Synthesis and Evaluation of a Mitochondria-Targeting Poly(ADP-ribose) Polymerase-1 Inhibitor

Article abstract of DOI:10.1021/acschembio.8b00423

The poly(ADP-ribose) polymerase (PARP) family of enzymes plays a crucial role in cellular and molecular processes including DNA damage detection and repair and transcription; indeed, PARP inhibitors are under clinical evaluation as chemotherapeutic adjunc

Full text of DOI:10.1021/acschembio.8b00423

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Article
Synthesis and Evaluation of a Mitochondria-
Targeting Poly(ADP-ribose) Polymerase-1 Inhibitor
Tanja Krainz, Andrew Lamade, Lina Du, Taber Maskrey, Michael Calderon, Simon
Watkins, Michael Epperly, Joel S. Greenberger, Hulya Bayir, Peter Wipf, and Robert Clark
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00423 • Publication Date (Web): 05 Sep 2018
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Synthesis and Evaluation of a Mitochondria-Targeting Poly(ADP-ribose) Polymerase-1
Inhibitor
†
Tanja Krainz, Andrew M. Lamade, Lina Du, Taber S. Maskrey, Michael Calderon,
,ǁ
‡,ǁ
‡
†
¶
¶
Simon C. Watkins, Michael W. Epperly, Joel S. Greenberger, Hülya Bayır,*
€
€
, ‡,§,¥
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Peter
,†
Wipf* and Robert S. B. Clark,*
, ‡,¥
†
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA
Department of Critical Care Medicine, Safar Center for Resuscitation Research, University
‡
of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA
¶
Department of Cell Biology, Center for Biologic Imaging, University of Pittsburgh,
Pittsburgh, Pennsylvania 15260, USA
€
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh,
Pennsylvania 15232, USA
§
Department of Environmental and Occupational Health, Center for Free Radical and
Antioxidant Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15224, USA
¥
Children’s Neuroscience Institute, Department of Pediatrics, Children's Hospital of
Pittsburgh of UPMC, University of Pittsburgh School of Medicine; University of Pittsburgh,
Pittsburgh, Pennsylvania 15224, USA
ǁAuthors contributed equally
*
Correspondence: Hülya Bayır, M.D., bayihx@ccm.upmc.edu
Robert S. B. Clark, M.D., clarkrs@ccm.upmc.edu
Peter Wipf, Ph.D., pwipf@pitt.edu
Keywords: ABT-888; Hypoxia-ischemia; mtPARP; PARP; Veliparib; XJB-veliparib
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Abstract
The poly(ADP-ribose) polymerase (PARP) family of enzymes plays a crucial role in cellular
and molecular processes including DNA damage detection and repair, and transcription;
indeed, PARP inhibitors are under clinical evaluation as chemotherapeutic adjuncts given
their capacity to impede genomic DNA repair in tumor cells. Conversely, overactivation of
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PARP can lead to NAD depletion, mitochondrial energy failure, and cell death. Since PARP
activation facilitates genomic but impedes mitochondrial DNA repair, non-selective PARP
inhibitors are likely to have opposing effects in these cellular compartments. Herein, we
describe the synthesis and evaluation of the mitochondria-targeting PARP inhibitor, XJB-
veliparib. Attachment of the hemigramicidin S pentapeptide isostere for mitochondrial
targeting using a flexible linker at the primary amide site of veliparib did not disrupt PARP
affinity or inhibition. XJB-veliparib was effective at low nanomolar concentrations (10-100
nM) and more potent than veliparib in protection from oxygen glucose deprivation (OGD) in
primary cortical neurons. Both XJB-veliparib and veliparib (10 nM) preserved mitochondrial
+
+
NAD after OGD; however, only XJB-veliparib prevented release of NAD into cytosol.
XJB-veliparib (10 nM) appeared to inhibit poly(ADP-ribose) polymer formation in
mitochondria and preserve mitochondrial cytoarchitecture after OGD in primary cortical
neurons. After 10 nM exposure, XJB-veliparib was detected by LC-MS in mitochondria- but
not nuclear-enriched fractions in neurons and was observed in mitoplasts stripped of the outer
mitochondrial membrane obtained from HT22 cells. XJB-veliparib was also effective at
preventing glutamate-induced HT22 cell death at micromolar concentrations. Importantly, in
2 2
HT22 cells exposed to H O to produce DNA damage, XJB-veliparib (10 ꢀM) had no effect
on nuclear DNA repair, in contrast to veliparib (10 ꢀM) where DNA repair was retarded.
XJB-veliparib and analogous mitochondria-targeting PARP inhibitors warrant further
evaluation in vitro and in vivo, particularly in conditions where PARP overactivation leads to
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mitochondrial energy failure and maintenance of genomic DNA integrity is desirable, e.g.
ischemia, oxidative stress, and radiation exposure.
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INTRODUCTION
Poly(ADP-ribosyl)ation (PARylation) plays a crucial role in cellular and molecular processes
including DNA damage detection and repair, transcription, and the maintenance of genomic
1
integrity. The currently identified 17 members of the poly(ADP-ribose) polymerase (PARP)
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family induce the cleavage of NAD into nicotinamide and ADP-ribose moieties and mediate
their polymerization on target proteins, with links to cellular redox homeostasis,
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, 3
inflammatory, and metabolic networks. PARP1 binds to nuclear DNA single-strand break
(SSBs) sites and recruits repair proteins to the DNA, subsequently dissociating itself from the
SSB. The most potent small-molecule inhibitors, rather than just off-setting PARP1’s
enzymatic activity, trap it at the site of DNA damage and stabilize PARP–DNA complexes,
4
ultimately causing DNA double-strand breaks that require more complex repair mechanisms.
DNA damage induced by irradiation or oxidative stress leads to overactivation of
+
PARP1 and induces depletion of cellular NAD and ATP levels, leading to cell dysfunction
+
and necrotic cell death. The primary location of NAD is in mitochondria, where it is utilized
for oxidative phosphorylation. Furthermore, mitochondrial DNA (mtDNA) is constantly
being exposed to damaging species such as reactive oxygen and nitrogen species and is
efficiently repaired through at least a subset of the mechanisms involved in nuclear DNA
5
repair, including regulation by PARP1. However, opposing roles for PARP1 have been
observed in genomic versus mtDNA repair with PARP1 facilitating DNA repair in the
6
nucleus and impeding DNA repair in mitochondria. Overactivation of PARP1 and NAD
+
depletion has been linked to the pathogenesis of central nervous system (CNS) disorders,
including ischemia, traumatic brain injury (TBI), neuroinflammation, and neurodegenerative
diseases such as Alzheimer’s and Parkinson’s diseases, which have a pronounced
1
mitochondrial component. Preserving NAD levels may reverse mitochondrial dysfunction
, 7
+
8
and prevent energy failure. If suitable PARP inhibitors could be targeted exclusively to the
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mitochondria, they might reduce NAD depletion-associated energy failure while allowing
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repair of damaged DNA in the nucleus and facilitating mtDNA repair.
