Identification of a novel ligand for the ATAD2 bromodomain with selectivity over BRD4 through a fragment growing approach

Article abstract of DOI:10.1039/c8ob00099a

ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (M_r < 200) that yield productive structure-activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.

Full text of DOI:10.1039/c8ob00099a

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Martin, S. Adhikari, A. Brennan, J. Endicott, B. Golding, I. Hardcastle, A. Heptinstall, S. J. Hobson, C.
Jennings, L. Molyneux, Y. Ng, S. R. Wedge, M. Noble and C. Cano, Org. Biomol. Chem., 2018, DOI:
10.1039/C8OB00099A.
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Identification of a Novel Ligand for the ATAD2 Bromodomain with Selectivity Over
BRD4 Through a Fragment Growing Approach.
Duncan C. Miller,1† Mathew P. Martin,2† Santosh Adhikari,1† Alfie Brennan,1 Jane A.
Endicott,2 Bernard T. Golding,1 Ian R. Hardcastle,1 Amy Heptinstall,1 Stephen Hobson,1
Claire Jennings,2 Lauren Molyneux,1 Yvonne Ng,2 Stephen R. Wedge,2 Martin E. M.
Noble,2 Celine Cano1
* *
1Newcastle Drug Discovery, Northern Institute for Cancer Research, School of Chemistry,
Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.
2Newcastle Drug Discovery, Northern Institute for Cancer Research, Paul O’Gorman
Building, Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH UK.
*To whom enquiries should be addressed. For inquiries concerning structural biology,
contact MEMN Tel. (44) 191 208 4466. Eꢀmail: martin.noble@ncl.ac.uk. For inquiries
concerning chemistry, contact CC Tel (44) 191 208 7060. Eꢀmail: celine.cano@ncl.ac.uk
† These authors contributed equally to the work described in this paper.
Abstract
ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with
a poor patient prognosis. This protein has been suggested to function as a cofactor for a
range of transcription factors, including the protoꢀoncogene MYC and the androgen
receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a
bromodomain which allows it to interact with acetylated histone tails. Dissection of the
functional roles of these two domains would benefit from the availability of selective, cellꢀ
permeable pharmacological probes. An in silico evaluation of the 3D structures of various
bromodomains suggested that developing small molecule ligands for the bromodomain of
ATAD2 is likely to be challenging, although recent reports have shown that ATAD2
bromodomain ligands can be identified. We report a structureꢀguided fragmentꢀbased

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approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our
findings indicate that the ATAD2 bromodomain can accommodate fragment hits (Mr<200)
that yield productive structureꢀactivity relationships, and structureꢀguided design enabled
the introduction of selectivity over BRD4.
Introduction
Histone proteins undergo several post translational modifications (PTMs) that affect
DNA transcription, replication and repair, and genomic architecture.1 Histone PTMs are
regulated by three groups of proteins known as writers, erasers and readers. Bromodomains
are a class of protein interaction modules, conserved across evolution, which recognise ɛꢀNꢀ
acetyl lysine PTMs.2 A total of 61 bromodomains in 46 diverse proteins have been
identified in the human proteome, and these are divided into eight structural classes. Within
subfamily IV in the structureꢀbased classification of bromodomainꢀcontaining proteins is
ATAD2 (ATPase Family, AAA Domain Containing 2). ATAD2 is overexpressed in a wide
range of human cancers, including breast, lung, and prostate carcinomas, and it is present in
low levels in normal nonꢀtumour cells.3ꢀ5 The overexpression of ATAD2 has been
associated with a poor patient outcome to treatment in breast6, lung7, ovarian8,
hepatocellular9, endometrial10 and gastric11 cancers. Functionally, ATAD2 has been shown
to act as a coactivator of various transcription factors including the androgen receptor (AR)4,
and the protoꢀoncogene MYC5, and studies examining gene silencing of ATAD2, using
siRNA or shRNA, report it to have a role in tumour cell proliferation and survival.3, 12 These
studies suggest that ATAD2 is a potential target for cancer drug discovery, and smallꢀ
molecule inhibitors would provide insight into the phenotypic response to the inhibition of
ATAD2.

