investigate whether a fragment screen
could potentially provide new insights and result in
chemical starting points for further optimization. We were
particularly interested in identifying ligands for allosteric
binding sites. p38a is a serine/threonine protein kinase and
a member of the mitogen-activated protein (MAP) kinase
family. Four isoforms of p38 are known (a, b, c, d), which
display different expression and activation patterns. They
are all part of signalling pathways that transduce extra-
cellular signals to trigger intracellular responses [4]. The
a-isoform is the most intensely investigated subtype and is
well known for its role in inflammatory processes; upon
stress-induced activation, p38a upregulates several down-
stream kinases and transcription factors and upregulates the
production of pro-inflammatory cytokines such as TNFa,
IL1-b and IL-6 [5].
The structures of different activation states of p38a have
been extensively investigated (for a landmark publication,
see Wang et al. [6]). To date, well over 150 crystal struc-
tures of p38a have been deposited in the Protein Data Bank
(PDB). By far the majority of p38a inhibitors studied thus
far bind either at or proximal to the ATP binding site of the
kinase; however, other biologically relevant binding pock-
ets have been reported. Diskin et al. [7] found that the
detergent n-octyl-b-glucopyranoside as well as arachidonic
acid and derivatives thereof bound to a lipid-binding site
within the MAP kinase insert region at the C-terminus of
p38a. Following this observation, Jefferson et al. [8] and
Hajduk et al. [9] identified the first fragments which bound
to this site. Their results will be compared with our findings.
The average molecular weight of the library is 198 Da
and compares favourably with other libraries that display
average molecular weights in the range of 180–250 Da
(analysis of commercially available libraries carried out
internally). Overall, the profile of calculated properties was
similar to that of many fragment libraries [12], although
there was a high proportion of compounds with H-bond
acceptors greater than three (the average number of H-bond
acceptors of other commercial libraries was two). In our
view, a higher number of H-bond acceptors is not neces-
sarily unfavourable as such compounds offer several
directed points of contacts.
Assessing the coverage of chemical space and, in
essence, the applicability of the library is not simple.
Quantifying the diversity of any library using computa-
tional algorithms can be performed. Such predictions
involve a calculation of molecular fingerprints of the
library members followed by generation of a matrix of
similarities which can be compressed and represented as a
plot of diversity. These approaches give an indication of
the internal diversity of the library and reveal chemical
space that is over or under represented. Generating a true
assessment of diversity is challenging: to perform such an
analysis, a framework or canvas of chemical space is
required upon which the library must be superimposed. We
used the ChEMBL database [13], from which we extracted
all compounds with a binding affinity of less than 1 lM to
their respective biological targets. These compounds were
considered as ‘drug-like’ and formed the test set on which
the canvas of chemical space was based. In total this
set comprised *190,000 compounds from which we
Results and discussion
Description of the fragment library
The current BioFocus fragment library consists of a mix-
ture of commercially available and proprietary compounds
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679
Fig. 1 Calculated
physiochemical properties of
the BioFocus fragment library:
(a) molecular weight,
(b) calculated logP, (c) number
hydrogen bond donors,
(d) number of hydrogen bond
acceptors, (e) number of
rotatable bonds
evaluated the coverage of our fragment library as sub-
structures. The internal diversity was assessed using pro-
tocols built in Pipeline Pilot [14] and in-house tools.
Initially, the FCFP-6 molecular fingerprints were calcu-
lated before the set was condensed into 1,500 clusters. A
clustering step was required to reduce the intense compu-
tational resource that would be necessary to perform a
diversity calculation on the entire test set. Using the
compound most central as the clusters’ representative in
chemical space, a cluster based plot of molecular diversity
was then generated. Each fragment was searched for as an
exact substructure present within the fully elaborated
members of the test set. As a result, 52% of the BioFocus
fragment library existed as a substructure of the test set and
these results were transferred to the plot giving a visual
representation of the diversity (Fig. 2). This exercise was
not used to generate a library consisting solely of fragments
that exist in known active compounds, but rather to give an
overview. In our view, a fragment library should have at
most around 50% representation as this ensures sufficient
novel starting points for further development.