We have previously reported evidence for the localization of PARP in mitochondria
and mitochondria-specific PARylation by immunocytochemistry, electron microscopy, and
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by western blot analysis of purified mitochondrial fractions.
Furthermore, we
demonstrated that the inhibition of mtPARP preserved mitochondrial transmembrane
potential and reduced neuronal cell death triggered by oxidative stress or excitotoxicity.
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However, to the best of our knowledge, the hypothesis that mitochondria-targeted PARP-
inhibitors would be therapeutically effective in neurodegeneration and other CNS and non-
CNS conditions associated with energy failure, and a desire to maintain genomic integrity,
has not yet been addressed.
While there are broader therapeutic implications for synthetic PARP modulators,
research and development of poly(ADP-ribose) polymerase-1 (PARP1) inhibitors in cancer
research has intensified since the regulatory approval of olaparib and rucaparib for patients
with BRCA1/2 mutant ovarian cancer, and niraparib for recurrent gynecologic cancers (Figure
11-13
1).
Veliparib (1, 2-[(S)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide,
ABT-888) is a promising PARP1 inhibitor that has entered clinical phase I/II/III trials for
several forms of cancer, including breast cancer and solid tumor neoplasm, either as a single
agent or as a combination with chemotherapeutics. A phase III trial is currently ongoing to
assess the efficacy, safety and tolerability of veliparib in patients with previously untreated
14
ovarian cancer. The (S)- and (R)-enantiomers of veliparib have been shown to have
15
identical biological activity, and relative to other FDA approved PARP1 inhibitors veliparib
16
seems to be the least potent in terms of trapping PARP at the site of DNA damage.
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7-20
The XJB pentapeptide isostere is a mitochondrial targeting unit
derived from the
cyclopeptide antibiotic gramicidin S (GS). The XJB moiety has been used successfully to
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deliver various payloads to mitochondria, including nitroxides, a nitric oxide synthase
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(NOS) antagonist (AMT), and a derivative of the natural anticancer product β-lapachone.
In line with these previous studies, we used XJB to deliver the payload veliparib to
mitochondria. Here we show that XJB-veliparib is enriched in mitochondria, and that the
compound potently inhibits PARP-mediated cell death in multiple models of cytotoxicity.
XJB-veliparib is particularly effective in preventing cell death from hypoxia-ischemia in
neurons, warranting further evaluation and development of this class of mitochondria-
targeting PARP inhibitors for neurological diseases.
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RESULTS AND DISCUSSION
Mitochondria-targeting Veliparib
The chemical synthesis of the mitochondria-targeting XJB-veliparib hybrid molecule is
shown in Scheme 1 and fully described in the Methods. Since (S)- and (R)-enantiomers of
15
veliparib have shown identical biological activity, we utilized racemic veliparib for the
preparation of the XJB-linked hybrid molecule. Furthermore, we hypothesized that the
attachment of a flexible linker at the primary amide would be unlikely to disrupt veliparib’s
high PARP affinity since, while energetically not as favored, the amide could adopt a cis-
configuration and engage Gly429 in PARP with two hydrogen bonds, or, in the trans-amide
configuration, a hydrophopic alkyl linker chain could reinforce the single remaining amide
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carbonyl hydrogen bond to Gly429. We selected the XJB pentapeptide isostere as the
17-20
mitochondrial targeting sequence (Figure 2).
is a surrogate of the leucyl-
antibiotic gramicidin S (GS), and its side chain-protected ornithylvalylproline tripeptide
subunit is taken directly from GS. The -Phe-Pro sequence is based on the reverse turn
The alkene peptide isostere segment in XJB
D
-phenylalanine dipeptide in the bacterial membrane-associating
D
inducing sequence of GS and folds into a type II′ β-turn structure that buries several polar
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4-27
amide groups inside the molecule and thus may facilitate membrane transport.
This
moiety has previously been used in combination with a nitroxide payload to generate XJB-5-
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31, a reactive oxygen species (ROS) scavenger that validated the targeting design and was
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found to be ca. 600-fold enriched in mitochondria over the cytosol. XJB-5-131 has shown
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8-30
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TBI, ischemia-
in vivo efficacy in rodent models of Huntington’s disease (HD),
32, 33
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and hemorrhagic shock. In the radiation protector XJB-AMT, a
reperfusion injury,
nitric oxide synthase (NOS) antagonist (AMT) was conjugated to the targeting sequence,
with the goal to counteract the activation of mitochondrial NOS by ionizing radiation, which
can lead to inhibition of the respiratory chain, a burst of superoxide and peroxynitrite, and
21
cellular damage. XJB-Lapachone introduced a derivative of the natural anticancer
compound β-lapachone into mitochondria and triggered extensive cellular vacuolization and
2
2
autophagy, as well as stimulating ROS generation in mitochondria.
XJB-veliparib and Veliparib PARP1 Enzyme Inhibition ex vivo
To determine whether the linkage to the mitochondria-targeting moiety on 15 affected
PARP1 inhibition, we tested the capacity of veliparib and XJB-veliparib to inhibit active
PARP1 enzyme (10 U) ex vivo. PARP1 inhibition was similar between untargeted veliparib
and the XJB-veliparib conjugate 15 (Figure 3; experiments performed in triplicate), and is
35
consistent with the reported K of veliparib of 5.2 nmol/L.
i
XJB-veliparib and Veliparib Cytotoxicity in Primary Cortical Neurons
To investigate the biological properties and potential cytotoxicity of XJB-veliparib, we
exposed rat primary cortical neurons to varying concentrations of XJB-veliparib and
veliparib. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release from dying
neurons at 24 h. Both XJB-veliparib and veliparib showed a concentration-dependent
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cytotoxicity profile; however, XJB-veliparib was significantly less toxic compared with
unconjugated veliparib (Figure 4; n = 6/group; mean ± standard deviation [SD]; *P < 0.05).
Neurotoxicity defined as >10% cell death was seen with veliparib at 1 ꢀM concentration, vs.
a 10 ꢀM concentration required for XJB-veliparib. Significant cytotoxicity has previously
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been reported when leukemia cells are exposed to micromolar concentrations of veliparib.
Cytotoxicity produced by PARP inhibitors in clinical use, including veliparib, have been
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associated with inability to efficiently repair DNA damage and genomic instability.
However, recent evidence further points to trapping of PARP1 enzyme itself in double-strand
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DNA (dsDNA) breaks by PARP1 inhibitors. Trapped PARP-dsDNA complexes retain
catalytic activity and enhance genotoxicity and lethality of chemotherapeutic agents.
