Discovery of a Selective Allosteric Inhibitor Targeting Macrodomain 2 of Polyadenosine-Diphosphate-Ribose Polymerase 14

Article abstract of DOI:10.1021/acschembio.7b00445

Macrodomains are conserved protein interaction modules that can be found in all domains of life including in certain viruses. Macrodomains mediate recognition of sequence motifs harboring adenosine diphosphate ribose (ADPR) modifications, thereby regulating a variety of cellular processes. Due to their role in cancer or viral pathogenesis, macrodomains have emerged as potential therapeutic targets, but the unavailability of small molecule inhibitors has hampered target validation studies so far. Here, we describe an efficient screening strategy for identification of small molecule inhibitors that displace ADPR from macrodomains. We report the discovery and characterization of a macrodomain inhibitor, GeA-69, selectively targeting macrodomain 2 (MD2) of PARP14 with low micromolar affinity. Co-crystallization of a GeA-69 analogue with PARP14 MD2 revealed an allosteric binding mechanism explaining its selectivity over other human macrodomains. We show that GeA-69 engages PARP14 MD2 in intact cells and prevents its localization to sites of DNA damage.

Full text of DOI:10.1021/acschembio.7b00445

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Article
Discovery of a selective allosteric inhibitor targeting macrodomain
2 of poly-adenosine-diphosphate-ribose polymerases 14
Marion Schuller, Kerstin Riedel, Ian Gibbs-Seymour, Kristin Uth, Christian Sieg, André P
Gehring, Ivan Ahel, Franz Bracher, Benedikt M Kessler, Jonathan M Elkins, and Stefan Knapp
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00445 • Publication Date (Web): 09 Oct 2017
Downloaded from http://pubs.acs.org on October 11, 2017
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Allosteric inhibitors for the second macrodomain of PARP14 were identified. The binding mode of the lead
compounds was determined using crystallographic methods. The inhibitors prevented recruitment of the
PAPP14 macrodomain to DNA damage sites induced by lase irradiation.
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Discovery of a selective allosteric inhibitor targeting macrodomain 2 of
poly-adenosine-diphosphate-ribose polymerase 14
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Marion Schuller1, 2, Kerstin Riedel3, Ian GibbsꢀSeymour4, Kristin Uth1, #, Christian Sieg1, §
,
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André P. Gehring3, Ivan Ahel4, Franz Bracher3, Benedikt M. Kessler2, *, Jonathan M.
Elkins1, * & Stefan Knapp1,5, 6, *
1Structural Genomics Consortium (SGC), Nuffield Department of Clinical Medicine, University of Oxford,
Oxford, OX3 7DQ, UK
2Target Discovery Institute (TDI), Nuffield Department of Clinical Medicine, University of Oxford, Oxford,
OX3 7FZ, UK
3Department of Pharmacy – Center for Drug Research, LudwigꢀMaximilians University of Munich,
81377 Munich, Germany
4Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
5Institute of Pharmaceutical Chemistry and Buchmann Institute for Molecular Life Sciences, Goethe University,
60439 Frankfurt, Germany
6German Cancer Network (DKTK), Frankfurt/Mainz site
#current address: Division of Experimental Pathology, Institute of Pathology, University of Bern, 3008 Bern,
Switzerland
§current address: LIMES Chemical Biology Unit, Rheinische FriedrichꢀWilhelmsꢀUniversität Bonn,
53121 Bonn, Germany
*
corresponding authors: B.M.K. (email: benedikt.kessler@ndm.ox.ac.uk), J.M.E. (email:
jon.elkins@sgc.ox.ac.uk) and S.K. (email: knapp@pharmchem.uniꢀfrankfurt.de)
Running Title: Selective inhibition of PARP14 macrodomain 2
Keywords: small molecule inhibitor, macrodomain, ADPꢀribosylation, PARP14, DNA damage,
allosteric inhibition
Author Contributions:
Conceptualization of study and experiments, J.M.E. and S.K.; Design and performance of
experiments, M.S.; Synthesis of GeAꢀ69 and derivatives, K.R., A.P.G. and F.B.; Performance of laser
microꢀirradiation experiments, I.G.S.; Protein crystallisation and structure determination, K.U. and
J.M.E.; Resources, M.S. and C.S.; Data interpretation – DNA damage experiments, I.A.; Writing –
Original Draft, M.S.; Writing – Review & Editing, J.M.E., B.M.K. and S.K. with support of all other
authors.
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Abstract
Macrodomains are conserved protein interaction modules that can be found in all domains of life as
well as in certain viruses. Macrodomains mediate recognition of sequence motifs harbouring
adenosine diphosphate ribose (ADPR) modifications, thereby regulating a variety of cellular
processes. Due to their role in cancer or viral pathogenesis, macrodomains have emerged as potential
therapeutic targets, but the unavailability of small molecule inhibitors has hampered target validation
studies so far. Here, we describe an efficient screening strategy for identification of small molecule
inhibitors that displace ADPR from macrodomains. We report the discovery and characterisation of a
macrodomain inhibitor, GeAꢀ69, selectively targeting macrodomain 2 (MD2) of PARP14 with low
micromolar affinity. Coꢀcrystallisation of a GeAꢀ69 analogue with PARP14 MD2 revealed an
allosteric binding mechanism explaining its selectivity over other human macrodomains. We show
that GeAꢀ69 engages PARP14 MD2 in intact cells and prevents its localisation to sites of DNA
damage.
