Label-free high-throughput assays to screen and characterize novel lactate dehydrogenase inhibitors

Article abstract of DOI:10.1016/j.ab.2013.07.003

Catalytic turnover of pyruvate to lactate by lactate dehydrogenase (LDH) is critical in maintaining an intracellular nicotinamide adenine dinucleotide (NAD⁺) pool for continuous fueling of the glycolytic pathway. In this article, we describe two label-free high-throughput assays (a kinetic assay detecting the intrinsic reduced nicotinamide adenine dinucleotide (NADH) fluorescence and a mass spectrometric assay monitoring the conversion of pyruvate to lactate) that were designed to effectively identify LDH inhibitors, characterize their different mechanisms of action, and minimize potential false positives from a small molecule compound library screen. Using a fluorescence kinetic image-based reader capable of detecting NADH fluorescence in the ultra-high-throughput screening (uHTS) work flow, the enzyme activity was measured as the rate of NADH conversion to NAD⁺. Interference with NADH fluorescence by library compounds was readily identified during the primary screen. The mass spectrometric assay quantitated the lactate and pyruvate levels simultaneously. The multiple reaction monitoring mass spectrometric method accurately detected each of the two small organic acid molecules in the reaction mixture. With robust Z' scores of more than 0.7, these two high-throughput assays for LDH are both label free and complementary to each other in the HTS workflow by monitoring the activities of the compounds on each half of the LDH redox reaction.

Full text of DOI:10.1016/j.ab.2013.07.003

Analytical Biochemistry 441 (2013) 115–122
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Label-free high-throughput assays to screen and characterize novel
lactate dehydrogenase inhibitors
a
b
a
a
b
a,
⇑
Erica VanderPorten , Lauren Frick , Rebecca Turincio , Peter Thana , William LaMarr , Yichin Liu
a
Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA 94080, USA
Life Science Solutions, Agilent Technologies, Wakefield, MA 01880, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Catalytic turnover of pyruvate to lactate by lactate dehydrogenase (LDH) is critical in maintaining an
intracellular nicotinamide adenine dinucleotide (NAD ) pool for continuous fueling of the glycolytic path-
Received 25 March 2013
Received in revised form 30 June 2013
Accepted 1 July 2013
+
way. In this article, we describe two label-free high-throughput assays (a kinetic assay detecting the
intrinsic reduced nicotinamide adenine dinucleotide (NADH) fluorescence and a mass spectrometric
assay monitoring the conversion of pyruvate to lactate) that were designed to effectively identify LDH
inhibitors, characterize their different mechanisms of action, and minimize potential false positives from
a small molecule compound library screen. Using a fluorescence kinetic image-based reader capable of
detecting NADH fluorescence in the ultra-high-throughput screening (uHTS) work flow, the enzyme
Available online 16 July 2013
Keywords:
High-throughput screen
Lactate dehydrogenase
Mass spectrometry
Fluorescence kinetic assay
+
activity was measured as the rate of NADH conversion to NAD . Interference with NADH fluorescence
by library compounds was readily identified during the primary screen. The mass spectrometric assay
quantitated the lactate and pyruvate levels simultaneously. The multiple reaction monitoring mass spec-
trometric method accurately detected each of the two small organic acid molecules in the reaction mix-
0
ture. With robust Z scores of more than 0.7, these two high-throughput assays for LDH are both label free
and complementary to each other in the HTS workflow by monitoring the activities of the compounds on
each half of the LDH redox reaction.
Ó 2013 Elsevier Inc. All rights reserved.
Cancer cells have been shown to have an altered metabolism to
meet the demands of faster cell division and higher energy con-
sumption [1–3]. The altered metabolism in cancer cells is often
accompanied either by a change in the expression level of an en-
zyme or its isoform or by an introduction of mutations or different
posttranslational modification patterns that can affect functional
activity of a given protein. For example, the biosynthetic pathways
involved in generating fatty acids or nucleotides can be up-regu-
lated to supply these building blocks in the fast proliferating cancer
cells [4,5]. In many tumor cells, an increase in the aerobic glyco-
lytic flux and a reduction in the oxidative phosphorylation of glu-
cose metabolism in mitochondria was observed; these phenomena
are known as the Warburg effect [6,7]. Several oncogenes such as
Akt, Myc, and Ras have been linked to the Warburg effect and
can activate the aerobic glycolysis pathways [8]. In addition, the
loss of tumor suppressor genes such as p53 can cause a switch
from cellular respiration to aerobic glycolysis, directly contributing
to the Warburg effect [9,10]. The increase in the glucose uptake
and glycolytic flux allows the tumor cells to quickly produce ATP
as an energy source through the conversion of glucose to pyruvate,
especially under hypoxic conditions that fast-growing solid tumor
cells often experience. For every glucose molecule that is con-
sumed, glycolysis reduces two molecules of nicotinamide adenine
+
1
dinucleotide (NAD ) to reduced nicotinamide adenine dinucleotide
(NADH) in order to generate two molecules of ATP. To continue fuel-
ing this glycolytic pathway in tumor cells, NADH needs to be effi-
+
ciently recycled back to NAD . The decrease in aerobic glucose
metabolism in the mitochondria of tumor cells also reduces NADH
oxidation by the TCA cycle; thus, an alternative reaction is required.
