A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransferase

Article abstract of DOI:10.1016/j.ab.2010.12.035

Nicotinamide adenine dinucleotide (NAD) plays a crucial role in many cellular processes. As the rate-limiting enzyme of the predominant NAD biosynthesis pathway in mammals, nicotinamide phosphoribosyltransferase (Nampt) regulates the cellular NAD level. Tumor cells are more sensitive to the NAD levels, making them more susceptible to Nampt inhibition than their nontumorigenic counterparts. Experimental evidence has indicated that Nampt might have proangiogenic activity and supports the growth of some tumors, so Nampt inhibitors may be promising as antitumor agents. However, only four Nampt inhibitors have been reported, and no high-throughput screening (HTS) strategy for Nampt has been proposed to date, largely limiting the drug discovery targeting Nampt. Therefore, the development of a robust HTS strategy for Nampt is both imperative and significant. Here we developed a fluorometric method for a Nampt activity assay by measuring the fluorescence of nicotinamide mononucleotide (NMN) derivative resulting from the enzymatic product NMN through simple chemical reactions. Then we set up an HTS system after thorough optimizations of this method and validated that it is feasible and effective through a pilot screening on a small library. This HTS system should expedite the discovery of Nampt inhibitors as antitumor drug candidates.

Full text of DOI:10.1016/j.ab.2010.12.035

Analytical Biochemistry 412 (2011) 18–25
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
A fluorometric assay for high-throughput screening targeting
nicotinamide phosphoribosyltransferase
Ruo-Yu Zhang a,1, Ye Qin a,1, Xiao-Qun Lv , Pei Wang , Tian-Ying Xu , Lei Zhang , Chao-Yu Miao
a
a
a
b
a,c,d,
⇑
a
Department of Pharmacology, Second Military Medical University, Shanghai 200433, China
Department of Pharmacognosy, Second Military Medical University, Shanghai 200433, China
Shanghai Key Laboratory of Vascular Biology, Ruijin Hospital, Shanghai 200025, China
Shanghai Institute of Hypertension, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Nicotinamide adenine dinucleotide (NAD) plays a crucial role in many cellular processes. As the rate-
limiting enzyme of the predominant NAD biosynthesis pathway in mammals, nicotinamide phosphoribo-
syltransferase (Nampt) regulates the cellular NAD level. Tumor cells are more sensitive to the NAD levels,
making them more susceptible to Nampt inhibition than their nontumorigenic counterparts. Experimen-
tal evidence has indicated that Nampt might have proangiogenic activity and supports the growth of
some tumors, so Nampt inhibitors may be promising as antitumor agents. However, only four Nampt
inhibitors have been reported, and no high-throughput screening (HTS) strategy for Nampt has been pro-
posed to date, largely limiting the drug discovery targeting Nampt. Therefore, the development of a
robust HTS strategy for Nampt is both imperative and significant. Here we developed a fluorometric
method for a Nampt activity assay by measuring the fluorescence of nicotinamide mononucleotide
(NMN) derivative resulting from the enzymatic product NMN through simple chemical reactions. Then
we set up an HTS system after thorough optimizations of this method and validated that it is feasible
and effective through a pilot screening on a small library. This HTS system should expedite the discovery
of Nampt inhibitors as antitumor drug candidates.
Received 26 September 2010
Received in revised form 25 December 2010
Accepted 28 December 2010
Available online 4 January 2011
Keywords:
Nampt
NAD
NMN
Enzyme activity assay
High-throughput screening
Ó 2011 Elsevier Inc. All rights reserved.
Nicotinamide adenine dinucleotide (NAD)2 plays a crucial role in
many cellular processes. It functions as a cofactor in more than 200
oxidation reduction reactions as well as a consumable substrate for
Nampt, also known as visfatin or PBEF, is highly conserved
throughout evolution [10]. Because Nampt is the rate-limiting en-
zyme in the NAD biosynthesis pathway in mammals, it regulates
the total cellular NAD level [11] and mitochondrial NAD levels
[12]. In addition, Nampt maintains an energy supply by synthesiz-
ing NAD and plays a critical role in cell survival [12,13]. Cancer
cells exhibit a high rate of NAD turnover due to the high level of
ADP-ribosylation activity required for DNA repair, genome stabil-
ity, and telomere maintenance, making these cells more dependent
on the cellular NAD level for rapid proliferation [14–16]. As Nampt
is a key enzyme in the NAD biosynthesis in mammals, more depen-
dence on NAD levels makes tumor cells more susceptible to Nampt
inhibition. Nampt has been shown to be up-regulated in several
cancers [17–19] as well as to be involved in angiogenesis and in-
duce vascular endothelial growth factor [20] and induce prolifera-
tion and capillary-like tube formation in human umbilical vein
endothelial cells [21]. These findings indicate that Nampt might
have proangiogenic activity and support the growth of some tu-
mors [22], making Nampt an attractive target for antitumor thera-
peutics during recent years [22,23].