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Introduction
Protein ADPꢀribosylation is catalysed by polyꢀADPꢀribose polymerases (PARPs) which transfer
either a single adenosine diphosphate ribose (ADPR) unit (MARylation) or multiple units
(PARylation) from NAD+ onto acceptor proteins 1. Macrodomains specifically recognise these
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modifications and serve therefore as “reader domains” of this posttranslational modification . In
addition to recognising MARylation and PARylation sites in proteins, some macrodomains control
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also the turnover of ADPR signalling through their ability to remove these modifications . In
comparison to other protein ADPꢀribosylation recognising modules such as the WWE or PBZ
domains, macrodomains recognise the entire ADPR moiety either directly at the protein attachment
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site or at the termini of polyꢀADPꢀribose (PAR) chains . This leads to the formation of protein
complexes or the recruitment to DNA lesions, i.e. sites of high PAR generation mediated by PARP1 4–
6. Macrodomains are thus directly linked to MARylation and PARylation pathways and dysregulation
of macrodomain function by overexpression or mutations have been associated with several diseases
including cancer, developmental defects and neurodegeneration 2,7
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Given their pathobiological importance, macrodomains were recently suggested as potential
therapeutic targets. Targeting components of ADPR signalling has already been a successful strategy
for cancer therapy where PARP inhibitors have been successful in clinical trials and have been
approved for treatment 8,9. Targeting macrodomains with small molecule inhibitors may provide an
attractive alternative to PARP inhibitors by disrupting ADPR signalling by a different mechanism and
without the difficulties of obtaining inhibitors specific for individual PARP enzymes. So far, inhibitor
development has focused on macrodomains that have enzymatic activity such as the enzyme
poly(ADPR) glycohydrolase (PARG) which, similarly to PARP1, performs a critical role in DNA
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damage repair 10–12. Only recently, a chemical probe targeting PARG hydrolase activity was released
which shows convincing onꢀtarget pharmacology and selectivity in contrast to early inhibitors such as
rhodamine or salicylanilide based compounds 13–15
.
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Macrodomain inhibitor development has been hampered due to the lack of suitable highꢀthroughput
screening (HTS)ꢀcompatible biochemical assays to identify chemical starting points for drug
discovery projects. Stowell et al. recently reported a HTS HTRF screening assay used for inhibitor
discovery targeting the glycohydrolase activity of PARG 16. This assay is, however, specific for
PARG and does not enable identification of inhibitors for other macrodomains that have reader
domain function.
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In this work, we developed an AlphaScreen based screening strategy suitable for highꢀthroughput
screening of most human macrodomains. Using this assay, we identified a selective, allosteric and
cellꢀactive macrodomain inhibitor, GeAꢀ69, targeting macrodomain 2 of PARP14 in vitro and in
intact cells where it prevents its localisation to the sites of DNA damage.
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Results
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Identification of the PARP14 MD2 inhibitor GeA-69.
The majority of the 16 human macrodomain proteins are reader domains of protein ADPꢀribosylation
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and only four of them show additional catalytic activity . We therefore adapted an AlphaScreen
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(amplified luminescence proximity homogenous assay) based displacement binding assay using a
peptide that was both biotinylated and monoꢀADPꢀribosylated together with His6ꢀtagged
macrodomains. This strategy led to the development of an AlphaScreen assay at low peptide
concentration creating an excellent signal (Figure 1A). Isothermal titration calorimetry (ITC) was
used for selected macrodomains (Figure S1) in order to determine binding affinities in solution. The
ITC data verified that the ADPꢀribosylated peptide was recognised by macrodomains although a
decrease of at least 2ꢀfold in binding affinities to the peptide in comparison to the free ADPR
monomer was observed. This decrease could be due to the method of conjugation to the peptide which
resulted in a ringꢀopened terminal ribose unit 17, or alternatively due to the presence of the attached
peptide sequence. The assay conditions were optimised for nine macrodomain proteins representing
different members of the phylogenetic tree of human macrodomains. A stable assay signal was also
obtained for the catalytically active macrodomains MacroD1, MacroD2 and TARG1 confirming that
the ADPR imitating part was not hydrolysed. Furthermore, the consistency of the assay signal and of
Z’ (on average 0.85) for all of the macrodomains tested demonstrated the robustness of the assay.
Using this assay, we then performed a screen of ~48,000 small molecules against PARP14 MD2
(Figure 1A). In an initial screen, PARP14 MD2 was screened along with four other macrodomains
against an inꢀhouse kinase inhibitor library of 3000 compounds at a compound concentration of
50 µM. Following this, the NDDI (NIBR Drug Discovery Incubator) compound library comprising
45,000 compounds provided by Novartis (Basel, Switzerland) was screened against PARP14 MD2 at
a compound concentration of 25 µM. The rationale behind a kinase inhibitor screen was that
macrodomains as binding domains of ADPR, which is structurally closely related to ATP, may show
activity towards ATPꢀcompetitive kinase inhibitors or fragments. Being aware of the susceptibility of
the AlphaScreen for assay interfering compounds 18, all initial hits showing more than 50 %
macrodomain inhibition were validated by counter screening using a biotinylatedꢀHis6 peptide, dose
response experiments to obtain IC50 values, and secondary assays including biolayer interferometry
(BLI) and ITC measurements.
