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ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 376 (2008) 122–130
www.elsevier.com/locate/yabio

A highly sensitive high-throughput luminescence assay

for malonyl-CoA decarboxylase
Mei-Chu Lo a,*, Minghan Wang b, Ki Won Kim b, James Busby b, Harvey Yamane c,
James Zondlo c, Chester Yuan d, Stephen W. Young a, Shou-Hua Xiao a,*
a
Chemistry Research and Discovery, Amgen, South San Francisco, CA 94080, U.S.A.
b
Metabolic Disorders, Amgen, Thousand Oaks, CA 91320, U.S.A.
c
Protein Science, Amgen, Thousand Oaks, CA 91320, U.S.A.
d
Chemistry Research and Discovery, Amgen, Thousand Oaks, CA 91320, U.S.A.

Received 17 December 2007

Available online 2 February 2008

Abstract

Malonyl-CoA decarboxylase (MCD) catalyzes the conversion of malonyl-CoA to acetyl-CoA and thereby regulates malonyl-CoA
levels in cells. Malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase-1, a key enzyme involved in the mito-
chondrial uptake of fatty acids for oxidation. Abnormally high rates of fatty acid oxidation contribute to ischemic damage. Inhibition of
MCD leads to increased malonyl-CoA and therefore decreases fatty acid oxidation, representing a novel approach for the treatment of
ischemic heart injury. The commonly used MCD assay monitors the production of NADH fluorometrically, which is not ideal for library
screening due to potential fluorescent interference by certain compounds. Here we report a luminescence assay for MCD activity. This
assay is less susceptible to fluorescent interference by compounds. Furthermore, it is 150-fold more sensitive, with a detection limit of 20
nM acetyl-CoA, compared to 3 lM in the fluorescence assay. This assay is also amenable to automation for high-throughput screening
and yields excellent assay statistics (Z0 > 0.8). In addition, it can be applied to the screening for inhibitors of any other enzymes that
generate acetyl-CoA.
Ó 2008 Elsevier Inc. All rights reserved.

Keywords: Malonyl-CoA decarboxylase; High-throughput screening; Luminescence assay; Coupled assay; IC50; Inhibitor; Km; kcat

The heart has a high energy demand that is met primarily
by the oxidation of fatty acids and secondarily by the metab-
olism of glucose and lactate [1]. Fatty acid oxidation is highly
regulated to ensure that energy supply closely matches
demand. Carnitine palmitoyltransferase-I is a key enzyme
involved in mitochondrial uptake of fatty acids and is inhib-
ited by malonyl-CoA [2]. As a result, a rise in cardiac malo-
nyl-CoA levels suppresses mitochondrial fatty acid
oxidation by decreasing fatty acid transport, whereas a
decrease in malonyl-CoA leads to increased fatty acid oxida-
tion [3]. The steady state level of malonyl-CoA is controlled
by its synthesis and degradation. It is synthesized from car-
*
Corresponding authors.
E-mail addresses: meil@amgen.com (M.-C. Lo), josh.xiao@amgen.
com (S.-H. Xiao).
boxylation of acetyl-CoA by acetyl-CoA carboxylase
(ACC)1 and degraded to acetyl-CoA by malonyl-CoA decar-
boxylase (MCD). Under ischemic conditions, limited oxy-
gen supply reduces both fatty acid and glucose oxidation
and thereby decreases subsequent ATP production. To
maintain ATP levels during ischemia, glycolysis is stimulated
to drive anaerobic ATP production. Whereas overall mito-
chondrial oxidative metabolism decreases during ischemia,
two factors cause excessively high rates of fatty acid oxida-
tion at the expense of glucose oxidation: (1) circulating fatty
acid levels increase during ischemia [4] and (2) malonyl-CoA
1
Abbreviations used: ACC, acetyl-CoA carboxylase; BSA, bovine serum
albumin; CoA, coenzyme A; DMSO, dimethyl sulfoxide; DTT, dithio-
threitol; HTS, high-throughput screening; MCD, malonyl-CoA decarbox-
ylase; PPi, pyrophosphate.