These results indicated that our library covered the
clusters well, with 1,308 of the 1,500 clusters represented,
translating into only 1,616 compounds from the 190,000
test set that lacked representation. Using ChEMBL as the
test set also gave us a top level indication of the likely
areas of success for our library. The hierarchical structure
Fig. 2 Plot of BioFocus fragment library diversity coverage showing
20,000 clusters compared with clusters representing the ChEMBL
database
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J Comput Aided Mol Des (2011) 25:677–687
of ChEMBL allowed us to extract the associated protein
classes and protein names for the test set. Such information
meant that every substructure hit could be traced back to a
protein and protein class. Whilst we appreciate that in
reality this may not translate to true fragment binding, we
found the results of this analysis extremely useful in
assessing the evolution of our fragment library.
Table 1 SPR results for the 24 confirmed selective fragment hits
(structures in Fig. 3)
Compound
Mw (Da)
Kd (mM)
% TRmax
1
220.2
211.3
185.2
240.0
194.6
210.0
208.3
214.3
234.0
235.3
246.3
200.0
243.4
230.0
208.2
218.2
238.0
236.2
212.2
245.3
244.3
237.6
187.2
244.3
1.80
3.87
1.27
0.78
1.59
1.38
[10
2.21
1.97
1.84
[10
4.89
2.45
[10
0.22
0.79
7.13
1.54
2.34
3.19
0.48
[10
[10
1.9
122
137
221
216
365
434
N/A
333
232
147
0
2
3
4
5
Description of conducted screens
6
7
Subsets of the BioFocus fragment library were selected for
screening in various biophysical and biochemical assay
formats. This selection process was driven by structural
diversity with no particular bias toward partial structures of
known kinase inhibitors.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
493
312
N/A
198
126
624
255
447
288
125
N/A
N/A
170
Surface plasmon resonance screen
A selection of 266 fragments from the BioFocus fragment
library was screened at 200 lM concentration for binding
to active p38a using SPR technology (BiacoreTM T100)
with MKK6 and CA as specificity controls. Hits against
p38a were selected on the basis of the following criteria: a
binding response greater than 50% of TRmax (102 of the
266 compounds or 38%), specificity for p38a compared to
the reference surfaces (binding responses equivalent to less
than 50% of TRmax against CA or MKK6) and sensogram
shapes consistent with fragment binding (rapid on and off
rates). Based on these criteria, 24 confirmed hit fragments
were selected. The binding affinities of these fragments
were determined by SPR to range from 0.2 to [10 mM
(data in Table 1, structures in Fig. 3). The estimated
binding stoichiometry of these compounds varied from
approximately 1:1 binding to superstoichiometric binding
(up to 6:1). Importantly, these ratios are only approxima-
tions due to variations in effects on refractive index
amongst compounds.
Figure 4 shows the data for four compounds which were
subsequently analyzed by X-ray crystallography (see
below), indicating a non-ATP competitive binding mode
for compound 6 and competitive binding by the other three
compounds.
Mobility shift assay
In order to gain insights about the binding mode, we
used SPR to conduct a competitive study of the 24 con-
firmed hits and SB203580, a known ATP site binding
inhibitor. Compounds were tested in the presence and
absence of SB203580 at a saturating concentration in order
to determine whether there is an additive binding response,
which would indicate non-competitive binding; i.e. binding
at an alternative site remote from the ATP binding site.
Under these conditions, compounds that bind at the ATP
binding site are expected to compete with SB203580 giv-
ing rise to non-additive responses. Eight of the 24 com-
pounds examined were determined to compete with
SB203580 for the ATP binding site, four compounds were
non-competitive and the mode of binding of the remainder
could not be determined conclusively due to poor affini-
ties or discrepancies between independent experiments.
To assess the biochemical activity of the fragments, a
larger portion (890 compounds) of the BioFocus fragment
library was screened in a mobility shift assay format using
the LC3000 (Caliper) [15] at 1 mM and 250 lM concen-
trations. The hit rate was expected to be lower compared to
the SPR analysis as only compounds blocking functionally
relevant sites can be detected under these conditions. In
this screen, the assay quality was high, with Z’ values for
all test plates above 0.5.
