A poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of PHIP(2), an atypical bromodomain

Article abstract of DOI:10.1039/c5sc03115j

Research into the chemical biology of bromodomains has been driven by the development of acetyl-lysine mimetics. The ligands are typically anchored by binding to a highly conserved asparagine residue. Atypical bromodomains, for which the asparagine is mutated, have thus far proven elusive targets, including PHIP(2) whose parent protein, PHIP, has been linked to disease progression in diabetes and cancers. The PHIP(2) binding site contains a threonine in place of asparagine, and solution screening have yielded no convincing hits. We have overcome this hurdle by combining the sensitivity of X-ray crystallography, used as the primary fragment screen, with a strategy for rapid follow-up synthesis using a chemically-poised fragment library, which allows hits to be readily modified by parallel chemistry both peripherally and in the core. Our approach yielded the first reported hit compounds of PHIP(2) with measurable IC₅₀ values by an AlphaScreen competition assay. The follow-up libraries of four poised fragment hits improved potency into the sub-mM range while showing good ligand efficiency and detailed structural data.

Full text of DOI:10.1039/c5sc03115j

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DOI: 10.1039/C5SC03115J
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EDGE ARTICLE
A Poised Fragment Library Enables Rapid Synthetic Expansion
Yielding the First Reported Inhibitors of PHIP(2), an Atypical
Bromodomain
Received 00th January 20xx,
Accepted 00th January 20xx
Oakley B. Cox,a,b,c Tobias Krojer,a Patrick Collins,c Octovia Monteiro,a,b Romain Talon,a Anthony
Bradley,a Oleg Fedorov,a,b Jahangir Amin,d Brian D. Marsden,a,e John Spencer,d Frank von Delft*a,c,f
and Paul E. Brennan*a,b
DOI: 10.1039/x0xx00000x
www.rsc.org/
Research into the chemical biology of bromodomains has been driven by the development of acetyl-lysine mimetics. The
ligands are typically anchored by binding to a highly conserved asparagine residue. Atypical bromodomains, for which the
asparagine is mutated, have thus far proven elusive targets, including PHIP(2) whose parent protein, PHIP, has been linked
to disease progression in diabetes and cancers. The PHIP(2) binding site contains a threonine in place of asparagine, and
solution screening have yielded no convincing hits. We have overcome this hurdle by combining the sensitivity of X-ray
crystallography, used as the primary fragment screen, with a strategy for rapid follow-up synthesis using a chemically-
poised fragment library, which allows hits to be readily modified by parallel chemistry both peripherally and in the core.
Our approach yielded the first reported hit compounds of PHIP(2) with measurable IC50 by AlphaScreen competition assay.
The follow-up libraries of four poised fragment hits improved potency into the sub-mM range while showing good ligand
efficiency and detailed structural data.
Most Brd inhibitors are anchored by a highly conserved
asparagine (Asn) residue and a network of four water
molecules in the KAc binding pocket. Together the Asn and
Introduction
Bromodomains are acetyl-lysine (KAc) reader domains
involved in the modulation of gene expression.1 The
druggability of bromodomains have made them attractive
targets for the treatment of diseases such as cancer and
inflammation, leading to the development of a range of
chemical probes for the investigation of bromodomain (Brd)
biology (Figure 1).2 In 2010, JQ-1 and I-BET were reported as
potent inhibitors of the BET bromodomains (subfamily II)3, 4
and subsequent academic and industrial research has focused
on targeting the BET bromodomains.5-8 However, recent
publications indicated other subfamilies of the Brd family tree
could be targeted by small molecule inhibitors,9, 10 including
CBP/p300 (subfamily III),11 BRD7/9 (IV),12, 13 BAZ2A/B (V),14, 15
CECR2 (I), BRPF1/2/3 (IV) and SMARCA2/4/PB1 (VIII), as well as
the pan-Brd inhibitor, bromosporine (Figure 1).16
first water in the chain of four donate two H-bonds to a pair of
H-bond acceptors on the inhibitor. The importance of the
conserved Asn residue in inhibitor binding reflects its critical
role in KAc recognition of the natural ligand (Supplemental
Figure 1A).1 However, only forty-eight of the sixty-one known
human Brds (79%) Brds have an Asn residue in the KAc binding
pocket. The remaining thirteen Brds (21%) have a tyrosine,
threonine or aspartic acid in place of the asparagine (Figure 1
and Supplemental Figure 1B). To date, no inhibitors of these
atypical, non-asparagine bromodomains have been reported.