Relevant here, veliparib concentrations of > 100 µM are tumoricidal in most cancer cell
3
8
lines. Importantly, mitochondria-targeting PARP inhibitors would avoid formation of
trapped PARP-dsDNA complexes, and accordingly may have lower toxicity and a therapeutic
advantage where prevention of cell death is desired.
Effect of XJB-veliparib and Veliparib after Oxygen-glucose Deprivation in Primary
Cortical Neurons
To determine whether mitochondria-targeting XJB-veliparib can promote neuronal survival
in ischemia-like conditions in vitro, we subjected primary cortical neurons at 12 DIV to
33
oxygen-glucose deprivation (OGD). Cultured neurons were exposed to a hypoxic and
glucose-depleted environment for 2 h, followed by normal culture conditions for 24 h to
mimic ischemia/reperfusion injury. As shown in Figure 5a, treatment with XJB-veliparib
significantly attenuated OGD-induced cell death at low nanomolar concentrations.
Specifically, 10 nM XJB-veliparib reduced cell death by 65% (LDH release 12.1 ± 3.1% vs.
34.2 ± 4.0%, 10 nM XJB-veliparib vs. vehicle; n = 30/group; mean ± SD; *P < 0.05).
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Treatment with concentrations >100 nM of either veliparib or XJB-veliparib conjugate
provided no additional protection. Both XJB-veliparib and non-targeting veliparib appear
more protective against OGD compared with other published PARP1 inhibitors, where
39, 40
protection is observed in micromolar ranges.
In vivo, PARP1 inhibition is highly
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effective at reducing neuronal death caused by ischemia-reperfusion injury.
To determine whether XJB-veliparib and veliparib related neuroprotection was
+
+
associated with preservation of intracellular NAD , NAD concentrations in mitochondria-
and cytosol-enriched fractions were measured 24 h after OGD in primary cortical neurons.
+
Ten nM XJB-veliparib or non-targeting veliparib preserved mitochondrial NAD stores after
OGD (Fig. 5b; n = 6/group; mean ± SD; *P < 0.05 vs. vehicle). However, XJB-veliparib, but
+
not veliparib, also prevented efflux of NAD from the mitochondria to the cytosol after OGD
(**P < 0.05 vs. vehicle and veliparib). These results are consistent with preservation of
+
mitochondrial NAD as one of the mechanisms behind PARP-mediated neuroprotection after
OGD.
To evaluate the impact of XJB-veliparib on mitochondrial structure after OGD, we
used stimulation emission depletion (STED) microscopy and immunohistochemistry. Primary
rat cortical neurons were treated with 10 nM XJB-veliparib or vehicle and then exposed to
OGD for 2 h. Vehicle treated neurons showed increased PARP activation at 1 h, assessed by
immunofluorescence staining with anti-PAR antibody, and swollen, circular mitochondria
(labelled with translocase of outer mitochondrial membrane (OMM) 20 [TOMM20]),
consistent with fission vs. coiling and compaction compared with control (no ischemia)
neurons (Figure 5c; performed in triplicate). In contrast, PAR immunoreactivity was reduced
in neurons treated with XJB-veliparib (vs. vehicle) and mitochondrial architecture appeared
partially preserved. The relative increase in PAR immunoreactivity observed in mitochondria
vs. nuclei in neurons after OGD may be explainable by more effective PAR metabolism by
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poly(ADP-ribose) glycohydrolase in cell nuclei vs. ADP-ribosylhydrolase 3 in
42
mitochondria.
To verify mitochondrial enrichment at a therapeutic dose, rat primary cortical neurons
were exposed to 10 nM XJB-veliparib for 24 h. Mitochondria- and nuclear-enriched
subcellular fractions were obtained as described and verified by western blot using antibodies
against the cytochrome oxidase subunit Vα (COX Vα) and histone H3, respectively (Figure
0
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5d). Liquid chromatography-mass spectrometry (LC-MS) analysis of XJB-veliparib showed
a concentration of 40.5 ± 15.8 pmol/10 ꢀg protein in mitochondria-enriched fractions (Figure
5d; mean ± SD; n = 3 independent experiments). XJB-veliparib was undetectable in nuclear-
enriched fractions.
Detection of XJB-veliparib in Mitoplasts
To verify that XJB-veliparib detected in mitochondria-enriched fractions shown in Figure 5d
was not simply associated with the OMM, we isolated mitoplasts stripped of the OMM
obtained from HT22 cells exposed to 10 nM XJB-veliparib for 45 min. Mitochondria were
isolated using differential centrifugation following a standardized protocol (ThermoFisher
#89874). The OMM was stripped from the inner mitochondrial membrane (IMM)/matrix
using digitonin (0.15 – 1.5 mg/mL). The purity of the mitoplast containing fractions was
verified by the absence of translocase of the outer mitochondrial membrane 40 (TOM40) and
presence of manganese superoxide dismutase (MnSOD) observed on western blot. LC-MS
analysis of XJB-veliparib showed a concentration of 62.6 ± 16.3 pM in mitoplast fractions
(Figure 6; mean ± SD; n = 8 samples from 3 independent experiments). For comparison, the
concentration of XJB-veliparib in whole mitochondria was 85.1 ± 5.3 pM in HT22 cells after
a 45 min exposure to 10 nM XJB-veliparib (n = 3).
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Effect of XJB-veliparib and Veliparib after Excitotoxic Injury in Primary Cortical
Neurons and Hippocampal Neuronal Cells
Previous studies have shown an important role for nuclear PARP1 activation in inhibiting
43
excitotoxic neuronal death in vitro. In addition, PARP1 inhibition in vivo is effective at
1
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reducing N-methyl-D-aspartate (NMDA) but not non-NMDA excitotoxicity. To determine
whether XJB-Veliparib was also effective in attenuating excitotoxic cell death in vitro,
primary cortical neurons were exposed to 10 ꢀM L-glutamate and 10 ꢀM glycine with
varying concentrations of XJB-Veliparib or naked veliparib for 24 h as previously described.
PARP inhibition reduced cell death after glutamate/glycine exposure in a concentration-
dependent manner (Fig. 7a; n = 6/group; mean ± SD; *P < 0.05 vs. vehicle). Differing from
9
in vitro ischemia/reperfusion, veliparib was more potent than XJB-veliparib in reducing LDH
release. The underlying basis for the differential effectiveness of targeting versus non-
targeting PARP inhibitors in models of OGD versus excitotoxicity is beyond the scope of the
present study. However, one possible explanation is that since glutamate stimulates DNA
repair via activation of transcription factors including cyclic AMP response element binding
45
protein and upregulation of apurinic endonuclease 1, any impact of non-targeting PARP
inhibitors on nuclear DNA repair and formation of trapped PARP-dsDNA complexes may be
mitigated.