0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2008.01.033
 A luminescence HTS assay for malonyl-CoA decarboxylase / M.-C. Lo et al. / Anal. Biochem. 376 (2008) 122–130
levels are reduced by ischemia-induced activation of AMP-
activated protein kinase, which phosphorylates and inhibits
ACC [5]. Fatty acid oxidation leads to elevated ratios of
NADH/NAD+ and acetyl-CoA/free CoA that further inhi-
bit glucose oxidation through inhibition of pyruvate dehy-
drogenase [6]. As a result, the pyruvate produced from
glycolysis is not readily oxidized in the mitochondria and is
instead reduced to lactate, resulting in accumulation of met-
abolic by-products (lactate and protons) that can lead to
impaired cardiac function, decreased cardiac efficiency,
and increased myocardial tissue injury [7].
Because the excessive use of fatty acids leads to contractile
dysfunction and ischemic injury, one approach to the treat-
ment of ischemia is to decrease fatty acid oxidation by
increasing malonyl-CoA through inhibition of MCD [8].
Potent MCD inhibitors have been developed [9–11] and
tested in a number of ischemia/reperfusion animal models
[12]. These MCD inhibitors significantly increased glucose
oxidation rates and reduced lactate production compared
with vehicle-treated hearts and showed a significant accom-
panying increase in cardiac work compared with controls.
However, no efficacious and safe MCD inhibitor has been
demonstrated clinically and approved for use in humans
yet. Therefore we are interested in the discovery of additional
MCD inhibitors, especially of different pharmacophores.
Currently, the commonly used MCD assay works
through coupling to malate dehydrogenase/citrate synthase
and measuring the production of NADH spectrophotomet-
rically [13,14]. Because this assay monitors the fluorescence
emission of NADH, interference from fluorescent com-
pounds may be an issue when screening compounds using
this assay. Here we report the development of a lumines-
cence-based MCD assay that is more sensitive and less sus-
ceptible to compound interference. We have characterized
the steady state kinetics of purified human MCD using this
assay and compared the results with previously reported
data from the literature. In addition, the assay was adapted
to a high-throughput screening (HTS) format with a robust
Z0 factor.
Materials and methods
General
AMP, acetyl-CoA, malonyl-CoA, L-malate, NAD+, and
PPi were purchased from Sigma–Aldrich (Milwaukee, WI).
Acetyl-CoA synthetase, L-malate dehydrogenase, and cit-
rate synthase were obtained from Roche Applied Science
(Indianapolis, IN). Kinase-Glo Luminescent Kinase Assay
was from Promega (Madison, WI). Compounds A and B
were synthesized in-house.
Cloning, expression, and purification of His-tagged human
MCD
A cDNA clone containing the coding region of
human MCD (residues 41–493) was obtained from
123
human liver cDNA using high-fidelity Pfu Ultra Poly-
merase (Stratagene, La Jolla, CA). The 50 and 30 PCR
primers used in the reaction were 50 -AAC ATA TGC
ATC ATC ACC ATC ACC ATG ACG AGC TGC
TGC GCC GCG-30 and 50 -AAC TCG AGT CAG
AGC TTG CTG TTC TTT TGA AAC-30 , respectively.
Two restriction digestion sites, NdeI and XhoI, were
introduced into the 50 and 30 primers outside the open
reading frame, respectively. In addition, an in-frame
6X His tag was introduced at the 50 terminus so that
the expressed protein started with the sequence
MHHHHHH followed by the residue 41 onward of
the native protein. The PCR product was subcloned
into a sequencing vector (PCR2.1) and verified by
sequencing. The insert in the PCR2.1 vector was
digested with restriction enzymes NdeI and XhoI and
subcloned into an Amgen proprietary plasmid,
pAMG21, for bacterial expression of the MCD protein.
This plasmid was derived from Amgen plasmid
pCFM1656 (ATCC 69576) to allow for chemical induc-
tion of recombinant proteins.
The recombinant pAMG21 His6-MCD plasmid was
transformed into Escherichia coli expression strain
BL21 [15] and cultured overnight at 37 °C. A fermentor
batched with Terrific Broth at 25 °C was inoculated 1%
v/v from the overnight culture and growth continued at
25 °C until an A600 of approximately 1.5 was reached.
Temperature was then dropped to 20 °C and expression
chemically induced. Cell paste was harvested by centrifu-
gation 16 h postinduction. SDS–PAGE analysis of cell
paste fractions indicated that approximately 50% of the
total recombinant protein appears in the soluble
fraction.
To purify the His-tagged human MCD, the cell
paste was resuspended in a buffer containing 20 mM
Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM imidazole,
10% glycerol, and 20 mM b-mercaptoethanol. Cells
were lysed by mechanical shearing in a Microfluidizer
(Microfluidics, Newton, MA). The resulting lysate was
clarified by centrifugation at 20,000g for 2 h at 4 °C,
followed by filtration through a 0.45-lm membrane.
The clarified lysate was mixed with Talon metal affinity
resin (Clontech, Mountain View, CA) for 1 h at 4 °C.
After washing with a buffer consisting of 20 mM Tris–
HCl, pH 7.5, 500 mM NaCl, 5 mM imidazole, 10%
glycerol, and 20 mM b-mercaptoethanol, bound pro-
teins were eluted with 20 mM Tris–HCl, pH 7.5, 150
mM NaCl, 100 mM imidazole, 10% glycerol, and 20
mM b-mercaptoethanol. The Talon pool was diluted
with four volumes of 20 mM Tris–HCl, pH 7.0, 10%
glycerol, and 5 mM DTT and applied to a column
of SP Sepharose HP (GE Healthcare, Piscataway, NJ)
equilibrated in the same buffer. Elution was performed
with a linear gradient of 0–0.5 M NaCl in the equili-
bration buffer. Peak fractions containing MCD were
pooled and aliquots were stored at 80 °C prior to
use.
124 A luminescence HTS assay for malonyl-CoA decarboxylase / M.-C. Lo et al. / Anal. Biochem. 376 (2008) 122–130
Luminescence-based MCD assay
In the luminescence-based MCD assay, MCD activity
was measured by coupling to the acetyl-CoA synthetase
and luciferase reactions as shown in Eqs. (1)–(3):
MCD
Malonyl-CoA ƒƒ! acetyl-CoA þ CO2 ;
acetyl-CoA Synthetase
Acetyl-CoA þ AMP þ PPi ( + acetate
þ ATP þ CoA and
luciferase
Beetle luciferin þ ATP þ 1=2 O2 ƒƒƒƒ! oxyluciferin
2þ
Mg
þ AMP þ PPi þ CO2 þ light:
Acetyl-CoA synthetase catalyzes the formation of ATP in
the presence of AMP, PPi, and the MCD product, acetyl-
CoA. ATP was subsequently quantified by measuring lumi-
nescence intensity generated by monooxygenation of beetle
luciferin catalyzed by luciferase in the presence of Mg2+,
ATP, and molecular oxygen.
The MCD reactions were performed in a white 384-
well Opti-plate (PerkinElmer, Shelton, CT) with a total
volume of 20 lL. To determine the Km of malonyl-CoA,
reactions were conducted with 0.1 nM MCD and various
malonyl-CoA concentrations at 1.9, 3.8, 7.5, 15, 30, 60,
and 90 lM in buffer containing 40 mM Tris, pH 8.0,
0.02% Tween 20, 0.1% BSA, and 1 mM DTT. Reactions
were stopped at different time points by adding 5 lL com-
pound A (Fig. 1), a MCD inhibitor [12], to a final concen-
tration of 10 lM (1000-fold above IC50); 25 lL of the
luminescence detection reagent containing 0.9X Kinase-
Glo Reagent, 20 lM AMP, 20 lM PPi, and 0.003 U/lL
acetyl-CoA synthetase was then added and 1X Kinase-
Glo Reagent was prepared by adding 10 mL of Kinase-
Glo Buffer to one vial of Kinase-Glo Substrate following
the manufacture’s instructions. After adding the lumines-
cence detection reagent, the plate was incubated at room
temperature for at least 5 min, and luminescence intensity
was measured using an Envision microplate reader (Perk-
O O
CF3
R/S
O N OH
H added to the negative control wells to match the DMSO
N CF3
Compound A O
O for 15 min and then stopped and detected in the same
O