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ATAD2 comprises a four helical bundle (αZ, αA, αB, αC) and two loops (ZA and BC).
The acetyllysine binding pocket, created by helices αB, αC and the ZA loop, is polar and
shallow compared to several other bromodomains.13 The ZA loop of ATAD2, which forms
a major part of the binding site is polar, whereas, the binding site in the bromodomain
BRD4 is mostly hydrophobic.14 The flexibility of the ZA loop coupled to the shallow and
polar nature of the binding site resulted in the druggability of the ATAD2 bromodomain
being classified as ‘difficult’.13, 14
Compounds 1 and 2aꢀb were recently disclosed as relatively potent inhibitors of the
bromodomain of ATAD2.15ꢀ17 Compound 1 was not selective for ATAD2 over BRD4.
Enhanced ATAD2 potency and selectivity over BRD4 was achieved in this series through
introduction of a cyclic sulfone (2a) or a difluorocyclohexane moiety (2b). These groups
form favourable interactions with the sidechains of Arg1007 and Arg1077 in the ATAD2
binding site, and achieve selectivity due to unfavourable interactions with lipophilic Trp81
and Met149 sidechains in BRD4. Compounds 3ꢀ5 were reported as ATAD2 inhibitors
arising from a fragment screen,18 but the low potency of these fragments makes them
unsuitable as chemical probes of ATAD2 function. More recently BAYꢀ850 (6) has been
reported as a potent ATAD2 inhibitor arising from screening of a DNAꢀencoded library
with an unusual dimerꢀinducing mode of action, although not BRD4 selectivity data was
reported.19
This work describes the development of ATAD2 inhibitors employing structureꢀ
guided optimization of a fragment hit, with potential for development into selective
chemical tools to investigate ATAD2 bromodomain function in biological systems.

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Synthesis
Screening of a small targeted fragment library identified 1,6ꢀdihydroꢀ2Hꢀpyrrolo[2,3ꢀ
c]pyridineꢀ2,5(3H)ꢀdione 7 as a promising scaffold for the design of inhibitors of the
ATAD2 bromodomain. Synthesis of the bicyclic template started with protection of
commercially available 6ꢀmethoxyꢀ4ꢀmethylpyridinꢀ3ꢀamine 8 as its tertꢀbutyl carbamate to
give 9 (Scheme 1). Deprotonation followed by quenching with carbon dioxide gave 10.
Acetic anhydride mediated ring closure in the presence of catalytic tetraꢀNꢀbutylammonium
acetate followed by bisꢀalkylation with iodomethane or iodoethane gave 12 and 13,
respectively. Deprotection and Nꢀalkylpyridone formation was achieved in a single step by

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heating 12 with iodomethane to give compound 7. Targets varying the N1 substituent (14aꢀf)
were synthesised by alkylation of 7. 14f was deprotected using TBAF to provide 14g.
Scheme 1: Reagents and conditions: (a) Boc2O, THF, Na2CO3, THF, r.t., 42 h, 95%; (b) (i)
sꢀBuLi, THF, ꢀ78 ˚C, 15 min; (ii) CO2 (dry ice), ꢀ78 ˚C to r.t., 45 min, 74%; (c) Ac2O,
tetrabutylammonium acetate, 65 ˚C, 1 h, 81%; (d) R1 = Me: MeI, Cs2CO3, MeCN, 60 ˚C, 3
h, 78%; R1 = Et: EtI, Cs2CO3, MeCN, 60 ˚C, 3 h, 51% (e) MeI, MeCN, 170 ˚C, µW, 1 h,
quant; (f) For compounds 14aꢀc (i) NaH, DMF, r.t., 15 min; (ii) ethyl iodide or benzyl
bromide or (2ꢀbromoethyl)benzene, r.t., 3 h, 61% (14a), 61%; (14b), 30% (14c); For
compounds 14dꢀf: Cs2CO3, DMF, 100 ˚C, µW, 30 min: 1ꢀ(2ꢀchloroethyl)pyrrolidine
hydrochloride 40% (14d), or 4ꢀ(2ꢀchloroethyl)morpholine hydrochloride, 82% (14e), or (2ꢀ
bromoethoxy)(tertꢀbutyl)dimethylsilane, 55%; (14f); (g) TBAF, THF, r.t., 18 h, 90%.
N6ꢀAlkylated target compounds were synthesised from 12 or 13 under microwave
irradiation using an array of alkyl halides to give 15aꢀp and 16aꢀb, respectively. Subsequent
N1ꢀmethylation gave compounds 17aꢀq and 18aꢀb (Scheme 2).

