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© MARY ANN LIEBERT, INC. • VOL. 8 NO. 2 • APRIL 2010 ASSAY and Drug Development Technologies 175
DOI: 10.1089/adt.2009.0249
D a v i d C . S m i t h s o n , 1,2 A n a n g A . S h e l a t , 2 J e f f r e y B a l d w i n , 3
M a r g a r e t A . P h i l l i p s , 3 a n d R . K i p l i n G u y 2
1 Graduate Program in Chemistry and Chemical Biology, University
of California, San Francisco, California.
2 Department of Chemical Biology and Therapeutics, St. Jude
Children’s Research Hospital, Memphis, Tennessee.
3 Department of Pharmacology, The University of Texas
S o u t h w e s t e r n M e d i c a l C e n t e r a t D a l l a s , D a l l a s , T e x a s .
A B S T R A C T
Herein, we describe the optimization of a linked enzyme assay suit-
able for high-throughput screening of decarboxylases, a target fam-
ily whose activity has historically been dif ficult to quantif y. Our
approach u ses a commerci ally available bicarbonate detection reagent
to measure decarbox ylase activity. The assay is performed in a fully
enclosed automated screening system under inert nitrogen atmo-
sphere to minimize perturbation by exogenous CO
2 . Receiver operat-
ing characteristic (ROC) analysis following a pilot screen of a small
library of ∼3,600 unique molec ules for inhibitors of Tr ypanosoma
brucei ornithine decarboxylase quantitatively demonstrates that the
assay has excellent discriminatory power (area under the curve =
0.90 with 95% confidence interval between 0.82 and 0.97).
INTRODUCTION
D ecarboxylase enzy mes represent a significant target fam-
ily for the development of therapeutic agent s. Ornithine
decarboxylase (ODC) and S -adenosylmethionine decar-
boxylase control the polyamine biosynthetic pathway,
a therapeutic target in both oncology and parasitology.
1,2 Other
medically relevant decarboxylases include histidine decarboxyl-
ase, a target in the inflammation pathway, DOPA decarboxylase,
a Parkinson’s disease target, and diaminopimelate decarboxylase,
a potential antibiotic target.
3–5 In spite of the importance of the
enzyme class, few decarboxylases have been subjected to large-
scale drug discovery efforts, in part due to difficulty in quantifying
thei r activities in a ma nner c ompatible with moder n high- th rough-
put approaches. Classical methods for assaying decarboxylase func-
tion often involve capture of
14 CO
2 from radiolabeled substrates or
de ri va ti zation of t he e nzy me p ro duc ts f ol lowed by HP LC a naly si s.
6,7
Neither of these techniques is suitable for high-throughput screen-
ing (H TS) efforts, as they involve either formation of radioactive
gas or leng thy, resource intensive detection procedures. Use of a
commercial low-throughput method linking the production of CO
2
to the consumption of NADH using phosphoenolpy ruvate carbox-
ylase (PEPC) and malate dehydrogenase (MDH) to track decarbox-
ylase activities has been reported
8 ( Scheme 1 ). Reaction progress
was measured by the decrease in NADH absorbance at 340 nm. We
believed this assay was a good candidate for adaptation to HTS.
The target chosen for optimization of this assay system was
ODC from Tr y pa no so ma br ucei , the causative agent of human
African trypanosomiasis. T his disease is fatal if left untreated
and is a major health problem in much of central Africa.
9 The
only clinica lly validated molecular target for treatment of T.
brucei infections is ODC, which catalyzes the f irst step in poly-
amine metabolism, the decarbox ylation of ornithine to produce
putrescine. Putrescine and the other polyamines, sper midine and
sper mine, are necessar y for cellular reproduction, making their
regulatory enzymes attractive d rug targets in both parasitology
and oncology. The biological roles of polyam ines are numerous,
and they have been implicated in the regulation of a wide range
of important genes, including P53 and c-myc .
10,11
Optimization of a Non-Radioactive
High-Throughput Assay for Decarboxylase
Enzymes
ABBREVIATIONS: DFMO, di uoromethyl ornithine; DTT, dithiothreitol; HTS, high throughput screening; MDH, malate dehydrogenase; ODC, ornithine decarboxylase;
PEPC, phosphoenol pyruvate carboxylase; PLP, pyridoxal-5′-phosphate; ROC, receiver operator characteristic.
adt.2009.0249.indd 175 4/19/2010 11:37:43 AM
SMITHSON ET AL.