N to equation [16],
Compound B
F3C CF3
OH
Fig. 1. Chemical structures of MCD inhibitors, compounds A and B.
inElmer) with 0.2 s per well integration time. The initial
velocities were determined from the slopes of the progress
curves in the linear range and the luminescence units were
converted to the acetyl-CoA concentrations based on the
standard curve of acetyl-CoA. Km and kcat were obtained
by fitting the initial velocities to the Michaelis–Menten
ð1Þ equation (Eq. (4)) using GraphPad Prism 4.02 (Graph-
Pad, San Diego, CA).
ð2Þ V max ½S
V ¼ ; ð4Þ
ðK m þ ½SÞ
where [S] is the substrate concentration, V is the initial
ð3Þ velocity at a given [S], Vmax is the maximum velocity, and
Km is the Michaelis–Menten constant. kcat, the enzyme
turnover number, was derived by dividing Vmax by [E],
the concentration of enzyme.
To determine the IC50 values of MCD inhibitors, reac-
tions were conducted at 9 lM malonyl-CoA, 0.1 nM
MCD, and various concentrations of compounds. After
15 min incubation at room temperature, reactions were
stopped by adding compound A to a final concentration
of 10 lM. The luminescence detection reagent was added
and luminescence intensity was measured as described
above. To obtain IC50 values, the luminescence intensities
at different compound concentrations were fitted to a sig-
moidal dose–response curve with a variable slope (Eq.
(5)) using GraphPad Prism 4.02 (GraphPad).
ðV max  V min Þ
V i ¼ V min þ Hillslope
; ð5Þ
1 þ ðIC½I50 Þ
where [I] is the concentration of the inhibitor, Vi is the ini-
tial velocity at a given [I], Vmin is the background of the as-
say, and Vmax is the initial velocity without inhibitor.
For Z0 factor determination, 48 wells of a 384-well
plate were used as positive controls and another 48 wells
were used as negative controls. Negative controls were
uninhibited MCD reactions containing 9 lM malonyl-
CoA and 0.1 nM MCD in buffer of 40 mM Tris, pH
8.0, 0.02% Tween 20, 0.1% BSA, and 1 mM DTT. Posi-
tive controls were completely inhibited reactions contain-
ing 10 lM compound A (dissolved in DMSO) in
addition to the same MCD reaction components present
in negative controls. A small amount of DMSO was
concentration in the positive control wells (final 5%
DMSO). Reactions were incubated at room temperature
ways as described above. Z0 was calculated according
ð3rcþ þ 3rc Þ
Z0 ¼ 1  ; ð6Þ
jlcþ  lc j
where rc+ and lc+ are the standard deviation and the mean
of the positive control signal, respectively, and rc- and lc-
are the standard deviation and the mean of the negative
control signal, respectively.
 A luminescence HTS assay for malonyl-CoA decarboxylase / M.-C. Lo et al. / Anal. Biochem. 376 (2008) 122–130
Fluorescence-based MCD assay
The Km of malonyl-CoA was also determined using a
protocol described by Zhou et al. [14] with slight modifica-
tions. In this assay, the MCD activity was measured by
coupling to the L-malate dehydrogenase and citrate syn-
thase reactions. The equilibration between L-malate/
NAD+ and oxaloacetate/NADH is catalyzed by L-malate
dehydrogenase (Eq. (7)). Acetyl-CoA, the product of
MCD, shifts the equilibrium to the right by reacting with
oxaloacetate in the presence of citrate synthase (Eq. (8)),
resulting in a continuous generation of NADH from
NAD+.
lmalate dehydrogenase
l  Malate þ NADþ ( + oxaloacetate þ NADH:
citrate synthase
Acetyl-CoA þ oxaloacetate ƒƒƒƒƒƒƒ! citrate þ CoA:
To determine the Km of malonyl-CoA, reactions were
performed in a black 96-well Microfluor plate (Thermo Sci-
entific, Waltham, MA). Each 50-lL reaction contained
2 mM L-malate, 2 mM NAD+, 0.786 U L-malate dehydro-
genase, 0.028 U citrate synthase, 0.1 nM MCD, and vari-
ous concentrations of malonyl-CoA ranging from 0 to
300 lM in buffer of 40 mM Tris, pH 8.0, 0.02% Tween
20, 0.1% BSA, and 1 mM DTT. Reactions were initiated
by adding MCD and fluorescence intensity was monitored
continuously using a Tecan Safire II (Tecan Systems, San
Jose, CA) with an excitation wavelength at 340 nm (band-
width 20 nm) and an emission wavelength at 460 nm (band-
width 20 nm). The initial velocities were obtained from the
slopes of the progress curves in the linear range and cor-
rected for the background rate determined in the absence
of MCD. Km and kcat were obtained from curve fitting of
the initial velocities as described above.
HPLC-based MCD assay
The HPLC-based MCD assay was used to monitor
the inhibition of MCD activity by L-malate and
NAD+. Both L-malate and NAD+ were reconstituted in
reaction buffer (40 mM Tris, pH 8.0, 0.1% BSA) and
reactions were performed in a final volume of 100 lL
containing 0.1 nM MCD, 5 lM [2-14C]malonyl-CoA
(GE Healthcare, Piscataway, NJ), and various concentra-
tions of L-malate or NAD+ in reaction buffer. After 30
min incubation at room temperature, reactions were
stopped by adding 10 lL of 100% cold methanol and
placing on ice for 10 min. The solutions were then trans-
ferred to HPLC vials and loaded onto an HPLC
machine for analysis. Acetyl-CoA was isolated on an
HPLC instrument using methods described previously
[17]. For Km determination, reactions were conducted
at 0.1 nM MCD and various concentrations of
[2-14C]malonyl-CoA ranging from 0.1 to 15 lM. Reac-
tions were stopped at different time points using the pro-
cedure stated above. Km and kcat were determined from
the initial velocities as described previously.
125
Results
Cloning, expression, and purification of human MCD
It is not uncommon for full-length proteins containing
membrane targeting sequences to be insoluble when
expressed in bacteria [17]. Amino acid analysis of human
MCD reveals a putative 39-residue mitochondrial targeting
domain at the N terminus [18]. These residues were shown
to have no effect on the catalytic activity [14]. Therefore, to
facilitate protein expression and purification, we cloned an
N-terminally truncated human MCD (residues 41–493, res-
idue 40 is methionine) lacking the putative mitochondrial
targeting domain. A 6X His tag was inserted after the
ð7Þ methionine start codon for purification convenience. This
ð8Þ His-tagged MCD expressed well as a soluble protein in
Escherichia coli (Fig. 2, lane 3). This is in contrast to
reports from Zhou and co-workers [14]. Their attempts
to express the soluble His-tagged full-length or N-termi-
nally truncated human MCD were unsuccessful because
overexpressed MCD was found predominantly in inclusion
bodies. These different results might be due to the difference
in the induction temperature of protein expression: we used
20 °C, while Zhou et al. [14] used 30 °C. We purified MCD
from the soluble fraction of the cell lysate by Talon metal
affinity chromatography followed by SP Sepharose HP
ion-exchange chromatography. The purity of the recombi-
nant protein improved after each step of purification, with
a final purity of 95% as shown in Fig. 2 (lane 5).
1 2 3 4 5 6 kDa
250
150
100
75
50
37
25
20
15
10
Fig. 2. SDS-PAGE analysis of the expression (lanes 1-3) and purification
(lanes 4-5) of human MCD. Lanes 1-3 are total lysate, the insoluble and
soluble fractions, respectively. Lanes 4 and 5 are pooled eluant fractions
containing MCD from Talon metal affinity and SP Sepharose HP
columns, respectively. Lane 6 is the molecular weight marker. 5 lg of
the soluble fraction, Talon pool, and SP Sepharose pool were loaded and
separated by 4–20% non-reducing SDS polyacrylamide gel and stained
with Coomassie blue. Loading of total lysate and the insoluble fraction
was normalized by volume to the amount of the soluble fraction. The
MCD band is indicated by an arrow.
126 A luminescence HTS assay for malonyl-CoA decarboxylase / M.-C. Lo et al. / Anal. Biochem. 376 (2008) 122–130

