Discovery of a PCAF Bromodomain Chemical Probe

Article abstract of DOI:10.1002/anie.201610816

The p300/CBP-associated factor (PCAF) and related GCN5 bromodomain-containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine-based L-45 (dubbed L-Moses) as the first potent, selective, and cell-active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)-(?)-norephedrine furnished L-45 in enantiopure form. L-45 was shown to disrupt PCAF-Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co-crystal structure of L-45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L-45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell-permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.

Full text of DOI:10.1002/anie.201610816

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201610816
German Edition: DOI: 10.1002/ange.201610816
Chemical Probes
Hot Paper
Discovery of a PCAF Bromodomain Chemical Probe
Moses Moustakim, Peter G. K. Clark, Laura Trulli, Angel L. Fuentes de Arriba,
Matthias T. Ehebauer, Apirat Chaikuad, Emma J. Murphy, Jacqui Mendez-Johnson,
Danette Daniels, Chun-Feng D. Hou, Yu-Hui Lin, John R. Walker, Raymond Hui,
Hongbing Yang, Lucy Dorrell, Catherine M. Rogers, Octovia P. Monteiro, Oleg Fedorov,
Kilian V. M. Huber, Stefan Knapp, Jag Heer, Darren J. Dixon,* and Paul E. Brennan*
Abstract: The p300/CBP-associated factor (PCAF) and
related GCN5 bromodomain-containing lysine acetyl trans-
ferases are members of subfamily I of the bromodomain
phylogenetic tree. Iterative cycles of rational inhibitor design
and biophysical characterization led to the discovery of the
triazolopthalazine-based L-45 (dubbed L-Moses) as the first
potent, selective, and cell-active PCAF bromodomain (Brd)
inhibitor. Synthesis from readily available (1R,2S)-(À)-nor-
ephedrine furnished L-45 in enantiopure form. L-45 was
shown to disrupt PCAF-Brd histone H3.3 interaction in cells
using a nanoBRET assay, and a co-crystal structure of L-45
with the homologous Brd PfGCN5 from Plasmodium falci-
parum rationalizes the high selectivity for PCAF and GCN5
bromodomains. Compound L-45 shows no observable cyto-
toxicity in peripheral blood mononuclear cells (PBMC), good
cell-permeability, and metabolic stability in human and mouse
liver microsomes, supporting its potential for in vivo use.
increasingly receiving the attention of small molecule inter-
vention efforts, with the disclosure of more than 10 new
chemical probes/inhibitors in 2016.[5]
The p300/CBP-associated factor, PCAF (KAT2B), is
a multi-domain protein containing a single Brd, an N-terminal
domain, and a histone acetyltransferase (HAT) domain.
Known to associate with CBP[6] and p300[6b] during tran-
scription, misregulation of PCAF has been linked to cancer,[7]
HIV infection,[7a,8] and neuroinflammation.[7a,9] Despite pre-
dictions of high druggability[10] and links with inflammatory
disease,[7a,11] the role of PCAF and, more specifically, con-
tributions of the Brd in such disease states are poorly
understood. The development of a small molecule modulator
of PCAF Brd would provide a useful tool for interrogating
this potential therapeutic target and allow for dissociation of
the roles of the Brd and enzymatic domains in disease. Initial
reports of PCAF Brd inhibitors were focused on disrupting
interactions between the HIV-1 peptide TAT-1 and PCAF
Brd.[8a,d] Wang et al. reported the first PCAF Brd inhibitor,
compound 1 (PCAF IC50 1.6 mm, Figure 1), which was
effective at disrupting HIV-1 replication (EC50 2.8 mm).[8c]
Further efforts made by Hu et al.[12] towards more potent
compounds such as 2 were described without significant
increases in potency or indication of selectivity (PCAF IC50
0.93 mm, EC50 11.5 mm, Figure 1). Additional chemotypes have
been disclosed from fragment-based screening by Chaikuad
Bromodomains proteins (Brds) bind to acetylated lysines
(KAc) through the Brd acetyllysine-binding site. Misregula-
tion of these proteins is linked to the onset and progression of
multiple disease states, such as cancer.[1] Significant efforts
have been made recently to interrogate the role of these
targets through the development of chemical probes and
inhibitors.[2] Considerable work has focused on the BET
family (Brd sub-family II),[3] however non-BET[4] Brds are