176 ASSAY and Drug Development Technologies APRIL 2010
In mammalian cells, ODC is highly regulated and possesses a
very short half-life of 10 to 20 min.
12 However, in protozoal para-
sites, par ticularly T. brucei brucei and T. b ru ce i g am bi ense , the
enzyme is longer lived (∼4 h).
13 This difference allows the use of
ir rever sible and non select ive inhibitor s of ODC such as difluorom-
ethylornithine (DFMO) in clinical treatments for African trypano-
somiasis.
14 Unfortunately, DFMO possesses poor pharmacokinetic
proper ties, resulting in the need for multi-day high-dosage intra-
venous treatment regimens that limit its use in the third world.
15
To date, most d rug discover y efforts directed toward ODC
inh ibitors have focused on analogs of or nith ine (such as DFMO),
putrescine, or pyridoxal-5′-phosphate (PLP), a cofactor necessar y
for ODC activity shared by many other enzymes.
16,17 None of these
have proved as effective as DFMO for treatment of T. b rucei infec-
tions. No large-scale efforts to discover inhibitors based on scaf-
folds other than substrate, product, or cofactor analogs have been
repor ted. The lack of large-scale drug discover y effor ts has been
due in part to the difficulty in assay ing ODC activity in a high-
throughput manner.
Therefore, we have optim ized a commercial enzy me-linked
bicarbonate detection system for use in H TS of ODC. We report
the results of this optimization and the performance of the assay
on a proof-of-concept screen of ∼3,600 unique molecu les.
MATERIALS AND METHODS
Materials
All chemicals assayed in this st udy were purchased from ven-
dors without further purification. DI water was filtered with a
MilliQ Synthesis Ultra-Pu re water system (Millipore, Billerica,
MA) immediately before use. Infinity™ Carbon Dioxide Liquid
Stable Reagent was purchased from Thermo Fisher Scientific
(Waltham, M A).
L -Ornithine, pyridoxal 5′-phosphate (PLP), and
dithiothr eitol (DTT ) were purchased from Sigma-Aldrich (St. Louis,
MO). DFMO-HCl was purchased from Chem-Impex International
(Wood Dale, IL). All plate-based enzymatic assays were perfor med
in 384-well black-sided, clear-bottomed polystyrene microplates
(#3,702) from Cor ning Life Sciences (Acton, M A). The bioac-
tive compound screening librar y at St. Jude Children’s Research
Hospital was assembled from commercially available collections
including the Prestwick Chemical Library (Prestwick Chemical,
Ill kirch, France); the LOPAC Collection (Sigma-Aldrich, St. Louis,
MO); and the Spectrum Collection, the N INDS Collection, the
Natural Product Collection, and t he Killer Plate Collection from
Microsource (Microsource Discovery systems, Gaylordsville, CT).
The tota l bioactive test set contains ∼7,300 compounds, including
many internal replicates. The total number of unique compounds
in the collection is ∼3,600 molecules.
Purification of T. br u ce i ODC
ODC was expressed as an N-terminal 6×His-tag fusion protein
in Escherichia coli BL21 (DE3) cells as described.
8 Protein was
purified by Ni
2+ -NTA-agarose column followed by Superdex 200
gel-filtration column chromatography. Fractions containing the
desired protein were identified by SDS-PAGE. Those conta ining
pure ODC were combined and concentrated using an Amicon-Ultra
centrifugal filter device (10 kDa cutoff, UFC901024; Millipore,
Bi ller ica, M A) to concentrat ions of ∼40 mg/mL. Yields of purified
ODC were generally 7 to 13 mg/L of cultured cells.