Under anabolic conditions, the conversion of the NADH back to NAD+
is catalyzed by lactate dehydrogenase (LDH), and this oxidation is
coupled to the reduction of the glycolytic product pyruvate to
lactate, which will then be secreted from the cells. In many
glycolytic-addicted tumor cells, the expression of LDH (especially
the isoform LDHA) is found to be elevated [11,12].
1
+
Abbreviations used: NAD , nicotinamide adenine dinucleotide; NADH, reduced
nicotinamide adenine dinucleotide; LDH, lactate dehydrogenase; LDHA, lactate
dehydrogenase A; uHTS, ultra-high-throughput screening; BGG, bovine gamma
globulin; TCEP, tris(2-carboxyethyl)phosphine; FDSS, Functional Drug Screening
System; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DTT, dithiothre-
itol; RFU, relative fluorescence unit; DMSO, dimethyl sulfoxide; MRM, multiple
reaction monitoring; SPE, solid-phase extraction; AUC, area under the curve.
⇑
Corresponding author. Fax: +1 650 467 8091.
E-mail address: liu.yichin@gene.com (Y. Liu).
0
003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ab.2013.07.003

1
16
Label-free LDHA assays / E. VanderPorten et al. / Anal. Biochem. 441 (2013) 115–122
The enzymatic reaction catalyzed by LDH has been character-
1536-Well kinetic LDHA FDSS assay for primary HTS and IC50
confirmation
ized since the 1960s, with numerous publications primarily using
the detection of NADH absorbance or intrinsic fluorescence. The
enzyme family is capable of carrying out both the forward (pyru-
vate to lactate) and reverse (lactate to pyruvate) reactions, with
the preference varying among different LDH isoforms [13,14]. Fur-
thermore, the forward enzymatic reaction has been shown to pro-
ceed as an ordered reaction, with the enzyme binding NADH first
followed by the substrate, pyruvate [13,14]. Because of the ability
to use NADH or NAD+ as substrate, LDH was used extensively as
The HTS screen was conducted on a BioCel 900 automated sys-
tem outfitted with a Direct Drive Robot (Agilent Automation Solu-
tions, Santa Clara, CA, USA). An FDSS (Functional Drug Screening
System) 7000 kinetic fluorescence reader with onboard dispenser
(Hamamatsu, Bridgewater, NJ, USA) was integrated with this sys-
tem, enabling a fully automated workflow, including reagent addi-
tions, incubations, and detection.
Prior to the start of the screening campaign, assay-ready plates
were prepared using a dedicated plate replication automation sys-
tem. Using an Echo 555 acoustic dispenser (Labcyte, Sunnyvale, CA,
USA), 50 nl per well of 1 mM compound was spotted onto 1536-
well low-base, clear-bottom black microplates (Brooks Life Science
Systems, Poway, CA, USA) and sealed using an Agilent PlateLoc
microplate sealer to prevent evaporation. The sealed plates were
then loaded onto the BioCel for processing. The plate seal was re-
moved using an XPeel automated microplate seal removal device
+
coupling enzymes for NADH or NAD detection. Even though the
elevated expression of LDH in cancer had been noted a few dec-
ades ago [15], it was not until 2006 when Fantin and coworkers
demonstrated the link between LDHA and tumor metabolism
[
16] that the LDH enzyme was transformed from a supporting role
of being used in coupling enzyme assays to center stage and being
used as a potential therapeutic target for cancer. This also explains
in part why there has been a lack of validated inhibitors for LDH
despite the fact that this enzyme has been well characterized for
decades. Until recent months, the only validated inhibitor known
to LDH is a small organic acid, oxamate, which is a pyruvate mi-
metic with a reported biochemical IC50 ranging from 17 to
(Brooks Life Science Systems), followed by the addition of 6 ll of
suspension buffer containing 50 mM Hepes buffer (pH 7.2), 0.01%
Triton X-100, and 2 mM dithiothreitol (DTT) using a MultiDrop
Combi dispenser (Thermo Scientific, Waltham, MA, USA). Next,
1
50 lM, depending on the assay conditions [17,18]. This weak
and nonselective inhibitor that has poor cell permeability is less
than an ideal tool compound for target validation in cells or
in vivo.