[*] M. Moustakim, Dr. C. M. Rogers, O. P. Monteiro, Dr. O. Fedorov,
Dr. K. V. M. Huber, Prof. Dr. P. E. Brennan
Dr. A. Chaikuad, Prof. Dr. S. Knapp
Johann Wolfgang Goethe-University, Institute for Pharmaceutical
Chemistry and Buchmann Institute for Life Sciences
60438 Frankfurt am Main (Germany)
Structural Genomics Consortium & Target Discovery Institute
University of Oxford, NDM Research Building
Roosevelt Drive, Oxford, OX3 7DQ and OX3 7FZ (UK)
E-mail: paul.brennan@sgc.ox.ac.uk
J. Mendez-Johnson, Dr. D. Daniels
Promega Corporation
M. Moustakim, Dr. A. L. Fuentes de Arriba, Prof. Dr. D. J. Dixon
Department of Chemistry, Chemistry Research Laboratory
University of Oxford, Mansfield Road, Oxford, OX1 3TA (UK)
E-mail: darren.dixon@chem.ox.ac.uk
2800 Woods Hollow Road, Madison, WI 153611 (USA)
Dr. C-F. D. Hou, Y.-H. Lin, Dr. J. R. Walker, Dr. R. Hui
Structural Genomics Consortium, MaRS South Tower, Suite 732
101 College Street, Toronto, Ontario, M5G 1LZ (Canada)
Dr. P. G. K. Clark
Department of Chemistry, Simon Fraser University
Burnaby V5A 1S6 (Canada)
Dr. H. Yang, Prof. Dr. L. Dorrell
Nuffield Department of Medicine and Oxford NIHR Biomedical
Research Centre, University of Oxford
Oxford, OX3 7FZ (UK)
L. Trulli
Dipartimento di Chimica
Universitꢀ degli Studi di Roma “La Sapienza”
Piazzale Aldo Moro 5, 00185 Roma (Italy)
Dr. J. Heer
UCB Pharma Ltd, Slough, SL1 3WE (UK)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under
http://dx.doi.org/10.1002/anie.201610816.
Dr. M. T. Ehebauer, Dr. E. J. Murphy, Prof. Dr. P. E. Brennan
ARUK Oxford Drug Discovery Institute, University of Oxford
Oxford, OX3 7FZ (UK)
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Figure 1. Reported PCAF bromodomain inhibitors.
Scheme 1. Synthesis of [1,2,4]triazolo[4,3-a]phthalazine derivatives.
Reagents and conditions: a) Acetohydrazide, DMF 1208C 16 h, 62%;
b) N2H4·H2O, EtOH, 1208C, 10 min, quant.; c) TFA, 1008C, 2 h, 43%;
d) R’2NH (1.5–2.0 equiv) KI (0.1 equiv), HCl (0.05 equiv), EtOH or
iPrOH, reflux, 3 days 8–94%.
et al.[5l] Concurrent to this work, Constellation/Genentech
reported compound 3[13] and others, which are potent PCAF
inhibitors (AlphaLISA IC50 13 nm) but lack reported selec-
tivity over other Brds (Figure 1).[7b,c] Despite recent develop-
ments of PCAF Brd inhibitors, a potent, selective, and cell-
active chemical probe has not been reported. The work herein
describes the discovery of such a probe.
Our first line of inquiry towards the first PCAF Brd
chemical probe was focused on the core of non-selective Brd
inhibitors, bromosporine[14] (PCAF isothermal titration calo-
rimetry (ITC) KD: 5 mm) and [1,2,4]triazolo[4,3-a]phthal-
azine[15] derivatives as starting points. Small amine substitu-
ents, as in compounds 7–9 (Table 1), were designed to extend
out of the narrow PCAF pocket and target glutamic acid
residues E750 and E756 at the edge of the KAc-binding
pocket through amine–acid salt bridge interactions (PDB:
5FE0).[5l] Commercially available 1,4-dichlorophthalazine 4
underwent a scalable (up to 20 g) tandem SNAr/condensation
reaction to furnish corresponding triazole intermediate 5 in
good yields (Scheme 1). Significant efforts were employed to
screen conditions using Pd-catalyzed couplings of 5 with
various amine nucleophiles; disappointing yields or lack of
reactivity were observed in all of these cases. It was found that
a KI/HCl-catalyzed SNAr reaction allowed for a tractable
divergent synthesis of various N-linked derivatives
(Scheme 1).
After the synthesis of a focused set of 20 compounds,
screening conducted using a differential scanning fluorimetry
(DSF) assay revealed two hits, dimethylamino compounds 7
and 10 (Table 1). It was found that compounds 8 and 9
featuring a longer amine chain were less potent. With the 2-
(dimethylamino)ethyl group of compounds 7 and 10 identi-
fied as optimal substituents, a virtual library of ~ 12k
compounds was constructed by in silico reaction of compound
5 with commercial compounds containing the 2-(dimethyla-
mino)ethyl motif.[16] Over 60 compounds bearing a tethered
1,2-diamine motif were chosen for synthesis based on docking
score, diversity, and potential for new interactions with the
PCAF Brd (Table 1, compounds 11–16 and Tables S1 and S2).