From this screen, we identified GeAꢀ69, which was part of the kinase inhibitor library, as initial hit
for PARP14 MD2. BLI and ITC experiments verified its binding to PARP14 MD2 with a KD of
1.4 µM and 860 nM, respectively, in a 1:1 binding stoichiometry; a closely related compound MnK2ꢀ
68 (developed during an inhibitor optimisation programme) did not bind and was used as a negative
control (Figure 1B ꢀ D).
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GeA-69 is an allosteric inhibitor of PARP14 MD2.
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To establish the binding mode of GeAꢀ69, we first attempted to determine the coꢀcrystal structure of
PARP14 MD2 with GeAꢀ69. Initial attempts at coꢀcrystallisation of GeAꢀ69 with wildꢀtype PARP14
MD2 gave crystals that diffracted Xꢀrays only to modest resolution. Five different surfaceꢀentropyꢀ
reduction (SER) mutants of PARP14 MD2 were prepared. In these SER constructs, up to five lysine
residues on the protein surface were truncated to serine residues, to improve the likelihood of forming
tighter crystal contacts 19. Each of these mutant proteins was used in crystallisation trials with either
GeAꢀ69 or closelyꢀrelated analogues. Finally, a construct of the PARP14 MD2K1048S, K1154S, K1158S, K1162S
mutant gave a crystal diffracting to 1.6 Å resolution (Table 1) in complex with compound MnK2ꢀ13
(Figure 2A). MnK2ꢀ13 is the methanesulfonamide analogue of the acetamide GeAꢀ69 and binds to
PARP14 MD2 with a KD of 2.1 µM (Figure S2A).
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Compound MnK2ꢀ13 was well resolved in the electron density (Figure S2B). Surprisingly MnK2ꢀ13
did not occupy the ADPR binding site but was deeply buried within the macrodomain with no contact
with the solvent phase (Figure 2A). Ligand binding was stabilised by hydrophobic and hydrogen
bonding interactions to the protein (Figure 2B). The planar carbazole itself was bound in a
hydrophobic pocket with several hydrophobic interactions including the Tꢀshaped πꢀπ interaction with
ring C and F1129 and with an additional hydrogen bond of the NH of the carbazole to the proline
carbonyl of P1130. The benzene ring of MnK2ꢀ13 was surrounded by hydrophobic residues including
F1182, L1171 and F1169. The sulfonamide moiety of MnK2ꢀ13 formed several hydrogen bonds to
the macrodomain including to the amide of I1132 and the primary carboxamide of the N1178 side
chain. Furthermore, the NH of the sulfonamide group was involved in a complex with a water
molecule together with the N1178 carbonyl group and K1141. Superimposition of the ADPRꢀbound
macrodomain structure (PDB 3Q71) and the MnK2ꢀ13ꢀbound structure furthermore showed that
F1144 was pushed aside by the carbazole of the inhibitor. Ring A of the carbazole may therefore be
crucial for enabling the binding of the inhibitor to the protein.
MnK2ꢀ13 bound adjacent to but not in the ADPR binding site as would have been expected based on
its carbazole scaffold that was used for the development of ATP mimetic inhibitors 20. The structure
superimposition revealed that upon inhibitor binding a loop from P1130 to P1140 was pushed into the
ADPR binding site displacing the bound ADPR (Figure 2C), and a portion of this loop consisting of
residues G1133ꢀG1135 has weaker electron density and may thus be present in multiple
conformations. ITC competition experiments confirmed GeAꢀ69 as an allosteric inhibitor of PARP14
MD2 able to prevent ADPR binding to the macrodomain. In the presence of 80 µM GeAꢀ69, binding
of ADPR to the macrodomain was not detected while in absence of the inhibitor recognition of the
ADPR with a KD of 7.8 µM was measured (Figure 2C).
GeA-69 is a highly selective macrodomain inhibitor.
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To assess GeAꢀ69 selectivity over the other human macrodomain family members, we tested the
inhibitor on eleven other representative human macrodomain proteins either by the macrodomain
AlphaScreen assay at a compound concentration of 200 µM, by BLI at a compound concentration of
50 µM or by ITC at a compound concentration of 30 µM (Figure 3A, S4). Binding was only detected
to PARP14 MD2 proving the very high target selectivity of GeAꢀ69 over the macrodomain family.
Although the macrodomain fold is highly conserved, at the amino acid level sequence divergence is
high, and structural and sequence comparison revealed a number of differences around the binding
site that provide a rationale for the excellent selectivity of GeAꢀ69 (Figure 3B, S5, S6). Since GeAꢀ69
was part of a kinase inhibitor library, originally synthesised in a programme aimed at the optimisation
of the activities of the alkaloid annomontine 21,22, the internal database was searched for experimental
thermal shift assay data of kinases that were screened against this compound. A number of 46 kinases
representing different branches of the phylogenetic kinase tree were screened (Figure S7), none of
them showing any significant stability shifts and thus no binding affinity for this inhibitor. Further in
vitro profiling showed that GeAꢀ69 exhibited, despite its allosteric binding mode, fast on/fast off
kinetics when binding to PARP14 MD2, with a mean dissociation rate constant of 2.4 × 10ꢀ2 sꢀ1
resulting in a halfꢀlife of binding (t1/2, residence time) of 42 seconds (Figure 3C). Performance of
parallel artificial membrane permeability assays (PAMPA) showed that GeAꢀ69 was highly cell
permeable with a logPe of ꢀ3.5 over three representative pH values (4.0, 6.8, 8.0) (Figure 3D). Finally,
metabolic activity assays showed that GeAꢀ69 has only moderate cytotoxicity with an EC50 of
~50 µM on different cell lines tested (HeLa, HEK293 and Uꢀ2 OS) (Figure 3E). Altogether, GeAꢀ69
demonstrated a biochemical profile to warrant the evaluation of cellꢀbased activities.