2 ll of 250 lM NADH with 10 nM C-terminally His-tagged full-
length LDHA enzyme in reaction buffer A (50 mM Hepes [pH
7.2], 0.01% Triton X-100, and 0.1% BGG) was added using a Bio-
RAPTR dispenser (Beckman Coulter, Indianapolis, IN, USA). The
full-length LDHA (A2-F332) was purified as a tetramer from an
Escherichia coli expression system using nickel–nitrilotriacetic acid
(Ni–NTA) and size exclusion chromatography and stored as ali-
quots in storage buffer (10 mM Tris [pH 8.5], 150 mM NaCl, 45%
glycerol, and 0.25 mM TCEP) at ꢀ80 °C. For control inhibition,
With an increasing interest in exploring metabolic pathways
for potential cancer therapeutic strategies, we sought to develop
assay platforms that are amenable to ultra-high-throughput
screening (uHTS) to identify chemical tool compounds with vari-
ous mechanisms to probe the effect of LDH inhibition on cancer
cell growth. LDHA has been implicated in disease progression
for tumor cells that rely on glycolytic flux to provide the requisite
fast energy source. LDHA is expressed to efficiently recycle NADH
250 lM oxamate was included in the NADH and LDHA mixture.
The plate was then centrifuged using a VSpin Microplate Centri-
fuge (Agilent Automation Solutions). Following a 10-min incuba-
tion at room temperature, the plate was loaded into the FDSS
7000 reader for an initial baseline read of 5 s. The onboard 1536-
well pipette head of the FDSS 7000 was then used to transfer
+
back to NAD to continue fueling glycolysis; thus, the enzyme has
been found to drive the reaction in the forward direction (i.e.,
+
pyruvate to lactate and NADH to NAD ) [16,19]. Using LDHA that
carries out predominantly the forward reaction as a model target,
here we describe a robust 1536-well NADH fluorescence kinetic
assay for screening and characterizing LDHA inhibitors. To avoid
the potential for fluorescence interference from the use of NADH
fluorescence as a readout, we optimized the protocol to readily
identify compound fluorescence interference observed at the
excitation and emission wavelengths of NADH. We also describe
a label-free mass spectrometric assay set up to monitor the con-
version of pyruvate to lactate by LDHA. This label-free assay was
complementary to the higher throughput NADH fluorescence as-
say because it detected pyruvate to lactate conversion and was
effective in further eliminating fluorescence artifacts as well as
in identifying additional false positive mechanisms of inhibition.
Finally, taking advantage of the well-characterized ordered
enzyme reaction, the primary screening assay was designed to
capture, and has successfully identified, uncompetitive or non-
competitive inhibitors with the NADH–LDHA complex in addition
to conventional inhibitors that are competitive with the
substrates.
2 ll of 250 lM pyruvate in reaction buffer A from an AutoFill refill-
ing reservoir (Acorn Instruments, South San Francisco, CA, USA) to
the assay plates. The final concentration of test compound in the
screen was 5 lM. Fluorescence was measured at an excitation
wavelength of 340 nm and an emission wavelength of 482 nm,
with one read taking place each second for a total of 3 min.
The kinetic traces were analyzed by in a Genedata Screener
Kinetic Analyzer (Basel, Switzerland) using the robust slope curve
fit. The slopes of the kinetic traces were calculated using a time-
frame of 60 to 120 s of the aggregated data. The baseline NADH
fluorescence intensity was typically measured at 300 to 400 rela-
tive fluorescence units (RFU). Fluorescent compounds with RFU
values greater than 500, which was the upper limit of the linear
detection range in the FDSS 7000, were excluded. The remaining
compounds with more than 50% inhibition were selected for IC50
confirmation. A hit rate of less than 0.1% was achieved after remov-
0
ing fluorescent artifacts. Average plate Z scores were 0.7 (% LDHA
activity with enzyme = 99.6 ± 5.8 and without enzyme = 0.3 ± 3.8),
with reproducible NADH fluorescence window and oxamate inhi-
bition from run to run.
Materials and methods
384-Well IC50 confirmation assay using a combination of FDSS 7000
Chemicals
and mass spectrometric methods
NADH was purchased from Roche Diagnostics (Indianapolis, IN,
USA). Oxamate, pyruvate, bovine gamma globulin (BGG), tris(2-
carboxyethyl)phosphine (TCEP), NaCl, and Triton X-100 were ob-
tained from Sigma–Aldrich (St. Louis, MO, USA).
IC50 follow-up by the mass spectrometric assay was conducted
with compounds serially diluted in an 8-point dose response in
384-well clear-bottom black microplates. In a final reaction vol-
ume of 50 ll, 0.25 nM LDHA was incubated with 75 lM pyruvate

Label-free LDHA assays / E. VanderPorten et al. / Anal. Biochem. 441 (2013) 115–122
117
and 50 lM NADH in reaction buffer B (20 mM Tris [pH 7.5], 0.005%
Triton, 0.005% BGG, and 2 mM DTT) and NADH fluorescence was
first measured for approximately 9 min in the FDSS 7000 to reach
approximately 30% conversion of pyruvate to lactate. To character-
ize a compound’s mode of inhibition, additional experiments were
conducted to determine whether compounds were pyruvate com-
petitive or NADH competitive. To identify pyruvate-competitive
inhibitors, 0.25 nM LDHA was reacted with 75
lM NADH and
5
7
00
l
M pyruvate, and the IC50 was compared with a reaction with
5
l
M NADH and 75 lM pyruvate. Reactions were read in the
NADH fluorescence kinetic assay for 4.5 min and also resulted in
+
3
0% conversion of NADH to NAD . Similarly, to identify NADH-
competitive inhibitors, 0.25 nM LDHA was reacted with 300
NADH and 75 M pyruvate and read for 4.5 min to achieve 30%
conversion of pyruvate to lactate. The 50- l reactions from the
lM
Fig.1. An overview of the LDHA enzyme reaction and the two assays detecting the
substrates and products. NADH was measured by its fluorescence (excitation at
l
340 nm and emission at 480 nm), whereas lactate and pyruvate were detected
l
using mass spectrometry with their respective MRM shown.