Derivatives were screened for PCAF Brd affinity by ITC,
leading to the discovery of compound 11 (Table 1). By ITC,
the stoichiometry of binding showed that all of the activity of
the racemate lay in a single enantiomer, later found to have
(S)-configuration after synthesis using enantiopure building
blocks (11 ITC KD 0.30 mm, Brd/11 2:1; (S)-11 KD 0.28 mm,
Brd/(S)-11 1:1). Groups larger than a methyl substituent at R2
were detrimental to activity (compounds 12, 13) as was
a bulkier N,N-diethyl substituent (compound 14). Although
a phenyl substituent at R3 conferred potency to compound 10,
compounds 15 and 16 with smaller methyl and ethyl groups
were less potent. Compound (S)-17 featuring a trifluoro-
methyl group at position R1 caused a loss in activity consistent
with previously reported Brd SAR of the [1,2,4]triazolo[4,3-
a]phthalazines.[15]
Table 1: Amino-substituted triazolophthalazine are potent PCAF Brd
inhibitors.
Compound
R1
R2
R3
R4
n
DTm [8C][a] KD [mm]
(ITC)
7
8
9
10
11
(S)-11
12
13
14
Me
Me
Me
Me
Me Me
Me Me
Me
Me iBu
Me Me
Me
Me
H
H
H
H
H
H
H
Ph
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Et
1
2
3
1
1
1
1
1
1
1
1
1
8.5[b]
ND
ND
1.7
5.6
7.4
3.3
0.85
0.0
4.6
ND
0.65
8.0Æ0.65
>30
>30
In a DSF panel of 48 human Brds, compound (S)-11
showed binding to PCAF and GCN5 with no observable
activity against other Brds (Figure S1). To improve the
potency of (S)-11, it was rationalized that a combination of
appropriate substituents at R2/R3 might improve the avidity of
binding interactions and addition of an aryl group at R3 would
serve as a chemical handle for introduction of new function-
ality. The R2/R3-substituted compounds would be a hybrid of
the most potent analogues 10 and (S)-11.
1.0
0.30Æ0.039
0.28Æ0.029
1.8Æ0.23
>30
Et
>30
15
16
(S)-17
H
H
Me Me
Et
H
7.3Æ1.1
6.9Æ1.4
>30
Me
Me
CF3 Me
Synthesis of aryl substituted compounds was achieved
through a non-selective aza-Henry reaction with p-substi-
tuted benzaldehydes (Scheme 2). p-Substituted benzalde-
[a] Compound concentration 10 mm, unless stated otherwise; [b] Com-
pound concentration 100 mm; ND: not determined.
2
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as an inactive control compound. Having achieved good
potency against PCAF Brd, L-45 was then screened for
selectivity against the panel of 48 human bromodomains using
DSF (Figure 2B). Homologous Brd of GCN5 was the only
other Brd that showed any affinity for L-45, confirmed by ITC
(DTm + 3.68C, KD 0.55 mm). L-45 competitively displaced
a biotinylated tool derivative, compound 46 (Supporting
Information) in a homogeneous time-resolved resonance
fluorescence (HTRF) assay (PCAF Ki 47 nm), corresponding
to exquisite selectivity over BRD4 (> 4500-fold selective).
Scheme 2. Synthesis of threo-substituted derivatives 39–45. Reagents
and conditions: a) NH4OAc (0.2 equiv), EtNO2, reflux, 1:1 E/Z, quant.;
b) Me2NH (5 equiv), THF, RT, 16 h, d.r. 4.6:1–33:1; c) H2 (1 atm), Pd/
C (10%), MeOH, RT, 16 h, 11–15% over two steps, single diastereo-
mer; d) H2 (1 atm), Ra/Ni (0.3 equiv), MeOH, RT, 16 h, 25–28%, over
two steps, single diastereomer; e) 5 (0.8 equiv) KI (0.1 equiv), HCl
(0.05 equiv), EtOH or iPrOH, reflux, 3 days 16–79%.
hydes were chosen as provisional in silico scoring of potential
inhibitors suggested that o- or m-substitutions would be less
tolerated for binding. Highly unstable olefins 18–24 were
telescoped through a diastereoselective (d.r. 4.6:1–33:1)
nitro–olefin conjugate addition furnishing racemic (S*,S*)-
configured[17] compounds 25–31, then reduced to correspond-
ing amines, 32–38, using either Pd/C- or Raney/Ni-catalyzed
hydrogenation. Compounds 32–38 were isolated as single
diastereomers and submitted to the aforementioned KI-
catalyzed SNAr reaction (Scheme 2) to produce compounds
39–45 in low to good yields (16–79%). Racemic compounds
were screened by ITC for PCAF-binding affinity (Table 2).
