Emissive Synthetic Cofactors: A Highly Responsive NAD+ Analogue Reveals Biomolecular Recognition Features

Article abstract of DOI:10.1002/chem.201805520

Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide (NAD⁺) has emerged as a crucial substrate for NAD⁺-consuming enzymes, including poly(ADP-ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD⁺ surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4-d]pyrimidine-based NAD⁺ surrogate, termed N^thAD⁺, are described. Its preparation was accomplished by enzymatic conversion of synthetic ^thATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD⁺ analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, N^thAD⁺ was reduced by alcohol dehydrogenase (ADH) to N^thADH and afforded ^thADP-ribose (^thADPr) upon hydrolysis by NAD⁺-nucleosidase (NADase). Furthermore, N^thAD⁺ was engaged in cholera toxin A (CTA)-catalyzed mono(^thADP-ribosyl)ation, but was found incapable in promoting PARP1-mediated poly(^thADP-ribosyl)ation. Due to its high photophysical responsiveness, N^thAD⁺ is suited for spectroscopic real-time monitoring. Intriguingly, and as an N7-lacking NAD⁺ surrogate, the thieno-based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD⁺) relative to its isothiazolo-based analogue. The distinct tolerance, displayed by diverse NAD⁺ producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1-mediated reactions.

Full text of DOI:10.1002/chem.201805520

A Journal of
Accepted Article
Title: Emissive synthetic cofactors: A highly responsive NAD+
analogue reveals biomolecular recognition features
Authors: Jonas Feldmann, Yao Li, and Yitzhak Tor
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Emissive synthetic cofactors: A highly responsive NAD+
analogue reveals biomolecular recognition features
Jonas Feldmann[a, b], Yao Li[a], and Yitzhak Tor*[a]
Abstract: Apart from its vital function as
a redox cofactor,
nicotinamide adenine dinucleotide (NAD+) has emerged as a crucial
substrate for NAD+-consuming enzymes, including poly(ADP-
ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association
with severe diseases, such as cancer, Alzheimer’s disease, and
depressions, necessitates the development of new analytical tools
based on traceable NAD+ surrogates. Here we describe the
synthesis, photophysics and biochemical utilization of an emissive,
thieno[3,4-d]pyrimidine-based NAD+ surrogate, termed NthAD+. Its
preparation was accomplished by enzymatic conversion of synthetic
Figure 1. Structural formula of native NAD+ and emissive analogues NtzAD+
and NthAD+. Purine N7 moieties are highlighted in blue.
thATP via nicotinamide mononucleotide adenylyltransferase
1
(NMNAT1). The new NAD+ analogue possesses useful
photophysical features including red-shifted absorption and emission
maxima as well as a relatively high quantum yield. Serving as a
versatile substrate, NthAD+ is reduced by alcohol dehydrogenase
(ADH) to NthADH and affords thADP-ribose (thADPr) upon hydrolysis
by NAD+-nucleosidase (NADase). Furthermore, NthAD+ can be
In this regard, three major classes of NAD+-consuming enzymes
are
commonly
identified,
including
sirtuins,
(ADP-
ribose)polymerases and cyclic ADP-ribose synthases.[2–10,11]
Malfunction or dysregulation of these enzymes have been found
to disrupt cellular NAD+ homoeostasis and, as a consequence,
could cause severe diseases such as cancer, diabetics,
atherosclerosis, Alzheimer’s disease, depressions, and
neurodegeneration.[2–10,12]
engaged in Cholera toxin
ribosyl)ation but is found incapable in promoting PARP1-mediated
poly(thADP-ribosyl)ation.
Due to its high photophysical
A
(CTA)-catalyzed mono(thADP-
responsiveness, NthAD+ is suited for spectroscopic real-time
monitoring. Intriguingly, and as an N7-lacking NAD+ surrogate, the
thieno-based cofactor shows reduced compatibility (i.e. functional
similarity compared to native NAD+) relative to its isothiazolo-based
analogue. The distinct tolerance, displayed by diverse NAD+
producing and consuming enzymes, suggest unique biological
recognition features and dependency on the purine’s N7 moiety,
which is found to be of importance, if not essential, for PARP1-
mediated reactions.
To restore cellular NAD+ levels and thus counteract
detrimental pathways, inhibition of NAD+-consuming enzymes,
most notably poly(ADP-ribose)polymerase
1 (PARP1) and
CD38/CD157, has shown promise.[13–17,18] The significance of
NAD+-mediated processes necessitates the development of
novel tools and probes to facilitate biophysical analyses and the
fabrication of effective discovery assays. Radioactive,[19]
clickable,[20,21] isotopically-labelled,[22] and fluorescent[23,24] NAD+
surrogates have been developed. Design of the latter has
employed the incorporation of emissive adenosine analogues,
including the perturbing 1,N6-ethenoadenosine and 8-(pyrrol-2-
yl)adenosine.[23,24] To exploit the safety and sensitivity of
fluorescence-based tools, which closely reflect the native system,
both isomorphicity and isofunctionality of a synthetic analogue
have to be attained by reducing structural and functional
perturbations, respectively, thus ensuring maximum biological
compatibility. Concurrently, photophysical responsiveness
towards environmental and structural changes is required to
enable real-time spectroscopic monitoring.[25]
Introduction
The most prominent role of nicotinamide adenine dinucleotide
(NAD+, Scheme 1) is as a redox cofactor in regulating central
metabolic pathways by catalyzing a variety of oxidoreductase-
mediated processes.[1] More recently, but as significantly, NAD+
has been found to act as a substrate for multiple enzymes that
catalyze glycosidic cleavage of the nicotinamide moiety and
release an ADP-ribose (ADPr) moiety, which is then transferred
onto acceptor molecules, such as proteins, DNA, and smaller
metabolites.[2–10]
In the last few years, a new generation of emissive
nucleoside analogues has been developed based on a novel set
of purine-mimicking scaffolds: thieno[3,4-d]pyrimidines[26] and
isothiazolo[4,3-d]pyrimidines.[27] Such isomorphic analogues
open a spectral window into processes involving nucleosides
and nucleotides derivatives, which is otherwise inaccessible with
the native counterparts.[28,29] A red shifted absorption and useful
fluorescence features in the visible range, which are non-
existent for the native counterparts, facilitate the fabrication of
biochemical and discovery assays with minimal spectral
interference by the native chromophores.[28,29]
[a]
[b]
Y. Li, Prof. Yitzhak Tor
Department of Chemistry and Biochemistry
University of California, San Diego
La Jolla, CA 92093-0358 (USA)
E-mail: ytor@ucsd.edu
J. Feldmann
Department of Chemistry
Ludwig-Maximilians-Universität München
Butenandtstr. 5–13, 81377 Munich (Germany)
Supporting information for this article is given via a link at the end of
the document.