NADH fluorescence kinetic assay were quenched with 5
l
l of 10%
formic acid at the end of the kinetic measurement. A 50-
l
l aliquot
of the final reaction mixture was transferred to a 384-well Greiner
polypropylene clear V-bottom plate and stored at ꢀ20 °C until
ready to be shipped in dry ice packages to Agilent Technologies
interference at the wavelengths of NADH detection, which poses
challenges in identifying true inhibitors among false positives
and negatives. A well-designed kinetic measurement, however,
can provide crucial information to determine how compound fluo-
rescence might have masked the true enzyme activity via either
the consumption or generation of NADH. Complementary to the
NADH fluorescence detection is the measurement of pyruvate
conversion to lactate (Fig. 1). Similar to NADH quantitation, a
few enzyme-coupled detection methods for either pyruvate or
lactate have been published [20]. Although some of the approaches
can be fairly sensitive, the inhibition of the coupling enzyme in a
small molecule library screen can significantly complicate the hit
triage and confirmation against the target enzyme. Instead, we
have taken the mass spectrometry approach to measure pyruvate
and lactate. Using MRM on a triple-quadrupole mass spectrometer,
pyruvate and lactate were detected by isolating parent negative
ions of 86.9 and 89.0 m/z, respectively, in quad 1 followed by frag-
mentation in quad 2 and detection of the 43.0 m/z daughter ions in
the third quad (Fig. 1).
(
Wakefield, MA, USA). The enzymatic activity measured by NADH
fluorescence was analyzed using a Genedata Kinetic Analyzer as
described above. Data were analyzed by normalizing to no-enzyme
control and dimethyl sulfoxide (DMSO) control and were repre-
sented as percentage inhibition.
SPE and mass spectrometric detection
Lactate and pyruvate levels in the LDHA samples were analyzed
on a RapidFire 300 High-Throughput Mass Spectrometry System
(
Agilent) coupled to an AB Sciex API 4000 triple-quadrupole mass
spectrometer (AB Sciex, Foster City, CA, USA). The API 4000 was fit-
ted with an electrospray ionization source and was run in negative
multiple reaction monitoring (MRM) mode for the detection of
lactate and pyruvate. The instrument aspirated aliquots of each
sample from microplates sequentially, removing sample until the
sip sensor determined that the 10-
ll loop was full (usually
ꢁ
200 ms). The contents of the loop were then applied to a
solid-phase extraction (SPE) cartridge packed with a hydrophilic
interaction chromatography (HILIC) material and washed with
Quantitative detection of pyruvate and lactate in mass spectrometry
The mass spectrometric assay that was developed to measure
LDHA activity and inhibition detected the conversion of pyruvate
to lactate. Pyruvate and lactate are both small organic acids that
differ from each other by only 2.1 Da. MRM methods were estab-
lished for each analyte for quantitative analysis as described above
9
0% acetonitrile supplemented with 10 mM ammonium acetate
for 2.5 s. The purified sample was then reverse-eluted in 40% ace-
tonitrile supplemented with 10 mM ammonium acetate in a 5-s
elution step, and the eluent was sent to the mass spectrometer,
which was already monitoring the mass transitions of interest. A
reequilibration of 2 s brought the total cycle time to approximately
(Fig. 1). The mass spectrometer (TripleQuad AB Sciex 4000) was
coupled to an Agilent RapidFire 300 system, which is an SPE plat-
form capable of processing samples every 10 to 12 s. The analytes
were first cleaned on the RapidFire system under normal-phase
chromatographic conditions prior to mass spectrometric detection.
The titration of each acid showed concentration-dependent inten-
sity of the daughter ions from the targeted fragmentation of their
respective parent ions as well as good detection linearity (Fig. 2A
and B). The limit of quantitation for both lactate and pyruvate
1
2 s. Data analysis was performed using RapidFire Integrator ver-
sion 3.4 software (Agilent), which generated an output file of inte-
grated peak areas (areas under the curve, AUCs) for each MRM
transition for each sample. Percentage conversion was calculated
as follows: 100 * [Product AUC/(Product AUC + Substrate AUC)].
