A colorimetric substrate for poly(ADP-ribose) polymerase-1, VPARP, and tankyrase-1

Article abstract of DOI:10.1002/anie.200603988

(Chemical Equation Presented) Color me yellow: Poly(ADP-ribose) polymerases (PARPs) play a major role in cellular survival and maintenance of energy stores after genotoxic insult. The colorimetric PARP substrate ADP-ribose-pNP can be used to monitor PARP

Full text of DOI:10.1002/anie.200603988

Communications
DOI: 10.1002/anie.200603988
Enzyme Kinetics
A Colorimetric Substrate for Poly(ADP-Ribose) Polymerase-1,
VPARP, and Tankyrase-1**
Amanda C. Nottbohm, Robin S. Dothager, Karson S. Putt, Mirth T. Hoyt, and
Paul J. Hergenrother*
Upon recognition of DNA breaks caused by various geno-
toxic insults, the enzyme poly(ADP-ribose)polymerase-1
(PARP-1; ADP = adenosine diphosphate)is able to bind
damaged DNA and initiate the repair process.[1] Once bound
to DNA, PARP-1 is activated and uses b-NAD+ (NAD+ =
nicotinamide adenine dinucleotide)to poly(ADP-ribosyl)ate
proteins such as histones, transcription factors, and itself (in
an automodification that leads to inactivation), thus markedly
altering the overall size and charge of the modified protein
(Scheme 1).[2] Sites for poly(ADP-ribose)(PAR)binding
have been identified in numerous DNA-damage checkpoint
proteins, including tumor suppressor p53, DNA-ligase III, X-
ray repair cross-complementing 1 (XRCC1), DNA-depen-
dent protein kinase (DNA-PK), nuclear factor-kB, and
telomerase, and are consistent with the role of PAR in the
DNA-repair pathway.[3–5] The rate of PAR synthesis is directly
proportional to the number of single- and double-strand
breaks found in DNA,[6] and although the levels of PAR may
increase more than 100-fold in minutes immediately following
DNA damage,[7] synthesis of such polymers is transient and
closely regulated by poly(ADP-ribose)glycohydrolase
(PARG), an enzyme that cleaves PAR to ADP-ribose
monomers.[8]
The cytoprotective role of PARP-1 in response to DNA-
damaging agents has been thoroughly examined and is
supported by studies with PARP-1-deficient cell lines.[9]
Accordingly, inhibition of PARP-1
with small molecules has proven to
potentiate anticancer drugs[10,11] and
initial studies have demonstrated
that some BRCA1-deficient tumor
cells (BRCA1 = breast cancer 1,
early onset)are quite sensitive to
PARP-1 inhibitors.[12,13] On the
other hand, extreme DNA damage
leads to PARP-1 overactivation and
a severe depletion in cellular b-
NAD+/ATP (ATP = adenosine tri-
phosphate)stores. The resulting loss
of cellular energy causes necrotic
cell death.[14] Thus, overactivation of
PARP-1 has a cytotoxic effect, and
PARP-1 inhibitors prevent cell
death caused by ischemia and
injury associated with reactive
oxygen species.[15–18]
Scheme 1. Synthesis of poly(ADP-ribose) on glutamic acid residues of protein acceptors as
catalyzed by PARP enzymes, producing nicotinamide as a by-product.
PARPs comprise a large family
of 18 putative isozymes,[19–21] and
although basic enzymatic function and biochemistry has been
characterized for at least six members of this family, much
work remains to be done in this area. Although PARP-1
accounts for more than 90% of PAR synthesis upon DNA
damage,[6,22] it is now known that PAR synthesis by various
other PARPs is critical in a variety of cellular processes.
Particularly intriguing are the functions of tankyrase-1 and
vault PARP (VPARP). Unlike PARP-1, tankyrase-1 is not
activated by DNA damage. About 10% of this protein is
recruited to telomeres and it has been shown that over-
expression of tankyrase-1 results in the lengthening of
telomeres through the tankyrase-1-mediated poly(ADP-ribo-
syl)ation of telomeric repeat binding protein-1 (TRF-1).[23,24]
VPARP is most commonly found associated with vault
[*] A. C. Nottbohm, M. T. Hoyt, Prof. P. J. Hergenrother
Department of Chemistry
University of Illinois
600 S. Mathews, Urbana, IL 61801 (USA)
Fax: (+1)217-333-8024
E-mail: hergenro@uiuc.edu
R. S. Dothager, K. S. Putt, Prof. P. J. Hergenrother
Department of Biochemistry
600 S. Mathews, Urbana, IL 61801 (USA)
[**] This work was supported by a grant from the Michael J. Fox
Foundation for Parkinson’s Disease Research and by the University
of Illinois. ADP=adenosine diphosphate; VPARP=vault poly(ADP-
ribose) polymerase.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
2066
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Chemie
particles, but can also localize to the nucleolus, nuclear
spindle, or nuclear pores.[25–27]
Even though members of the PARP family have
fascinating cellular functions, little progress has been
made in developing isozyme-specific PARP inhibitors.