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Table 1. Photophysical properties of NAD+ and its emissive analogues
λabs [nm]
260
ɛ [M-1 cm--1
]
λem [nm]
310
Φ
A[a]
14.9 x 103
7.8 x 103
7.4 x 103
5 x 10-5
0.053
0.21
tzA[a]
thA[a]
338
410
341
420
Scheme 1. Synthesis of NthAD. Reagents and conditions: (a) i) TMP, POCl3,
0 °C; ii) tris(tetrabutylammonium) hydrogen pyrophosphate, Bu3N, DMF, 0 °C,
6.1% over two steps; (b) Tris-HCl pH 7.8, KCl, NaCl, MgCl2, NMN, PPase,
NMNAT1, ddH2O, 37 °C, 81%. NMN, β-nicotinamide mononucleotide; PPase
Pyrophosphatase; NMNAT1, mononucleotide adenylyltransferase 1.
NAD+[a]
259
338
341
16.9 x 103
7.2 x 103
7.0 x 103
-
-
NtzAD+[a]
NthAD+[b]
411
431
0.044
0.071
[a] Previously reported values, see corresponding references.[26,27,29,40,41] [b]
ɛ determined at the end of NMNAT1-mediated conversion in relation to
respective substrates, assuming complete consumption in 50 mM Tris-HCl
buffer. All photophysical properties reflect the average of two independent
measurements.
While the thieno[3,4-d]pyrimidine family of nucleosides displays
outstanding emission features, the isothiazolo[4,3-d]pyrimidine-
based analogues, while somewhat less emissive, provide
enhanced isomorphicity and isofunctionality.[30,31] Having access
to both purine analogues, offers, however, a unique opportunity
to assess biomolecular recognition features using sensitive
fluorescence-based tools. Comparing the native NAD+ to its
emissive analogues, NthAD+ and its single-site mutant NtzAD+
(Figure 1), can provide mechanistic insight into the biomolecular
contacts mediated via the imidazole ring, and particularly its N7
nitrogen. While the purines’ N7 moiety is known to be crucial for
a variety of biological recognition events, including enzyme–
substrate binding (e.g., adenosine–adenosine deaminase),[32]
Hoogsteen base pairing,[33] and G-quadruplex formation,[34] its
significance in NAD+-dependent processes is poorly explored
and largely limited to crystallographic studies.[35–39] Unlike N7-
deaza-NAD+, which has been employed as an N7-lacking
substrate, we hypothesized that NthAD+ will likely be emissive
and responsive, and thus will likely facilitate fluorescence
spectroscopic monitoring of enzymatic activities in real time.[37–40]
Furthermore, its red-shifted absorption spectra, compared to the
native NAD+, facilitates selective excitation and opens a spectral
window that is unavailable for the native cofactor. Herein, we
therefore describe the synthesis and photophysics of NthAD+
(Figure 1), an NAD+ analogue containing the isomorphic and
highly emissive thieno[3,4-d]pyrimidine core. We report its
enzymatic synthesis by NMNAT1, and its responsiveness to
ADH-mediated reduction to NthADH, NADase-catalyzed
hydrolysis yielding thADP-ribose (thADPr) and consumption by
then enzymatically reacted with β-nicotinamide mononucleotide
(NMN) using recombinant nicotinamide mononucleotide
adenylyl-transferase1 (NMNAT1).[26] This overcomes the final
phosphodiester bond formation in the non-enzymatic syntheses
of NAD+ analogues, which is commonly accomplished via
coupling reagents (e.g., Staab’s reagent) and is associated by
long reaction times and low yields.[30] The optimized enzymatic
approach can be adapted to preparative scales.[31]
Practically, thA was first treated with POCl3 and trimethyl
phosphate, followed by the addition of tris(tetrabutylammonium)
hydrogen pyrophosphate and tributylamine to yield thATP
(Scheme 1).[41] It was then treated with β-nicotinamide
mononucleotide (NMN) and NMNAT1 (Tris pH 7.8, 37°C).
Addition of inorganic pyrophosphatase (PPase) was required to
avoid product inhibition of NMNAT1 by the released
pyrophosphate. As assessed by HPLC, quantitative conversion
was achieved after 2.5 h and a good isolated yield (81%) was
obtained on a 1 mg scale.