Out of 890 compounds, 39 demonstrated [50% inhibi-
tion at 1 mM and [30% inhibition at 250 lM concentra-
tion. All of the 39 fragments that exhibited activity in the
biochemical assay have also been detected in the initial
SPR screen (1 mM), while SPR yielded additional hits that
were not identified in the biochemical screen. In order to
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681
H
N
OH
NH2
O
O
N
N
O
N
O
N
N
N
HN
N
N
Cl
H
N
N
N
HN
H
S
H
Cl
F
N
H
O
N
1
2
3
4
5
6
O
O
OH
N
N
O
O
H
N
N
N
N
N
S
N
N
S
N
NH2
N
H
O
S
H
N
OH
F
F
N
N
7
8
9
10
11
12
N
OH
F
F
O
N
O
OH
H
N
OH
O
H2N
N
N
HN
N
NH
N
N
N
N
O
F
N
H
O
N
14
15
16
17
18
13
OH
S
O
O
O
N
N
Cl
NH
N
HN
N
HN
HN
N
N
N
O
N
O
N
N
N
N
N
N
O
N
H
OH
19
20
21
22
23
24
Fig. 3 Structures fragment hits described in Table 1
compare the results of the two technologies, we focused on
the 266 fragments which were selected for the earlier
screen by SPR. This set included 10 out of the 39 hits
identified in the LC3000 MSA screen and 102 fragments
that fulfilled the criterion for a primary hit in SPR. The
overlap of hits obtained by the two orthogonal technologies
is shown in Fig. 5. Nine out of 10 LC3000 fragment hits
were also identified in the binding assay.
For FLT, a weak cut-off criterion of 30% inhibition
(70% residual activity, % Ar) was chosen for hit definition
due to the higher ATP concentration applied in the assay.
A reasonable overall correlation between Caliper
LC3000 MSA and FLT screening data was observed for the
42 compound fragments that were tested in both assay
formats. Of the 42 fragments, 20 were confirmed hits in the
LC3000 MSA testing (i.e. part of the hit list of 39 frag-
ments). Sixteen of those were confirmed by FLT. Only one
fragment of the 24 SPR confirmed hit fragments was
confirmed in FLT and MSA biochemical assays with
around 49% inhibition in both biochemical assays.
From the top 24 compounds which were selected based
on affinity, binding stoichiometry and competition deter-
minations in the SPR screen (Table 2), 11 did not show
significant activity at 1 mM concentration in the bio-
chemical assay, whereas 13 showed at least 50% inhibition.
Figure 7 summarizes the overlap of hit fragments for all
three technologies. For this comparison, we considered the
26 fragments that were assessed in all three assays. Due to
the selection process, all of those were primary hits in the
SPR assay and included the 24 confirmed hits selected
based on selectivity, binding affinity and stoichiometry
analysis. Four fragments were included in the 39 hit frag-
ments identified in the LC3000 MSA screen, three of which
were also confirmed in the FLT assay.
Fluorescence lifetime assay
Fluorescence Lifetime Technology was employed as an
additional method to assess biochemical activity of com-
pound fragments. A p38a FLT assay was developed using
the FLEXYTETM technology from Almac utilising a
9-aminoacridine labelled substrate tailored for p38a
[16, 17]. A subset of 42 fragments was assayed in this
format at 250 lM concentration. The test set included the
24 confirmed hit fragments identified in the SPR screen
(Table 1); in addition, we tested fragments that showed
activity in the LC3000 MSA assay. As illustrated in Fig. 6,
the FLT activity data was compared to the screening data
obtained from the mobility shift assay (LC3000).
Potency determination in both biochemical assay formats
In a next step, ten fragments inhibiting p38a activity in the
MSA and the four fragments selected for X-ray crystal-
lography were further tested in concentration response
analysis experiment in the MSA. A good correlation of
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J Comput Aided Mol Des (2011) 25:677–687
Fig. 4 SPR analysis of binding
competition between SB203580
and the four fragments analyzed
by X-ray crystallography
of the assay was confirmed by testing serial dilutions of
reference compounds SB202190 and BIRB796 in parallel.