This search for potent and selective compounds is
hampered by the lack of suitable high-throughput
assays. Most commonly, PARP activity is detected with
radiolabeled NAD+,[18,28] but other assays have been
developed that employ antibodies,[29] biotinylated
NAD+,[18] or fluorescence-based quantization of
NAD+.[30] Ideally, a PARP assay should be inexpensive,
sensitive, rapid, and logistically simple, but methods
involving specialized/radioactive reagents can be cost-
prohibitive and/or time-consuming when testing a large
number of compounds. Herein we report the develop-
ment of a continuous assay that utilizes a colorimetric
Scheme 2. Synthesis of PARP substrate from commercially available b-NAD+
and p-nitrophenol.
PARP substrate, easily synthesized from b-NAD+, to kineti-
cally monitor PARP-1, tankyrase-1, and VPARP activity.
All PARP isozymes utilize b-NAD+ to synthesize ADP-
ribose polymers, producing nicotinamide as a by-product.
Exchanging the nicotinamide moiety of NAD+ for a colori-
metric leaving group would provide a substrate suitable for a
continuous kinetic PARP assay. As the use of p-nitrophenoxy
(pNP)-substituted colorimetric substrates has been well-
established with various enzyme classes, and as PARP-1 has
been known to utilize biotinylated NAD+ to synthesize
poly(ADP-ribose),[31] it seemed that ADP-ribose-pNP
would make an acceptable colorimetric substrate. The use
of commercially available b-NAD+ as a starting material
would greatly simplify the synthesis of this substrate. Very
little literature precedent exists for the chemical modification
of NAD+; basic methanolysis is possible and yields a 3.7:1
mixture of b/a anomers,[32] whereas methods employing the
NADase/NAD+ enzymatic system provide N- and O-(ADP-
ribosyl)ation products, albeit in low yield and limited
substrate scope.[33–35] As syntheses of cyclic ADP-ribose
analogues have utilized nucleophilic metal halides in the
presence of triethylamine to successfully and stereoselectively
cyclize NAD+,[36] this strategy was adopted in the preparation
of ADP-ribose-pNP, 1. ADP-ribose-pNP was synthesized
directly from commercially available b-NAD+ by stirring with
sodium bromide and triethylamine in dimethyl sulfoxide
(DMSO)at 70 8C for 2 h (Scheme 2). This reaction can easily
be performed on the 250–500-mg scale, with isolated product
yields of 35%. Purification involves removal of excess DMSO
under reduced pressure and reverse-phase column chroma-
tography. The absolute configuration at the anomeric position
was assigned on the basis of coupling constants and NOE
experiments, both of which indi-
PARP assay conditions (50 mm Tris, 10 mm MgCl2, pH 8.0,
RT; Tris = tris(hydroxymethyl)aminomethane) for at least
24 h, and is generally stable between pH 4 and 8 (see the
Supporting Information). With ADP-ribose-pNP in hand, a
continuous colorimetric assay for PARP activity was devel-
oped and the kinetic parameters for three PARP isozymes
were determined. In a 96-well plate, a range of concentrations
of ADP-ribose-pNP in PARP assay buffer solution were
incubated with either PARP-1 (DNase-digested DNA was
added to activate PARP-1), tankyrase-1 (refers to “active
domain,” see the Supporting Information), or VPARP (also
refers to the “active domain”), and the optical density at
405 nm was measured every 60 seconds over a time period of
2 h. Changes in absorbance were assessed in triplicate, and
blanks containing 0–700 mm ADP-ribose-pNP in PARP assay
buffer solution were also measured over the same time
period. The absorbance of a range of p-nitrophenol concen-
trations was determined at 405 nm, and the slope of this
calibration curve (see the Supporting Information)was used
to convert the absorbencies to moles of product generated; in
this way the kinetic parameters for PARP-1, tankyrase-1, and
VPARP were calculated (see Table 1 and Figure 1a). Con-
sistent with data in the literature, PARP-1 has the largest KM
and Vmax values (151 mm and 1.30 ” 10À3 mmolminÀ1 mgÀ1,
respectively)followed by tankyrase-1 (82 mm and 1.81 ”
10À5mmolminÀ1 mgÀ1, respectively)and VPARP (46 mm and
2.03 ” 10À6mmolminÀ1 mgÀ1, respectively). Although exact
kinetic parameters for PARP-1 vary with the assay used, for
PARP-1, the KM value with ADP-ribose-pNP is consistent
with that of the natural b-NAD+ substrate and the Vmax value
is approximately 100-fold lower with the colorimetric sub-
strate (see Table 1). For tankyrase-1, the KM value is
cate that the product is the b ano-
Table 1: Comparison of kinetic data for PARP-1, tankyrase-1, and VPARP as reported in the literature and
with the ADP-ribose-pNP substrate.[a]
mer of ADP-ribose-pNP (see the
Supporting Information). In addi-
tion, similar reactions are known to
produce products of the b configu-
ration.[36]
Control experiments revealed
that ADP-ribose-pNP is stable in
aqueous buffer solution under
PARP-1
PARP-1
(Refs. [42–44])
tankyrase-1
tankyrase-1
(Ref. [45])
VPARP
k
cat [sÀ1
KM [mm]
max [mmolminÀ1 mgÀ1
]
0.025
0.41
59–278
0.2–2.4
1.88”10À5
82
0.71
1500
–
2.18”10À6
46
151
V
]
1.30”10À3
1.81”10À5
2.03”10À6
[a] No literature data is available for the kinetic parameters of VPARP.