HO
NH2
HCO3–
–O
–O–O
O–
–OO
O
OOO
OOH
OO
P
HO–
NH2
NH2+CO2
H2N
O
ODC
PEPC
Monitored via Decrease in
Absorbance at 340 nm
MDH
NADH NAD+
O–O–
S c h e m e 1 . Linked a ssay mechanism. Ornithine decarbox ylase (ODC) catalyzes the dec arboxylation of or nithine, releasing CO
2 , which is then
captured by the basic buffer (pH 8.05) as bicarbonate. Phosphoenolpyruvate carboxylase (PEPC) uses this bicarbonate to generate oxa-
loacetate from phosphoenolpyruvate. The oxaloacetate is then reduced by malate dehydrogenase (MDH) to malate in a NADH-dependent
fashion. There is a 1:1 relationship between the amount of CO
2 produced by ODC and t he amount of NADH ox idized by MDH, a llowing kinetic
parameters for ODC to be calculated from obser ving NADH levels as a function of time. The assay system has been optimized such that ODC
is the rate-limiting step.
adt.2009.0249.indd 176 4/19/2010 11:37:44 AM
DECARBOXYLASE ENZYME HTS
© MARY ANN LIEBERT, INC. • VOL . 8 NO. 2 • APRIL 2010 ASSAY and Drug Development Technologies 177
Assay Automation and Nitrogen Atmosphere Generation
All screening data were generated on a High Resolution
Engineering (Woburn, MA) integrated screening system using
Liconic plate incubators (Woburn, MA) and a Staubli T60 robotic
arm (Staubli, SC). This s ystem is enclosed in a gas-tight Plex iglas
enclosure allowing a nitrogen atmosphere to be generated by
continual purging with ∼30 psi nitrogen through t win 8-mm
inner diameter tubes. Nitrogen was obtained by boil-of f from liq-
uid nitrogen usi ng an in-house dry nitrogen system supplied by
NexAir (Memphis, TN). Nitrogen consumption during screening
was estimated to be ∼200 L/min (12,000 L/h or 1,200 L per plate
for a kinetic read). Percent oxygen within the enclosure was mon-
itored using an Air Aware oxygen detector (model 6810-0056;
Industrial Scientific, Oakdale, PA) and maintained at <2.5%
throughout all high-t hroughput assays. Percent carbon diox-
ide was below detectable levels (0.5%) as measured by Liconic
STR240 series incubators (Liconic Instruments, Woburn, MA).
Assay solutions were dispensed using Mat rix Wellmates (Matrix
Technologies, NH) equipped with 1 μL rated tubes. Plates were
centrifuged after all liquid additions using a Vspin plate cen-
trifuge (Velocit y11, Menlo Park, CA). Compound transfers were
performed using a 384-well pin tool equipped wit h 10 nL slot-
ted hydrophobic surface-coated pins (V&P Scientific, San Diego,
CA). This allowed delivery of ∼25 nL of DMSO stock solution
with CVs of <10%. All absorbance data were measured using an
EnVision Multilabel Plate Reader equipped with a 340 nm narrow
bandwidth fi lter (Perkin Elmer, 2100-5740). During automation,
the screening system was operated offline and individual instru-
ments were accessed using manual operation of the robot arm.
ODC-PEPC-MDH-Linked Assay
This assay was performed under nitrogen atmosphere. Assay
buffers were prepared under normal atmosphere while f lushing
with a stream of nitrogen and transferred to enclosed nitrogen
atmosphere upon complet ion. Assay buffer (66 mM Tris, 25 mM
NaCl, 8 mM MgSO
4 , 0.01% Triton-X, pH 8.05) was prepared daily.
The assay reaction was prepared using 2 master mixes A and B,
which were prepared immediately before use. Mix A contained
ornithine (0–10 mM), PLP (0–937 μM), and 5.7 mM DTT in assay
buffer. Mix A was prepared immediately before testing from fro-
zen stocks prepared in water (0.5 M Orn, pH 7.5, 20 mM PLP, and
1 M DTT). Mix B contained I nfinity Carbon Dioxide Liquid Stable
Reagent (Infinity CO
2 , Thermo Fisher Scientific) and ODC (0–300
nM). Mix B was prepared immediately prior to testing from fresh
Inf init y Carbon Dioxide Liquid Stable Reagent and frozen ODC
stocks. For testing, 15 μL Mix B was added to appropriate wells in a
384-well clear-bottomed plate followed by compounds transferred
by pin. T he 10 μL Mix A was then added to star t the reaction.