Microscale thermophoresis, nanotemper
Fig. 5 Comparison of hits identified in the SPR and LC3000 MSA
screens. Two hundred and sixty-six fragments were tested in SPR at
200 lM and in the LC3000 MSA screen at 250 lM and 1 mM. Of
those fragments, 102 were primary hits from SPR with TRmax [ 50%,
10 were (confirmed) hits in LC3000 (\50% Ar at 1 mM and\70% Ar
at 250 lM)
Three fragments that were identified in the SPR based
fragment screen and one inactive negative control com-
pound within the same chemical series were selected for
analysis by microscale thermophoresis. The activities of
the fragments determined by different biophysical and
biochemical assay technologies correlated well and are
summarized in Table 3.
Table 2 Activities for 14 fragments and two known control com-
pounds obtained in the LC3000 MSA screen: percent inhibition (PIN)
at 1 and 0.25 mM concentrations (primary screen), IC50 and pIC50
values (dose response experiment), and ligand efficiency (LE) [3]
Using microscale thermophoresis, binding to p38a was
detected for compounds 4, 5 and 6. While the binding
curves for compounds 4 and 6 had positive amplitudes, the
curve for compound 5 surprisingly displayed a negative
amplitude. The relevance of the inverse amplitude of the
binding curve observed for 5 in the MST assay is not yet
understood, but may reveal new information regarding the
molecular interaction between the compound and protein
that could not be obtained by the use of alternative
technologies.
Compound
PIN
(1 mM)
[%]
PIN
(0.25 mM)
[%]
IC50
(lM)
pIC50
LE
SB 202190
0.014
0.028
778
7.86
7.55
3.11
3.39
2.58
2.49
4.18
3.82
3.65
3.58
3.26
3.18
3.16
3.12
3.02
2.81
0.31
0.19
0.22
0.19
0.20
0.18
0.23
0.15
0.20
0.22
0.15
0.14
0.15
0.17
0.20
0.22
BIRB 796
3
55.4
67.6
19.4
7.9
28.5
50.0
10.9
-0.8
81.3
71.9
41.8
79.2
40.5
42.1
69.6
33.2
33.4
36.0
4
411
Due to solubility limits, the fragments could not be
tested at concentrations above 2 mM and thus saturation
binding could not be determined. Nevertheless, for com-
pounds 4 and 5 Kd values of 893 and 1,152 lM were
estimated based on fitted sigmoidal binding curves. No
significant interaction with p38a was observed for the
inactive control compound. Table 3 summarizes the IC50
values obtained in the LC3000 MSA and FLT assays as
well as the Kd values obtained in the SPR and MST assays
for four fragments.
5
2,600
3,250
65
6
25
26
27
28
29
30
31
32
33
34
90.0
89.1
62.3
92.3
67.0
69.4
76.1
57.3
57.6
58.9
150
224
263
555
655
686
766
955
X-ray analysis of selected hits
1,536
Selection of hits for crystallization
activities in the primary screen and potency testing was
observed as shown in Table 2 (Fig. 8).
Of the hits identified by SPR, 12 molecules were taken
forward into crystallization/soaking trials and crystal
structures were obtained for the four compounds shown in
Fig. 9 (for experimental details, see Supplementary mate-
rials and references [18–25]).
For all 14 compounds tested in this experiment, IC50
values could be determined. The weakest activity was
observed for the non ATP-site binding compound 6, for
which an IC50 of about 3 mM was observed. The sensitivity
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J Comput Aided Mol Des (2011) 25:677–687
683
As illustrated in Fig. 10, three of these compounds were
shown to bind within the ATP-binding site (compounds 3–
5), whereas one of the compounds (6) unexpectedly
occupied a distal, potentially allosteric site within the
C-terminal MAP kinase insert region. This binding site has
recently been described as a lipid binding pocket [7–9].