In addition to rat primary cortical neuron cultures, we also determined the effect of
veliparib and XJB-veliparib in a stable mouse hippocampal neuronal cell line HT22, in which
46
ferroptotic cell death is induced by high concentrations of glutamate. HT22 cells grown to
confluence were exposed to 5 mM glutamate and various concentrations of veliparib or XJB-
veliparib with LDH release measured at 24 h. Both veliparib and XJB-veliparib similarly
inhibit glutamate-induced ferroptosis in HT22 cells, with XJB-veliparib slightly more
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effective than veliparib at higher (10 µM) concentrations (Fig. 7b; n = 5/group; mean ± SD;
*P < 0.05 vs. vehicle).
Effect of XJB-veliparib and Veliparib on DNA Repair in Hippocampal Neuronal Cells
Exposed to H O
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To determine whether XJB-veliparib and/or veliparib impacted DNA repair, we evaluated
DNA damage serially in HT22 cells exposed to 200 ꢀM H using the comet tail intensity
produced DNA damage with evidence of
2
O
2
47
2 2
assay as previously described. Exposure to H O
DNA repair over 30 min (Fig. 8). Veliparib, but not XJB-veliparib (both 10 ꢀM), impeded
DNA repair (Fig. 8; *P < 0.05 vs. veliparib; **P < 0.05 vs. vehicle; n = 52-60 cells/group;
mean ± SEM). These data suggest that mitochondria-targeting PARP inhibitors at a dose that
protects HT22 cells from glutamate toxicity (Fig. 7b) do not impact repair of damaged DNA.
Role for Mitochondria-targeting PARP Inhibitors
An undisputed role for mitochondrial PARP, and in particular mitochondrial PARP1, in
48
health and disease is controversial; however, at least a partial role for mitochondrial PARP
5
in mtDNA repair and cellular metabolism is becoming more apparent. Several early studies
4
have shown the presence of ADP-ribosylation in mitochondria from testes, liver,
9
50, 51
and
52
brain. More recently, inhibition of mitochondrial PARP1 using genetic approaches was
shown to increase the efficiency of mtDNA base excision repair via interaction with
6
exo/endonuclease G and DNA polymerase γ; as opposed to inhibition of nuclear PARP1
53
which decreases the efficiency of genomic DNA repair. Here we show that mitochondria-
2 2
targeting XJB-veliparib has no effect on nuclear DNA repair after H O exposure in neuronal
cells, in contrast to non-targeting veliparib (Fig. 8).
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PARP1 inhibition has been shown to increase mitochondrial metabolism via silent
5
mating type information regulation 2 homolog 1 (SIRT1), and directly preserve
4
+ 55
mitochondrial function and prevent cell death via maintenance of NAD stores. Here we
show that mitochondria-targeting XJB-veliparib is very effective at preserving mitochondrial
1
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NAD levels and preventing efflux of NAD into cytosol in neurons after OGD (Fig. 5b). We
+
1 0
have previously reported PARylation of the electron transport chain proteins F F ATPase,
cytochrome c oxidase, and cytochrome c reductase, and preservation of respiration in isolated
brain mitochondria under conditions of nitrosative stress with PARP inhibition or addition of
10
poly(ADP-ribose) glycohydrolase. In addition, we have shown that mitochondrial PARP
inhibition using PARP1 gene deletion or relatively selective PARP1 inhibitors protects
9
primary cortical neurons and fibroblasts from oxidative stress and excitotoxicity.
Powerful neuroprotection using non-mitochondria targeting PARP inhibitors or PARP
4
1
56, 57
gene deletion has been demonstrated in pre-clinical models of stroke, TBI,
and
42, 58
neurodegenerative disease.
In these and other CNS diseases, an important role for
mitochondrial energy failure is implicated. XJB-veliparib (and similar compounds) that
concentrate in mitochondria would be expected to directly inhibit mitochondrial PARP,
+
reduce NAD depletion, prevent energy failure, and reduce cell death while having minimal
impact on genomic DNA repair and integrity (please refer to Abstract Graphic). Furthermore,
6
XJB-veliparib (and similar compounds) may also enhance mtDNA repair when used to treat
these diseases. Mitochondria-targeting PARP inhibitors may also reduce PARylation of
apoptosis-inducing factor (AIF), potentially mitigating AIF release from mitochondria,
59
translocation of AIF into nuclei, and parthanatos-related cell death.
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Conclusions
Mitochondria-targeting PARP inhibitors may have unique and important advantages over
PARP inhibitors that are non-selective to cellular compartments, including directly
preserving mitochondrial function and viability and preventing mitochondrial energy failure.
Moreover, mitochondria-targeting PARP inhibitors could have clinical efficacy in diseases
where the pathophysiology includes mitochondrial dysfunction and/or energy failure, but
efficient nuclear DNA repair is desired—such as ischemia-reperfusion injury, radiation
toxicity, and other diseases involving oxidative stress. As PARP inhibitors are being
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reconsidered for many clinical indications, further development and in vitro and in vivo
evaluation of mitochondria-targeting PARP inhibitors appears warranted.
METHODS
Synthesis of XJB-veliparib
For the synthesis of mitochondrially targeted veliparib, XJB-Veliparib, N-Cbz-L-proline
methyl ester (7) was treated with NaHMDS and MeI, followed by saponification, to give the
acid 9 in 98% yield over 2 steps (Scheme 1). One-pot EDCl coupling to methyl 2,3-
diaminobenzoate (10) and acid-catalyzed cyclization provided the benzimidazole 11 in good
overall yield. Hydrolysis of the methyl ester followed by acylation with the N-Boc-1,3-
diaminopropane spacer group afforded 12. Removal of the Cbz-group via hydrogenation
followed by Boc-deprotection provided 13, which was coupled to the mitochondrial targeting
61
sequence 14 to afford the desired XJB-Veliparib conjugate 15.
General Experimental Conditions. All moisture- and air-sensitive reactions were
performed in oven dried glassware under a positive pressure of argon. All reagents and
solvents were used as received unless otherwise specified. THF and Et O were distilled over
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sodium/benzophenone ketyl; CH
2
Cl
2
2
was distilled over CaH , MeCN and DMF were dried
over molecular sieves. Reactions were monitored by TLC analysis (pre-coated silica gel 60
254 plates, 250 µm layer thickness) and visualization was accomplished with a 254/280 nm
UV light and/or by staining with KMnO solution (1.5 g KMnO and 1.5 g K CO in 100 mL
F
4
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of a 0.1% NaOH solution), a ninhydrin solution (2 g ninhydrin in 100 mL EtOH), a PMA
solution (5 g phosphomolybdic acid in 100 mL EtOH), or a p-anisaldehyde solution (2.5 mL
2 4
p-anisaldehyde, 2 mL AcOH and 3.5 mL conc. H SO in 100 mL EtOH). Flash
chromatography was performed on silica gel (40-63 µm). Melting points were determined on
a Mel-Temp II capillary melting point apparatus fitted with a Fluke 51 II digital thermometer.