Results and discussion
was 1 lM. The replicates of each titration also demonstrated solid
Complementary assay strategies for detecting LDHA enzymatic activity
reproducibility of the mass signals. Furthermore, the daughter ion
mass signal from lactate was shown to be negligible during a titra-
tion of pyruvate (Fig. 2C), suggesting that in spite of the small mass
difference between lactate and pyruvate, the detection of the mass
for each acid was specific. There was also a lack of background sig-
nal in the pyruvate detection channel when a lactate titration was
performed (Fig. 2D), further validating our mass spectrometry
detection method. A time course of the enzymatic reaction carried
LDHA catalyzes the glycolytic interconversion of pyruvate and
lactate, a transformation that is coupled to the redox reaction be-
+
tween NADH and NAD (Fig. 1). Because NADH is intrinsically fluo-
rescent, with a characteristic excitation maximum at 340 nm and
an emission maximum at 480 nm (Fig. 1), it has been historically
used to monitor LDH activity. Despite NADH fluorescence being a
common detection method in characterizing NADH-dependent en-
zyme reactions, it is generally an undesirable method in screening
small molecule inhibitors due to the high incidence of fluorescence
out at 50 lM NADH and 75 lM pyruvate demonstrated good line-
arity of the reaction progress curve (Fig. 2E). Using the mass spec-
trometric assay allowed us to measure the potency of compounds

1
18
Label-free LDHA assays / E. VanderPorten et al. / Anal. Biochem. 441 (2013) 115–122
Fig.2. (A,B) Titration of pyruvate (A) and lactate (B) in replicates in mass spectrometric assay. (C,D) Mass spectrometric signal of pyruvate MRM (C) and lactate MRM (D). The
titrations of pyruvate (closed circles) and lactate (open circles) were linear, and the MRM detection was specific to each acid. The linearity is shown in the insets for lower
concentrations of the analytes. (E) The LDHA reaction (0.25 nM LDHA with 75
lM pyruvate and 50 lM NADH) showed good reaction kinetic linearity up to 20 min in mass
spectrometric assay. Approximately 30% conversion was achieved after 10 min of reaction, consistent with the conversion observed in NADH fluorescence kinetic assay.
that had mild fluorescence interference with NADH, to confirm
true hits, and to characterize mechanism of action.
determined against LDHA (Fig. 3B and C). The apparent K
of NADH were 6 M in the NADH fluorescence assay (in the pres-
ence of 200 M pyruvate) and 14 M in the mass spectrometric as-
say (in the presence of 500 M pyruvate). The apparent K values
for pyruvate were 56 M in the NADH fluorescence assay (with
M in the mass spectrometric assay (with
M NADH). Overall, the apparent K values obtained from
m
values
l
l
l
l
m
NADH fluorescence kinetic assay
l
5
5
0 lM NADH) and 120 l
The LDHA fluorescence kinetic assay was set up in a 1536-well
format for the primary library screen. Because LDHA preferentially
00
l
m
+
the two assays are within the range of reported values for LDHA
pyruvate K = 55–350 M and NADH K = 5–35 M) [21–25].
converts pyruvate and NADH to lactate and NAD with very mini-
(
m
l
m
l
mal activity observed in the reverse direction (data not shown), the
NADH fluorescence assay is a signal loss assay. The change in
NADH fluorescence was monitored using a Hamamatsu FDSS
Primary screen using NADH kinetic fluorescence assay
7
000, which is a kinetic fluorescence/luminescence reader typi-
cally employed for G-protein-coupled receptor (GPCR) and ion
channel cell-based assay detection. In combination with the use
of assay-ready plates, the integration of an FDSS 7000 with an Agi-
lent Biocel 900 that automated the reagent addition created an effi-
cient linear plate flow that permitted the extreme HTS of 320,000
wells per day. The absence of separate compound plates also dou-
bled the effective storage capacity for assay plates for this fairly
It is known that LDHA binds to its cosubstrates in a sequential
manner, with NADH binding first followed by pyruvate [13,14].
The NADH concentration in cancer cells is generally elevated and
has been reported to be from 168 to 870
lM [26,27], which is sig-
nificantly higher than the reported K value [21–25]. These data
m
suggest that LDHA is likely bound to NADH in cancer cells. There-
fore, a competitive inhibitor with NADH, such as those reported by
Ward and coworkers, might generally yield poor cellular activity
2
small footprint system (ꢁ3 m ). The baseline fluorescence of the
assay plates that contained 8 ll of test compound and LDHA pre-
[
28] (WO/2012/061557). Similar to pyruvate where binding to
bound to NADH in assay buffer was measured for 5 s after the
plates were loaded into the FDSS 7000. Compounds that altered
the NADH fluorescence baseline were flagged as potential fluores-
the enzyme is dependent on the presence of NADH, oxamate (a
pyruvate-mimetic inhibitor) exhibited higher inhibitory potency
with increasing concentrations of NADH (Fig. 3D), suggesting an
uncompetitive inhibitory mechanism with the LDHA–NADH com-
plex. These collective data led us to design our kinetic fluorescence
HTS assay strategy to maximize the opportunity of finding either
uncompetitive or noncompetitive inhibitors with NADH by screen-
cent artifacts. Using a 1536-well dispensing tip head, 2 ll of con-
centrated pyruvate (5ꢂ) was added to initiate the enzymatic
reaction and the change in NADH fluorescence was recorded for
3
min. The activity of the enzyme was analyzed in a Genedata
Kinetic Analyzer and defined by the slope of NADH fluorescence
decay between 60 and 120 s, which was the linear range of the ob-
served enzyme reaction (Fig. 3A).