The structure and exact binding mode of these compounds
is shown in Fig. 11. The benzylaminopyrazine (3) undergoes
a direct interaction with the hinge element of p38a: a
hydrogen bond is observed from N(1) of the pyrazine ring of
the fragment to the backbone of Met109 on the hinge (resi-
dues 107–109). A lipophilic contribution between the side
chain of Lys53 and the phenyl ring stabilizes the rear of the
fragment. The OÁÁÁN distance between the side chain of
gatekeeper Thr106 and the aniline linker is measured at
˚
3.6 A, providing a key hydrogen bond interaction.
In contrast, the bicyclic pyrimidinone (4) displays an
indirect, water-mediated interaction to Met109 and Lys53/
Asp168 at the rear of the pocket. Val38 provides a lipo-
philic interaction to the plane of the bicyclic core. The
hydroxypyrazole (5) does not interact with the hinge region
of the kinase. Here, the electron density maps show clear
features of ligand binding near the DFG motif of the
activation loop (residues 168–170). The hydroxyl group of
compound 5 serves as an H-bond donor to Glu71 and
acceptor from Phe169, whereas the pyrazole nitrogen
interacts with the backbone amide group of Asp168. There
is a p-cation interaction between the chloro-phenyl ring
and the side chain of Lys53 (the invariant lysine). Notably,
additional peaks in the electron density between this
binding site and the hinge region of the kinase indicate
additional fragment binding in two distinct parts of the
pocket, possibly due to disorder and low ligand occupancy
as the electron density is not well defined and the additional
sites could not be included in the model. This observation
Fig. 6 Comparison of activities obtained for 42 fragments tested in
the LC3000 MSA and FLT assays. Fragment concentration was
250 lM in both assays. ATP concentration was 10 lM in the MSA
and 150 lM in FLT. % Ar are relative activities with regards to no
enzyme (0%) and full reaction (100%) control raw data
Fig. 7 Comparison of the three screen technologies. Twenty-six
compounds tested in all technologies were considered. Due to the
selection process followed, all were primary hits in the SPR
screening. Hit criterion SPR: TRmax [ 50% (includes the 24
confirmed hits), Hit criteria LC3000 MSA: [ 50% inhibition at
1 mM and [30% inhibition at 250 lM, hit criterion FLT: [ 30%
inhibition at 250 lM
O
Fig. 8 Structures fragments
described in Table 2
OH
H
N
N
N
N
N
H
N
N
O
N
H
O
F
SB202190
N
BIRB 796
H
N
O
N
O
N
O
N
O
O
N
N
HN
N
N
O
O
HN
N
N
N
N
O
NH2
25
26
27
28
29
F
Cl
O
N
N
O
N
NH
O
N
HN
O
Br
N
O
O
N
F
N
H
S
O
O
HN
O
30
31
32
33
34
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J Comput Aided Mol Des (2011) 25:677–687
MAP kinase insert region. Importantly, the 4-chlorophenyl
triazole (6) displayed a slightly different binding mode
compared to the known ligands as it induced a conforma-
tional change of Trp197 in a different manner than has
been observed previously. A ligand with improved affinity
to this lipophilic pocket may interfere more specifically
with the biological function of p38a. For these reasons, we
commenced an optimization process aiming at improved
affinity of analogs of compound 6.
Table 3 Comparison of assay results for compounds 4–6
Compound
Binding
site
SPR Kd
(mM)
MSA
IC50
(mM)
FLT
IC50
MST
Kd
(mM)
(mM)
4
ATP
ATP
0.78
1.59
0.41
2.6
0.52
n.a.b
n.a.
0.89
1.15
[0.3a
5
6
Distal site 1.38
3.24
Neg. control
–
TRmax 25.4% 2.2% inh. Not tested [2
(0.2 mM) (1 mM)
a
Concentration dependent binding for compound 6 has been detected by MST,
but the Kd could not be exactly determined due to a high Hill slope; an esti-
mated Kd for 6 is within the range of those determined for compounds 4 and 5
The triazole hit 6 and close analogs were prepared
according to a method described by Webb and Labaw [26]
(Scheme 1). Conversion of the appropriate aniline with
diphenyl cyanocarbonimidate gave the cyano intermedi-
ates, which upon treatment with hydrazine cyclised to the
corresponding diamino-triazoles (6, 35–43). All final
compounds were purified either by crystallization, column
chromatography or reverse-phase HPLC and showed purity
levels in excess of 95% as determined by analytical HPLC
and H-NMR spectroscopy.
b
Dose response curves were started at 1.5 mM maximal concentration and
IC50 values are not applicable
is consistent with the SPR analysis that indicates super-
stoichiometric binding for this fragment.