Photophysical Features
To assess the basic photophysical features of NthAD+, its
absorption and emission spectra were recorded and compared
to the native cofactor and the previously synthesized NtzAD+
(Table 1). In comparison to NAD+ (λabs = 259 nm) and similarly
to NtzAD+ (λabs = 338 nm), NthAD+ features a bathochromically
shifted absorption maximum at λabs = 341 nm (Table 1). Notably,
excitation near the absorption maximum is found to cause photo
bleaching, which can be avoided by excitation at λex = 360 nm,
yielding a well-defined emission band with a maximum at 431
(ADP-ribosyl)ating enzymes including Cholera toxin
A and
PARP1. Comparison with both native NAD+ and emissive
NtzAD+ provides insight into its putative recognition features via
the basic N7 within the purine skeleton
.
nm. Compared to NtzAD+ (Φ
= 0.044), and as initially
Results and Discussion
hypothesized, NthAD+ exhibits a higher quantum yield of Φ =
0.071.
Synthesis
The preparation of NthAD+ was accomplished in two steps
starting from the previously synthesized thA.[26] The
corresponding triphosphate was first chemically synthetized and
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NMNAT1- and ADH-Mediated Conversion
NADH by oxidizing ethanol to acetaldehyde (Scheme 2).
To compare the performance of thATP, tzATP and native ATP,
the conversion of each nucleotide in this coupled enzymatic
process was followed by HPLC, real-time spectroscopic
monitoring and steady-state emission and absorption
spectroscopy. Treatment with NMN and NMNAT1 facilitated
quantitative conversion of both ATP and tzATP within 30 min and
complete consumption of thATP within 100 min as assessed by
HPLC (Figure 2a–c). Spectroscopic assessment highlighted the
differences between the analogues. While the emission intensity
decreased upon formation of both NtzAD+ and NthAD, it
remained unchanged during the formation of “fluorescently silent”
NAD+ (Figure 2d–f). Notably, all substrates were reduced rapidly
within a few minutes in the presence of ethanol and ADH.
Spectroscopically, reduction of native NAD+ shows an increase
in emission intensity, whereas decreasing emission intensity
was observed upon reduction of both tzA- and thA-based
synthetic emissive cofactors. Steady state spectra recorded
before treatment and after each enzymatic conversion (Figures
2g–i) reveal phosphodiester bond formation to have little or no
effect on the absorption spectra of neither substrate but show a
diminished fluorescence for both synthetic cofactors. In all cases,
ADH-mediated cofactor reduction results into an increased
optical density between 300 and 400 nm, accompanied by a
further decrease in emission. This two-step decrease, however,
is found to be distinct for each emissive cofactor: While the initial
emission level of thATP is diminished by 70% upon NthAD+
formation, ADH-mediated reduction causes an additional
decrease of only 8%. In
Scheme 2. NMNAT1-mediated synthesis of native NAD+ and emissive
analogues followed by ADH-mediated reduction in the presence of EtOH.
Purine and derived ring systems (blue), pyridinium (purple) and 1,4-
dihydropyridine (red) moieties are highlighted. Triphosphate groups are
represented by PPPi. NMN, nicotinamide mononucleotide; NMNAT1,
nicotinamide mononucleotide adenylyl-transferase 1; ADH, alcohol
dehydrogenase.
To evaluate the compatibility of NthAD+, and gain access to
NthADH, its reduction equivalent,
a
sequential enzymatic
conversion assay employing both Homo sapiens recombinant
NMNAT1
and
Saccharomyces
cerevisiae
alcohol
dehydrogenase (ADH) was performed.[31,42] While NMNAT1
naturally accomplishes phosphodiester bond formation between
NMN and ATP to generate NAD+,[43] ADH reduces NAD+ to
contrast, the conversion of
tzATP shows a diminution of
40% after the first and 30%
after the second reaction.
Figure 2. Monitoring of the NMNAT1-mediated synthesis and ADH-catalyzed reduction of native NAD+ and emissive
analogues. Top row: HPLC traces of (a) ATP, (b) tzATP and (c) thATP (blue each), corresponding NAD+ species
(purple) and their reduced forms (red) monitored before conversion, 30 min (thATP: 60 min) after treatment with
NMNAT1 and 30 min after the addition of ADH at λabs = 260, 330 and 340 nm, respectively. Mid row: Normalized
emission intensity signals over time recorded for the conversion of (d) ATP at 465 nm (λex = 335), (e) tzATP at 410 nm
(λex = 330) and (f) thATP at 410 nm (λex = 360) before (blue) and upon (purple) treatment with NMNAT1 and after the
addition of ADH (red). Bottom row: Steady state absorption (dashed lines) and emission (solid lines) spectra for the
conversion of (g) ATP (λex = 335), (h) tzATP (λex = 330) and (i) thATP (λex = 360) recorded before conversion (blue), 30
min (thATP: 60 min) after treatment with NMNAT1 (purple) and 10 min (thATP: 20 min) after the addition of ADH (red).