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NAD+ to synthesize PAR as well,[31] although no direct
evidence has been presented in the literature. Interestingly, in
addition to cleaving the glycosidic bond of ADP-ribose-pNP
to produce p-nitrophenolate, PARP-1 is able to synthesize
PAR from the colorimetric substrate. After incubation of
PARP-1 with activated DNA and ADP-ribose-pNP for one
hour, a nitrocellulose dot blot treated with an anti-PAR
antibody revealed accumulation of the polymer (Figure 1c).
Furthermore, the same experiment completed in the presence
of the PARP inhibitor EB-47 (concentration required for
50% inhibition, IC50 = 45 nm for PARP-1)[38] greatly reduced
the amount of PAR formed (Figure 1c).
Next, to demonstrate the potential of this assay to identify
isozyme-specific PARP inhibitors, we utilized ADP-ribose-
pNP to determine the IC50 value of the known PARP-1
inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-iso-
quinolinone (DPQ)[39] with PARP-1, tankyrase-1, and
VPARP. For these measurements, concentrations of DPQ
ranging from 0.05 nm to 10 mm were added to a 96-well plate
containing PARP assay buffer solution and ADP-ribose-pNP,
and the absorbance at 405 nm was measured in triplicate. As
shown in Figure 2, DPQ has similar IC50 values for all of the
Figure 1. a) Kinetic data for tankyrase-1 obtained by using the ADP-
ribose-pNP PARP substrate. Analogous curves for PARP-1 and VPARP
can be found in the Supporting Information. b) Relative absorbance at
405 nm versus time for 250 mm ADP-ribose-pNP, 250 mm ADP-ribose-
pNP incubated with 20 mm BSA, and 250 mm ADP-ribose-pNP incu-
bated with 99 nm PARP-1. All experiments were completed in PARP
assay buffer solution with absorbance readings every minute for 2 h.
c) Nitrocellulose dot blot illustrating the buildup of PAR after a 1 h
incubation of PARP-1, activated DNA, and ADP-ribose-pNP. The
addition of 100 mm EB-47, a PARP inhibitor, greatly reduces the
amount of PAR buildup. PAR levels were assessed with an anti-PAR
antibody by using the procedure described in the Supporting Informa-
tion.
Figure 2. IC50 curves for DPQ with PARP-1, tankyrase-1, and VPARP.
PARP isozymes tested, though it has a slightly lower IC50
value for PARP-1 (23 nm)and tankyrase-1 (33 n m)as
compared with VPARP (281 nm). Literature reports of the
IC50 value for DPQ with PARP-1 range from 40 nm to
3500 nm.[39–41] Notably, IC50 values have never been deter-
mined for any compounds with VPARP and tankyrase-1.
Utilizing ADP-ribose-pNP as a colorimetric substrate, we
have developed a simple, sensitive, and inexpensive kinetic
assay for assessing activity of the PARP family of enzymes.
This novel substrate has been used to determine the kinetic
parameters for PARP-1, tankyrase-1, and VPARP, and it has
been employed to obtain IC50 values for a small-molecule
inhibitor. This type of continuous assay has considerable
advantages over standard discontinuous PARP assays. With
this new tool for elucidating PARP activity, we now have the
ability to gain further understanding of the kinetic activity of
the diverse PARP family. As ADP-ribose-pNP lends itself
easily to milligram-scale synthesis, testing of large libraries to
find isozyme-specific inhibitors of these enzymes should be a
straightforward task that will provide even more information
approximately 18-fold lower and the Vmax value is significantly
lower as compared with the values reported in the literature
with b-NAD+ as a substrate. To our knowledge, no data is
available on the kinetic parameters of VPARP with b-NAD+.
Control experiments in which bovine serum albumin (BSA)
was incubated with the ADP-ribose-pNP substrate produced
no increase in signal at 405 nm, indicating that this substrate is
not processed by proteins in a nonspecific manner (Fig-
ure 1b). It has been postulated that PARP-1 is capable of
producing PAR from alternative substrates; ADP-ribose
chains of no more than four residues in size were observed
with 3’-deoxy-NAD+ as a substrate,[37] and thin-layer chro-
matography has suggested that PARP-1 can use biotinylated
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Keywords: enzyme assays · enzyme kinetics · enzymes ·
polymerases
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