ing our compounds in the presence of 50
higher than its experimental K ). Equimolar pyruvate to NADH,
which was equivalent to the pyruvate K value, was used.
lM NADH (ꢁ10-fold
m
m
Using the NADH kinetic fluorescence assay, a screen was suc-
cessfully conducted against more than 1 million HTS compounds.
The plate view of the 1536-well fluorescence kinetic traces is
shown in Fig. 4A with a magnified view of representative controls
m
Apparent K determination in both mass spectrometric and NADH
fluorescence assays
0
Using both the NADH fluorescence kinetic assay and mass
and inhibitory reactions. The average Z of the 1536-well fluores-
spectrometric detection, the apparent substrate K
m
values were
cence kinetic assay screen was 0.7 (Fig. 4B). The primary hit rate

Label-free LDHA assays / E. VanderPorten et al. / Anal. Biochem. 441 (2013) 115–122
119
Baseline fluorescence
LDH+NADH+compound
A
C
No Enzyme
+
oxamate
LDH
Pyruvate
addiꢀon
+
Time (s)
Kmapp
NADH
Kmapp
Pyruvate
B
D
Fluorescence
MS/MS
6 μM
56 μM
14 μM
120 μM
NADH
Oxamate IC (μM)
50
2
1
2
5 μM
88
57
28
00 μM
00 μM
Fig.3. (A) NADH fluorescence kinetic assay. The sequence of kinetic reaction measurement and reagent addition is shown with the kinetic traces of no-enzyme, plus LDHA,
and plus LDHA and oxamate controls. The slope of the reaction was measured between 60 and 120 s highlighted in green using a Genedata Kinetic Analyzer. (B,C) Apparent
K determination of pyruvate and NADH in the mass spectrometric and NADH fluorescence assays. MS/MS, tandem mass spectrometry. (D) IC50 determination of oxamate in
m
reactions titrated with NADH.
corresponding to the number of compounds that yielded at least
reaction or detection. Even though this primary screen approach
can effectively lower the number of compound hits that interfere
with assay format-specific detection, it does not prevent other
types of undesirable inhibitors such as covalent modifiers and re-
dox reactive compounds. To help prioritize validated hits that will
likely yield evaluable SAR (Structural-Activity Relationship), a
post-HTS triage cascade was put into place (Fig. 5). On completion
of IC50 confirmation in the NADH fluorescence kinetic assay, the
confirmed hits were further tested in the mass spectrometric assay
that detects the other half of the reaction (conversion of pyruvate
to lactate) and a few selected hits also underwent mechanistic
characterization to probe their reversibility and substrate competi-
tion. In the next sections, we describe the characterizations of
various inhibitors identified from the screen.
5
0% inhibition in the rate of LDHA-mediated NADH fluorescence
decrease was 0.3%. This preliminary hit rate was high considering
that this enzyme class is not known to be a highly druggable target
by HTS against a small molecule library [28]. When the baseline
fluorescence of NADH in the presence of compounds was
compared with the apparent decrease of the NADH depletion, as
expected, a significant number of primary hits fluoresced at wave-
lengths that overlapped with NADH fluorescence (Fig. 4C). In fact, a
series of compounds were found to have a positive correlation of
fluorescence intensity with apparent percentage inhibition. The
overall high fluorescence intensity of those compounds put the
NADH fluorescence out of the linear detection range of the FDSS
7
000, which resulted in a smaller than anticipated change in NADH
fluorescence intensity during the enzymatic reaction. The primary
hits that had a combined baseline fluorescence of less than 500
RFU were prioritized for IC50 confirmation, and the hit rate after
applying this fluorescence cutoff decreased 5-fold to 0.06%, a value
expected for this enzyme target class when screened against the
Genentech HTS library.