sirtuins, poly(ADP-ribose) polymerase 1 (PARP1), and ADP cyclases
+
[
1], which are linked to Ca2 mobilization, genomic stability, apopto-
sis, metabolism, and other critical biological processes [2–5]. Trypto-
phan, nicotinamide riboside, nicotinic acid (NA), and nicotinamide
(
NAM) are precursors of NAD biosynthesis [6–9], and NAM is
predominantly used in mammals [8]. In the predominant NAD
biosynthesis pathway, NAM is converted to nicotinamide mononu-
cleotide (NMN) by nicotinamide phosphoribosyltransferase (Nampt)
and further to NAD by nicotinamide/nicotinic acid mononucleotide
adenylyltransferase (Nmnat).
⇑
Corresponding author at: Department of Pharmacology, Second Military
Medical University, Shanghai 200433, China. Fax: +86 21 65493951.
E-mail address: cymiao@smmu.edu.cn (C.-Y. Miao).
These authors contributed equally to this work.
Abbreviations used: NAD, nicotinamide adenine dinucleotide; PARP1, poly(ADP-
1
2
ribose) polymerase 1; NA, nicotinic acid; NAM, nicotinamide; NMN, nicotinamide
mononucleotide; Nampt, nicotinamide phosphoribosyltransferase; Nmnat, nicotin-
amide/nicotinic acid mononucleotide adenylyltransferase; NAPRT, nicotinic acid
phosphoribosyltransferase; HTS, high-throughput screening; SDS–PAGE, sodium
dodecyl sulfate–polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide; PRPP,
phosphoribosylpyrophosphate; BSA, bovine serum albumin; DTT, dithiothreitol; SD,
standard deviation; S/N, signal-to-noise; CV, coefficient of variation.
A persisting challenge in cancer therapy is to achieve a suffi-
cient therapeutic window between inducing tumor cell death
and cytotoxic effects in somatic cells to obtain efficient antitumor
therapy [24]. The experimental evidence indicates that some
0
003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2010.12.035

Assay for high-throughput screening targeting Nampt / R.-Y. Zhang et al. / Anal. Biochem. 412 (2011) 18–25
19
tumors, such as glioblastomas and neuroblastomas, are deficient in
nicotinic acid phosphoribosyltransferase (NAPRT), an enzyme that
catalyzes NA to nicotinic acid mononucleotide. So, NA can be used
as the antidote to reduce the toxicity of Nampt inhibitor on normal
cells, and coadministration of NA provides an opportunity to widen
the therapeutic index of Nampt inhibitors [24–26]. NA can be cat-
alyzed by NAPRT to nicotinic acid mononucleotide [27,28] and
eventually converted to NAD through the sequential reactions of
Nmnat and NAD synthetase [29]. NAD from this pathway bypasses
the inhibition of Nampt and serves to protect NAPRT-proficient
normal cells from inhibitor cytotoxicity while not compromising
the antitumor effect of inhibitor on NAPRT-deficient tumor cells
phate (PRPP, Sigma), 2 mM ATP, 0.02% bovine serum albumin
(BSA), 2 mM dithiothreitol (DTT), 12 mM MgCl , and 50 mM Tris–
HCl (pH 7.5). PRPP was unstable at low concentration or after a
few cycles of freezing and thawing, so it would be better to store
small-volume aliquots of 100 mM PRPP stock at ꢁ80 °C and freshly
thaw for use.
2
Determination of IC50 values for Nampt inhibitors
To determine the IC50 values of inhibitors, 5-ll compound solu-
tions (containing 10% DMSO) with various concentrations were
added into a 96-well plate. The plate was incubated at 37 °C for
[
26]. In addition, the activity of Nampt inhibitors is cell cycle
5 min after the addition of 16.5
Nampt. The enzyme reactions were initiated by 4.5
(1.11 M) and allowed to proceed for 15 min before the NMN
l
l of reaction buffer containing
independent, and they are not subject to commonly known mech-
anisms of multidrug resistance [30]. These advantages make
Nampt inhibitors very promising antitumor agents.