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Scheme 2: Reagents and conditions: (a) RX, MeCN, 170˚C, µW, 45 min, 22ꢀ93%; (b) MeI,
Cs2CO3, DMF, 100 ˚C, µW, 30 min, 74ꢀ94%; (c) NaOH, EtOH, H2O, 100 ˚C, 23 h, 32%;
Structures of 17aꢀq and 18aꢀb are given in Tables 2 and 3.
Diversification of the C3ꢀposition could be achieved using Knoevenagel condensation
of aldehydes with 11 to provide 19aꢀj in high yield (Scheme 3). Subsequent alkene
reduction, followed by methylation provided 21aꢀf. Treatment of methoxypyridines 21aꢀf
with benzyl bromides at high temperature provided the target pyridones (22aꢀf) in moderate
yields.
Scheme 3: Reagents and conditions: (a) RCHO, piperidine, THF, 100 ˚C, 30 min, 50ꢀ88%;
(b) H2, 10% Pd/C, THF, MeOH, r.t., 2 h; (c) MeI, Cs2CO3, DMF, 50 ˚C, 1.5 h, 55ꢀ56% over

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2 steps; (d) 4ꢀchlorobenzyl bromide, MeCN, 170 ˚C µW, 45 min, 50ꢀ64%. Structures of
22aꢀf are given in Table 5.
Results and Discussion
A crystallographic screen of a small targeted library identified pyrrolidinopyridone 7
as a fragment which bound in the Nꢀacetyllysine histone binding site of the bromodomain of
ATAD2 (Figure 1A and 1B). Isothermal titration calorimetry (ITC) was used to infer the
binding affinity of 7 for the bromodomain of ATAD2, with an apparent Kd of 2.4 mM
(Figure 1C). The pyridone carbonyl forms a hydrogen bond with the carboxamide sidechain
of Asn1064, and another through a waterꢀmediated hydrogen bond to Tyr1021, with the N6ꢀ
methyl group projecting towards a pocket containing four conserved water molecules. This
interaction network mimics the acetylated lysine of the histone, and is consistent with small
hydrophobic groups that occupy the equivalent pocket in other bromodomain family ligands
such as the BRD4 inhibitors JQ120 and IꢀBET762.21 Selectivity over other bromodomain
family members is important in the development of a tool compound to investigate the role
of the ATAD2 bromodomain in biological systems.22 Minimizing the BET activity of an
ATAD2 chemical probe is particularly necessary due to the known effects of BET
inhibitors. Thus BRD4 was selected as representative BET family member to assess
compound selectivity. The binding orientation of 7 in ATAD2 was designated as binding
mode 1 for this template. The residues that line the acetylated lysine binding pocket of
BRD4 and ATAD2 are highly conserved (Figure 1D), with greater divergence in the more
solvent exposed residues including the shelf region of the protein. Achieving selectivity for

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ATAD2 over BRD4 would thus require identification of fragments that extend away from
the conserved core and towards the less conserved areas.
Figure 1: Binding of initial fragment hit 7. (A) Binding mode of fragment hit 7 (yellow ball
and stick) bound to ATAD2 (white). ATAD2 active site residues shown in cylinder and
conserved water molecules shown as red spheres. Potential hydrogen bonding interactions

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shown in black dash. (B) 2D Interaction map of 7 with the active site residues of ATAD2
represented by Lidia within Coot. (C) Isothermal titration calorimetry of 7 binding with
saturation to ATAD2. (D) ATAD2 represented in solid surface and coloured through
conservation of sequence identity between ATAD2 and the first bromodomain of BRD4
Methylation of the N1 position (17a) resulted in a change in binding mode (Figure
2A), whereby the fragment core rotates and the pyrrolidinone carbonyl now forms a
hydrogen bond with the carboxamide sidechain of Asn1064, and a second through a waterꢀ
mediated hydrogen bond to Tyr1021. In this binding mode the N1ꢀmethyl group projects
towards the conserved water molecules. Rotation of the pyrrolidinone core allows the
pyridone carbonyl to interact through a water molecule with the backbone NH of Asp1014 on
the ZA loop. One of the C3ꢀgeminal methyl groups occupies a small hydrophobic pocket
formed between the sidechains of ZAꢀloop residues Tyr1021, Val1018 and the Tyr1063 residue
that reside next to the conserved asparagine. Additional hydrophobic interactions are formed
with the base of the binding pocket, where Val1008 and Ile1074 interact with the second C3
ꢀ
geminal methyl group and pyrrolidinone core, respectively. This binding orientation was
designated as binding mode 2. Intriguingly, when the N1 substituent was further elaborated
to an ethyl group (14a: Figure 2B) the compound reverted to binding mode 1, suggesting the
conserved waterꢀrich pocket was unable to accommodate the larger ethyl group.