Irreversible inhibitory hits
Among the primary hits that were confirmed in the dose–re-
sponse study using the NADH kinetic fluorescence assay, a signifi-
cant number of compounds were found to contain a carboxylic acid
moiety and, therefore, were predicted to bind to the pyruvate sub-
strate site. Among the acid-containing hits, compound A, which
shared a similar compact structure as the pyruvate-mimic inhibi-
tor oxamate, yielded similar inhibitory potency as oxamate in the
With a primary screen conducted using a kinetic NADH fluores-
cence assay, we believe that we have effectively eliminated the
majority of fluorescence artifacts that could create false positives
+
in HTS. By directly measuring NADH to NAD conversion instead
of using coupling enzyme detection, the HTS assay we have chosen
also had prevented another major source of false positives—those
compounds that may preferentially inhibit the coupling enzyme
presence of 50
fluorescence kinetic assay (Fig. 6A). Surprisingly, however, at an
elevated level of pyruvate (500 M), the IC50 value of compound
lM pyruvate and 50 lM NADH using the NADH
l

1
20
Label-free LDHA assays / E. VanderPorten et al. / Anal. Biochem. 441 (2013) 115–122
A
B
Plate number
C
Time
Fluorescent
compounds
Iniꢀal RFU (compound+NADH)
Fig.4. LDHA primary screen. (A) Representative kinetic traces of LDHA reactions carried out in a 1536-well microplate. Examples of no-enzyme controls (rows A and B and
columns 1–4), positive enzyme controls (rows C–E and columns 1–4), control compound oxamate inhibition (rows F and G and columns 1–4), and library compounds (rows
0
A–G and column 5) are shown in the magnified view. (B) Z values of all plates in the screen. (C) Correlation of LDHA percentage inhibition and the baseline fluorescence
intensity of NADH with compound measured at initial 5 s: no-enzyme control (red), oxamate control inhibition (yellow), enzyme positive control (green), and library
compound (blue). A significant number of fluorescent compounds were found to have positive correlation with the apparent LDHA inhibition.
LDHA and 62.5
lM NADH were incubated with 100 lM compound
A or 200 M oxamate, concentrations approximately 10-fold high-
l
HTS: single point NADH
fluorescence Kineꢀcs
er than their respective IC50 values, and the reaction mixture
showed the expected full inhibition measured by the NADH fluo-
rescence assay (Fig. 6B and C). After 30 min of incubation, the en-
zyme–inhibitor complexes were diluted 50-fold into a buffer
IC : NADH
50
containing only 62.5 lM NADH to reach the compound concentra-
fluorescence Kineꢀcs
tions 5-fold below their IC50 values, and the inhibitory potencies
were immediately measured on the addition of pyruvate substrate.
The enzyme that was incubated with oxamate showed full
recovery of catalytic activity (to the level of the DMSO control)
IC : lactate
Reversibility
5
0
&
pyruvate
mass spec
& substrate
compeꢀꢀon
(Fig. 6D), whereas the reaction that contained the compound A
dilution yielded full inhibition, on par with the level seen with
predilution (Fig. 6E). Similar enzyme activities were observed at la-
ter time points of 1 and 2 h postdilution (data not shown), suggest-
ing that compound A is indeed an irreversible inhibitor. This result
is consistent with the observed noncompetitive mechanism of
compound A with the pyruvate substrate. It is hypothesized that
the thiophene might not be stable because discoloration was ob-
served in a 10-mM DMSO stock solution over a few weeks and
might present a reactive moiety to LDHA. Together, the data repre-
sent a good case study illustrating the importance of mechanistic
follow-up in confirming hits obtained from an HTS campaign even
if the hits share similar structures with known inhibitors and are
presumed to interact with the protein in a similar binding mode.
Validated HTS hits
Fig.5. HTS primary screen and hit triage cascade for LDHA.
A was the same as that observed at 50
ues = 13 and 14 M, respectively), whereas the IC50 value of oxam-
ate was shifted weaker on an increase in pyruvate concentration
IC50 values = 18 M at 50 M pyruvate and 153 M at 500
lM pyruvate (IC50 val-
l
(
l
l
l
lM
pyruvate), as would be expected for a pyruvate-competitive inhib-
itor. This noncompetitive mechanism of compound A with respect
to pyruvate suggested that compound A may bind to a different
site in spite of the structural similarity. An alternative hypothesis
would be that compound A might be an irreversible inhibitor that
can covalently modify the enzyme. This is a distinct possibility be-
cause it has been previously reported that the transient dissocia-
tion of the LDH tetramer can render the protein susceptible to
irreversible inactivation by iodoacetamide [29]. To investigate
the hypothesis, compound A was tested side by side with oxamate
to assess the reversibility of its inhibitory activity. Both 100 nM
HTS follow-up triage using the mass spectrometry assay to validate
LDHA inhibitors
As described in previous sections, a primary screen was con-
ducted using an NADH fluorescence kinetic assay against the
Genentech HTS library. Primary hits were selected for IC50 confir-
mation based on percentage inhibition, and those hits that showed
minimal fluorescence interference with NADH fluorescence were

Label-free LDHA assays / E. VanderPorten et al. / Anal. Biochem. 441 (2013) 115–122
121
compounds reaching sub-micromolar potency. Among the con-
firmed hits, there were a few compounds showing a moderate de-
gree of fluorescence interference at concentrations significantly
higher than the reported IC50 values; these compounds were in-
cluded in the set prioritized for further follow-up assays. When this
set of hits was tested in the mass spectrometric assay, a solid cor-
relation of IC50 values was observed for compounds that demon-
strated inhibitory potency in both assays (Fig. 7A). Among those
compounds that were flagged to have fluorescence interference
at a high compound concentration (Fig. 7A, red circles), only 18%
of them were confirmed in the mass spectrometric assay, suggest-
ing that many were indeed false positive compounds due to fluo-
rescence interference. This number was in contrast to the 72%
confirmation rate among compounds that did not show fluores-
cence interference at all concentrations tested (Fig. 7A, blue cir-
cles). In addition to identifying fluorescent false positive hits, the
mass spectrometric data also revealed a new class of artificial
inhibitors of the LDHA reaction. For an on-target enzyme inhibi-
tion, the titration of the compound is expected to yield a decrease
in the enzyme-mediated lactate production as well as a concomi-
tant accumulation of the pyruvate substrate (Fig. 7B). However, a
set of structurally related compounds decreased generation of lac-
tate production and at the same time resulted in a depletion of
detectable pyruvate substrate using the specific MRM method
when they were incubated in the enzyme reaction (Fig. 7C). Even
though the mechanism of the substrate depletion by this class of
compounds is unknown, the decrease in the lactate generation
was likely due to the decrease in the substrate available for enzy-
matic catalysis.