PHIP(2), an atypical bromodomain
The second bromodomain of the Pleckstrin homology
domain-interacting protein (PHIP(2)) is one of the atypical
bromodomains. PHIP is believed to mediate the activity of
insulin-receptor (IRS) proteins,17 and has been identified as a
possible marker in melanoma prognosis.18 More recently, PHIP
has been observed to be overexpressed in metastatic
melanomas.17 The PHIP protein contains two bromodomains,
PHIP(1) and PHIP(2). The first bromodomain, from family VII of
the bromodomain phylogenetic tree, has no reported crystal
or NMR structures. However, in 2010 the crystal structure of
the second PHIP bromodomain was deposited in the Protein
Data Bank (PDB) (NMP, PDB ID 3MB3) (Figure 2), bound to N-
methyl pyrrolidone but in a flipped conformation compared to
typical Brds, such as CREBBP (PDB ID 3P1D).1
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DOI: 10.1039/C5SC03115J
Fig. 1 Bromodomain family tree with sub-families (I-VIII) as defined by Filippakopoulos et al.1 Brds for which SGC Chemical Probes have been released are highlighted
and the structures depicted: SGC-CBP30,11 I-CBP112, (+)-JQ1,3 PFI-1,7 PFI-3, BAZ2-ICR,15 GSK cmpd 3, OF-1, LP99,12 I-BRD9 and the pan-Brd inhibitor bromosporine.16
Atypical bromodomains are highlighted for which the conserved asparagine residue in the KAc binding site is replaced by tyrosine (blue), threonine (green) or
aspartic acid (purple).
PHIP(2) belongs to the bromodomain subfamily III alongside
CREBBP (Figure 1).1 The atypical binding site of PHIP(2) is
characterised by a threonine (Thr) residue (T1396) in place of
the conserved asparagine (Figure 2). Because the Thr side
chain is one atom shorter than Asn, there is space for an
additional water molecule, dubbed the ‘PHIP water’, to
mediate an interaction between T1396 and the typical water
network at the back of the binding pocket. The water-network
is otherwise comparable to the network observed in most Brd
binding sites, consisting of four water molecules and anchored
by a tyrosine residue (Y1353). Despite being identified as a
druggable Brd,19 to date no small molecule inhibitors of
PHIP(2) have been reported. An unpublished screening
campaign by the Structural Genomics Consortium (SGC) did
not yield any hits from a library of approximately seven
thousand Brd-focused compounds.
as a hit-finding method and has enjoyed widespread success in
both academia and industry.22
Fragment screening has been especially effective in
discovering Brd inhibitors using both focused and diverse
24
libraries.23,
Fragments have been optimized into more
potent chemical probes but in all cases the Brds targeted
contained the key Asn residue to anchor an acetyl lysine
mimetic fragment.25,26
A major challenge of fragment screening is that, although
fragment sized compounds make high quality interactions with
their targets and bind with high ligand efficiency, overall
binding affinity is typically weak due to their small size.27
Therefore the techniques used to detect fragment binding
must be sensitive on the µM to mM scale. Biophysical and
biochemical solution-based techniques are convenient and
widely used but drawbacks include insufficient sensitivity, high
rates of false positives and screening concentrations limited to
low mM.28 In contrast, NMR and X-ray crystallography are ideal
for detecting weak binding by yielding direct structural
information, although historically both techniques have been
labour intensive and low-throughput. However, in recent years
X-ray crystallography has seen an order-of-magnitude speed-
up thanks to robotics, improved algorithms and detectors and
Fragment based drug discovery by X-ray crystallography
Fragment based drug discovery (FBDD) has become an
increasingly important tool for finding hit compounds for
difficult targets.20 The technique utilises smaller than drug-like
compounds to identify low potency, high quality leads.
Libraries containing hundreds to thousands of compounds
achieve similar coverage of chemical space as the millions
required for traditional high throughput screening (HTS)
campaigns.21 As a result, FBDD is considerably more affordable
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technical advances at synchrotron facilities.29, 30 Moreover, at acid chloride and an amine. Upon discovery of a poised amide
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beamline I04-1 at Diamond Light Source (DLS)31, other recent hit, purchase of similar synthons to those found in the hit
DOI: 10.1039/C5SC03115J
developments around soaking, harvesting and data analysis allows the synthesis of a library of analogous amides, which
have further reduced the effort to such an extent that a 1000- can be soaked in crystals just days after the initial hit has been
compound fragment screen by crystallography can be obtained. The result is detailed structural information of the
completed within
crystallography is now a viable primary screen for academic activity large enough to allow measurement using
and industrial researchers.33
biophysical or biochemical assay. Poised chemistry requires
a
week.