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Figure 2: Overlay of the crystal structures of fragment 17a, 14a, 14b, 17c, 17d, 17f, 17k,
22e and 22c bound to ATAD2. Compounds represented in yellow as ball and stick. Binding
pocket of ATAD2 shown in transparent surface, with neighbouring residues of compound
shown in stick representation. Conserved waters are represented by red spheres.
Fragments 7, 17a, and 14a bound very weakly and were unable to displace the
acetylated histone ligand sufficiently in a homogeneous timeꢀresolved fluorescence (HTRF)
assay to enable a Kd to be determined, although some inhibition was observed at the highest
concentration assayed (1000 µM; Table 1). Surface plasmon resonance (SPR) was used to
assess the direct binding of the fragments and provide Kd values for binding to both ATAD2
and BRD4. SPR can yield artefactual data where the concentration of a smallꢀmolecular
analyte exceeds ~250 µM, and accordingly, this was the highest concentration used in the

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assays. Where the Kd exceeded this limit, projected Kds were determined by extrapolation,
using the observed response units for compound binding (RU) relative to the molecular
weight of a known control compound.23 Compounds 7 and 17a bound too weakly to
ATAD2 to allow Kd values to be calculated. However, 17a did show detectable binding to
BRD4. Compound 14a showed a remarkable increase in affinity towards BRD4, reporting a
Kd of 12.7 µM and a binding constant 100ꢀfold weaker towards ATAD2 (Table 1).
The two binding modes offered different vectors to explore structure activity
relationships (SARs) of substituents directed towards the RVF shelf and the ZA loop. Thus
a set of N1ꢀalkylated pyrrolidinopyridones was prepared to attempt to access this region
while maintaining binding mode 1 (Table 1). Lipophilic benzyl (14b) and phenethyl (14c)
substituents exhibited subꢀmillimolar HTRF IC50 values, and showed the first detectable
signs of direct binding to both ATAD2 and BRD4 when analysed by SPR. Coꢀcrystallisation
confirmed that 14b retained binding mode 1 (Figure 2C). Hydrophilic substituents (14dꢀg)
gave no measurable ATAD2 inhibition or binding in HTRF or SPR format experiments.
ID
R
ATAD2 IC50
(µM)a
ATAD2
LE
ATAD2 Kd BRD4 Kd
M)b M)c
(µ
(µ
H
>1000
N/A
>2000
2400 d
>2000
>2000
7
Me
>1000
N/A
1500
17a
Et
Bn
>1000
810±57
N/A
0.21
1300
>2000
12.7
1050
14a
14b
ꢀCH2CH2Ph
868±33
>1000
0.19
N/A
1400
800
14c
14d
>2000
1700

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>1000
>1000
N/A
N/A
>2000
>2000
>2000
14e
14g
ꢀCH2CH2OH
>2000
Table 1: ATAD2 inhibition data for compounds 7, 14aꢀg and 17a. a HTRF format assay, b, c
SPR format assay, d Isothermal titration calorimetry. Ligand efficiency (LE) = 1.4(ꢀlogIC50)/N,
where N is number of nonꢀhydrogen atoms.
To attempt to exploit binding mode 2, a set of N6ꢀalkylated pyrrolidinopyridones were
also prepared (Table 2) with N6ꢀbenzyl (17c) and N6ꢀphenethyl (17d) analogues giving subꢀ
millimolar ATAD2 IC50 values. As expected, 17c and 17d retained binding mode 2 (Figures
2DꢀE), with the benzyl group sitting above the RVF shelf, forming a hydrophobic
interaction with the sidechain of Lys1011. Pairwise comparison with N1ꢀdesꢀmethyl analogue
15c confirmed the importance of a methyl group in the conserved water pocket. 17c and 17d
bound more tightly to BRD4 than to ATAD2 in the SPR experiments.
ID
R1
R2
ATAD2
IC50 (µM)a
>1000
ATAD2
ATAD2 Kd BRD4 Kd
M)b M)c
(µ
(µ
LE
N/A
H
Pr
>2000
1000
17b
15c
N/A
H
Bn
>1000
2000
1400
0.21
0.20
Me
Me
Bn
681±120
741±74
700
101
161
17c
17d
ꢀCH2CH2Ph
1000
Table 2: ATAD2 inhibition, SPR and BRD4 binding data for compounds 15c and 17bꢀd.
aHTRF format assay, b, c SPR format assay. Ligand efficiency (LE) = 1.4(ꢀlogIC50)/N, where
N is number of nonꢀhydrogen atoms.