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The enhanced photophysical features of the thieno-based
cofactor reveal NthAD+ to be a suited fluorophore for real-time
spectroscopic monitoring of NAD+ synthesis and reduction
processes. Its high photophysical responsiveness may be
attributed to intramolecular ground and excited state interactions
between the modified purine surrogate and the nicotinamide
moiety, interactions that have been evoked for the native
cofactor but remain insufficiently explored .[30,44] The relatively
slow conversion of thATP by NMNAT1 suggests thA to be less
compatible than both the native adenosine and its isothiazolo-
based purine analogue. We therefore suggest that the imidazole
ring and especially contacts through its N7 nitrogen are of
significance for NMNAT1–substrate recognition, although the
exact contribution of the N7 nitrogen has not been studied in
great detail and remains somewhat elusive.[45]
NADase-Mediated Hydrolysis
Figure 3. Monitoring of the NADase-mediated hydrolysis of emissive NAD+
analogues. Top row: HPLC traces of (a) NtzAD+ (purple) and tzADPR (yellow)
monitored before and 30 min after treatment with NMNAT1 at λabs = 330 nm;
(b) NthAD+ (purple) and thADPR (yellow) monitored before and 120 min after
treatment with NMNAT1 at λabs = 340 nm. Mid row: Normalized emission
intensity signals over time recorded for the conversion of (c) NtzAD+ at 410 nm
(λex = 330) and (d) NthAD+ at 410 nm (λex = 360) before (purple) and upon
(yellow) treatment with NADase. Bottom row: Steady state absorption (dashed
Scheme 3. NADase-mediated hydrolysis of native NAD+ and emissive
analogues, yielding corresponding ADP-ribose (ADPr) species. Pyridinium
moiety (purple) and anomeric hydroxy group (yellow) are highlighted. NADase,
NAD+ nucleosidase.
lines) and emission (solid lines) spectra for the hydrolysis of (e) NtzAD+ (λex
=
330) and (f) NthAD+ (λex = 360) recorded before (purple) treatment and 25 min
(NthAD: 120 min) after the addition of NADase (yellow).
To take advantage of the intramolecular photophysical
interaction between the nicotineamide moiety and the emissive
purine surrogate and to evaluate enzymatic cleavage of NthAD+,
an NADase-based assay was applied.[30] This hydrolase
catalyzes the glycosidic cleavage of the nicotinamide moiety and
release of ADP-ribose (ADPr, Scheme 3), which naturally
(Figures 3e,f), where treatment of NtzAD+ and NthAD+ with
NADase
yielded
a
40%
and
60%
enhancement,
respectively,upon enzymatic hydrolysis.
The findings presented above reveal NthAD+ to be highly
photophysically responsive towards glycosidic cleavage and the
associated enhanced emission supports the nicotinamide-
mediated quenching effects. The comparatively slow hydrolysis
of NthAD+ compared to NtzAD+ indicates, however, somewhat
reduced substrate compatibility for NADase and, as before,
suggests the purine’s N7 moiety to be involved in enzyme –
substrate interactions in this enzymatic pocket.
serves as
a secondary messenger and is involved in
intramolecular calcium signaling.[46]
Both emissive cofactors were treated with porcine brain
NADase. Quantitative conversion was observed after 30 min for
NtzAD+ and 120 min for NthAD+, and was accompanied by the
formation of tzADPr and thADPr, respectively (Figures 3a,b).
Assuming similar extinction coefficients for the starting materials
and products, tzADPr was obtained in a 2.8-fold higher amount
compared to thADPr. The latter was isolated, identified and
characterized by steady state spectroscopy to reveal
CTA-Mediated Mono(ADP-ribosyl)ation
To further explore the compatibility of the emissive NthAD+
and gain insight into more complex and challenging modification
processes, enzymatic conversions by the Cholera toxin were
examined. As a hexameric protein complex, this Vibrio cholerae
toxin consists of five receptor-binding B subunits (CTB) and an
enzymatically active A subunit (CTA).[47] The latter catalyzes the
glycosidic cleavage of NAD+ and formation of ADP-ribose
bathochromically shifted absorption and emission maxima (λabs
=
340 nm, λem = 431 nm; Figure S1). Real-time spectroscopic
monitoring of NtzAD+ hydrolysis showed an increasing emission
signal within 25 min (Figure 3c). In contrast, a four-time longer
period was required for quantitative NthAD+ hydrolysis (Figure
3d). The corresponding steady state spectra reveal enhanced
optical density and emission intensity within the visible range
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(ADPr), which can be transferred onto arginine (Arg) side chains
treated with CTA. This cost-intensive assay and small reaction
and other guanidine-containing derivatives.[48] This process,
volumes,
however,
disfavored
real-time
spectroscopic
known as mono(ADP-ribosyl)ation, classifies CTA as
mono(ADP-ribosyl)transferase. Furthermore, CTA is found to
modify and intrinsically inhibit the guanine nucleotide-binding GS
a
monitoring and necessitated sequential records of steady state
spectra. Samples were thus taken after 0, 20, 50, 90, and 180
min and analyzed both spectroscopically and by HPLC.
Conversion of native NAD+ was exclusively monitored via HPLC.
Upon treatment with CTA, quantitative conversion was
detected by HPCL after 90 min for NAD+ and 180 min for both
NtzAD+ and NthAD+ (Figures 4a–c). In addition, thADPr-Agm was
isolated, identified and spectroscopically characterized,
revealing bathochromically shifted absorption and emission
maxima (λabs = 341 nm, λem = 425 nm, Figure S3). Along with the
formation of Agm adducts, hydrolysis to the corresponding ADPr
analogues was observed as a major side reaction in all three
cases. Assuming similar extinction coefficients for the starting
materials and Agm aducts, the by-product/product ratios amount
to 0.20, 0.37, and 0.59 for the native-, isothiazolo-, and thieno-
based cofactors, respectively. Furthermore, treatment of either
fluorescent cofactor with CTA causes a significant increase in
emission intensity, as assessed by steady state spectroscopy
(Figures 4e,f). CTA-mediated conversion of NthAD+ yielded an
emission enhancement of 360%, whereas conversion of NtzAD+
yielded a 70% increase only.
alpha subunit (Gα ) of heterotrimeric GS Proteins, which causes
S
severe dehydration and other cholera-associated symptoms.[49]
Scheme 4. CTA-mediated conversion of native NAD+ and emissive analogues,
including (a) hydrolysis and (b) mono(ADP-ribosyl)ation of agmatine. Anomeric
hydroxy group (yellow), pyridinium (purple) and agmatine (green) moieties are
highlighted. CTA, cholera toxin A; Agm, agmatine.