To date, only four Nampt inhibitors have been reported. FK866,
CHS-828, and CB30865 were found as antitumor agents [31–33]
whose targets were subsequently proved to be Nampt [26,34–
l
l of NAM
l
detection reaction. The IC50 values were determined by nonlinear
fitting of the concentration-dependent curves with the four-
parameter IC50 logistic equation.
3
6]. IS001 was an analogue of FK866 with an additional ribose ring
Library screening
[
37]. CHS-828 is in phase I clinical trials [38], and FK866 is in phase
II clinical trials [30,39], whereas FK866 exhibits low bioavailability,
rapid intravenous clearance, and substantial drug binding to plas-
ma proteins [30]. To find more Nampt inhibitors as potential anti-
tumor drug candidates, an effective high-throughput screening
First, 1 mM DMSO stock of approximately 550 compounds was
prepared, including FK866 (also known as APO866, donated by
Apoxis, Switzerland), gallotanine (Sigma), and other natural prod-
ucts from an in-house compound collection or National Compound
(
HTS) system targeting Nampt is imperative. In the current study,
Resource Center (Shanghai, China). Then a 0.5-ll stock of each
we have developed and optimized an HTS system targeting Nampt
and validate it through a pilot screening on a small compound
library. So far as we know, this is the first HTS study targeting
Nampt. Our results show that this HTS system is simple, sensitive,
and cost-effective. It is a feasible and robust platform by which
novel potent and specific Nampt inhibitors can be discovered.
compound was added into 96-well plates and followed by the
sequential Nampt enzyme reaction and NMN detection reaction.
The final fluorescence was recorded for further data analysis.
Data analysis
All experiments except library screening were performed at
least twice. Each final value is presented as the mean ± standard
deviation (SD). Each signal-to-noise (S/N) ratio was calculated
using the following equation [40]: (Meansignal ꢁ Meanbackground)/
SDbackground [40]. Each coefficient of variation (CV) was the ratio
of the SD to the mean. The Z’ factor was determined by Eq. (1) [41]:
Materials and methods
Protein expression and purification
The Nampt/pET28a+ plasmid was a gift from S. Imai (Washington
University School of Medicine) [11]. His-tagged Nampt proteins
were expressed in BL21–CondonPlus(DE3)–RIL cells (Stratagene)
3
ðSDhigh signal þ SDlow signalÞ
:
Z0 ¼ 1 ꢁ
ð1Þ
Meanhigh signal ꢁ Meanlow signal
at 28 °C in 2ꢀ YT medium containing 100
lg/ml kanamycin and
3
7
l
g/ml chloramphenicol and then purified with nickel–nitrilo-
Relative enzyme activity (Activity%) was calculated by Eq. (2):
triacetic acid resin (Qiagen). The purity of the protein was verified
more than 90% by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) and Coomassie staining.
F ꢁ FC0
Activity% ¼
;
ð2Þ
F
100% ꢁ F
0
0
where F was the fluorescence of background control (representing
NMN measurement
zero activity from a simulated enzyme reaction with Nampt but no
NAM or compound), F100% was the fluorescence of the reference
control (representing 100% activity from the intact enzyme reaction
without compound perturbation), FC0 was the fluorescence of an-
other simulated enzyme reaction with Nampt and compound but
no NAM (used to eliminate intrinsic fluorescence of compound or
fluorescent derivative occasionally converted from compound by
Nampt), and F was the fluorescence after the aforementioned pro-
cess described in the ‘‘Library screening’’ section above.
First, 10
and 10 l of 2 M KOH were added into 25
ous solution (for NMN quantification) or Nampt reaction solution
for enzymatic product NMN detection). Then the mixture was
incubated in an ice bath for 2 min before the addition of 45 l of
8% formic acid. After incubation at 37 °C for 10 min, the solution
ll of 20% acetophenone in dimethyl sulfoxide (DMSO)
l
l
l of NMN (Sigma) aque-
(
l
8
was transferred into a flat-bottom 96-well black plate (Greiner).
The fluorescence was measured using a Tecan Infinity M200 plate
reader (Tecan Group), setting the excitation and emission wave-
lengths to 382 and 445 nm, respectively.
Results
Development of fluorometric method for Nampt activity assay
Enzyme assay
Nampt converts NAM to NMN by the enzyme reaction depicted
in Fig. 1A. By quantifying the product NMN, relative Nampt activity
can be determined. Based on the fact that N-alkylpyridinium
compounds can be converted to fluorescent derivatives through
reactions with ketone followed by heating in excess acid [42–45],
The mixture of purified Nampt and its substrate NAM (Sigma)
was incubated at 37 °C for a given time, and the product NMN
was measured as described above to determine Nampt activity.