Interestingly, the aminotriazole 6 binds in a pocket
within the C-lobe formed by an opening of the helix-loop-
helix motif comprising residues Ala244 to Ser261 and
induced a translation of the Trp197 side chain. Crystal
structures of other small molecules binding to this pocket
have recently been published [7–9]. In contrast to the
previously reported structures, the aminotriazole 6 induces
a unique conformation of Trp197 which modifies the pro-
tein surface within this region. The fragment undergoes key
interactions with the protein, including a hydrogen bond
with the backbone of Ser293 and another with Leu291.
Aromatic ring binding is driven by a lipophilic interaction
with the Cb atom on the side chain of Glu192.
Triazole 6 demonstrated a binding affinity of about
1.4 mM and a ligand efficiency of 0.34 as determined by
SPR. As illustrated in Fig. 11, one of the triazole ring
nitrogens and the NH group provide an ideal hydrogen
bond donor/acceptor motif, which locks the compound into
its binding position, whereas the chlorophenyl group is
embedded into a small hydrophobic pocket. The unsub-
stituted amino group appeared to point toward the solvent.
In a first approach, we started exploring whether the ligand/
protein interaction can be improved within the hydrophobic
pocket. Hence, we investigated analogs with differing
substitution patterns at the phenyl moiety as given in
Table 4. Reducing the size of the C(4) substituent led to
diminished affinity whilst replacement of the chloro- by the
larger bromo-substituent resulted in an improvement
(612 lM). Nitrile 38 displayed similar affinity (635 lM)
whereas larger groups at C(4) such as an ethyl group (40)
were not well tolerated.
Initial ligand optimization
Whereas many p38a inhibitors that bind proximal to the
ATP-binding site have been described, only a few small
molecules are known that interact within the C-lobe of the
Fig. 9 Structures of
N
N
OH
Cl
H
Cl
O
N
N
compounds (3–6) analyzed by X
ray crystallography with
binding mode
N
N
N
N
NH2
N
N
H
H
H
F
N
3
4
5
6
direct hinge interaction
H2O-mediated
hinge interaction
activation loop
interaction
binding at distal site
Scheme 1 Synthesis of triazole
analogs. R is defined in Table 4
CN
N
CN
H
Ph
Ph
N
N
N
O
O
H2NNH2
Ph
R-NH2
RHN
O
RHN
NH2
iPrOH , 16 - 75 h, RT
N
MeOH, RT, 1h
or NaH/ THF/ 65°C, 1-2 h
6, 35-43
123

J Comput Aided Mol Des (2011) 25:677–687
685
In a second approach we investigated meta-substituted
biphenyl triazoles. We hypothesized that this additional
phenyl group may directly interact with Trp197. Indeed,
biphenyl 41 displayed a threefold improved binding affin-
ity. The 4-fluorobiphenyl 42 and the disubstituted 4-chloro-
3-phenyl (43) analogs interacted with similar affinities
(0.12 and 0.22 mM, respectively). To confirm the binding
mode of these analogs, we solved a crystal structure of
p38a in complex with biphenyl 41. While this compound
still bound to the same lipophilic pocket at the C-terminus
of the kinase, as shown in Fig. 12, an additional weak peak
in the electron density map was observed at the hinge
region indicating that the compound may bind simulta-
neously to both sites (not observed with initial hit 6). The
overall ligand occupancy at the hinge remains significantly
lower than at the allosteric site. Consistent with this find-
ing, super-stoichiometric binding of this fragment has been
observed in the SPR experiments.
Relative to the position of the initial ligand (6), the
triazole ring of the biphenyl compound 41 is shifted
towards Asp294: the ring nitrogen and the bridging NH
function of 6 still provide an H-bond donor/acceptor motif
and interact with the protein backbone, whereas the sec-
ond phenyl moiety roughly adopts the position of the
chlorine atom in 6. Binding of the biaryl ring is driven by
Fig. 10 Overlay of fragments binding to p38a; hinge (magenta) and
activation loop (orange). Compounds 3, 4 and 5 bound to the active
site and compound 6 bound to a distal site
Fig. 11 Binding mode of
compounds 3–6 in complex
with p38a
123

686
J Comput Aided Mol Des (2011) 25:677–687
BioFocus fragment library has been screened for binding
affinity and potency for p38a kinase using several assay
platforms.