To compare the chromatographic and spectroscopic
monitoring methods, the integrated HPLC product peak areas
and emission intensities were plotted against time. The HPLC-
derived plots reveal a faster formation of ADPr-Agm compared
to both tzADPr-Agm and thADPr-Agm, of which the latter is the
slowest to form (Figure 4d, solid lines). The normalized emission
intensities (Figure 4d, dashed lines) confirm the kinetics
observed for NthAD+ conversion but suggest NtzAD+ to convert
slightly slower than indicated by HPLC. The slight deviation
While Arg-containing proteins serve as major intracellular
CTA targets, the less bulky and easily detectable agmatine
(Agm), is commonly employed as an Arg surrogate for CTA-
based in vitro assays.[50] By utilizing this approach, we recently
found CTA to consume NtzAD+ and form mono(tzADP-
ribosyl)ated agmatine (tzADPr-Agm, Scheme 4). To compare
the suitability of NthAD+, solutions of the cofactors and Agm were
between
spectroscopically
and
HPLC-derived
kinetic
measurements has been
seen before[30] and could be
attributed to the formation
of by- or decomposition
products that affect the
overall emission intensity.
Despite
limitations, NthAD+ can be
used as powerful
these
minor
a
fluorophore due to its high
spectroscopic
responsiveness
upon
mono(thADP-ribosyl)ation.
Its slow conversion, as well
as the excessive formation
of thADPr, reveal, however,
a
poorer recognition by
CTA compared to both
NthAD+ and NAD+. These
findings further support
substrate recognition via
Figure 4. Monitoring of the CTA-mediated mono(ADP-ribosyl)ation accomplished by NAD+ and emissive analogues.
Top row: HPLC traces of (a) NAD+, (b) NtzAD+ and (c) NthAD+ (purple), agmatine adducts (green) and hydrolysis
products (yellow) monitored after 0, 20, 50, 90 and 180 min at λabs = 260, 330 and 340 nm, respectively. Bottom row:
(d) Normalized emission intensity signals (dashed lines) and HPLC peak area (solid lines) over time for the conversion
of NAD+ (grey), NtzAD+ (black) and NthAD+ (purple); Error bars refer to standard deviations of the normalized mean. the purine’s N7 nitrogen.
Steady state absorption (dashed lines) and emission (solid lines) spectra for the conversion of (e) NtzAD+ (λex = 330)
Although
X-ray-based
and (f) NthAD+ (λex = 360) recorded after 0 min (purple), 20 min (violet), 50 min (blue), 90 min (green) and 180 min
(bright green).
studies of Fieldhouse et al.
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have revealed multiple intermolecular interactions between
NAD+ and CTA in detail, the involvement of the N7 nitrogen has
remained crystallographically elusive.[36]
Visualization of anti-pADPr (blue channel) confirms the
formation of pADPr- and ptzADPr-modified PARP1, respectively,
which is manifested in smeared higher MW bands. NAD+-
induced formation of auto-modified PARP1 is detected as early
as after 2 min and found to increase for 60 min. A reduced
amount of pADPR-modified PARP1 was detected after 180 min.
In comparison, ptzADPr-related bands arise at lower MW, are
less intense and show a regression already after 10 min. In
contrast, no pthADPr was detected upon treatment with NthAD+.
An important consideration when assessing the
observations articulated above is the biochemical specificity of
the antibodies, particularly the anti-pADPr one. Designed to bind
native pADPr, anti-pADPr is known to interact with 3–4 ADPr
units of linear pADPr, which can be associated with proteins
both covalently and non-covalently.[55,56] Oligomers consisting of
10 ADPr units or less are not efficiently captured, whereas
polymers of > 20 ADPr units facilitate proper binding as revealed
by competitive binding assays.[55] Beside these known limitations,
neither the exact epitopes of anti-pADPr nor its interaction with
PARP1-Mediated Poly(ADP-ribosyl)ation
In contrast to CTA, which acts as
a
mono(ADP-
ribose)transferase, PARP1 mediates poly(ADP-ribosyl)ation by
transferring multiple ADPr units to itself or other target proteins,
forming poly(ADP-ribose) linkages (pADPr, Scheme 5).
Depending on its chain structure and length, such
posttranslational modifications can affect the activity and
localization of associated proteins and their non-covalent
interaction with other binding partners.[51] Hence, PARP1
functions as a regulatory key enzyme, which is involved in DNA
damage response, metabolic regulation and transcriptional
activation processes, among others.[52] Association with severe
diseases makes PARP1 an attractive therapeutic target, which is
driving the development of novel inhibitors and treatment of
breast, prostate, and ovarian cancer, among others.[13–17]
ptzADPr or pthADPr, respectively, are known.[57]Hence,
a
Given that PARP1 can act as both the enzyme and the
target, we studied its auto-poly(ADP-ribosyl)ation using all
cofactors and calf thymus DNA as an activating agent.[21,53,54]
Accordingly, a solution of PARP1 was treated with calf thymus
DNA before inducing the reaction with NtzAD+ or NthAD+.
Positive controls included native NAD+ and negative controls
included reaction mixtures without the cofactors. The conversion
was then monitored at different time points both
spectroscopically and via SDS-PAGE. To facilitate adequate
detection and differentiation between modified and unmodified
PARP1, western blotting based on anti-PARP1 and anti-pADPr
antibodies was performed.[21]
reduced or even no affinity of anti-pADPr towards synthetic
pADPr analogues cannot be excluded but will be neglected in
the following evaluation.