Infrared spectra were recorded on an ATR spectrometer. NMR spectra were recorded on 300,
400, 500 or 700 MHz instruments. Chemical shifts were reported in parts per million (ppm)
1
and referenced to residual solvent. H NMR spectra are tabulated as follows: chemical shift,
multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet),
13
coupling constant(s), number of protons. C NMR spectra were obtained using a proton-
decoupled pulse sequence and are tabulated by observed peak. LC-MS analyses were
performed on a Shimadzu UFLC instrument equipped with an Applied Biosystem MDS
SCIEX API 2000 mass spectrometer (ESI), under the following conditions: column: Varian
Polaris C18-A (100 x 4.6 mm, 5µm) equilibrated at 40 °C; buffer A: 0.1% aqueous AcOH,
buffer B: 0.1% AcOH in MeCN; 30 min gradient: 5% buffer B in buffer A for 1 min, then 5
to 95% buffer B in buffer A over 13 min, then 95% buffer B in buffer A for 4 min, then 95-
5% buffer B in buffer A over 7 min, then 5% buffer B in buffer A for 5 min; flow rate: 0.2
mL/min; detection: TIC and/or UV λ = 254/280 nm. Intracellular distribution and HRMS data
were obtained on a Thermo Scientific Exactive Orbitrap LC-MS (ESI positive ion mode)
coupled to a Thermo Scientific Accela HPLC system using a 3.5 ꢀM Water XTerra C18
column (2.1 x 50 mm; 10 min gradient elution with MeCN/H O/ MeOH containing 0.1%
2
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formic acid at a flow rate of 500 ꢀL/min from 3:92:5 at 0-0.5 min to 93:2:5 at 4.0 min, back
to 3:92:5 from 6.0 to 7.5 min).
1-Benzyl 2-methyl 2-methylpyrrolidine-1,2-dicarboxylate (8). To a -78 °C solution of N-
Cbz-L-proline 7 (0.43 mL, 1.9 mmol) and iodomethane (0.24 mL, 3.8 mmol) in THF (3.5
mL) was added dropwise NaHMDS (1 M in THF, 3.8 mL, 3.8 mmol). The resulting mixture
0
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9
0
was warmed to -20 °C, stirred for 3 h, quenched with H
extracted with EtOAc (3 x). The combined organic layers were washed with brine (1x), dried
over MgSO , filtered and concentrated to dryness. The residue was purified by
chromatography on SiO (30% EtOAc/hexanes) to give 8 as a pale yellow oil (0.52 g, 99%).
2
O, acidified with 2N HCl and
4
2
54 1
Spectral data of the mixture of rotamers are in accordance with literature values. H NMR
300 MHz, CDCl ) δ 1.54 (s, 1.5 H), 1.61 (s, 1.5 H), 1.86-1.99 (m, 3 H), 2.13-2.24 (m, 1 H),
.46 (s, 1.5 H), 3.56-3.69 (m, 2 H), 3.71 (s, 1.5 H), 4.99-5.23 (m, 2 H), 7.24-7.40 (m, 5 H).
(
3
3
1-((Benzyloxy)carbonyl)-2-methylpyrrolidine-2-carboxylic acid (9). Methyl ester 8 (1.53
g, 5.52 mmol) was dissolved in THF (10.5 mL) and H O (5.4 mL) and treated with a solution
2
2
of LiOH (265 mg, 11.0 mmol) in H O (5.4 mL). MeOH (1.5 mL) was then added and the
resulting homogenous solution heated to 60 °C overnight. The organic solvents were
removed and the aqueous layer acidified to pH 2 using 2 N HCl and extracted with EtOAc
4
(3x). The combined organic layers were washed with water (1x), dried (MgSO ) filtered and
concentrated to dryness to give 9 as a colorless solid (1.42 g, 98%). Spectral data of the
54 1
mixture of rotamers are in accordance with literature values. H NMR (500 MHz, DMSO-
6
d ) δ 1.44 (s, 1.5 H), 1.45 (s, 1.5 H), 1.80-1.96 (m, 3 H), 2.02-2.19 (m, 1 H), 3.43-3.53 (m, 2
H), 4.94-5.11 (m, 2 H), 7.24-7.41 (m, 5 H), 12.51 (br s, 1 H).
Methyl 2-(1-((benzyloxy)carbonyl)-2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-
carboxylate (11). To a solution of 9 (200 mg, 0.760 mmol) and methyl diaminobenzoate 10
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(189 mg, 1.14 mmol) in CH
2
Cl
2
(15 mL) was added DIPEA (0.13 mL, 0.76 mmol) followed
by addition of EDCI (218 mg, 1.14 mmol), HOAt (155 mg, 1.14 mmol) and DMAP (9.3 mg,
.076 mmol), and the resulting solution was stirred at room temperature overnight. The
reaction mixture was quenched with sat. aqueous NH Cl solution and extracted with CH Cl
0
4
2
2
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(2x). The combined organic layers were washed with brine (1x), dried (MgSO ), filtered and
4
concentrated to give the product as a brown oil. The crude residue was redissolved in AcOH
(5 mL) and heated at reflux for 2 h. The solvent was evaporated, the crude residue poured
3
onto sat. aqueous NaHCO and extracted with EtOAc (3x). The combined organic layers
were washed with brine (1x), dried (MgSO ) and concentrated. The crude residue was
4
purified via chromatography on SiO (30-90% EtOAc/hexanes) to give 11 as a pale yellow
2
+
oil (198 mg, 65% over 2 steps): HRMS (ESI ) m/z calcd for C22
H
24
N
3
O
4
[M+H] 394.1761,
found 394.1758.
2-(1-((Benzyloxy)carbonyl)-2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxylic
acid. Methyl ester 11 (280 mg, 0.712 mmol) was dissolved in THF/H O (14:1, 21 mL) and
2
cooled to -5 °C. A 40% n-Bu NOH (aqueous) solution (4.6 mL, 7.1 mmol) was added slowly
4
and the reaction mixture was stirred for 30 min at -5 °C and at room temperature over night.