Development of a luminescence-based MCD assay
To address the compound interference limitation associ-
ated with the reported fluorescence assay, we developed a
novel luminescence assay for MCD activity. In this assay,
acetyl-CoA, one of the products of MCD (Eq. (1)), is used
as a substrate for the coupled acetyl-CoA synthetase reac-
tion (Eq. (2)), which generates ATP. The production of
ATP is proportional to the amount of acetyl-CoA. Subse-
quently, ATP is quantified employing the firefly luciferase
reaction (Eq. (3)), which has been used widely to detect
ATP levels [19].
To develop a luminescence-based MCD assay, we first
optimized the detection of acetyl-CoA using coupled reac-
tions. We varied the concentrations of AMP, PPi, and
acetyl-CoA synthetase to identify the optimal concentra-
tions for acetyl-CoA detection. Under optimized condi-
tions (20 lM AMP, 20 lM PPi, and 0.003 U/lL acetyl-
CoA synthetase in the luminescence detection reagent),
luminescence intensity is linear (R2 = 0.9985) with respect
to the acetyl-CoA concentration from 20 nM to 20 lM,
with a signal range spanning 3 orders of magnitude
(Fig. 3A).
1000000
A 0.15 nM
100000
RLU
Next we monitored the MCD reaction using this cou-
pled system. We found that MCD can be stabilized by
the inclusion of 0.1% BSA and 0.02% Tween 20 in the reac-
tion buffer. To determine reaction linearity with respect to
time and enzyme concentration, reactions were performed
at various concentrations of MCD and stopped at different
time points. As shown in Fig. 4A, the reactions are linear
up to 16 min using a range of MCD concentrations up to
0.25 nM. In addition, the initial velocities are linear with
respect to the amounts of MCD used (Fig. 4B), indicating
that the velocities measured by this coupled system are
indeed the initial velocities. Based on the signal and the
reaction rate, 0.1 nM MCD was selected as the amount
used in the MCD assay for determination of Km and IC50
values.
Kinetic characterization of MCD by three different assays
Having established the assay conditions for luminescent
detection of MCD activity, next we determined the kinetic
parameters of MCD using this assay. The MCD reactions
70000
A
0.05 nM
60000 0.10 nM
50000 0.20 nM
0.25 nM
40000
RLU