As a consequence, X-ray ligand binding site and, potentially, an improvement in binding
a
In particular, this makes it realistic to screen by soaking reliable, robust reactions which tolerate a range of substrates
single compounds per crystal, rather than cocktails, so that and can be performed using a wide range of commercially
concentrations >100 mM can be achieved, one hundred to one available starting materials. The reaction products should be
thousand times higher than possible in solution-based formed in high yield and contain drug-like functionalities with
techniques, with accordingly increased sensitivity.32
no known toxicological moieties.
In 2011, Roughley and Jordan published an analysis of the
most commonly used reactions in drug discovery.33 The
authors propose the reactions are regularly used because
experienced medicinal chemists know they provide reliable
methods to synthesise drug-like molecules. The
top
ten
reactions identified by the analysis, including amide coupling,
reductive amination and Suzuki-type aryl-aryl coupling, can be
performed using standardised procedures for a wide range of
substrates. The starting materials for each reaction contain
common functionalities which are well represented in
commercial supplier space and are compatible with this
diverse range of commercial compounds. The ten reactions
were selected to create poised scaffolds with which to perform
substructure searches. The poised fragment chemical space
was further augmented with twelve heterocycle forming
reactions as defined by Hartenfeller et al.34 and an oxazole
formation developed in our own lab.35 The complete list of
poised scaffolds is depicted in Figure 4A.
Fig. 2 PHIP(2) (green sticks and ribbons) in complex with NMP (cyan sticks). The
substitution of an Asn for the smaller Thr allows an extra water molecule, the
PHIP water (red CPK, PW) to fill the space between T1396 and the usual four Brd
waters (red CPK I-IV) (PDB ID: 3MB3).1
The very weak hits identified by X-ray screening pose a
practical problem, in that they are in general too weak to be
verified by orthogonal solution assays; yet such information is
a common requirement in medicinal chemistry operations,
especially if the crystallographic evidence is ambiguous.
Optimising weak compounds by traditional SAR is equally
undermined without reliable assay readout in the range of the
compounds’ binding affinity. In any case, progressing weak
compounds to potency in general requires changes not only to
the periphery, where conventional SAR operates, but also to
the core of the compound.
To overcome the problem of optimising weak compounds
discovered by high concentration fragment soaking, we report
the design and use of a poised fragment screening library to
identify hits and the parallel synthesis of analogues to deliver
inhibitors with measurable activity. Furthermore, we
demonstrate how the method is effective on the previously
intractable target, PHIP(2).
Fig. 3 Schematic depicting how a poised fragment library can be used to rapidly
synthesise a follow-up library of analogues. If the orange fragment is identified
as a hit, a library of similar compounds can be rapidly constructed using parallel
synthesis with commercial analogues of synthons 1 and 2.
Design of Poised Fragment Libraries
Results and Discussion
To construct an initial poised fragment library, the
Diamond and SGC Poised Fragment Library 1.0 (DSPL1), the
large collection of SGC fragments assembled in-house and
from collaborator collections (11,677 in total), was analysed
for the presence of poised substructures and prevalent
synthons. Of the eleven thousand SGC fragments, 2,347 could
be considered poised by our definition. As screening the entire
set of >2,000 poised SGC fragments would not have been
possible and as some of the compounds were very similar to
Poised Fragments
We define a poised fragment as a fragment synthesised
from a robust and general synthetic reaction such that rapid
elaboration of the fragment hit into a library of analogues can
be done using parallel chemistry. Identification of a poised
bond (or bonds) in a fragment allows the compound to be
deconstructed into at least two synthons (Figure 3). As a
simple example, an amide bond can be deconstructed into an
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each other, a subset of 406 compounds was selected for DSPL1 characteristics (Supplemental Figure 2). From the resulting
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ensuring diversity of chemotype and poised classification. All library of 41,271 compounds, 28,438 (68D%O)I:w10e.r1e03f9o/Cu5nSdC0t3o11b5eJ
compounds were clustered by fingerprints using the default poised by our definition. While initially surprising that such a
method in MolSoft’s ICM to give 233 clusters. The compounds large number of commercial fragments could be synthesised
in DPSL1 were selected based on diversity of structure and by only twenty-one different chemical reactions, we believe
diversity of poised motif (Figure 4).