The reaction buffer contained 0.4 mM phosphoribosylpyrophos-

2
0
Assay for high-throughput screening targeting Nampt / R.-Y. Zhang et al. / Anal. Biochem. 412 (2011) 18–25
Fig.1. Two reactions involved in fluorometric assay for Nampt activity: (A) enzyme reaction and (B) detection reaction. PPi, pyrophosphate.
we tried to determine Nampt activity using a fluorometric ap-
proach by converting its enzymatic product NMN to a fluorescent
derivative through sequentially reacting with acetophenone/KOH
and formic acid (Fig. 1B, detection reaction).
We detected the maximum fluorescence intensity of NMN
derivative at a 382-nm excitation wavelength and a 445-nm emis-
sion wavelength (Fig. 2A), and we obtained the standard curve at
these wavelengths using a series of concentrations of NMN in the
detection reaction. As shown in Fig. 2B, the fluorescence was linear
with the concentration of NMN up to 80 M and was sensitive to at
l
least 60 nM NMN. Moreover, normal enzyme reaction and three
control reactions (without both or either of Nampt and NAM) were
carried out and respectively followed by the detection reaction,
and the results are shown in Fig. 2C. It was clear that in the reac-
tion buffer, NAM or Nampt did not enhance the fluorescence; only
NMN from the intact enzyme reaction resulted in fluorescence
Fig.2. Validation of fluorometric method for Nampt activity assay. (A) Fluorescence excitation and emission spectra of NMN derivative. (B) Standard curve of fluorescence
versus NMN. (C) Fluorescence from detection reaction based on normal enzyme reaction and three control reactions. (D) Standard curve of fluorescence versus Nampt
concentration. Various concentrations of Nampt reacted with 4 lM NAM for 6 min, followed by the detection reaction.

Assay for high-throughput screening targeting Nampt / R.-Y. Zhang et al. / Anal. Biochem. 412 (2011) 18–25
21
Fig.3. Optimization of detection reaction with 1 lM NMN. (A and B) step 2 was carried out at different temperatures (A) or for different times at 37 °C (B). (C) Effect of
reaction time and temperature in step 1 on NMN final fluorescence.
enhancement, verifying that the fluorescence could be used as a
measurement of the enzymatic product NMN without any interfer-
ence from other components of the enzyme reaction. Furthermore,
we checked the fluorescence response to the enzyme reaction to
confirm that the fluorescence can quantify the Nampt activity. Dif-
none was insoluble in the reaction buffer and that introducing
DMSO would help to emulsify the mixtures and achieve
reproducibility.
Optimization of enzyme reaction
ferent concentrations of Nampt (up to 40 lg/ml) and a sufficient
amount of NAM were used in the enzyme reaction, and the fluores-
cence after the same detection reaction was also linear with the
concentration of Nampt (Fig. 2D). All of these results demonstrated
that this fluorometric method could be used to determine the
Nampt activity conveniently and sensitively.
Because high temperatures (e.g., 95 °C) will denature proteins
and eliminate enzyme activity, heat treatment was tested to termi-
nate the enzyme reaction and make the reaction time controllable.
As shown in Fig. 4A, the enzyme treated at 95 °C for 1 min lost
activity and could not catalyze NAM to NMN. Most important,
NMN still remained intact after being treated at 95 °C for 1 min,
and the fluorescence intensities were nearly the same between
derivatives from NMN with and without heat treatment (Fig. 4B).
Optimization of detection reaction
We first optimized the reaction conditions in step 2 of the
detection reaction. After step 1, 45 ll of 88% formic acid was added,
the mixtures were incubated at 37, 70, and 90 °C for 6 min, respec-
tively, and the reaction was terminated by cooling on ice. As shown
in Fig. 3A, there was almost no difference in fluorescence at the
three temperatures, indicating that higher temperatures did not in-
crease the production of NMN derivation, so we selected a more
mild condition of 37 °C and went on to optimize the reaction time
of step 2. As shown in Fig. 3B, this reaction was approximately 90%
complete by 1 min and fully completed by 10 min, and after that
time the fluorescence could remain stable for even a few hours.
In summary, the optimal reaction condition in step 2 was: reacting
at 37 °C for 10 min.