Table 4 Structures and binding affinities of a first generation of tri-
azole analogs
The biochemical assay formats employed in this case
study were chosen as detection methods that are relatively
insensitive to compound interference [27] while SPR was
used as a well-established biophysical method for exam-
ining binding interactions. Overall, a good correlation of
the data from the different assays was found. As expected,
the hit rate observed by SPR was higher than in the bio-
chemical assays; with one exception, all active compounds
identified by the biochemical methods were detected by
SPR. In addition, SPR identified fragment hits that were
not detected in the biochemical screens. This discrepancy
is likely due to binding of the compounds at sites that have
no influence on the catalytic activity of the enzyme or non-
specific binding at the protein surface. The affinity obtained
for few compounds that were tested in microscale ther-
mophoresis correlated well with the other technologies,
indicating that this may be an attractive alternative bio-
physical method for fragment testing. However, further
validation studies will be critical to understand the positive
and negative amplitudes of the individual ligands.
affinity
(mM)
Compound
R
LE
Although the four hit fragments that were followed up
further by X-ray crystallographic analysis would not have
been selected with the LC3000 MSA assay alone, their
inhibitory activity on p38a was confirmed in a concentra-
tion–response experiment when higher concentrations of
the fragments were used, underlining the value of both
technologies for fragment-based drug discovery. Neither of
the two biochemical assays appeared to be superior to the
¨
other. Similar conclusions were drawn by Bottcher et al.
[28] who compared NMR- and SPR-based assay formats
with activity assays using fluorescence intensity, fluores-
cence lifetime or MSA readouts. Purely biophysical tech-
niques such as SPR have the advantage of being able to
identify unique low affinity fragment hits, as exemplified in
the current study, representing potential starting points for
the development of compounds with measurable bio-
chemical inhibition of the target. They could also serve as
‘diagnostic tools’ to identify novel binding sites with yet
unknown function.
Fig. 12 Crystal structure of biphenyl 41 bound to the allosteric
pocket
lipophilic interaction in the form of two hydrophobic
clamps between each ring that feature pairs Ile250/Glu192
and Pro191/Ile259. Trp197 adopts the same conformation
as in the p38a/6 complex and alters the protein surface in
this area.
Crystal structures were obtained for four hit fragments,
all of which adopted unique binding modes. Three mole-
cules bound at the ATP binding site; compound 5 bound
close to the activation loop, while compounds 3 and 4
underwent either a direct or a water-mediated interaction
with the hinge element. In contrast, triazole 6 was found to
bind at a distal lipid binding pocket and induced a novel
conformation of the Trp197 residue. With a first set of
analogs, a structure-affinity relationship within this chem-
ical series has been established and the binding affinity was
improved by about tenfold. At this stage, it remains unclear
Conclusions
A brief description of the BioFocus fragment library is
given, which includes key physicochemical properties as
well as a novel measure of diversity relative to an arbi-
trarily defined ‘drug-like molecular space’. A subset of the
123

J Comput Aided Mol Des (2011) 25:677–687
687
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allosteric effect or if at high concentrations and in solution,
this compound simultaneously binds and blocks the ATP
binding site. Crystallographic analysis confirmed the
interaction of biphenyl analog 41 at the same distal binding
site.
Further optimization of the binding affinity will be
required to achieve molecular tools that will allow an
interrogation of the biological function of this distal bind-
ing site. Overall this study illustrates the power of com-
bining a range of biophysical and biochemical techniques
to the discovery of fragments that facilitate the develop-
ment of novel modulators of other kinase and non-kinase
drug targets.
Acknowledgments The authors would like to acknowledge the
contributions of Marieke Lamers (X-ray crystallography) and Richard
Martin (data handling), Almac for providing the FLEXYTETM peptide
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