Figure 5. (a) Coomassie stained SDS-PAGE and (b) western blot of PARP1-
mediated auto-poly(ADP-ribosyl)ation reactions in presence of native NAD+
and emissive analogues, respectively. Marker (lane 1); untreated sample
(negative control, lane 2); treatment with NAD+ (positive control, lanes 3-6),
NtzAD+ (lanes 7-10) and NthAD+ (lanes 11-13); samples were taken after 2, 10,
60 and 180 min, respectively.
Scheme 5. PARP1-mediated auto-poly(ADP-ribosyl)ation, facilitated by both
native NAD+ and emissive NtzAD+. Phosphate groups are represented by Pi.
To further analyze the observations discussed above, two
opposite and kinetically divergent processes have to be
considered: First, the increasing amount of modified PARP1 as
well as the shift to higher MW reveal an ongoing polymerization
reaction upon treatment with NAD+ and NtzAD+. Secondly, the
subsequent regression of modified PARP1 indicates
decomposition and cleavage of the pADPr–protein linkages,
which are known to be fragile (t1/2 < 1 h, pH 7.5)[58,59] Notably,
this decomposition process is favored in the presence of
nicotinamide, which is released upon poly(ADP-ribosyl)ation.
Previous studies under similar conditions (pH 8.0, 20 µg rat liver
PARP, 100 nM NAD+, 23 °C) showed a regressing amount of
pADPr-modified PARP after 50 min, which is consistent with the
kinetics obtained for NAD+-induced PARP1 auto-modification.[59]
The lower amount of ptzADPr and premature regression of the
Initial attempts to spectroscopically monitor this transformation in
real time did not reveal any change in emission intensity, neither
upon treatment with NtzAD+ nor with NthAD+. The Coomassie-
stained SDS-PAGE (Figure 5a) did show, however, PARP1-
related bands to decrease in intensity over time for both
cofactors. This decrease is most striking upon adding NAD+ and
still substantial upon NtzAD+ treatment, whereas addition of
NthAD+ shows only little effect. Modified PARP1, in contrast,
remains undetectable by Coomassie staining and necessitated
western blotting for further analysis. The respective blot (Figure
5b) confirms a diminishing concentration of unmodified PARP1
(green channel) upon addition of NAD+ or NtzAD+, respectively.
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A suspension of thA (50 mg, 0.18 mmol, 1.0 eq) in trimethyl phosphate
(1.8 mL) was treated with POCl3 (41 µL, 0.44 mmol, 2.5 eq.) at 0 °C
under stirring. After 1 h stirring at 0 °C, additional POCl3 (41 µL, 0.44
mmol, 2.5 eq.) was added and the suspension was stirred at 0 °C for
another 2 h. After treatment with tris(tetrabutylammonium) hydrogen
pyrophosphate (164 mg, 0.88 mmol, 5.0 eq) dissolved in dry DMF (1.8
mL), tributyl amine (0.21 mL, 0.88 mmol, 5.0 eq) was added drop-wise
and the reaction mixture was stirred at 0 °C for 2 h. The reaction mixture
was quenched with ice-cold 0.05 M TEAB (5. 0 mL), stirred at 0 °C for 15
min and washed with EtOAc (10 mL). After extracting the organic phase
with 0.05 M TEAB (3 × 5 mL), the aqueous phases were combined and
concentrated in vacuo. The crude product was purified via ion exchange
chromatography (Sephadex DEAE A25 column; 0.05 M à 0.5 M TEAB;
4 °C). After concentration in vacuo and lyophilization, the resulting solid
was dissolved in ddH2O (1.00 mL) and purified via HPLC (70 µL
injections, method see SI). Lyophilization of the collected fraction
afforded a white solid of thATP (0.011 mmol, 6.1 %), which was used
without further purification. 1H-NMR (300 MHz, D2O): δ (ppm) = 8.55 (s,
1H), 8.22 (s, 1H), 5.44 (d, J = 7.43 Hz, 1H), 4.42 (dd, J = 5.10, 3.11 Hz,
1H), 4.35 (dd, J = 7.43, 5.10 Hz, 1H), 4.30 (m, 1H), 4.22 (m, 2H); 31P-
NMR (202 MHz, D2O): δ (ppm) = −9.92 (d, J = 20.0 Hz), −10.83 (d, J =
19.5 Hz), −22.57 (m); ESI-HRMS [C11H16N3O13S–H+] calculated
521.9544, found 521.9536.
correspondingly modified PARP1 suggest reduced compatibility,
which could reflect less effective oligomerization or faster
degradation of the shorter oligomers formed. On the other hand,
the apparent absence of pthADPr-modified PARP1 suggests
poly(thADP-ribosyl)ation to be severely limited with NthAD+, if not
impossible, and illustrates its diminished recognition as
a
PARP1 substrate. This is supported by crystallographic studies
of NAD+-bound PARP1, which illustrate hydrophobic interactions
between isoleucine (Ile872) and the adenosine’s imidazole ring.
Additionally, the purines N7 nitrogen is proposed to interact with
the protein via an aspartic acid residue.[60] Such enzyme–
substrate interactions, while possible with NtzAD+ cannot be
accommodated by NthAD+, the thiopheno-based system lacking
a basic N at the corresponding position.
Conclusions
This study set out to prepare NthAD+, an N7-lacking, thieno[3,4-
d]pyrimidine-based NAD+ surrogate, and assess its utility for
monitoring NAD+-dependent reactions by spectroscopic means.