All of the compounds showed an increase in potency
compared to compound (S)-11, with the simple unsubstituted
derivative 45 having highest potency.
Figure 2. A) Profile of L-45. B) L-45 is selective in a DSF assay panel of
48 Brds (black text). C) Displacement of PCAF-Brd from H3.3-nanoLuc
in live HEK-293 cells using the nanoBRET assay. [a] clogD was
calculated using ChemAxon.[18] [b] Ligand efficiency.[19]
In a cellular context, L-45 was shown to displace nano-
Luciferase-tagged PCAF-Brd from halo-tagged-H3.3 in
a nanoBRET target engagement assay at a single digit mm
concentration (Figure 2C).[20] Inactive enantiomer D-45 had
no effect in the same assay.
Compounds DL-45 and p-fluoro derivative 39 were then
tested for liver microsomal stability in vitro. DL-45 showed
good metabolic stability in both human (t1/2 40 min) and
mouse (t1/2 38 min) liver microsome preparations. para-F
derivative 39 showed a slightly increased metabolic stability in
both human (t1/2 48 min) and mouse (t1/2 65 min) liver
enzymes, likely due to metabolic protection of the para-
substituted aryl ring. DL-45 showed good kinetic solubility
(> 200 mm) and permeability in MDCK-MDR1 cells with low
efflux (Figure 2A). L-45 was also tested in peripheral blood
mononuclear cells and showed no observable cytotoxicity
after treatment at 10 mm for 24 hours.
Pleasingly, it was found following resolution by prepara-
tive chiral stationary phase HPLC, that active enantiomer L-
45, which was dubbed L-Moses, showed good binding affinity
for PCAF Brd (PCAF KD 126 nm, ITC). The other enantio-
mer D-45 showed no observable binding, implying its utility
Table 2: PCAF Brd-binding affinity of compounds 39–45 measured by
ITC.
Although attempts to obtain a co-crystal structure of
recombinant PCAF with L-45 were unsuccessful, which was
surprising given that numerous structures of less potent
PCAF fragments have been reported recently.[5l] A structure
using highly homologous (64% identity) Brd from Plasmo-
dium falciparum, PfGCN5, of which L-45 is also a potent
ligand (ITC KD 280 nm), was successfully obtained (PDB:
5TPX, Figure 3). L-45 bound as expected in the KAc-binding
site of PfGCN5 with key interactions that include a salt bridge
between E1389 (conserved in PCAF as E756) and the
dimethylamino motif of L-45 (Figure 3A). Additional con-
tacts are also observed in the form of an edge-to-face p-p
stacking interaction between W1379 (conserved in PCAF as
Compound
R
Configuration
KD (nm) (ITC)
39
40
41
42
43
44
45
F
(1S*, 2S*)
(1S*, 2S*)
(1S*, 2S*)
(1S*, 2S*)
(1S*, 2S*)
(1S*, 2S*)
(1S*, 2S*)
(1S, 2S)
195Æ23
133Æ15
160Æ54
223Æ78
163Æ117
179Æ48
168Æ27
126Æ15
Inactive
CO2Me
Me
Cl
CF3
OMe
H
L-45/L-Moses
D-45
H
H
(1R, 2R)
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In conclusion, we report the discovery of L-45, the first
nanomolar, selective, and cell-active chemical probe of the
PCAF bromodomain. Iterative cycles of rational inhibitor
design, in silico docking studies, and synthesis furnished L-45
after generation of a focused PCAF inhibitor library. L-45
shows a clean toxicity profile in primary PBMCs, and disrupts
interactions between PCAF Brd and H3.3 in HEK293 cells,
indicating cellular target engagement.
Good cell permeability in a MDCK-MDR1 assay and
stability to metabolism in both human and mouse liver
microsomes indicate that L-45, dubbed L-Moses, may also
have utility in vivo. L-Moses will allow for robust inter-
rogation of PCAF Brd inhibition and pharmacological effects
in relevant diseases models. Future work will investigate the
use of L-Moses in functional assays pertaining to PCAF-
associated diseases.
Figure 3. Co-crystal structure of L-45 with PfGCN5 (PDB ID 5TPX).