this reflects the very limited coverage of potential synthetic
Encouraged by the success of soaking DSPL1 in PHIP(2) (see compounds by commercial vendors. Further filters were
below), a second generation poised library was designed applied to ensure each compound is compatible with the
(DSPL2). DSPL2 was designed using similar methodology as reaction by which it was found to be poised. For example an
DSPL1 but was assembled entirely from commercial fragments amine substituted amide would be removed from the library
to ensure that anyone could purchase and use the library. The as an asymmetric diamine synthons could give a mixture of
ZINC Fragment-Like library was downloaded36 and filtered amides if used to synthesise a poised fragment. Compounds
based on vendor ID for suppliers from which we had routinely without commercially available starting materials were also
sourced compounds, yielding ~192k compounds. Filters were removed.
applied to the compound library to ensure drug-like
Fig 4 Design of DSPL1 (A) Scaffolds used to identify poised fragments. Core scaffolds based upon the most commonly used medicinal chemistry reactions as described
by Roughley and Jordan.33 Heterocycle scaffolds based on reactions described by Hartenfeller et al.37 (B) Poised reaction motif vs cluster. Circle size represents the
number of compounds in each group. 407 compounds for DSPL1 were selected from all SGC poised fragments based on diversity and poised reaction motif.
In theory, the resulting library of 10,448 compounds covers design in comparison to traditional chemical diversity
the whole of commercially accessible poised fragment space measures (MACCS, Morgan fingerprints, etc.). Although DSPL1
using our chosen reactions. The library is dominated by and 2 do not contain the exact same compounds, they were
amides, with 55.1% of fragments containing a poised amide constructed using similar principles and occupy similar
bond; ether bonds (18.2%) and reductive amination products chemical space (Supplemental Figure 4). The full identities of
(10.2%) were also heavily represented. The least represented DSPL1 and DSPL2 can be downloaded as sdf files from the
chemistries were Sonogashira products (0.2%) and electronic supplementary information.
sulfonamides (2.0%) (Supplemental Figure 3). Unfortunately,
poised heterocyclic reactions were observed in just 2.2% of the
Soaking PHIP(2) with DSPL1
compounds, despite thirteen reactions (only eight of which
To test the utility of the poised fragment library
were found to be present) being used to identify such methodology, the PHIP(2) Brd was screened using high
fragments and the importance of such scaffolds in drugs and concentration crystal soaking. The PHIP(2) Brd was crystallised
chemical tools.38 The finding demonstrates the bias of in its apo-form to generate enough crystals to screen DSPL1.40
commercially available compounds towards the most Due to the relatively potent binding of DMSO to Brds,41
commonly used reactions identified by Roughley and Jordan.33 compounds were dissolved in ethylene glycol at a nominal
Finally, the USRCAT algorithm was used to ensure chemical concentration of 400 mM. The supernatant of compounds
diversity was maintained upon selecting 1000 compounds for which did not fully dissolve was used with the assumption it
DSPL2 from the total of >10,000.39 USRCAT was selected was a saturated solution. The solutions were soaked into
because it was believed the additional conformation protein crystals in crystal buffer in a 1:1 volume ratio to give an
information makes it a superior measure of diversity for library approximate final compound concentration of 200 mM. All 406
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compound 1 displaces water I at the back of the binding
pocket allowing the formation of hydrogeDnOI:b1o0n.1d0Vs3i9eww/CiAt5rhtSicClYe01O331n1l5i5n3Je
and water II. The secondary thiourea nitrogen forms an H-
bond to T1396 via the structural PHIP water (PW). The primary
thiourea nitrogen lies in the hydrophilic pocket made by
poised fragments in DSPL1 were soaked into PHIP(2) Brd
crystals and when the structures were solved, compounds 1-4
were found to have bound to the protein (1.2% hit rate).
All of the poised fragment hits 1-4 bound to PHIP(2) Brd in
the KAc recognition pocket (Figure 5). The thiourea sulfur of
Fig. 5 PHIP(2) (green sticks, ribbons and mesh (2Fo-Fc)) in complex with fragment hits. (A) Thiourea 1 (cyan sticks and mesh (2Fo-Fc)) displaces waters I. (B) N-
Benzylacetamide 2 (purple sticks and mesh (2Fo-Fc)) binds with all water molecules intact and forms an edge-face interaction with Y1350. (C) Oxazole 3 (cyan sticks
and mesh (2Fo-Fc)) forms H-bonds to PW (black dashed line). PHIP(2) orientation has been changed for clarity. (D) Overlay of compounds 1-3 in PHIP(2). In (C) and (D)
water II is hidden behind water I.
waters II, III and IV. The phenyl ring of compound 1 makes an
Rapid synthesis of follow-up libraries
edge-face aromatic interaction with Y1350, which is shifted
As each of the hit compounds, 1-4, is a poised fragment, the
three distinct series were rapidly populated with analogues via
parallel, solution-phase synthesis.
significantly towards the ligand compared to the NMP-bound
conformation (Figure 2). The ortho-methyl substituent on the
phenyl ring makes additional interactions with the
hydrophobic residues I1403 and P1340.