The solution was acidified with aqueous AcOH and extracted with EtOAc (3x). The
4
combined organic layers were washed with brine, dried (MgSO ), filtered and concentrated
under reduced pressure. The crude residue was purified by chromatography on SiO (0-10%
2
MeOH/CH Cl ) to give 2-(1-((benzyloxy)carbonyl)-2-methylpyrrolidin-2-yl)-1H-
2
2
1
benzo[d]imidazole-4-carboxylic acid as a pale-yellow foam (215 mg, 80%): H NMR (400
MHz, DMSO-d ) δ 7.86 (br s, 1 H), 7.78 (d, J = 7.5 Hz, 1 H), 7.41 – 7.36 (m, 4 H), 7.33 –
.30 (m, 1 H), 7.30 – 7.25 (m, 1H), 7.06 (t, J = 7.4 Hz, 0.5 H), 6.91 (t, J = 7.0 Hz, 1H), 6.70
br s, 1 H), 5.15 – 5.09 (m, 0.5 H), 5.07 – 5.04 (m, 1H), 3.87 – 3.54 (m, 3 H), 2.76 (br s, 1 H),
6
7
(
13
1.92 (s, 3 H), 1.87 (br s, 2 H), 1.86 (br s, 1 H); C-NMR (700 MHz, DMSO-d ) δ 167.5,
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160.5, 159.8, 154.4, 154.1, 137.4, 136.8, 128.9, 128.2, 127.9, 127.7, 127.1, 124.4, 124.3,
121.7, 66.3, 66.2, 62.9, 55.4, 48.8, 48.3, 42.7, 40.5, 24.4, 24.2, 22.9, 22.4; IR (ATR, neat)
-
1
+
682.8, 1407.6, 1351.5, 1255.8, 746.4 cm ; Mp 102.3 – 104.6 °C; HRMS (ESI ) m/z calcd
1
22 3 4
for C21H N O
[M+H] 380.1605, found 380.1610.
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8
9
0
Benzyl 2-(4-((3-((tert-butoxycarbonyl)amino)propyl)carbamoyl)-1H-benzo[d]imidazol-2-
yl)-2-methylpyrrolidine-1-carboxylate (12). To a solution of N-Boc-1,3-propanediamine
(0.28 mL, 1.6 mmol) and 2-(1-((benzyloxy)carbonyl)-2-methylpyrrolidin-2-yl)-1H-
benzo[d]imidazole-4-carboxylic acid (342 mg, 1.05 mmol) in CH Cl (12 mL) at 0 °C was
2
2
added DIPEA (0.46 mL, 2.6 mmol), followed by the dropwise addition of T P (50 wt% in
3
EtOAc, 0.92 mL, 1.6 mmol). The resulting mixture was stirred at room temperature
overnight, washed with 5% aqueous Na
2
CO
3
and brine, dried (MgSO
4
), filtered and
2 2
(100% CH Cl to
concentrated. The crude residue was purified via chromatography on SiO
2
1
) to give 12 as a pale-yellow foam (503 mg, 89%): H NMR (600 MHz,
1
0% MeOH/CH
2
Cl
2
DMSO-d ) δ 12.76 (br s, 0.5 H), 12.69 (br s, 0.5 H), 9.93 (br s, 1H), 7.86 – 7.80 (m, 1 H),
6
7.64 (d, J = 7.7 Hz, 0.5 H), 7.60 (d, J = 7.7 Hz, 0.5 H), 7.39 – 7.34 (m, 2 H), 7.33 – 7.27 (m,
2
3
2
H), 6.94 – 6.81 (m, 2 H), 6.73 – 6.65 (m, 1 H), 5.09 – 4.97 (m, 1 H), 4.94 – 4.79 (m, 1 H),
.86 – 3.77 (m, 1 H), 3.70 – 3.60 (m, 1 H), 3.47 – 3.37 (m, 2 H), 3.11 – 3.00 (m, 2 H), 2.28 –
.10 (m, 2 H), 2.02 – 1.94 (m, 2 H), 1.91 (br s, 1.5 H), 1.89 (br s, 1.5 H), 1.73 – 1.65 (m, 3
1
3
H), 1.38 (s, 9 H); C-NMR (600 MHz, DMSO-d ) δ 165.1, 160.4, 160.2, 156.1, 154.1, 153.7,
6
140.7, 140.6, 137.5, 136.7, 135.3, 135.2, 128.8, 128.2, 127.8, 127.7, 127.0, 122.5, 122.4,
122.3, 122.2, 122.1, 115.2, 115.1, 77.9, 66.2, 62.6, 62.2, 55.4, 49.0, 48.2, 43.4, 42.1, 38.2,
36.9, 30.3, 30.3, 28.7, 24.4, 23.3, 23.0, 22.5 (mixture of rotamers); Mp 112.7 – 116.2 °C;
+
HRMS (ESI ) m/z calcd for C29
H
38
N
5
O
5
[M+H] 536.2867, found 536.2867.
tert-Butyl (3-(2-(2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-
carboxamido)propyl)carbamate. A mixture of 12 (500 mg, 0.933 mmol) and 10% Pd/C (99
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mg, 0.093 mmol) in MeOH (5 mL) in a high-pressure hydrogenator was purged with argon
and flushed with hydrogen (3x). The pressure was set to 6 bar and the mixture was stirred at
this pressure at room temperature overnight. The solution was filtered through a plug of
Celite® and washed with CH Cl . The filtrate was concentrated and the crude residue was
2
2
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
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2
2
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0
1
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1
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purified via chromatography on SiO (100% CH Cl to 10% MeOH/CH Cl ) to give tert-
2
2
2
2
2
butyl (3-(2-(2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-
1
carboxamido)propyl)carbamate as a white foam (375 mg, 85%): H NMR (600 MHz,
DMSO-d ) δ 12.50 (br s, 2 H), 9.94 (br s, 1 H), 7.78 (d, J = 7.5 Hz, 1 H), 7.62 (d, J = 7.5 Hz,
H), 7.25 (t, J = 7.7 Hz, 1 H), 6.92 – 6.87 (m, 1 H), 3.45 – 3.38 (m, 2 H), 3.12 – 3.04 (m,
6
1
3
1
7
1
H), 2.93 – 2.86 (m, 1 H), 2.45 – 2.38 (m, 1 H), 1.89 – 1.80 (m, 2 H), 1.72 – 1.64 (m, 3 H),
13
.59 (br s, 3 H), 1.38 (s, 9 H); C-NMR (600 MHz, DMSO-d ) δ 165.3, 156.2, 122.1, 121.8,
6
9.7, 79.4, 79.2, 77.9, 62.6, 60.2, 55.4, 49.1, 46.4, 38.1, 36.8, 30.4, 28.7, 27.6, 25.7, 21.3,
4.6; IR (ATR, neat) 3269, 2973, 1694, 1645, 1612, 1523, 1406, 1365, 1245, 1166, 1046,
-
1
+
988, 758 cm ; Mp 79.8 – 83.2 °C; HRMS (ESI ) m/z calcd for C H N O [M+H] 402.2500,
21
32
5
3
found 402.2499.