30000
10000
20000
1000
10000

100 0
0 5 10 15
0.01 0.1 1 10 100
Time (min)
Acetyl-CoA (μM)
B 0.4
B 100000
50000
40000
30000
0.3
RFU

20000
V 0 (μM/min)

10000
RFU

0
0 20 40 60 80 100 0.2
Acetyl-CoA (μM)

10000
0.1

0.0
1 10 100 0.00 0.05 0.10 0.15 0.20 0.25 0.30
Acetyl-CoA (μM) MCD (nM)

Fig. 3. Standard curve of acetyl-CoA from (A) the luminescence assay
and (B) the fluorescence assay. In (A), the luminescence signal shows a
linear correlation with the concentration of acetyl-CoA from 20 nM to 20
lM. The dashed line represents the 95% confidence band of the regression
line. In (B), only the data with filled squares were used in linear regression
and the dashed line represents the 95% confidence band of the regression
line. The inset in (B) shows the plot of the same data in the linear scale.
Fig. 4. (A) Linear sections of the progress curves at 0.05 to 0.25 nM
MCD. The reactions were monitored by the luminescence assay as
described under Materials and methods. (B) Initial velocities plotted as a
function of the MCD concentrations. The initial velocities were obtained
from the slopes of the corresponding progress curves in (A), followed by
converting the luminescence signals to the acetyl-CoA concentrations
according to the standard curve in Fig. 3A.
 A luminescence HTS assay for malonyl-CoA decarboxylase / M.-C. Lo et al. / Anal. Biochem. 376 (2008) 122–130 127

were conducted at various malonyl-CoA concentrations
ranging from 1.9 to 90 lM. A plot of the initial velocities
as a function of the malonyl-CoA concentrations is shown
in Fig. 5A. Km and kcat values obtained from fitting of the
Michaelis–Menten equation (Eq. (4)) were 8.7 lM and 47.3
s1, respectively. The kcat value obtained from this lumines-
cence assay is comparable to the value (28 s-1) reported by
Zhou and co-workers [14] but the Km is much lower than
the value they reported (330 lM). To determine whether
the Km discrepancy is due to the difference in the assay for-
A 0.3
0.2
V 0 (μM/min)
mats, we also determined the Km of malonyl-CoA using the
fluorescence assay described by Zhou et al. [14].
The fluorescence assay measures MCD activity by cou-
pling to L-malate dehydrogenase and citrate synthase
(Eqs. (7) and (8)). This assay was described originally by
Kim and Kolattukudy [13] and modified by Zhou et al.
[14]. To determine Km using the fluorescence assay, we first
generated a standard curve of acetyl-CoA (Fig. 3B). Sur-
prisingly, the limit of detection in the linear range is above
3 lM, 150-fold higher than our newly developed lumines-
cence assay (20 nM). Nevertheless, it is sensitive enough
for use in Km determination. To determine the Km of mal-
onyl-CoA, we conducted the MCD reactions at various
malonyl-CoA concentrations ranging from 0 to 300 lM
and monitored the NADH fluorescence continuously.
The Km and kcat values obtained from fitting of the initial
velocities (Fig. 5B) were 31 lM and 48.4 s1, respectively.
The kcat value is similar to that obtained from the lumines-

cence assay, while the Km value is 3.5-fold higher.