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GeA-69 engages PARP14 MD2 in intact cells.
Since PARP14 macrodomains have been reported to localise to sites of DNA damage 23, we tested the
ability of GeAꢀ69 to interfere with PARP14 MD2 recruitment to laserꢀinduced DNA damage sites. In
order to confirm that PARP14 MD2 is actively recruited to DNA damage sites, BrdU sensitised Uꢀ2
OS cells were transfected with YFPꢀPARP14 MD2 and subjected to laser microꢀirradiation coupled to
liveꢀcell imaging. We observed that PARP14 MD2 is recruited to these DNA lesions within seconds
after induction of DNA damage, while the corresponding ADPR binding deficient PARP14
MD2G1044E mutant was not able to localise to these sites (Figure 4A, S8). Furthermore, PARP14 MD2
recruitment depends on both the presence and activity of PARP1. In the absence of PARP1, in
PARP1-/- cells, and after inhibition of PARP1 catalytic activity using the PARP inhibitor Olaparib,
recruitment of the macrodomain could not be detected in comparison to the corresponding controls
(Figure 4B, C). Finally, we evaluated if GeAꢀ69 could interfere with the PARP14 MD2 recruitment
and observed that preꢀtreatment of Uꢀ2 OS cells with GeAꢀ69 for 2 hrs affected PARP14 MD2 reꢀ
localisation in this setꢀup. The macrodomain recruitment was significantly decreased in cells preꢀ
treated with 50 µM GeAꢀ69 and completely prevented in cells exposed to 250 µM GeAꢀ69. As
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expected, the negative control MnK2ꢀ68 did not interfere with PARP14 MD2 recruitment and further,
GeAꢀ69 did not affect the recruitment of an alternative macrodomain containing protein, YFPꢀALC1.
Although a high inhibitor concentration had to be applied to completely prevent PARP14 MD2
recruitment to DNA damage sites, these experiments proved that GeAꢀ69 engages PARP14 MD2 in
intact cells.
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Discussion
Although representative members of human, viral or bacterial macrodomains have so far been well
studied regarding their molecular structure, substrate recognition and in vitro biochemical activity,
very limited data is available on their in vivo characterisation which would provide a detailed
understanding of their (pathoꢀ)physiological functions. Those studies would be aided by inhibitors
that specifically target members of the macrodomain family. Great progress has been made on the
development of cellꢀactive and potent inhibitors of the catalytic activity of PARPs and of the
hydrolase PARG, however, to the best of our knowledge inhibitors of other macrodomains have not
been reported so far. To address this challenge, we have developed an AlphaScreen based assay
suitable for highꢀthroughput screening of several human (catalytically active or inactive)
macrodomains. We anticipate that this assay is also applicable for screening most nonꢀhuman
macrodomains that bind ADPR. Using this assay, we identified a small molecule inhibitor, GeAꢀ69,
targeting PARP14 MD2 with low micromolar affinity. GeAꢀ69 is a carbazoleꢀbased compound with
an acetylaminophenyl substituent attached to ring A. This, and very few related compounds, e.g. the
methanesulfonamide analogue MnK2ꢀ13 showed significant inhibition of PARP14 MD2. An
allosteric binding mode was proven by an Xꢀray crystal structure analysis. A detailed structureꢀ
activityꢀrelationship (SAR) study showing our attempts to improve the scaffold and potency of this
inhibitor will be published elsewhere.
PARP14 is described as proꢀsurvival protein and is associated with the development of inflammatory
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diseases and various types of cancer including Bꢀcell lymphoma and hepatocellular carcinoma
.
PARP14 has therefore emerged as a therapeutic target and there are concurrent approaches that focus
on the development of inhibitors targeting its catalytic PARP domain 27–29. PARP domain inhibitors
are however challenging to optimise in regard to their selectivity over PARP1. Targeting PARP14
macrodomains is an alternative strategy to interfere with PARP14 recruitment to ADPꢀribosylated
target sites through its macrodomains and consequently prevent MARylation of target proteins by its
catalytic domain. GeAꢀ69 shows good ligand efficiency and excellent selectivity over other human
macrodomains, obviously as a consequence of its allosteric binding mode. More importantly, GeAꢀ69
is cellꢀactive and prevents PARP14 MD2 recruitment to DNA damage sites. To what extent these
laser microꢀirradiation assays already reflect endogenous PARP14 activity in DNA damage repair
mechanisms is under current investigation.