Similar optimizations were also performed for step 1. The reac-
tion was carried out at 0 or 25 °C for different times, followed by
the step 2 reaction under optimal conditions, and the fluorescence
was measured and is represented in Fig. 3C. Reactions at 0 °C were
more favorable than those at 25 °C with approximately 1.2-fold
fluorescence intensity, and the elongation of reaction time did
not increase the fluorescence. Moreover, we found that acetophe-
Fig.4. The heating method could terminate the enzyme reaction and keep the
enzymatic product NMN intact for the subsequent detection reaction. (A) The
enzyme reactions were performed using Nampt with and without heat treatment at
95 °C for 1 min, and the fluorescence was measured after the same detection
reaction. (B) Fluorescence after detection reaction using heat-treated and untreated
NMN at 95 °C for 1 min.

2
2
Assay for high-throughput screening targeting Nampt / R.-Y. Zhang et al. / Anal. Biochem. 412 (2011) 18–25
Fig.5. The enzyme reaction was optimized to be suitable for HTS. (A) Michaelis–Menten curve. (B) Enzyme kinetics curve under optimal enzyme reaction conditions. (C)
Effects of DMSO on Nampt activity. (D) Effects of preincubation on the following enzyme reaction.
That is, heating at 95 °C for 1 min could completely terminate the
enzyme reaction and keep the enzymatic product NMN intact for
the subsequent detection reaction.
The concentrations of NAM and Nampt in the enzyme reaction
were also optimized. First, we determined the Michaelis constant
Because compound libraries were usually prepared in DMSO
stock, the tolerance to DMSO in the enzyme assay was tested. As
shown in Fig. 5C, the Nampt activity displayed a negligible change
through DMSO concentrations ranging from 1% to 8%. On the other
hand, to allow slow-binding compounds to have enough time to
interact with the enzyme, a preincubation step is necessary before
the enzyme reaction is started, so the stability of Nampt and other
components during the preincubation was examined. Nampt in the
reaction buffer incubated at 37 °C for different time was used in
the enzyme reaction, and the fluorescence (Fig. 5D) indicated that
Nampt and other components in the reaction buffer remained in-
tact at 37 °C within 30 min. The preincubation time was set to
5 min in the screening experiments.
(K
m
) of NAM, and the classical Michaelis–Menten curve is shown
in Fig. 5A. The fluorescence increased linearly with concentrations
of NAM below 0.31
fitting the curve to the Michaelis–Menten equation, the K
was determined to be 0.16
of NAM (0.2, 1, and 2 M) with Nampt (2, 4, and 8
l
M and reached a plateau above 1.25
l
m
M. By
value
l
M. Through different combinations
g/ml), a series
l
l
of S/N ratios were measured and are listed in Table 1. S/N ratios un-
der higher concentrations of NAM and Nampt were always better,
but considering that all S/N ratios were greater than 45 and met
the minimal requirement of 8 [46] as well as the cost factor for
Nampt, NAM, and compound in HTS, we fixed substrate at a near
Screen assay
m
K concentration of 0.2 lM and fixed Nampt at 2 lg/ml in the
Based on the results from all optimizations, the final protocol
subsequent activity assay. Under these conditions, we were able
to get a classical enzyme kinetics curve (Fig. 5B). The linear
relationship remained well until 30 min, so the optimal enzyme
reaction time was set to 15 min with consideration of the trade-
off between fluorescence intensity and time.
for HTS was as follows. First, 0.5
pound was transferred into a 96-well PCR plate, and with the addi-
tion of 20 l of reaction buffer containing Nampt, the plate was
incubated at 37 °C for 5 min. Then 4.5 l of NAM stock was added
to initiate the reaction, resulting in a final concentration of 2%
DMSO, 2 g/ml Nampt, 0.2 M NAM, and 20 M compound. After
reacting at 37 °C for 15 min, the reaction was terminated by heat-
ing at 95 °C for 1 min and cooling in an ice bath. Then 10 l of 20%
acetophenone in DMSO and 10 l of 2 M KOH were added, and the
mixture was vortex mixed and kept in an ice bath for 2 min. After
that, 45 l of 88% formic acid was added, and the mixture was
incubated at 37 °C for 10 min. Finally, 85- l mixtures in each well
ll of 1 mM DMSO stock of com-
l
l
l
l
l
Table 1
S/N ratios at different combinations of substrate and enzyme
l
l
[
NAM] (
l
M)
[Nampt] (lg/ml)
2
4
8
l
0
1
2
.2
46
208
197
48
247
458
45
214
485
l
were transferred into a flat-bottom 96-well black plate for fluores-
cence detection on a plate reader.