Towards this end, we employed recombinant NMNAT1 that
facilitates the preparative scale synthesis of NthAD+ from the
emissive thATP. When compared to our previously developed
analogue NtzAD+ (λabs = 338 nm, λem = 411 nm, Φ = 0.044),
NthAD+ shows improved photophysical features including red-
Synthesis of NthAD
A solution of Tris (50 mM), KCl (50 mM), NaCl (50 mM), MgCl2 (12 mM),
thATP (1.5 mM), NMN (2.0 mM) and PPase (10 U) in ddH2O (1.50 mL)
was left for equilibration at 37 °C for 5 min. After addition of NMNAT1
(Novus Biologicals, 0.42 U[60]), the reaction mixture was gently mixed and
left for 2.5 h at 37 °C. The reaction was monitored via HPCL (method see
SI) and stopped by freezing the mixture to –78 °C. The reaction mixture
was concentrated to a volume of 0.3 mL by lyophilization. Purification via
HPLC (100 µL injections, method see SI) and subsequent lyophilization
afforded a white solid of NthAD (1.82 mM, 81 %). 1H-NMR (300 MHz,
D2O): δ (ppm) = 9.30 (s, 1H), 9.10 (d, J = 6.19 Hz, 1H), 8.76 (d, J = 8.20
Hz, 1H), 8.28 (s, 1H), 8.14 (dd, J = 7.89, 6.46 Hz, 1H), 8.00 (s, 1H), 4.53
(d, J = 2.48 Hz, 1H), 4.46 (t, J = 5.23 Hz, 1H), 4.41 (m, 2H), 4.36 (m, 2H),
4.26 (m, 1H), 4.21 (m, 1H), 4.14 (m, 1H); 31P-NMR (202 MHz, D2O): δ
(ppm) = −10.63 (d, J = 18.73 Hz), −11.07 (d, J = 19.91 Hz); ESI-HRMS
[C22H29N5O14P2S–H+] calculated 678.0678, found 678.0679.
shifted absorption and emission maxima (λabs = 341 nm, λem
=
431 nm) and a higher emission quantum yield (Φ = 0.071).
Moreover, NthAD+ serves as a viable substrate for ADH-
mediated reduction affording NthADH, NADase-catalyzed
hydrolysis yielding thADPr and CTA-mediated mono(ADP-
ribosyl)ation.. Photophysical analysis revealed NthAD+ to be
substantially more responsive towards enzymatic conversions
than its isothiazolo-based analogue and thus suited as a
fluorescent probe for their spectroscopic real-time monitoring.
Spectroscopic and HPLC-based analyses found NthAD+ to be
less isofunctional than both NAD+ and NtzAD+, which is
manifested in lower conversion rates or increased by-product
formation. When assessing PARP1-mediated auto-poly(ADP-
ribosyl)ation, no conversion was observed upon treatment with
NthAD+ at all, whereas NtzAD+ is converted successfully ⎯ if
only to a lower extent compared to its native cofactor, indicating
an inherent PARP1-N7 dependency. These studies demonstrate
the utility of both NtzAD+ and NthAD+ , particularly when used
side by side, for investigating biomolecular recognition features
that involve purine N7 interactions. Even though being quenched
within pADPr, both emissive analogues can be easily employed
to monitor enzymatic reactions including NAD+ formation,
oxidation/reduction, hydrolysis, and, potentially, mono(ADP-
ribosyl)ation in real time.
Steady State Absorption and Emission Spectroscopy
Steady state absorption spectra were measured on a Shimadzu UV-2450
spectrophotometer setting the slit at 1 nm and using a resolution of 0.5
nm. Steady state emission spectra were measured on
a Horiba
Fluoromax-4 setting both the excitation and the emission slits at 3 nm,
the resolution at 1 nm and the integration time at 0.1 s. Both instruments
were equipped with a thermostat-controlled ethylene glycol-water bath
fitted to specially designed cuvette holders and the temperature was kept
at 25. 0 ± 0.1 °C or 37.0 ± 0.1 °C, respectively. Native, tzA-, and thA-
based probes were excited at 335, 330, and 360 nm, respectively. All
spectra were corrected for the blank. Further assay-dependent
specificities can be found in the SI.
Spectroscopic Real-Time Monitoring
Experimental Section
Enzymatic conversion was followed by monitoring the emission intensity
at a fixed wavelength as function of time. Emission was measured on a
Horiba Fluoromax-4 equipped with a cuvette holder with a built-in stirring
system, setting both the excitation and emission slits at 3 nm and taking
a point at regular time intervals. The temperature was kept at 25. 0 ±
Synthesis of thATP
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10.1002/chem.201805520
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FULL PAPER
0.1 °C or 37.0 ± 0.1 °C, respectively. Emission was monitored at 410 nm
(λex = 360 nm), 410 nm (λex = 330 nm) and 465 nm (λex = 335 nm) for
thATP, tzATP, and ATP, respectively. Further assay-dependent
specificities can be found in the SI.
After resolving the reaction mixtures via SDS-PAGE, the gel was rinsed
in ddH2O once, immersed in staining solution (0.15% Coomassie Brilliant
Blue R-250, 45% MeOH, 10% acetic acid in ddH2O), microwaved for 50 s
and shaken for 20 min at RT. After rinsing with ddH2O, the gel was
covered with destaining solution (5.0% MeOH, 7.5% acetic acid in
ddH2O), microwaved for 50 s and shaken for 24 h at RT. The destaining
solution was replaced frequently. To estimate molecular weights,
PageRuler Plus Prestained Protein Ladder (Thermo Fisher) was used as
a reference.