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With the aim of improving binding affinity towards ATAD2 the sidechain of Arg1007
was targeted through substitution of the 4ꢀposition of the N6ꢀbenzyl group. Compound 17f
showed similar ATAD2 affinity to 17c, and the coꢀcrystal structure with ATAD2 (Figure
2F) confirmed that the carboxylate did not form the projected chargeꢀcharge interaction with
Arg1007. Other analogues bearing functionality with the potential to form hydrogen bonds
with Arg1007 gave no further improvement in inhibition (17gꢀi). Lipophilic substituents at
the 4ꢀposition of the benzyl group (17jꢀm, Figure 2G) provided the most potent benzylic
analogues, but continued to be selective for BRD4. Addition of a second chloro substituent
at the 3ꢀposition (17n) resulted in a modest drop in potency. N6ꢀBenzyl analogues with
substituents at the 2ꢀ and 3ꢀ positions of the benzylic ring (17oꢀq) were prepared to attempt
to introduce further interactions with the ZA loop, or to favourably influence the
conformation of the ligand, but no further improvement in ATAD2 affinity was observed.
ID
R
ATAD2
IC50 (µM)
651±1
ATAD2
LE
ATAD2 Kd BRD4 Kd
(µM)
900
(µM)
141
1500
142
300
200
40
4ꢀCO2Me
4ꢀCO2H
4ꢀCN
0.18
0.19
0.20
0.18
0.19
0.24
0.23
0.23
0.21
0.21
N/A
0.19
N/A
17e
17f
533±40
562±67
680±66
359±37
190±63
236±36
274±75
179±43
299±28
>1000
600
900
17g
17h
17i
4ꢀCONH2
4ꢀSO2Me
4ꢀCl
700
600
200
17j
4ꢀBr
138
48
17k
17l
4ꢀMe
400
58
4ꢀCF3
400
124
65
17m
17n
17o
17p
17q
3,4ꢀdiCl
2ꢀCN
159
>2000
800
300
184
300
3ꢀCN
668±28
>1000
2ꢀCO2H
>2000

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Table 3: ATAD2 inhibition, SPR and BRD4 binding data for compounds 17eꢀq a HTRF
format assay, b, c SPR format assay. Ligand efficiency (LE) = 1.4(ꢀlogIC50)/N, where N is
number of nonꢀhydrogen atoms.
Attention turned to exploring substitution at the C3ꢀgeminal dimethyl group. The proꢀ
S methyl group occupies a small lipophilic pocket in the ligand binding site of ATAD2,
whereas the proꢀR methyl provides opportunities for fragment growth. It was envisioned
that larger substituents at the pro-R position may occupy the cleft between the ZA and BC
loops where the substrate acetylated lysine sidechain enters the active site, and may also
provide improved selectivity over the more sterically demanding BRD4. Synthetic
chemistry to access chiral C3 analogues that retained one methyl but differed in the second
C3 substituent proved challenging, with monoꢀalkylation strategies found to be low yielding.
Knoevenagel condensation was successful when the aldehyde coupling partner did not
contain an enolisable proton, allowing the synthesis of 22aꢀf (Table 4) as racemic mixtures.
Symmetrical C3ꢀdiethyl analogues 18a and 18b were also prepared.
ID R1
R2
X
ATAD2
IC50 (µM)
>1000
ATAD2
LE
N/A
ATAD2
BRD4 Kd
(µM)
>2000
K
d (µM)
Me
Me
Bn
Cl
Cl
500
22a
22b
>1000
N/A
300
1700
Me
Cl
352
0.17
110
1500
22c
Me
Me
CH2C(CH3)3
Cl
Cl
489±18
214
0.18
0.19
125
500
>2000
>2000
22d
22e
Me
Cl
304±83
0.20
113
800
22f

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Et
Et
Et
Et
Cl
163±23
163±37
0.22
0.20
107
300
1000
18a
18b
CF3
>2000
Table 4: ATAD2 inhibition, SPR and BRD4 binding data for compounds 22aꢀf and 18aꢀb.a
HTRF format assay, b, c SPR format assay. Ligand efficiency (LE) = 1.4(ꢀlogIC50)/N, where
N is number of nonꢀhydrogen atoms.
The crystal structure of 18a bound to ATAD2 (Figure 3A) showed that the diethyl
analogues delve further into the hydrophobic pockets formed within the ZAꢀloop residues
Tyr1021, Val1018 and with Ile1074 in the base of the binding site. Gratifyingly, these analogues
resulted in a significant improvement in selectivity over BRD4 while retaining ATAD2
binding and inhibition (Table 4) (Figure 3B).
Superimposition of the crystal structure of 18a with the first bromodomain of BRD4
(PDB entry 2OSS) indicates that the selectivity for ATAD2 over BRD4 may be due to a
steric clash of one of the ethyl groups with Leu94 and the other with Ile146 in the BRD4
structure (Figure 3C).