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Figure 1. Structure and identification of the PARP14 MD2 inhibitor GeA-69. (A) Performance of a
48,000 compound screen against PARP14 MD2 by AlphaScreen. The graph represents the screening
of two libraries and displays the AlphaScreen activity of 961 compounds that showed more than 50 %
macrodomain inhibition. 0.09 % of the 48,000 compounds showed more than 80 % inhibition of the
macrodomain and less than 25 % activity in the counterscreen (red lines). GeAꢀ69 is highlighted with
a red circle. (B) Molecular structure of the macrodomain inhibitor GeAꢀ69 and its inactive analogue
MnK2ꢀ68. (C) Schematic representation of the PARP14 domain architecture. Residue numbers refer
to the human PARP14 (NCBI ref: NP_060024). The macrodomain targeted by GeAꢀ69 is highlighted
with a red star. (D) Isothermal titration calorimetry (ITC) confirmed binding of GeAꢀ69 to PARP14
MD2 with a KD of 860 nM. Raw injection heats are shown for a blank titration of PARP14 MD2 into
buffer (top), and titrations of PARP14 MD2 into solutions of the inactive analogue MnK2ꢀ68 (middle)
and the active compound GeAꢀ69 (bottom). The inset shows normalized binding enthalpies corrected
for the heat of dilution as a function of binding site saturation (symbols as indicated in the inset).
Solid lines represent a nonlinear least squares fit using a singleꢀsite binding model.
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Figure 2. Crystal structure of PARP14 MD2 in complex with MnK2-13 reveals an allosteric
binding mode. (A) Surface representation of PARP14 MD2 in complex with the macrodomain active
GeAꢀ69 analogue MnK2ꢀ13. The macrodomain inhibitor (atomꢀcoloured sticks) is deeply bound
within the macrodomain. The solvent accessible surface of residues 1234ꢀ1234 (white loop) was
removed to visualise the inhibitor. (B) The binding site of MnK2ꢀ13 in PARP14 MD2. Residues
involved in direct contacts with the inhibitor are shown in atomꢀcoloured sticks. Hydrogen bonds
between ligand and protein or structural waters are indicated by dotted lines. Superimposition of the
ADPR bound macrodomain structure (PDB 3Q71) and inhibitor bound macrodomain structure shows
that F1144 (magenta) is pushed aside by the carbazole ring system of MnK2ꢀ13 (arrow). The part of
the loop (P1130ꢀP1140) that is rearranged by comparison to the ADPR bound structure is shown in
green. (C) GeAꢀ69 is an allosteric inhibitor of ADPR binding to PARP14 MD2. (Left) GeAꢀ69 was
docked into PARP14 MD2 using the PARP14 MD2ꢀMnK2ꢀ13 coꢀcrystal structure as template (PDB
5O2D). Superimposition of the resulting docking structure with the ADPRꢀbound macrodomain
structure (PDB 3Q71, green) revealed GeAꢀ69 as allosteric inhibitor. Binding of GeAꢀ69 (or the
macrodomain active analogue MnK2ꢀ13) to the macrodomain results in movement of the loop P1130ꢀ
P1140 (highlighted in magenta) into the ADPR binding site displacing the bound ADPR. (Right) ITC
experiments confirmed GeAꢀ69 as an inhibitor of ADPR binding to PARP14 MD2. Raw injection
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heats are shown resulting from titration of a 1 mM ADPR solution into the cell containing either
buffer (top) or 40 µM PARP14 MD2 only (bottom) or 40 µM PARP14 MD2 in presence of 80 µM
GeAꢀ69 (middle). Only in absence of the inhibitor, the expected KD for ADPR of 7.8 µM was
observed.
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Figure 3. GeA-69 is a selective and highly cell permeable macrodomain inhibitor. (A) GeAꢀ69
shows target selectivity over human macrodomain family members. Selectivity was assessed for 12
out of 16 human macrodomains (shown by circles) by the macrodomain AlphaScreen assay at a
compound concentration of 200 µM (top right) or by biolayer interferometry (BLI) at a compound
concentration of 50 µM (bottom right). Binding was only detected with PARP14 MD2 (red circle).
The numbers in the circles correspond to macrodomains shown on SDSꢀPAGE (bottom left). PARP14
MD1, 2 and 3 proteins were prepared with different tags. Details are provided in Figure S3. (B)
Multiple sequence alignment of human macrodomains confirming no other human macrodomain has
conserved all the residues that bind MnK2ꢀ13 and therefore also GeAꢀ69. Residues of PARP14 MD2
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involved in MnK2ꢀ13 interaction are highlighted in green, numbers on top of the alignment refer to
the human PARP14 sequence. Box colour legend: (blue) identical amino acid, (light blue) similar
amino acid, (brown) amino acid likely to sterically interfere with inhibitor binding, (light brown)
amino acid not favouring the inhibitor binding like the corresponding PARP14 MD2 residue based on
its physicochemical properties. (C) Binding kinetics of GeAꢀ69 to PARP14 MD2. The BLI
sensorgram shows the kinetics of GeAꢀ69 association and dissociation to PARP14 MD2. The
inhibitor concentration range and steady state analysis are shown next to the traces. Mean ka is
1.35 × 104 Mꢀ1sꢀ1 while the mean kd is 2.4 × 10ꢀ2 sꢀ1 resulting in a halfꢀlife of binding (t1/2, residence
time) of 42 seconds. (D) GeAꢀ69 is highly cell permeable. Cell permeability was assessed by PAMPA
(parallel artificial permeability assay) performance. The highest effective permeability (logPe) of ꢀ3.5
is identical over the three representative pH values tested. The fraction of absorbed drug (FA) was
calculated to 100 % with 2 % passing paracellular and 98 % transcellular over the membrane. Active
transport mechanisms are not considered in this model. (E) GeAꢀ69 shows moderate cytotoxicity.