Assay for high-throughput screening targeting Nampt / R.-Y. Zhang et al. / Anal. Biochem. 412 (2011) 18–25
23
of DMSO stock of each compound was added to each well of a
6-well plate except the last row. In the last row, the former six
wells were set for reference controls (F100%) and the latter six wells
were set for background controls (F ). A duplicate plate without
9
0
NAM in each well was used for compound controls (FC0). To mon-
itor the quality of screening, means of background and reference
0
controls from each assay plate were obtained and analyzed. Z fac-
tors during our screening ranged from 0.60 to 0.85, and S/N ratios
were greater than 11. The relative enzyme activity under com-
pound perturbation was calculated using Eq. (2), and the results
are shown in Fig. 6. Six compounds resulting in less than 60% rel-
ative activity were subjected to a secondary screen, and four of
them were validated as hits: FK866 and three novel compounds
named gallotanine, Jin Song biflavone, and amentotaxus biflavone
(
Fig. 7). These compounds were further confirmed as Nampt inhib-
Fig.6. Relative activity of screened compound library.
itors by a concentration-dependent inhibition assay. All of them
showed conventional inhibition curves (Fig. 7). The IC50 values
were calculated by fitting the curves with the four-parameter
IC50 logistic equation, and they were 0.9 nM, 1.3
and 6.6 M for FK866, gallotanine, Jin Song biflavone, and ament-
The robustness of this HTS system was validated before real
screening of a compound library. First, we simulated the screening
process by adding 0.5 ll of DMSO without compounds in 96-well
lM, 17.5 lM,
l
otaxus biflavone, respectively.
plates. All of the CV values were less than 8%, and the S/N ratios
0
were greater than 13. The Z factor was calculated from the assay
both with and without FK866. A total of 64 assays were run simul-
taneously on a 96-well PCR plate, representing 32 inhibited and 32
uninhibited reactions. The calculated Z factor was 0.67. All of these
Discussion
0
Inhibition of Nampt activity has been shown to be a promising
antitumor strategy. However, only four Nampt inhibitors have been
reported until now; therefore, more Nampt inhibitors with good
pharmacological and therapeutic potential need to be identified
and characterized as potential candidates for cancer treatment.
HTS now plays a pivotal role in molecular mechanism-based cancer
indexes met the requirements of HTS and verified the robustness of
this system.
As a preliminary test of this method, we carried out screening
experiments on a small library of approximately 550 compounds
that purposely included the known inhibitor FK866. First, 0.5 ll
Fig.7. Dose–response curves of FK866 and novel Nampt inhibitors identified from the screening.

2
4
Assay for high-throughput screening targeting Nampt / R.-Y. Zhang et al. / Anal. Biochem. 412 (2011) 18–25
drug discovery [47], but no appropriate HTS system targeting Nampt
has been reported. There are three methods used to measure Nampt
activity. First, the radioactive substrate NAM is converted to NMN,
which is precipitated in acetone and quantified by scintillation
counting [48,49]. Second, the enzymatic product NMN is separated
and measured by high-performance liquid chromatography [50].
Third, the enzymatic product NMN is catalyzed to NAD by Nmnat,
and NAD is further reduced to NADH by alcohol dehydrogenases.
NADH can be measured by excitation at 340 nm and emission at
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their use for HTS because the radiolabeling method involves radio-
active operations, the chromatography method needs an additional
separation process, and the enzyme-coupled method involves
additional two enzymes, which will increase the cost of HTS and
the possibility of an off-target inhibitor.
Here we have described a new fluorometric Nampt activity assay
that is robust and suitable for HTS. The enzymatic product NMN is
converted to a fluorescent derivation through two simple chemical
reactions, and the fluorescence is linear with the concentration of
NMN. The fluorescence response is more sensitive than that of the
reported fluorometric method [11] (data not shown), and the lower
limit of detection for NMN is at least 60 nM. The reaction tempera-
tures are below 37 °C, the time of whole assay is within 1 h, and fur-
ther separation is not required. The volume of the enzyme reaction is
[
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(
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This work was supported by grants from the National Basic
Research Program of China (2009CB521902 to C.-Y.M.), the
National Science and Technology Major Project (2009ZX09303-002
to C.-Y.M.), the Program of Shanghai Subject Chief Scientist (10XD14
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thank Dr. S. Imai (Washington University School of Medicine) for
the recombinant Nampt plasmid. We thank S.A. Apoxis for FK866
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