Fig.6. Characterization of LDHA inhibition by oxamate and compound A in the
NADH fluorescence assay. (A) Oxamate and compound A were tested at two
different concentrations of pyruvate. (B–E) Inhibitory reversibility study of oxamate
After confirming IC50 values using the primary mass spectro-
metric assay, a limited set of validated inhibitors with good po-
tency and physical–chemical properties was selected for further
characterization to elucidate the mode of inhibition using the mass
spectrometric assay. The compounds were tested at three different
NADH and pyruvate concentrations to assess their potency in the
range of NADH concentration approximate to the cellular level:
and compound A. Both 200
lM oxamate (B) and 100 lM compound A (C)
(
concentration 10-fold higher than their respective IC50 values) were incubated
with 100 nM LDHA, and both showed 70% to nearly 100% inhibition of the LDHA
activity relative to the DMSO control. After rapid dilution to reach a concentration
5
-fold below their respective IC50 values, oxamate (D) showed immediate recovery
of LDHA activity, whereas compound A (E) showed less than 10% residual LDHA
activity.
(i) 75 and 75 lM, (ii) 300 and 75 lM, and (iii) 75 and 500 lM,
respectively. A few compounds from this set showed a similar
inhibitory profile, and an example is depicted in Fig. 7D for com-
pound D. In the presence of an increased amount of pyruvate,
prioritized. Numerous selected hits were confirmed to have dose-
dependent inhibition in the fluorescence kinetic assay, with some
A
B
D
C
Mass spec log 10 IC 50 (M)
Fig.7. (A) Better IC50 correlation of LDHA inhibitors between the NADH fluorescence kinetic assay and mass spectrometric assay was observed for compounds that had no
fluorescence interference (blue) relative to compounds that showed modest fluorescence interference at high concentration (red). (B) A representation of an LDHA inhibitor
(
compound B) that showed a concentration-dependent decrease in lactate level with a concomitant increase in pyruvate substrate. (C) An example of a substrate-depleting
compound C that inhibited LDHA by decreasing the available pyruvate substrate concentration. (D) IC50 values of compound D at different concentrations of pyruvate and
NADH showed this compound to be competitive with pyruvate but not with NADH.

1
22
Label-free LDHA assays / E. VanderPorten et al. / Anal. Biochem. 441 (2013) 115–122
the potency of the compound was shifted weaker (IC50 val-
ues = 1.3 ± 0.2 and 4.5 ± 0.4 M for 75 and 500 M pyruvate,
respectively), suggesting that compound D might be occupying
the pyruvate binding site. At an elevated concentration of NADH,
compound D did not lose potency and in fact might have exhibited
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l
l
[
[
[
a very modest increase in inhibition (IC50 = 0.9 ± 0.2 lM). These
data confirmed an inhibitory mechanism that is competitive only
with pyruvate and not NADH. Furthermore, the result does not rule
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[
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A strategically designed kinetic high-throughput screen mea-
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ric assay that readily provides quantitative analysis of lactate and
pyruvate was able to efficiently capture both sides of the LDHA
reaction, eliminate fluorescent artifacts and pyruvate-depleting
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either noncompetitive or uncompetitive with the LDHA–NADH
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cells compared with an NADH-competitive inhibitor will be the fo-
cus of future studies. If demonstrated to be more effective in cells
than previously reported NADH site LDHA inhibitors, the elabora-
tion of these HTS hits will also provide valuable tool compounds
to aid the validation of LDHA’s role in glycolytically addicted can-
cer cells and tumor metabolism.
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Ultraviolet-induced autofluorescence characterization of normal and tumoral
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We thank the entire baculovirus expression group and protein
chemistry department at Genentech for cloning, expressing, and
purifying many of the enzymes used in the study, particularly Kris-
ta Bowman, Yvonne Franke, Mike Elliott, and Jiansheng Wu. We
also thank Mark Ultsch for providing initial protein samples for
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