In the fluorescence assay, the L-malate dehydrogenase
0.1 substrates, L-malate and NAD+, bear some structural sim-
ilarities to malonyl-CoA. In the coupled reaction, 2 mM of
both L-malate and NAD+ were used. We hypothesized that
the higher Km obtained from the fluorescence assay might
0.0
0 20 40 60 80 100 be, at least in part, due to competitive inhibition of
Malonyl-CoA (μM) MCD by L-malate and NAD+. To test this hypothesis,
we monitored the effects of L-malate and NAD+ on
B 0.3 MCD activity using an HPLC assay because the high con-
centration of L-malate or NAD+ inhibited acetyl-CoA
detection by the luminescence assay (data not shown).
The dose-response curves of L-malate and NAD+ are
0.2
shown in Fig. 6. The IC50 values are 4.1 and 2.2 mM for
V 0 (μM/min)

+
L-malate and NAD , respectively. This result indicates that
+
L-malate and NAD used in the coupled step of the fluores-
0.1 cence assay inhibit MCD activity and supports our hypoth-
esis that the Km from the fluorescence assay is artificially
elevated.
0.0
0 50 100 150 200 250 300
Malonyl-CoA (μM)

120
C 0.10
100
Percent of Activity

0.08
80 L-malate
NAD+
V0 (μM/min)

0.06 60

40
0.04
20
0.02
0
-6 -5 -4 -3 -2
0.00 Log (M)
0 2 4 6 8 10 12 14 16
Malonyl-CoA (μM) Fig. 6. Inhibition of the MCD reaction by L-malate and NAD+ monitored
by the HPLC assay. The MCD reactions were carried out in the presence
Fig. 5. Michaelis–Menten plots of the MCD reactions monitored by (A) of various concentrations of L-malate or NAD+. The percentage of
the luminescence assay, (B) the fluorescence assay, and (C) the HPLC activity is derived by dividing the MCD activity at a given concentration
assay. of L-malate or NAD+ by the MCD activity in the absence of inhibitors.
128 A luminescence HTS assay for malonyl-CoA decarboxylase / M.-C. Lo et al. / Anal. Biochem. 376 (2008) 122–130

We also determined kinetic parameters of MCD using
the HPLC assay (Fig. 5C). The Km and kcat obtained from
the HPLC assay are 3.5 lM and 16.9 s1, respectively,
which are slightly (two to three fold) lower than the values
determined using the luminescence assay, further support-
ing our notion that the higher Km from the fluorescence
assay is artificial.
Determination of the IC50 values of MCD inhibitors by the
luminescence assay
To validate the utility of the luminescence assay, we
determined IC50 values of two MCD inhibitors reported
by Cheng et al. [9,10]. The structures of these two inhibitors
are shown in Fig. 1. The IC50 values of compounds A and
B from the luminescence assay are 9 and 814 nM (Fig. 7),
respectively, which are similar to the reported values (7 and
931 nM, respectively) from the fluorescence assay [9,10].
The luminescence system that we used showed a glow-type
luminescent signal with a half-life greater than 4 h. There-
fore, the same reaction plate was read repeatedly over a 4-h
period after adding the luminescence detection reagent.
The IC50 value remained similar (within 10%) over this per-
iod (data not shown), indicating good signal stability.
Because the compounds in our screening library are
invariably dissolved in DMSO and the DMSO concentra-
tion in the final assay mixture can be up to 5%, it is impor-
tant to determine the DMSO tolerance of the assay. To test
the DMSO effect on the luminescence assay, we performed
dose–response experiments with compound A and final
Determination of the Z0 factor of the luminescence-based
MCD assay
Z0 is a statistical value that assesses dynamic range and
variability of an assay to indicate assay quality [16]. A Z0 P
0.5 indicates a good assay and a Z0 P 0.75 indicates an
excellent assay. To determine the Z0 of the luminescence-
based MCD assay, the MCD reactions were conducted in
a 384-well plate; 48 wells of a 384-well plate were used as
positive controls (100% inhibition) and another 48 wells
were used as negative controls (0% inhibition). The lumi-
nescent signals of the control wells are shown in Fig. 8A.
The Z0 factor is 0.81 when the plate was read at 5 min after
adding the luminescence detection reagent, indicating
excellent assay performance under these conditions. The
same reaction plate was read repeatedly over a 4-h period,
and the Z0 factor remained similar (Fig. 8B).
Discussion
When choosing an HTS assay format, there is a growing
preference for assays with luminescence readouts over fluo-
rescence formats due to considerations of sensitivity, linear
A 35000
30000
25000
20000
RLU

DMSO concentrations of 0 and 5%. Our results (not

shown) indicate that the DMSO effect is minimal on the 15000
MCD activity, with 5% DMSO increasing MCD activity 10000
slightly (10%), and not significant on the IC50 values of
compound A (11 and 14 nM with 0 and 5% DMSO, respec- 5000
tively). Therefore, the luminescence assay is tolerant of at 0
least 5% DMSO and is consequently amenable to HTS 0 10 20 30 40 50
operation. Well Number

B 1

0.8
Z ' Factor

0.6
30000
Compound A
25000 Compound B 0.4

20000 0.2
RLU

15000 0
0 50 100 150 200 250
10000 Time (min)