N-(3-Aminopropyl)-2-(2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxamide
hydrochloride (13). To a solution of tert-butyl (3-(2-(2-methylpyrrolidin-2-yl)-1H-
2 2
benzo[d]imidazole-4-carboxamido)propyl)carbamate (32.8 mg, 0.0817 mmol) in CH Cl (1
mL) was added 4 M HCl in dioxane (0.2 mL, 0.8 mmol). The resulting mixture was stirred at
room temperature for 2 h. The product was filtered off, washed with hexanes, and the
resulting white solid (27.1 mg, 98%) was used in the next step without further purification:
+
HRMS (ESI ) m/z calcd for C16
H
24
N
5
O [M+H] 302.1975, found 302.1974.
tert-Butyl ((4S,7S,E)-7-benzyl-2-methyl-8-((2S)-2-(((2S)-3-methyl-1-(((8S)-15-(2-(2-
methylpyrrolidin-2-yl)-1H-benzo[d]imidazol-4-yl)-3,9,15-trioxo-1-phenyl-2-oxa-4,10,14-
triazapentadecan-8-yl)amino)-1-oxobutan-2-yl)carbamoyl)pyrrolidin-1-yl)-8-oxooct-5-
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en-4-yl)carbamate (“XJB-Veliparib”, 15). To a solution of XJB-acid 14 (36.9 mg, 0.0458
mmol) and 13 (23.2 mg, 0.0687 mmol) in DMF (0.92 mL, 0.05M) at 0 °C was added DIPEA
3
(40 µL, 0.23 mmol), followed by the dropwise addition of T P (50% in DMF, 35 µL, 0.060
mmol). The reaction mixture was stirred at 0 °C for 1 h, quenched with aqueous NH Cl, and
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extracted with CH Cl (3x). The combined organic layers were washed with 5% aqueous
2
2
LiCl solution (1x), dried (MgSO
pale yellow oil that was purified by chromatography on SiO
MeOH/CH Cl
4
), filtered and concentrated to give the crude product as a
2
2 2
(100% CH Cl to 15%
1
2
2
) to afford 15 as a colorless solid (18.5 mg, 37%): H NMR (400 MHz,
DMSO-d , 100 °C) δ 9.32 (br s, 1 H), 7.88 (d, J = 7.44 Hz, 1 H), 7.72 (d, J = 7.96 Hz, 1 H),
6
7.69 – 7.59 (m, 1 H), 7.47 – 7.38 (m, 1 H), 7.37 – 7.31 (m, 5 H), 7.30 – 7.26 ( m, 1 H), 7.25 –
7.12 (m, 5 H), 6.79 (br s, 1 H), 6.12 (d, J = 7.72 Hz, 1 H), 5.52 – 5.42 (m, 2 H), 5.02 (s, 2 H),
4.44 – 4.37 (m, 1 H), 4.30 – 4.23 (m, 1 H), 4.18 – 4.09 (m, 1 H), 3.93 – 3.84 (m, 1 H), 3.53 –
3.35 (m, 6 H), 3.29 – 3.21 (m, 3 H), 3.10 – 3.03 (m, 4 H), 2.72 – 2.00 (m, 3 H), 1.98 – 1.88
(m, 2 H), 1.84 (br s, 3 H), 1.80 – 1.71 (m, 5 H), 1.66 – 1.57 (m, 1 H), 1.55 – 1.43 (m, 3 H),
1
3
1
1
1
1
4
.38 (s, 9 H), 1.30 – 1.25 (m, 4 H), 0.90 – 0.78 (m, 12 H); C NMR (700 MHz, DMSO-d ) δ
6
72.7, 172.5, 172.2, 171.9, 171.8, 171.7, 171.4, 171.3, 171.2, 164.8, 156.6, 155.3, 155.2,
39.7, 139.5, 137.7, 135.0, 134.6, 129.6, 128.8, 128.4, 128.3, 128.2, 127.8, 126.4, 126.2,
22.8, 78.0, 77.8, 65.6, 64.9, 64.6, 59.7, 59.6, 59.3, 58.2, 53.1, 53.0, 50.7, 50.5, 49.3, 48.8,
7.2, 47.1, 45.5, 38.9, 38.2, 36.5, 36.4, 35.2, 32.4, 30.9, 30.7, 30.2, 29.8, 28.7, 26.5, 25.0,
24.7, 24.4, 24.3, 23.8, 23.0, 22.8; Mp 147.2 – 152.6 °C; IR (ATR, CH Cl ) 3291.7, 2955.8,
2
2
-
1
1
647.1, 1528.8, 1439.1, 1365.7, 1248.0, 1167.2, 1028.3, 758.2, 698.1 cm ; HRMS (ESI) m/z
85 10 9
calcd for C60H N O 1089.6496, found 1089.6495.
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PARP1 Activity
The capacity to inhibit PARP1 was determined using a commercial assay (Trevigen,
Gaithersburg, MD) as per manufacturer’s direction. Various concentrations of XJB-veliparib
+
or veliparib were added to histone-coated wells containing active PARP1 enzyme and NAD
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0
in surplus.
Cell Cultures
Primary cortical neuron-enriched cultures were prepared from 16-17 day old Sprague-Dawley
9
rat embryos as described. Dissociated cell suspensions were filtered through a 70 µm nylon
4
cell strainer and seeded in 96-well plates (5×10 cells/well) or on poly-
D-lysine coated glass
coverslips, and maintained in Neurobasal medium with B27 supplements (Life Technologies,
Carlsbad, CA). Experiments were performed 12 days in vitro (DIV).
HT22 cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM)
(
Invitrogen Inc., Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Thermo
Fisher Scientific, San Jose, CA) and 1% penicillin-streptomycin (ATCC, Manassas, VA) in
an atmosphere containing 5% CO . Cells were cultured for 24 to 48 h before use.
2
Separation of Subcellular Proteins
Separation of cytosol-, nuclear-, and mitochondria-enriched protein fractions was performed
6
2
using differential centrifugations as previously described. Samples were homogenized in
lysis buffer then centrifuged at 1025 g for 15 min at 4°C to separate nuclei. Pellets containing
nuclei were resuspended in buffer and centrifuged at 16,000 g at 4°C for 20 min with these
supernatants containing nuclear-enriched fractions. Supernatants after separation of nuclei
containing mitochondria and cytosolic proteins were then centrifuged at 20,000 g for 50 min
at 4°C to separate cytosol-enriched protein fractions. The pellets were lysed, sonicated until
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frothy, and re-suspended in lysis buffer then centrifuged at 16,000 g for 25 min at 4°C. These
mitochondria-enriched pellets were then washed in 5 ꢀM CaCl and centrifuged at 1,600 g for
10 min. Western blots were performed using antibodies against cytochrome c oxidase (BD
Biosciences, San Jose, CA) and histone H3 (Abcam, Cambridge, MA) to verify purity of
subcellular fractions.