NMNAT1- and ADH-Mediated Conversion
In a 125 µL cuvette, buffer containing Tris-HCl (50 mM, pH 7.8), NaCl (50
mM), KCl (50 mM), MgCl2 (12 mM), NMN (15 µM), PPase (0.3 U), EtOH
(17 µM), and thATP, tzATP, or ATP (15 µM) was added to set up a total
reaction volume of 500 µL. The solution was left to stabilize at 37.0 ±
0.1 °C for 30 minutes before NMNAT1 (14 mU) was added. The mixture
was promptly mixed and left at 37.0 ± 0.1 °C for 6000 s (thATP) or 1800 s
(tzATP, ATP), respectively. To start the second reaction, the mixture was
treated with ADH (5 µL, 58 U / µM substrate), promptly mixed, and left at
37.0 ± 0.1 °C for 600 s (thATP) or 300 s (tzATP, ATP), respectively. All
reactions were done in duplicates.
Western Blot
After resolving the reaction mixtures via SDS-PAGE, gels were
electrophoretically transferred onto a PVDF membrane (GE HealthCare)
in 1× NuPAGE Transfer Buffer (ThermoFisher) + 10% MeOH at 30 V for
70 min. The membrane was blocked in Odyssey Blocking Bu er (LI-
COR) at RT for 30 min under agitation. After blocking, the membrane
was incubated with anti-PAR [10H] antibody (Tulip BioLabs, 10 µg/mL) in
1× TBST buffer at 4 °C o/n. The membrane was washed three times with
1× TBST buffer for 5 min each and incubated with anti-mouse IRDye
680RD antibody (LI-COR, diluted 1:15,000) at RT for 1 h. Subsequently,
the membrane was washed three times with 1× TBST buffer + 0.2 %
SDS for 5 min each and then three times with 1× PBS-buffer for 5 min
each. The membrane was incubated with anti-PARP1 antibody (Tulip
BioLabs, 1 µg/mL) in 1× TBST buffer at RT for 1 h. The membrane was
washed three times with 1× TBST buffer for 5 min each and incubated
with anti-chicken secondary antibody (diluted 1:15,000) at RT for 1 h.
Subsequently, the membrane was washed three times with 1× TBST
buffer + 0.2% SDS for 5 min each and then three times with 1× PBS-
buffer for 5 min each. Finally, the membrane was stored in PBS buffer
until imaged and quantitatively analyzed via Image Studio (LI-COR).
NADase-Mediated Hydrolysis
In a 3 mL cuvette equipped with a stirring bar, buffer containing Tris-HCl
(50 mM, pH 7.8), NaCl (50 mM), KCl (50 mM), and NthAD+ or NtzAD+ (7.5
µM) was added to set up a final reaction volume of 3 mL. The solution
was left to stabilize at 25.0 ± 0.1 °C for 30 min before an aliquot of
suspended NADase (30 mU) was added. The suspension was promptly
mixed and left at 25.0 ± 0.1 °C for 6300 s (NthAD+) or 1500 s (NtzAD+),
respectively. All reactions were done in duplicates..
CTA-Mediated Mono(ADP-ribosyl)ation
In a 600 µL sterilized conical vial, buffer containing potassium phosphate
(pH 7.2; 160 mM), DTT (20 mM), agmatine sulfate (15 mM), MgCl2 (20
mM), ovalbumine (4 g/L) and NAD+, NthAD+ or NtzAD+ (7.5 µM) was
added to set up a final reaction volume of 25.5 µL. The solution was left
to stir at 25.0 ± 0.1 °C for 5 minutes to equilibrate. To start the reaction,
CTA (255 µg) is added. All reactions were done in duplicates.
Acknowledgements
We thank the National Institutes of Health for generous support
(GM 069773) and UCSD’s Chemistry & Biochemistry MS Facility.
We are grateful to Dr. Alexander Rovira for helpful discussions
and to the DAAD and Bavarian State for supporting J.F. with the
PROSA LMU scholarship.
PARP1-Mediated Poly(ADP-ribosyl)ation
In a 600 µL sterilized conical vial, 4× buffer (30 µL) containing (Tris-HCl
pH 8.0, 400 mM), MgCl2 (40 mM), DTT (4 mM) and calf thymus DNA (15
mg/L) was added and left to stabilize at 37 °C for 3 min. Subsequently,
PARP1 (4.1µL, 150 nM final conc.) was added, the solution was gently
mixed and left at 37 °C for another 3 min to activate PARP1. After
treatment with NthAD+, NtzAD+, or NAD+ (60 µL, 500 µM final conc.), the
mixture was gently mixed and left at 37 °C for 180 min. All reactions were
done in duplicates.
Keywords: cofactors • NAD+ • spectroscopy • ADP-ribosylation •
PARP1
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10.1002/chem.201805520
Chemistry - A European Journal
FULL PAPER
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Jonas Feldmann,[a, b] Yao Li,[a] and
Yitzhak Tor*[a]
Page No. – Page No.
Emissive synthetic cofactors: A
highly responsive NAD+ analogue
reveals biomolecular recognition
features
A photophysically responsive, N7-lacking, NAD+ analogue (NthAD+) has been
enzymatically synthesized and employed to study biomolecular recognition features
by real-time fluorescence monitoring. NthAD+ serves as a substrate for multiple
NAD+-dependent enzymes, including ADH, NADase, CTA, and NMNAT1 but not
PARP1. Poorer recognition compared to an N7-containing analogue (NtzAD+)
suggests key biomolecular contacts via the purine’s N7 moiety.
This article is protected by copyright. All rights reserved.