5000 Fig. 8. Assay signal window (A) and Z0 factor evaluation (B) of the
luminescence assay. In (A), open circles are positive controls containing 10
0 lM compound A, and filled circles are negative controls without
-11 -10 -9 -8 -7 -6 -5 -4 -3 inhibitors. Solid lines indicate the means, and dotted lines indicate ±3
Log (M) SD of these populations. In (B), the same reaction plate was read
repeatedly over a 4-h period and Z0 factors at different time points were
Fig. 7. Dose–response curves of MCD inhibitors (compounds A and B) in plotted. The calculation of Z0 factor is described under Materials and
the luminescence assay. methods.
 A luminescence HTS assay for malonyl-CoA decarboxylase / M.-C. Lo et al. / Anal. Biochem. 376 (2008) 122–130
range, and robustness to assay artifacts arising from inter-
ference by fluorescent compounds. We have developed a
highly sensitive luminescence assay to detect MCD activity.
As shown in Fig. 3, the linear detection range for acetyl-
CoA is from 20 nM to 20 lM in the luminescence assay.
In contrast, in the fluorescence assay, the linear response
for acetyl-CoA is above 3 lM. Therefore, the luminescence
assay is about 150-fold more sensitive than the fluorescence
assay for acetyl-CoA detection. In fact, the concentration
of MCD used in the luminescence assay is about 100-fold
less than that in the reported fluorescence assay [9], making
the luminescence assay much more cost effective. Using
only 0.1 nM MCD in the luminescence assay, we obtained
a 10-fold assay window with a Z0 > 0.8.
We characterized the steady state kinetics of MCD using
the luminescence assay and compared our results with the
reported data. The kcat that we obtained from the lumines-
cence assay is comparable to the values reported for MCD
from humans and other species [14,20]. However, the Km
obtained from the luminescence assay is 20–40 fold lower
than the values reported for human MCD [14,21]. How-
ever, this large discrepancy was not solely due to the differ-
ence in the assay formats. When our purified MCD was
characterized using the fluorescence assay described in
the literature, the Km was only 3.5-fold higher than that
obtained using the luminescence assay. The remainder dif-
ference in Km may, at least in part, stem from the differ-
ences in protein constructs used: the reported kinetic
characterization used human MCD overexpressed as a
MBP fusion construct with or without postpurification
cleavage of the MBP tag because the soluble His-tagged
MCD could not be produced [14,21]. However, when we
overexpressed MCD via a His-tagged construct, the pro-
tein expressed well in the soluble fraction. It’s possible that
His-tagged MCD has slightly higher affinity for malonyl-
CoA than MBP-tagged MCD, resulting in smaller Km in
our hand even with the same fluorescence assay format.
When studying our purified MCD using three different
assays, the Km of malonyl-CoA obtained from the fluores-
cence assay was higher than the values obtained with the
luminescence (3.5-fold) and HPLC (8.9-fold) assays. In
the fluorescence assay, the coupling reagents were added
along with the MCD reaction components. We hypothe-
sized that L-malate and NAD+ used in the coupled reaction
might compete with malonyl-CoA to bind MCD and con-
sequently the Km measured is not the true Km. By using an
HPLC assay, we found that L-malate and NAD+ inhibited
the MCD reaction by 30–40% at the concentrations used in
the coupled reaction, which may explain the higher Km
observed in the fluorescence assay. In the luminescence
assay, the MCD reaction is separated from the detection
reactions, and therefore the Km obtained from this assay
should be closer to the true Km. When comparing the Km
values obtained from the luminescence and HPLC assays,
they were only 2.5-fold different. Because different sources
of malonyl-CoA (one radiolabeled and the other not) were
used in these two assays, the 2-fold difference in Km may be
129
due to experimental errors in determining the concentra-
tion of [2-14C]malonyl-CoA. If this is the case, the kcat
would also be affected. This is supported by the observa-
tion that the kcat value obtained from the HPLC assay
was about 2.8-fold lower than that from the luminescence
assay. Nevertheless, these differences are relatively small.
Using the luminescence assay, we determined the IC50
values of two known MCD inhibitors. The IC50 values
that we obtained from this assay were close to those
reported before. We also found that the IC50 and Z0 val-
ues remained similar over a 4-h period, making this for-
mat highly suitable for automated HTS operation.
Furthermore, luminescence detection is more sensitive
and less susceptible to interference by fluorescent com-
pounds than is fluorescence detection. Therefore, the
luminescence assay is a better choice for HTS. However,
as with all coupled assays, including the reported fluores-
cence assay, the luminescence assay does require a second-
ary assay to identify the false positives due to inhibition
of the coupled enzymes used in the detection reactions.
This can be easily performed using a counter assay in
which the MCD reaction components are replaced with