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Figure 3: Binding interactions of 18a. (A) Crystal structure of 18a (yellow ball and stick)
within the histone binding site of ATAD2 shown in white ribbon, active site residues shown
in cylinder and conserved water molecules shown in red sphere. Key potential hydrogen
bonding interactions shown in black dash. (B) Direct binding data using SPR of 18a with
ATAD2 (blue) and BRD4 (green). (C) Superimposition of 18a bound to ATAD2 with
BRD4 showing potential steric clashes of 18a within the binding site of BRD4 (red). (D)
HTRF data of improved potency within the series 17a pink, 17c blue, 18a green.
Conclusions

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Pyrrolidinopyridone 7 was identified through crystallographic fragment screening as a
novel ligand for the ATAD2 bromodomain. Assessment in HTRF, ITC and SPR assays
confirmed that this was a low affinity fragment. Structureꢀguided fragment growing at the
N1, N6 and C3 substituents led to the identification of two binding modes for this template in
ATAD2. For binding mode 1, methylation of N1, and incorporation of substituted benzyl
groups at N6 led to compounds with ATAD2 binding affinities below 200 µM (Figure 3D),
but these compounds bound more tightly to BRD4 than to ATAD2. Increasing steric
demand at the C3 position led to compounds 18a and 18b, retaining ATAD2 binding affinity
and activity in the HTRF biochemical assay. These C3ꢀsubstituents conferred selectivity
over BRD4 through the introduction of unfavorable interactions with lipophilic amino acid
sidechains in the more sterically demanding BRD4 Nꢀacetyllysine binding site.
Overall, we have demonstrated that structureꢀbased optimisation of ATAD2 fragments
was able to bring millimolar hits into a micromolar potency range. Although inhibition of
ATAD2 in the pyrrolidinopyridone series reached a plateau at 100ꢀ200 µM, we have
succeeded in identifying interactions that can enhance potency while introducing selectivity
versus BRD4. Application of these lessons might contribute to the development of
improved chemical probes of ATAD2’s role and structureꢀfunction relationship, if applied
to fragments with superior initial ligand efficiency.
Conflicts of Interest
There are no conflicts of interest to declare.
Acknowledgements

Organic & Biomolecular Chemistry
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DOI: 10.1039/C8OB00099A
The authors thank Cancer Research UK (Grant Reference C2115/A21421) and Astex
Pharmaceuticals for financial support. We thank Phil Day and Tom Heightman, Astex
Pharmaceuticals for helpful discussions and advice on the crystallography of ATAD2.The
use of the EPSRC Mass Spectrometry Service at the University of Wales (Swansea) is also
gratefully acknowledged. We also thank beamline staff at The Diamond Light Source who
provided excellent facilities for data collection.
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Author Contributions
†These authors contributed equally to the work described in this paper.

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DOI: 10.1039/C8OB00099A
† Duncan C. Miller – Experimental design and interpretation, data analysis, synthesis and
characterization of compounds and manuscript preparation
† Mathew Martin – Protein expression, crystallisation and structure determination, assay
development and manuscript preparation
†Santosh Adhikari – Synthesis and characterization of compounds
Alfie Brennan – Synthesis and characterization of compounds
Jane A. Endicott – Project conception, experimental design and interpretation
Bernard T. Golding – Experimental design and interpretation, data analysis
Ian R. Hardcastle – Experimental design and interpretation, data analysis
Amy Heptinstall – Synthesis and characterization of compounds
Stephen Hobson – Synthesis and characterization of compounds
Claire Jennings – Protein expression, crystallisation and structure determination
Lauren Molyneux – Synthesis and characterization of compounds
Yvonne Ng – Assay development
Stephen R. Wedge – Project conception, experimental design and interpretation
Martin E. M. Noble – Project conception, experimental design and interpretation, data
analysis and manuscript preparation
Céline Cano – Project conception, experimental design and interpretation, data analysis and
manuscript preparation (submitting author)