Compound 2 binds like a classical acetyl-lysine mimetic
(Figure 5B). The acetyl group lies deep in the binding pocket
allowing the methyl group to sit in the hydrophobic pocket
created by the four structural waters in a manner analogous to
inhibitors of typical Brds and acetyl lysine. The carbonyl oxygen
interacts with T1396 through a hydrogen bond mediated by
the PHIP water. Like compound 1, the phenyl ring of
(a) Thioureas. The thioureas were synthesised by the
sequential addition of the relevant amines to 1,1’-
thiocarbonyldiimidazole (thio-CDI) (Scheme 1). Analysis of the
crystal data obtained for compound 1 indicated a lipophilic
pocket adjacent to the 3-position of the benzene ring so
emphasis was placed on exploring this position. Substitution of
the terminal thiourea nitrogen was also proposed in an
attempt to displace more of the structural waters at the back
of the binding site. In total thirty-five compounds were
proposed for synthesis and twenty-nine (83%) were
synthesised and purified in moderate to good yield within two
weeks (Table 1, compounds 5-11 and Supplemental Table 1,
compounds s1-s22).
The thiourea series was tested for inhibition of PHIP(2)
ligand binding using an AlphaScreen assay based upon a
previously reported method.41 Ligand efficiencies (LE) were
calculated using the relationship described by Hopkins et al. in
which LE is taken to be proportional to the IC50 contribution
per non-hydrogen atom in the ligand.27
compound
2 appears to make an edge-face aromatic
interaction with the shifted Y1350 side chain, allowing the 2,6-
dichloro substituents to occupy the hydrophobic pockets
formed by V1345 and I1403 above and below the ring.
Oxazoles 3 and 4 share the same binding mode. The
primary amine and oxazole oxygen form a donor and acceptor
interaction with T1396 (Figure 5C, compound 4 not shown for
clarity). Y1350 forms an H-bond to the oxazole nitrogen. The
orientation of the oxazole rings of compounds 3 and 4 is
striking, and allows for the respective lipophilic benzyl and
isobutyl groups to lie in the methyl-binding pocket created by
the four structural waters. Furthermore, the 4-cyano and 4-
amino groups of compounds 3 and 4 interact with a
neighbouring protein in the crystal lattice. The cyano group
acts as an H-bond acceptor with the backbone N-H of D1352,
while the amino group forms a salt bridge with the D1352
carboxylate group.
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(b) N-Benzyl amides. The N-benzyl amVieiwdeArsticlewOnelirnee
synthesised using amine derivatisationDwOIi:t1h0.1a0c3y9l/Cc5hSClo0r3i1d1e5Js
(Scheme 1). Substitution of the benzene ring was proposed as
well as extension of the acetyl group with the aim of displacing
structural waters. In total twelve analogues were proposed for
synthesis and eleven (92%) were synthesised in good to
excellent yield within one week (Table 2, compounds 12-17
and Supplemental Table 2, compounds s23-s27).
Compounds 2 and 12-17 were tested for inhibition of PHIP(2)
ligand binding using the AlphaScreen assay. The original
fragment hit 2 was inactive against PHIP(2) at the highest
possible assay concentration and would not have been
detected as a hit if AlphaScreen had been used as the primary
screen. Attempts to modify the acetyl amide group or the
benzene ring gave no improvements in potency. The exception
was the 2,6-dimethoxy analogue 12 with pIC50 3.72 and LE of
0.35.
The benzyl amide compounds 12-17 were soaked into
PHIP(2) crystals. Surprisingly the inactive compounds 13 and
14 were found to bind in the KAc binding site as well as the
active compound 12. Compound 12 has the same binding
mode as fragment hit 2, with the 2,6-dimethoxy substitution
Scheme 1 Reagents and conditions: (a) Amine 1, Thio-CDI, DCM, rt, 30 min, then
amine 2, rt, 12 h. (b) Triethylamine, DCM, rt, 3 h. (c) 4-Toluenesulfonic acid,
NMP, MW irradiation, 120 °C.
The original fragment hit 1 had pIC50 of 3.11 in the
AlphaScreen assay. Addition of a substituent at the 3-position
of the phenyl ring to give compounds 5-8 increased potency.