HeLa, Uꢀ2 OS and HEK293 cells were incubated for 72 h in the presence of different GeAꢀ69
concentrations (25 nM ꢀ 250 µM) and cell viability was measured using the AlamarBlue™ reagent.
EC50 values were calculated from three independent experiments (EC50_HeLa = (58 ± 3.9) µM, EC50_Uꢀ2
OS = (52 ± 1.1) µM, EC50_HEK293 = (54 ± 2.2) µM. Results are expressed as mean ± SEM, n = 3).
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Figure 4. GeA-69 engages PARP14 MD2 and prevents localisation to the sites of DNA damage.
(A) PARP14 MD2 wildꢀtype is recruited to sites of laserꢀinduced DNA damage in contrast to the
corresponding ADPR binding deficient mutant G1044E. Uꢀ2 OS cells were transfected with YFPꢀ
PARP14 MD2 wt or G1044E mutant, subjected to laser microꢀirradiation (white arrows) and imaged
at the indicated times. (B) PARP14 MD2 recruitment depends on the presence of PARP1. Uꢀ
2 OS/PARP1+/+ and Uꢀ2 OS/PARP1-/- cells were transfected with YFPꢀPARP14 MD2 wt, subjected to
laser microꢀirradiation and imaged at the indicated times. (C) PARP14 MD2 recruitment depends on
the activity of PARP1. Uꢀ2 OS cells transiently expressing YFPꢀPARP14 MD2 wt were preꢀtreated
for 1 hour with DMSO or with 10 µM of the PARP1 inhibitor Olaparib, subjected to laser microꢀ
irradiation and imaged at the indicated times. (D) GeAꢀ69 prevents recruitment of PARP14 MD2 to
sites of laserꢀinduced DNA damage. Uꢀ2 OS cells transiently expressing YFPꢀPARP14 MD2 or fullꢀ
length YFPꢀALC1 were preꢀtreated with DMSO, GeAꢀ69 or the negative control inhibitor MnK2ꢀ68,
subjected to laser microꢀirradiation and imaged at the indicated times.
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Table 1. Data collection and refinement statistics.
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5O2D
PDB ID
Space group
C2
9
No. of molecules in the
asymmetric unit
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Unit cell dimensions
a, b, c (Å), β (°)
131.4, 35.7, 37.1, 94.8
Data collection
27.65ꢀ1.60
(1.63ꢀ1.60)
22712 (1036)
Resolution range (Å)a
Unique observationsa
Average multiplicitya
Completeness (%)a
3.8 (2.7)
98.9 (90.7)
0.05 (0.22)
a
Rmerge
Mean (I)/σ(I)a
Mean CC(1/2)
Refinement
12.2 (3.6)
0.998 (0.925)
Rꢀvalue, Rfree (%)
13.2, 17.4
0.011
r.m.s. deviation from ideal
bond length (Å)
r.m.s. deviation from ideal
bond angle (°)
1.48
a Values within parentheses refer to the highest resolution shell.
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Experimental Procedures
Materials and plasmids
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Bacterial expression constructs encoding PARP14 MD1 (G789–K979), PARP14 MD2 (A994–
N1191) and PARP14 MD3 (F1208–G1388) were a kind gift from Herwig Schüler 30. Fullꢀlength
PARP14 cDNA was obtained from Genecopoeia. The macrodomain AlphaScreen™ peptide was
synthesized by Cambridge Peptides (Birmingham, UK) and has the following sequence:
ARTK(Bio)QTARK(AoaꢀRADP)S (M.W.: 1986.8; purity: 92 %, peptide code: 19254). Dꢀbiotin and
adenosine 5′ꢀdiphosphoribose sodium salt (purity: 95 %) were purchased from Sigma Aldrich.
Macrodomain AlphaScreen™ assay
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Assays were performed with minor modifications from the manufacturer’s protocol (PerkinElmer).
All reagents were diluted in buffer containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 0.5 mM
TCEP, 0.1 % bovine serum albumin and 0.05 % CHAPS and allowed to equilibrate to room
temperature before addition to plates. The assays were run in 20 µL volumes in lowꢀvolume 384ꢀwell
plates (ProxiPlate™ꢀ384 Plus, PerkinElmer, USA) at RT. To determine ideal assay concentrations of
the corresponding macrodomain protein and peptide (a biotinylated and ADPꢀribosylated 11 residue
sequence, Figure S1A), 4 µL volumes of peptide (0ꢀ16 ꢁM; final assay concentration: 0ꢀ3.2 ꢁM) were
incubated with 4 µL volumes of His6ꢀtagged macrodomain protein (0ꢀ16 ꢁM; final assay
concentration: 0ꢀ3.2 ꢁM) in 4 µL buffer for 30 min at RT in foilꢀsealed plates. For compound
screening, 12 µL of a solution containing 25 nM peptide and 400 nM His6ꢀtagged PARP14 MD2
protein in assay buffer were incubated with 50 nL or 100 nL compound solution (10 mM; final assay
concentration: 25 ꢁM or 50 µM) for 30 min at RT in foilꢀsealed plates. Then, 8 µL of streptavidinꢀ
coated donor beads (7 ꢁg/ml) and nickel chelate acceptor beads (7 ꢁg/ml) (Perkin Elmer
AlphaScreen™ Histidine (Nickel Chelate) Detection Kit) were added under low light conditions and
plates were incubated for 60 min at RT protected from light. Plates were read on a PHERAstar FS
plate reader (BMG Labtech, Germany) using an AlphaScreen™ 680 excitation/570 emission filter set.