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Isolation of Mitoplasts
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Mitochondria from HT22 cells (25 x 10 cells/mL) were isolated using differential
centrifugation following a standardized protocol using a commercial isolation kit (#89874;
ThermoFisher Scientific, San Jose, CA). A low spin speed (3,000 g) was used to collect only
heavy mitochondria for OMM removal. Mitochondria were resuspended in 325 ꢀL
mitochondrial isolation reagent C, then 100 ꢀL of suspension was centrifuged at 3,000 g. The
pellet was then digested with 0.15 – 1.5 mg/mL freshly dissolved digitonin in 125 ꢀL
volume. The samples were agitated at 750 rpm for 15 min at 4ºC, then digestion was stopped
by adding an equal volume of reagent C. Suspensions were then centrifuged at 10,000 g for
10 min at 4ºC with the pellet containing mitoplasts (IMM and matrix) and the supernatant
containing OMM and protein from the intermembrane space. The purity of the mitoplast
containing fractions was verified by the absence of TOM40 and presence of MnSOD on
western blot.
Quantification of XJB-veliparib
The mitochondrial fraction (50 ꢀL) or nuclear fraction (50 ꢀL), treated with XJB-veliparib
2 2
was added to a 5:1 ratio of CH Cl :MeOH (950 ꢀL) and vortexed (30 sec). Water (150 ꢀL)
was added and the solution was vortexed (15 sec) and set aside to equilibrate at room
temperature (30 min). The resulting suspension was placed in an Eppendorf Centrifuge 5702
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(4400 rpm, 20 °C) for 12 min. The organic layer was extracted and filtered through a 0.45 ꢀm
filter for analysis.
XJB-veliparib was quantified on a Thermo Scientific Exactive Orbitrap LC-MS (ESI
positive ion mode) coupled to a Thermo Scientific Accela HPLC system using a 3.5 ꢀM
1
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Water XTerra C18 column (2.1 x 50 mm; 20 min gradient elution with MeCN/H O
2
containing 0.1% formic acid at a flow rate of 500 ꢀL/min from 5:95 at 0-1.0 min to 95:5 at
12.0 min, back to 5:95 from 16.0 to 16.1 min). Calibration curves for XJB-veliparib were run
in duplicate from 102 nM to 5.7 nM. Samples (10 ꢀL) were injected in triplicate and Thermo
Xcalibur software was used to determine the concentration of XJB-veliparib in mitochondrial
and nuclear fractions (n = 3). The concentration was reported as pM concentration of XJB-
veliparib per 10 ꢀg of protein with corresponding standard deviation values.
Oxygen-glucose Deprivation
To model ischemia-reperfusion in vitro, culture medium was replaced with a pre-equilibrated
low glucose (0.5 mM) medium. Neurons were transferred into a sealed hypoxic chamber
2
(Coy Laboratory Products Inc., Grass Lake, MI) set to an atmosphere of 95% N with 5%
∘
CO
2
at 37 C for 2 h. After OGD neurons were removed from the chamber and returned to the
incubator.
Excitotoxicity
To model excitotoxicity in primary cortical neurons, cells were exposed to 10 ꢀM
L-
glutamate with 10 ꢀM glycine. Neuronal cells from HT22 cell line were exposed to 5 mM
L-
glutamate.
Assessment of Cell Death
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Cell death was quantified by measuring lactate dehydrogenase (LDH) released into
9
supernatant using a colorometric assay. LDH values were normalized to 100% cell death
caused by 0.5% Triton X-100 exposure. Data are reported as the percentage of dead cells
relative to total cells and presented as mean ± standard deviation (SD).
0
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+
Measurement of NAD
+
NAD levels were quantified in subcellular fractions by the enzymatic cycling method using
9
alcohol dehydrogenase as described. A standard curve was generated using known
+
concentrations of NAD and levels were calculated.
STED Imaging
Neurons grown on poly-D-lysine coated glass coverslips were fixed in 2% paraformaldehyde
and permeabilized with TritonX-100. Coverslips were then incubated in a 1:200 dilution of
mouse monoclonal antibody against PAR (SA216, ENzo Life Sciences, Inc., Farmingdale,
NY) and an antibody against TOMM20 (Abcam, Cambridge, MA) followed by incubation in
the appropriate secondary antibodies. STED imaging was performed using a Leica TCS SP8
super resolution STED microscope with a pulsed white light laser and AOBS detection
system was used (Leica Microsystems, Wetzlar, Germany). Images were collected using the
775 nm STED laser line with 30% 3D STED using the Leica STED WHITE oil objective
lens (HC PL APO 100x/1.40 OIL) with a 200 Hz scan speed and 2× line averaging. Pixel size
was set to 45 nm/pixel, step size was set to 160 µm, and pinhole was set at 132.8 µm (0.875
AU). TOMM20 was visualized using Alexa Fluor 555, exciting at 553 nm and detecting
between 558-599 nm and temporally gate between 0.83-4.33 nm. PAR was visualized with
Alexa Fluor 594, exciting at 598 nm and detecting between 603-666 nm and temporally gated
between 0.3-6.0 nsec. DRAQ5 (ThermoFisher Scientific, Waltham, MA) used to label cell
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nuclei was excited at 662 and detected between 667 to 780 nm and temporally gated between
0.3-6.0 nsec. Channels were collected between stacks, sequentially.
COMET Tail Assay
1
1
1
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1
1
1
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HT22 cells were incubated for 60 min at 37°C in growth media (DMEM) with vehicle
(
DMSO, 0.1%), 10 µM XJB-veliparib, or 10 µM veliparib. Cells were then washed with PBS
and exposed to 200 µM H in the presence of vehicle, XJB-veliparib, or veliparib for 30
min on ice. Cells were rewashed to remove H , and media containing vehicle, XJB-
2
O
2
2 2
O
veliparib, or veliparib was replaced for specified times before collection via rubber
policeman. Cells were washed in 4°C PBS, counted, resuspended in DMEM (10% DMSO,
40% FBS), and frozen (-80°C) for batch analysis. The comet tail intensity assay was
47
performed as previously described.
Acknowledgements
We thank D. Franicola for technical assistance with the comet assay. HT22 cells were a
generous gift from D. Schubert (The Salk Institute, La Jolla, CA).
Funding Sources
NIH grants U19 AI068021 (HB, ME, JG, PW) and R01 NS084604 (HB, RC, PW).
Disclosures
United States Patent Application No. 62/407,639 and International Patent Application
PCT/US2017/056503 have been filed for Mitochondrially Targeted PARP Inhibitors.
References
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