The methyl derivative 6 and the trifluoromethyl derivative 7 pattern on the benzene ring occupying the V1345 and I1403
had improved pIC50 ~3.8. The addition of a methyl group at
the6-position of the phenyl ring to give compound 9 slightly
increased potency to pIC50 3.32 as did methylation of the
primary thiourea nitrogen to give compound 10 (pIC50 3.38).
The SAR improvements were not cumulative (see
supplemental material, compounds s1-s20), but replacement
of 2-methylphenyl with 2-chlorophenyl on the methylated
thiourea, giving compound 11, improved PHIP(2) activity to
lipophilic pockets above and below the ring (Figure 6A).
Alcohol 13 binds in the same position as fragment hit 2,
with the additional hydroxyl group making an H-bond
interaction to the protein backbone at P1340 (Figure 6B). In
contrast, the N-methyl group of the tertiary amide 14 forms a
hydrophobic interaction with the P1340 sidechain (Figure 6C).
Table 2 Synthesis and Screening of N-Benzylamides 2,12-17.
Table 1 Synthesis and Screening of Thioureas 1,5-11.
a
h
Cmpd
2
12 2,6-(OMe)2
13
14
R1
R3
Yieldb X-tal Soakf PHIP(2) pIC50
LE
d
Cmpd
R1
R3 Yielda X-tal Soakb PHIP(2) pIC50
LE
2,6-Cl2
C-Me
C-Me
44%
79%
Success
<2.30 (2)
<0.25
Success 3.72 ± 0.04 (2) 0.35
1
5
6
7
8
9
10
11
2-Me
2-Me-3-OMe
2,3-Me2
2-Me-3-CF3
2-Me-3-Cl
2,6-Me2
2-Me
H
H
H
H
H
H
66%
71%
12%
33%
26%
8%
Success 3.11 ± 0.06 (2)
Error 2.97 ± 0.18 (2)
Successc 3.78 ± 0.06 (2)
NL 3.85 ± 0.21 (2)
Successc 3.59 ± 0.03 (2)
0.40
0.32
0.45
0.36
0.42
0.39
0.39
0.45
2,6-Cl2
2,6-Cl2 N-Me, C-Me 83%
C-CH2OH 15%c
Success
Success
Successg
NL
<2.30 (2)
<2.30 (2)
<2.30 (2)
<0.23
<0.23
<0.20
15 2,6-(OMe)2 C-CH2OH 23%d
16 2,6-(OMe)2 5-lactam
17 2,6-(OMe)2 6-lactam 18%e
6%e
3.25 ± 0.04 (2) 0.27
3.51 ± 0.04 (2) 0.27
NL
NL
NL
NL
3.32 ± 0.08 (2)
3.38 ± 0.30 (2)
3.89 ± 0.05 (2)
aR3 = N-H unless stated. bSynthesised using procedure described in scheme 1
unless stated. cReagents and conditions: EDC, DMAP, triethylamine, DMF, rt.
dOver three steps. See supplementary material for details. eTriethylamine, AcN,
Me 14%
Me 19%
2-Cl
aSynthesised using procedure described in scheme 1. NI: not isolated. bNL: No
ligand found in model. Error: Experiment failure during soaking, at beamline or
f
rt. NL: No ligand found in model. gBinding pose uncertain as a result of partial
occupancy. hBy AlphaScreen peptide displacement assay.
c
during data processing or model refinement. Binding pose uncertain as a result
of partial occupancy. dBy AlphaScreen peptide displacement assay.
pIC50 3.89. The LE of fragment hit 1 was calculated to be 0.40.
The LE of fragment hit 1 was calculated to be 0.40. The value
was increases to 0.45 for the most potent thioureas 6 and 11.
When the thiourea compounds 5-11 were subjected to
soaking experiments with PHIP(2) crystals, two of the most
potent compounds, 6 and 8, appeared to bind in the PHIP(2)
KAc recognition site (Supplemental Figure 5A,B). The apparent
poses are similar to compound 1, displacing structural water.
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showed a pIC50 of 3.48, comparable to the isobutyl hit 4.
View Article Online
DOI: 10.1039/C5SC03115J
However the isopropyl analogue 19 showed complete loss of
activity in the AlphaScreen. The 4-chlorobenzyl derivative 20
showed the best activity with pIC50 3.95, although the bulky
phenyl ring ensured LE was kept to just 0.35. In comparison,
the lighter compounds 4 and 18 have LEs of 0.42 and 0.44
respectively. The cyclohexyl analogue 21 showed good activity
(pIC50 = 3.31) but substitution of the 5-amino group with N-
pyrrolidine to give compound 22 increased the pIC50 by 0.4.