Alternatively for counter screening of the compounds, 12 µL of 75 nM biotinylated and hexahistidineꢀ
tagged linker peptide (PerkinElmer) was added to 50 nL or 100 nL of the compounds (10 mM) and
plates were processed as described above.
Isothermal titration calorimetry (ITC)
Binding experiments were carried out on a VPꢀITC microcalorimeter (MicroCal). All experiments
were performed in 50 mM HEPES (pH 7.4), 300 mM NaCl, 5 % glycerol, 0.5 mM TCEP at 12 °C, a
reference power of 12 ꢁCal/sec and a stirring speed of 307 rpm. The titrations were conducted using
an initial injection of 2 µL followed by 28 identical injections of 10 µL with duration of 4 sec (per
injection) and a spacing of 240 sec between injections. Competition experiments were performed on
an iTC200 instrument (MicroCal) at 20 °C, a reference power of 12 ꢁCal/sec and a stirring speed of
1000 rpm. Following an initial injection of 0.2 µL, 20 identical injections of 2 µL were run with a
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duration of 4 sec (per injection) and a spacing of 150 sec between injections. Data analysis was
carried out using Origin software with the MicroCal plugin. Thermodynamic parameters were
calculated using ꢂG = ꢂH − TꢂS = −RTlnKB, where ꢂG, ꢂH, and ꢂS are the changes in free energy,
enthalpy, and entropy of binding, respectively. In all cases, a single binding site model was employed.
Biolayer interferometry (BLI)
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Kinetic ligandꢀbinding measurements were performed using an Octet RED384 BLI instrument
(fortéBio). Superstreptavidin (SSA) biosensors were loaded with biotinylated macrodomain protein
and equilibrated for 120 sec in assay buffer (25 mM HEPES (pH 7.4), 100 mM NaCl, 0.01 %
Tween 20). Association and dissociation were monitored for 240 sec each in assay buffer at 25 °C.
GeAꢀ69 was prepared as seven 1:1 serial dilutions starting from 10 µM. Binding to the reference
sensors (no protein attached) was subtracted before calculations and data was processed using the
fortéBio analysis software provided by the manufacturer.
Protein crystallisation and data collection
Surface entropy reduction mutations were introduced into PARP14 MD2 (A994–N1191) by the
overlapping PCR method. Several mutants were prepared of which PARP14 MD2SER3 with K1048S,
K1154S, K1158S, and K1162S mutations could be crystallised with MnK2ꢀ13. For protein
crystallisation, purified PARP14 MD2SER3 was bufferꢀexchanged into 20 mM HEPES (pH 7.4),
500 mM NaCl, 5 % glycerol, 0.5 mM TCEP, and concentrated to 16 mg/ml, using 10 kDa MWCO
centrifugal concentrators (Millipore). MnK2ꢀ13 inhibitor dissolved to 50 mM in DMSO was added to
a final concentration of 1.0 mM (2 % DMSO) and incubated on ice for approximately 30 min. The
sample was centrifuged at 14,000 rpm for 10 min at 4 °C prior to setting up 150 nL volume sitting
drops at three ratios (2:1, 1:1 or 1:2 proteinꢀinhibitor complex to crystallisation solution). Drops were
equilibrated at two temperatures (4 °C or 20 °C). A crystal was obtained with a 1:2 ratio of protein to
a crystallisation solution consisting of 0.8 M sodium phosphate monobasic, 0.8 M potassium
phosphate dibasic, 0.1 M HEPES pH 7.5, and was cryoprotected in mother liquor supplemented with
25 % ethylene glycol before flashꢀfreezing in liquid nitrogen for data collection. Diffraction data were
collected at the Diamond Light Source beamline I02.
Structure solution and refinement
The diffraction data was processed using MOSFLM 31 and AIMLESS 32 (Table 1). The structure was
solved by molecular replacement using PHASER 33 and a published structure of PARP14 MD2 (PDB
ID 3Q71) as a search model. There was one molecule of PARP14 MD2 in the asymmetric unit. Coot
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and REFMAC5 were used for building the model and refinement. MOLPROBITY was used
for model validation and analysis.