A) L-45 (pale sticks) binds in the KAc-binding pocket of PfGCN (blue
ribbon and sticks) and makes H-bonds (dotted lines) through the
triazole to N1436 and the first of a network of four water molecules
(red spheres). The dimethylamino group forms a salt bridge with
E1389. Blue mesh: 2Fo Fc omitted map contoured at 2.5 s. B) Surface
view of complex of PfGCN5 (surface, blue sticks) and L-45 (pale
sticks). The phenyl group of L-45 lies in a hydrophobic groove between
W1379 and the alkyl linker of K1383. The structure of PCAF (orange
sticks, PDB ID 5FTZ) is superimposed to show key residue similarities
(black text PfGCN5/PCAF) and differences (blue text PfGCN5, red text
PCAF).
Acknowledgements
The authors gratefully acknowledge Cyprotex for providing
in vitro metabolism data and Charles Rivers Laboratories for
providing MDCK-MDR1 cell permeability data. Authors
thank S. Velupillai for aiding the crystallographic analysis.
SGC is a registered charity (number 109773 7) that receives
funds from AbbVie, Bayer Pharma AG, Boehringer Ingel-
heim, 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,
S¼o Paulo Research Foundation-FAPESP, Takeda, and Well-
come Trust [092809/Z/10/Z]. M.M. is supported by the
EPSRC Centre for Doctoral Training in Synthesis for Biology
and Medicine (EP/L015838/1). P.G.K.C. gratefully acknowl-
edges the Woolf Fisher Trust. We would also like to extend
our gratitude to Tiger M. Frystone and Maximilian H. K.
Brennan for preparing compounds 8 and 9.
W746) and the phenyl substituent of L-45 (average distance
4.5 ꢀ); a p–p stacking interaction between Y1442 (conserved
in PCAF as Y809) and pyridazo ring of the triazolophthal-
azine motif (average distance 3.7 ꢀ); and characteristic H-
bonds from the triazolophthalazine group and N1436 residue
(conserved in PCAF as N803) and a water molecule. Intol-
erance of substitution of L-45 in R2 and R3 positions
(compounds 12–16, Table 1) was rationalized by the tight fit
of the alkyl amine chain of L-45 (Figure 3B). Interestingly,
K1383 in PfGCN5 is substituted with E750 in human PCAF,
and as such the Plasmodium homologue features a slightly
open KAc-binding site (Figure 3B). Targeting this difference
may allow for design of Plasmodium-selective Brd inhibitors.
As previously supported by SAR, the absolute configuration
of L-45 was confirmed to be (1S,2S).
For the asymmetric synthesis of L-45, commercially
available (1R,2S)-(À)-norephedrine was Boc-protected and
cyclized to a sulfamidite and then directly oxidized using
sodium periodate to boc-protected sulfamidate 46 in reason-
able yields (Scheme 3). Subsequent treatment with dimethyl-
amine facilitated regio-selective ring opening of sulfamidate
46,[21] extruding SO3 and furnishing protected diamine 47 as
a single diastereoisomer with inversion of configuration at the
benzylic centre. Following a deprotection of 47 to the free
amine and SNAr with aryl chloride 5, L-45 was furnished in six
steps as a single stereoisomer.
Conflict of interest
The authors declare no conflict of interest.
Keywords: bromodomains · chemical probes · epigenetics ·
medicinal chemistry · structure-based design
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Manuscript received: November 5, 2016
Revised: November 28, 2016
Final Article published: && &&, &&&&
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Chemical Probes
Let my PCAF go: The first potent, selec-
tive, and cell-active inhibitor of PCAF
bromodomains (Brd) is reported. L-
Moses was shown to disrupt the PCAF-
Brd/histone H3.3 interaction in cells
using a nanoBRET assay, and a co-crystal
structure of L-Moses with the homolo-
gous Brd PfGCN5 helps explain the high
selectivity for PCAF and GCN5 bromo-
domains.
M. Moustakim, P. G. K. Clark, L. Trulli,
A. L. Fuentes de Arriba, M. T. Ehebauer,
A. Chaikuad, E. J. Murphy,
J. Mendez-Johnson, D. Daniels,
C-F. D. Hou, Y.-H. Lin, J. R. Walker, R. Hui,
H. Yang, L. Dorrell, C. M. Rogers,
O. P. Monteiro, O. Fedorov,
K. V. M. Huber, S. Knapp, J. Heer,
D. J. Dixon,*
P. E. Brennan*
&&&& — &&&&
Discovery of a PCAF Bromodomain
Chemical Probe
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!