The observation indicates the amine group interacts with
T1396 as an H-bond acceptor and a tertiary amine can be
tolerated at the 5-position of the oxazole ring. Despite the
good activity of the oxazole series, no further crystal structures
of the bound ligand were observed following soaking
experiments.
Fig. 6 PHIP(2) (green sticks and ribbons) in complex with N-benzylacetamides 12-
14 (cyan sticks). Electron density shown as green and blue mesh (2Fo-Fc). (A)
The amide 12 binds in the same pose as fragment hit 2. (B) The
hydroxyacetamide 13 forms an H-bond to P1340. (C) Tertiary amide 14 appears
to displace structural water II.
Despite binding in a similar way to the other amide
compounds, tertiary amide 14 appears to displace structural
water II.
X-rays Structures in Screening and Follow-up
The need for screening at very high-concentrations has
been borne out by the hits identified for PHIP(2) through the
X-ray approach, where conventional solution screening using
far more compounds had yielded no hits. Moreover, as we
have now shown at DLS beamline I04-1 that crystal-based
screening is achievable with throughput comparable to NMR,
we foresee a re-emergence of its popularity over the
historically more practical but less sensitive solution-based
techniques.
The activity and crystallographic data resulted in three new
compounds being proposed for synthesis. All three were
synthesized in low yield within two weeks (Table 2,
compounds 15-17). The 2,6-dimethoxy analogue of alcohol 13
(compound 15) is inactive against PHIP(2) but appears to bind
to the protein when subjected to soaking experiments
(Supplemental Figure 5C).
Overlay of fragment hit 2 with the previously reported
NMP-bound structure (Figure 2) showed good overlap
between the amide binding modes for each compound. A
In contrast, the assessment of the follow-up compounds
requires different criteria. Our observation that some of the
more potent compounds such as 11 are not visible in the
crystal structure upon soaking is line with common anecdotal
experience, that crystallographic clarity is not a useful ranking
criterion for micromolar compounds. In the fragment field,
this manifests as the widely-reported lack of hit overlap
between techniques.42 A variety of factors influence whether
soaking is successful, including compound solubility in the
crystallization buffer.
Table 3 Synthesis and screening of oxazoles 3-4,18-22
d
Cmpd
3
4
18
19
20
21
22
R1
Bn
Yielda
62%
42%
93%
54%
46%
71%
30%b
X-tal Soakc
Success
Success
NL
NL
Error
NL
PHIP(2) pIC50
3.23 ± 0.10 (2)
3.57 ± 0.10 (2)
3.48 ± 0.03 (2)
<2.30 (2)
LE
0.30
0.42
0.44
<0.29
0.35
0.33
0.29
iBu
cPr
iPr
4-Cl-Bn
cHex
cHex
3.95 ± 0.03 (2)
3.31 ± 0.06 (2)
3.71 ± 0.16 (2)
Conclusions
We have designed and assembled a library of poised
fragments to enable rapid elaboration of fragment hits weak
enough to be identified only by X-ray crystallography. The
identity of the library, called the Diamond SGC Poised Library
2.0 (DSPL2), is freely available (electronic supplementary
material). The library will be regularly updated to reflect
findings regarding compound suitability for soaking, storage
and chemical elaboration.
Our in-house poised fragments were used in combination
with a novel medium-throughput crystallographic screen to
identify the first reported inhibitors of PHIP(2), an atypical
bromodomain. By exclusively utilising poised fragments in our
screen, we have rapidly generated follow-up leads and shown
increases in compound activity alongside highly detailed
structural data.
NL
aSynthesised using procedure described in scheme 1 unless stated. bOver three
steps. See Spencer et al. for details.35 cNL: No ligand found in model. Error:
Experiment failure during soaking, at beamline or during data processing or
model refinement. dBy AlphaScreen peptide displacement assay.
scaffold merging approach was adopted and the lactam
containing compounds 16 and 17 proposed and synthesised.
Both lactams were found to inhibit the activity of PHIP(2).
pIC50s of 3.25 and 3.51 for compounds 16 and 17 respectively
demonstrate good activity but LE of only 0.27. The best
compound of the amide series, compound 12, has a calculated
LE of 0.35.