Synthesis of GeA-69, MnK2-13 and MnK2-68
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GeAꢀ69, Nꢀ[3ꢀ(9Hꢀcarbazolꢀ1ꢀyl)pyridinꢀ4ꢀyl]acetamide, was prepared from known
1ꢀ
bromocarbazole by a Suzuki crossꢀcoupling procedure as follows: 74 mg (0.30 mmol) 1ꢀbromoꢀ9Hꢀ
carbazole, 61 mg (0.31 mmol) 2ꢀacetamidophenylboronic acid, and 39 mg (0.034 mmol) Pd(Ph3P)4
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were placed in a microwave vial under nitrogen atmosphere. A mixture of 3 mL 1,2ꢀdimethoxyethane
und 2 mL ethanol, previously degassed by purging with for 10 min with nitrogen, was added. The
suspension was stirred for 10 min at room temperature. Then 1.0 mL 2 M degassed sodium carbonate
solution was added. The reaction was conducted for 10 min under microwave irradiation at 70 °C, 150
Watt and a maximum pressure of 6 bar. After cooling of the reaction mixture in an ice bath, water (50
mL) was added, followed by extraction with diethyl ether (3 x 40 mL). The combined organic layers
were dried over sodium sulfate and concentrated in vacuum. The residue was purified by flash column
chromatography (dichloromethane/ethyl acetate 2:1). Yield: 55 mg (0.18 mmol / 60 %) light grey
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solid. Melting point: 173ꢀ175 °C. HꢀNMR (500 MHz, CD2Cl2): δ (ppm) = 8.31 (br s, 1H, 9ꢀNH),
8.26 (d, J = 8.2 Hz, 1H, 6´ꢀH), 8.15 (dd, J = 6.1 Hz, 2.8 Hz, 1H, 4ꢀH), 8.12 (d, J = 7.7 Hz, 1H, 5ꢀH),
7.47 – 7.41 (m, 2H, 4´ꢀH, 5´ꢀH), 7.41 – 7.39 (m, 2H, 7ꢀH, 8ꢀH), 7.37 – 7.34 (m, 2H, 2ꢀH, 3ꢀH), 7.29
(d, J = 6.8 Hz, 1H, 3´ꢀH), 7.25 (ddd, J = 8.0 Hz, 7.8 Hz, 2.6 Hz, 1H, 6ꢀH), 7.11 (br s, 1H, 1´ꢀNH), 1.76
(s, 3H, CH3). 13CꢀNMR (100 MHz, CD2Cl2): δ (ppm) = 169.2 (C=O), 140.1 (Cꢀ8a), 138.2 (Cꢀ9a),
136.1 (Cꢀ1´), 131.0 (Cꢀ5´), 129.4 (Cꢀ2´), 129.2 (Cꢀ4´), 127.0 (C2), 126.7 (Cꢀ7), 125.2 (Cꢀ3´), 124.1
(Cꢀ4a), 123.6 (Cꢀ4b), 122.9 (Cꢀ6´), 120.9 (Cꢀ1), 120.8 (Cꢀ5), 120.7 (Cꢀ4), 120.2 (Cꢀ3), 120.1 (Cꢀ6),
111.3 (Cꢀ8), 24.6 (CH3).
Methanesulfonamide analogue MnK2ꢀ13 was prepared in a similar manner, starting from 1ꢀ
bromocarbazole, by Suzuki crossꢀcoupling with 2ꢀaminophenylboronic acid, followed by Nꢀ
sulfonylation with methanesulfonyl chloride. For details see Supplemental Information. The synthesis
of the inactive analogue MnK2ꢀ68, a novel approach, including SEMꢀprotection of 1ꢀbromocarbazole,
Masuda borylation at Cꢀ1, Suzuki crossꢀcoupling with 4ꢀaminoꢀ3ꢀbromopyridine, and subsequent Nꢀ
acetylation and SEM deprotection was worked out. For details, see Supplemental Information.
Laser micro-irradiation assays
Laser microꢀirradiation was performed as described previously 38. Uꢀ2 OS wildꢀtype or PARP1-/- cells
were preꢀsensitised for 24 h with 10 ꢁM BrdU (Sigma), then the media was changed to Live Cell
Imaging Solution (Thermo Fisher) before laser microꢀirradiation and imaging. Laser microꢀirradiation
was performed on an Olympus Fluoview FV1200 confocal microscope equipped an inverted IX83
motorised stage with a 37 ºC humidified chamber and 60x/1.40 oil UPlanSApo objective and 405 nm
laser.
Acknowledgements:
The authors are grateful for financial support by the SGC, 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 through Ontario Genomics Institute,
Innovative Medicines Initiative (EU/EFPIA) [ULTRAꢀDD grant no. 115766], Janssen, Merck & Co.,
Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo
Research FoundationꢀFAPESP, Takeda, the Centre of Excellence (CEF) Macromolecular Complexes
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at Frankfurt University, and the Wellcome Trust. Funding for M.S. was also provided by the EPSRC
(award reference: 1368548) and Novartis Pharma AG through the Systems Approaches to Biomedical
Sciences – Industrial Doctoral Centre (SABSꢀIDC) programme of the University of Oxford.
We very much would like to thank Novartis Institutes for BioMedical Research (Basel) for providing
the resources for the macrodomain highꢀthroughput screen and are grateful for support and helpful
advice in HTS performance and data analysis provided by J. Trappe and team members (NIBR,
Basel). Furthermore, we would like to thank S. Jacob, C. Scheufler and H. Widmer (NIBR, Basel) for
supporting the project. We thank N. Domage (NIBR, Basel) for generating the PAMPA data. We are
grateful to O. Fedorov and team members (SGC, Oxford) for assistance with the macrodomain assay
development. We thank Diamond Light Source for access to the beamline I02 for data collection and
processing.
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Supporting information comprising additional experimental data methods and figures is available
online: This material is available free of charge via the internet at http://pubs.acs.org.
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