acetyl-CoA at a concentration generated in the uninhib-
ited MCD reaction (2 lM in our assay, corresponding
to 22% substrate conversion). We have tested com-
pounds A and B in this counter assay and they do not
inhibit the coupled enzymes in the same dose range tested
(data not shown), supporting the notion that these two
compounds are true MCD inhibitors. Finally, because
this luminescence assay detects acetyl-CoA, it can be
applied to other enzymes with acetyl-CoA as a substrate
or a product as long as those enzymes do not require
ATP as a cosubstrate.
References
[1] W.C. Stanley, G.D. Lopaschuk, J.L. Hall, J.G. McCormack, Regu-
lation of myocardial carbohydrate metabolism under normal and
ischaemic conditions. Potential for pharmacological interventions,
Cardiovasc. Res. 33 (1997) 243–257.
[2] J.D. McGarry, N.F. Brown, The mitochondrial carnitine palmitoyl-
transferase system. From concept to molecular analysis, Eur. J.
Biochem. 244 (1997) 1–14.
[3] J.R. Dyck, G.D. Lopaschuk, Malonyl CoA control of fatty acid
oxidation in the ischemic heart, J. Mol. Cell. Cardiol. 34 (2002) 1099–
1109.
[4] G.D. Lopaschuk, R. Collins-Nakai, P.M. Olley, T.J. Montague, G.
McNeil, M. Gayle, P. Penkoske, B.A. Finegan, Plasma fatty acid
levels in infants and adults after myocardial ischemia, Am. Heart J.
128 (1994) 61–67.
[5] N. Kudo, A.J. Barr, R.L. Barr, S. Desai, G.D. Lopaschuk, High rates
of fatty acid oxidation during reperfusion of ischemic hearts are
associated with a decrease in malonyl-CoA levels due to an increase in
5’-AMP-activated protein kinase inhibition of acetyl-CoA carboxyl-
ase, J. Biol. Chem. 270 (1995) 17513–17520.
[6] W.C. Stanley, H.N. Sabbah, Metabolic therapy for ischemic heart
disease: the rationale for inhibition of fatty acid oxidation, Heart Fail.
Rev. 10 (2005) 275–279.
[7] C.D. Folmes, G.D. Lopaschuk, Role of malonyl-CoA in heart disease
and the hypothalamic control of obesity, Cardiovasc. Res. 73 (2007)
278–287.
130 A luminescence HTS assay for malonyl-CoA decarboxylase / M.-C. Lo et al. / Anal. Biochem. 376 (2008) 122–130
[8] G.D. Lopaschuk, W.C. Stanley, Malonyl-CoA decarboxylase inhibi-
tion as a novel approach to treat ischemic heart disease, Cardiovasc.
Drugs Ther. 20 (2006) 433–439.
[9] J.F. Cheng, M. Chen, D. Wallace, S. Tith, M. Haramura, B. Liu, C.C.
Mak, T. Arrhenius, S. Reily, S. Brown, V. Thorn, C. Harmon, R.
Barr, J.R. Dyck, G.D. Lopaschuk, A.M. Nadzan, Synthesis and
structure-activity relationship of small-molecule malonyl coenzyme A
decarboxylase inhibitors, J. Med. Chem. 49 (2006) 1517–1525.
[10] J.F. Cheng, Y. Huang, R. Penuliar, M. Nishimoto, L. Liu, T.
Arrhenius, G. Yang, E. O’leary, M. Barbosa, R. Barr, J.R. Dyck,
G.D. Lopaschuk, A.M. Nadzan, Discovery of potent and orally
available malonyl-CoA decarboxylase inhibitors as cardioprotective
agents, J. Med. Chem. 49 (2006) 4055–4058.
[11] D.M. Wallace, M. Haramura, J.F. Cheng, T. Arrhenius, A.M.
Nadzan, Novel trifluoroacetophenone derivatives as malonyl-CoA
decarboxylase inhibitors, Bioorg. Med. Chem. Lett. 17 (2007) 1127–
1130.
[12] J.R. Dyck, J.F. Cheng, W.C. Stanley, R. Barr, M.P. Chandler, S.
Brown, D. Wallace, T. Arrhenius, C. Harmon, G. Yang, A.M. Nadzan,
G.D. Lopaschuk, Malonyl coenzyme A decarboxylase inhibition
protects the ischemic heart by inhibiting fatty acid oxidation and
stimulating glucose oxidation, Circ. Res. 94 (2004) e78–e84.
[13] Y.S. Kim, P.E. Kolattukudy, Purification and properties of malonyl-
CoA decarboxylase from rat liver mitochondria and its immunolog-
ical comparison with the enzymes from rat brain, heart, and
mammary gland, Arch. Biochem. Biophys. 190 (1978) 234–246.
[14] D. Zhou, P. Yuen, D. Chu, V. Thon, S. McConnell, S. Brown, A.
Tsang, M. Pena, A. Russell, J.F. Cheng, A.M. Nadzan, M.S.
Barbosa, J.R. Dyck, G.D. Lopaschuk, G. Yang, Expression, purifi-
cation, and characterization of human malonyl-CoA decarboxylase,
Protein Expr. Purif. 34 (2004) 261–269.
[15] F.W. Studier, B.A. Moffatt, Use of bacteriophage T7 RNA polymer-
ase to direct selective high-level expression of cloned genes, J. Mol.
Biol. 189 (1986) 113–130.
[16] J.H. Zhang, T.D. Chung, K.R. Oldenburg, A Simple Statistical
Parameter for Use in Evaluation and Validation of High Throughput
Screening Assays, J. Biomol. Screen. 4 (1999) 67–73.
[17] K.W. Kim, H. Yamane, J. Zondlo, J. Busby, M. Wang, Expression,
purification, and characterization of human acetyl-CoA carboxylase
2, Protein Expr. Purif. 53 (2007) 16–23.
[18] J. Gao, L. Waber, M.J. Bennett, K.M. Gibson, J.C. Cohen, Cloning
and mutational analysis of human malonyl-coenzyme A decarboxyl-
ase, J. Lipid Res. 40 (1999) 178–182.
[19] F. Fan, K.V. Wood, Bioluminescent assays for high-throughput
screening, Assay Drug Dev. Technol. 5 (2007) 127–136.
[20] G.Y. Lee, Y.Y. Bahk, Y.S. Kim, Rat malonyl-CoA decarboxylase;
cloning, expression in E. coli and its biochemical characterization, J.
Biochem. Mol. Biol. 35 (2002) 213–219.
[21] K.A. Sacksteder, J.C. Morrell, R.J. Wanders, R. Matalon, S.J.
Gould, MCD encodes peroxisomal and cytoplasmic forms of malo-
nyl-CoA decarboxylase and is mutated in malonyl-CoA decarboxyl-
ase deficiency, J. Biol. Chem. 274 (1999) 24461–24468.