(c) Oxazoles. The aminooxazoles 3-4, 18-22 and s38-46
were synthesised by addition of aminomalononitrile p-
toluenesulfonate to the relevant acyl chloride following a
previously published method (Scheme 1).35 The fragment hits
3 and 4 demonstrated good activity against PHIP(2) with pIC50
3.23 and 3.57 respectively. The cyclopropyl analogue 18
The thiourea 1 displaces one of the structural waters at the
back of the PHIP(2) binding site, something rarely seen in other
bromodomain ligand-protein complexes. The series was
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Chemical Science
Page 8 of 9
ARTICLE
Journal Name
2. M. Brand, A. M. Measures, B. G. Wilson, W. A. VCieowrtAortpicalesOsin,linRe.
Alexander, M. Hoss, D. S. Hewings, T. P.DCO.I:R1o0o.1n0e3y9,/CR5.SSC.0P3a1t1o5nJ
and S. J. Conway, ACS Chem. Biol., 2015, 10, 22-39.
expanded to give highly ligand efficient inhibitors with IC50’s of
100 - 200 µM and calculated LE values greater than 0.40
(compounds 6, 7, and 11).
3. P. Filippakopoulos, J. Qi, S. Picaud, Y. Shen, W. B. Smith, O.
Fedorov, E. M. Morse, T. Keates, T. T. Hickman, I. Felletar, M.
Philpott, S. Munro, M. R. McKeown, Y. Wang, A. L. Christie, N.
West, M. J. Cameron, B. Schwartz, T. D. Heightman, T. N. La, C.
French, O. Wiest, A. L. Kung, S. Knapp and J. E. Bradner, Nature
(London, U. K.), 2010, 468, 1067-1073.
4. E. Nicodeme, K. L. Jeffrey, U. Schaefer, S. Beinke, S. Dewell, C.-
w. Chung, R. Chandwani, I. Marazzi, P. Wilson, H. Coste, J.
White, J. Kirilovsky, C. M. Rice, J. M. Lora, R. K. Prinjha, K. Lee
and A. Tarakhovsky, Nature (London, U. K.), 2010, 468, 1119-
1123.
5. R. Gosmini, V. L. Nguyen, J. Toum, C. Simon, J.-M. G. Brusq, G.
Krysa, O. Mirguet, A. M. Riou-Eymard, E. V. Boursier, L. Trottet,
P. Bamborough, H. Clark, C.-w. Chung, L. Cutler, E. H. Demont,
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Amide 2 was found to be inactive in an AlphaScreen assay
against PHIP(2), yet one round of poised synthesis yielded
compound 12 with IC50 of 190 µM and LE 0.35. Structural data
from AlphaScreen-inactive compounds show displacement of
structural waters and allowed further development of the
series. The 5- and 6- member lactams, 16 and 17, showed sub-
millimolar activity and provide a number of additional vectors
for elaboration into unexploited regions of the PHIP(2) binding
pocket.
The binding mode of the aminooxazoles 3 and 4 appears to
be driven by interactions with T1396, with no displacement of
the structural waters or the PHIP water deeper in the binding
pocket. The 2-isobutyl and 2-cyclopropyl oxazoles, 4 and 18,
show excellent ligand efficiency, with IC50’s 226 µM and 329
µM respectively and LE > 0.40.
Ongoing studies are focusing on improving the affinities of
the PHIP(2) hit series reported here with the aim of developing
a viable PHIP(2) chemical probe to help further explore the
role of this Brd in disease, notably cancer. In parallel, through
the SGC’s network of synthetic chemistry collaborators, we are
actively expanding our poised fragment library beyond the
simple chemistry described here with compounds derived
from sp3 rich, stereochemically controlled poised reactions.
We submit that the use of a crystallographic primary
screen followed by rapid poised chemistry to generate a
follow-up library offers a new, powerful method in hit
discovery and lead series selection which can be utilised by
both academic and industrial researchers. The success in
finding efficient hits for the low druggability PHIP(2)
bromodomain indicates the power of this method in
addressing difficult protein-protein interaction targets.
6. D. S. Hewings, O. Fedorov, P. Filippakopoulos, S. Martin, S.
Picaud, A. Tumber, C. Wells, M. M. Olcina, K. Freeman, A. Gill,
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Acknowledgements
The SGC is a registered charity (number 1097737) that receives
funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim,
Canada Foundation for Innovation, Eshelman Institute for
Innovation, Genome Canada, Innovative Medicines Initiative
(EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck &
Co., Novartis Pharma AG, Ontario Ministry of Economic
Development and Innovation, Pfizer, São Paulo Research
Foundation-FAPESP,
[092809/Z/10/Z].
Takeda,
and
Wellcome
Trust
The EPSRC National Mass Spectrometry Facility at Swansea
University is thanked for HRMS data (JS group).
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