Final optim ized assay concentrations were 2.3 mM DT T, 600 μM
ornithi ne, 60 μ M PLP, 60% I nfinit y Carbon Dioxide Liquid Stable
Reagent, 150 nM ODC, 10 μM test compound, and 0.01% Triton-X,
in a 25-μ L final volume unless otherwise specified. Reaction pro-
gress was monitored by following absorbance at 340 nm using
an Env ision plate reader (Perk in Elmer) equipped with a narrow
bandwidth 340 nm filter (Perkin Elmer, 2100-5740). Absorbance
was monitored for 20 min, with time points taken every minute.
Data from 15 to 20 min after addition of Mix A were fit to a linear
model using statistical methods described below. The resulting
slope of this fit was taken as the rate of the reaction and used as
the endpoint for the assay.
Compounds for screening were placed in 384-well poly propyl-
ene plates (Corning Life Sciences, Acton, MA) at 10 mM concen-
tration in DMSO with columns 1, 2, 13, and 14 empty. Positive
controls (DFMO, 1 M, 10 μL) were placed in a separate 384-well
polypropylene plate in wells A2, B2, C2, D2, E14, F14, G14, H14, I2,
J2, K2, L2, M14, N14, O14, and P14. Negative controls (DMSO, 10
μL) were placed in the same plate as positive controls in wells A14,
B14, C14, D14, E2, F2, G2, H2, I14, J14, K14, L14, M2, N2, O2, and
P2. All microplate compound transfers were accomplished using
a 384-well pin tool equipped with 10 nL hydrophobic surface-
coated pins (V&P Scientific, San Diego, CA). This allowed deliv-
ery of ∼25 nL of DMSO stock solution with CVs of <10%.
Cuvette assays were performed as described above with the
following minor mod ifications; the f inal assay volume in cuvettes
was 500 μL at 40% Infinity Carbon Dioxide Liquid Stable Reagent,
50 μM PLP, 50 μM DTT, 1% DMSO, and varied ornithine concen-
tration s from 10 mM to 100 μM. As with microplate assays, assay
buffer (66 mM Tris, 25 mM NaCl, 8 mM MgSO
4 , 0.01% Triton-X,
pH 8.05) was prepared fresh daily.
PEPC-MDH-Linked Assay—Microplate
For assay of the linking enzymes, assay buffer (66 mM Tris, 25
mM NaCl, 8 mM MgSO
4 , 0.01% Triton-X, pH 8.05) was prepared
daily using water. The assay reaction was prepared in 2 master
mixes. Mix A contained 1.25 mM sodium bicarbonate (Sigma-
Aldrich), 100 μM PLP, and 5.7 mM DTT in assay buffer. Mix B was
100% Infinity Carbon Dioxide Liquid Stable Reagent. For testing
15 μL, Mix B was added to appropriate wells of a 384-well clear-
bottomed microplate followed by pin-transfer compound DMSO
stocks. Compounds were allowed to equilibrate in the presence of
enzymes for 20 min before substrate was added. The reaction was
star ted by addition of 10 μL Mix A and reaction progress was mon-
itored by absorbance at 340 nm. Positive controls were wells with
no sodium bicarbonate added, negative controls were DMSO.
Final assay concentrations were 2.3 m M DTT, 60 μM PLP, 0.75
mM sodium bicarbonate, 60% Infinity Carbon Dioxide Liquid
adt.2009.0249.indd 177 4/19/2010 11:37:44 AM
SMITHSON ET AL.
178 ASSAY and Drug Development Technologies APRIL 2010
Stable Reagent, and 0.01% Triton-X. Reaction progress was mon-
itored by decrease in absorbance at 340 nm using an Envision
plate reader (Perkin Elmer) equipped with a nar row bandwidth
340 nm f ilter (Perkin Elmer, 2100-5740) for 10 min with time
points taken every 1 min. Data from 1 to 5 min were fit to a linear
model using statistical methods as descr ibed below and normal-
ized to the positive and negative controls.
Primary Screening Data Analysis and Reaction
Rate Calculation
Pr imar y screening data analysis was perfor med using cus-
tom protocols w ritten in Pipeline Pilot (v. 6.1.1, Accelr ys) and the
R program (htt p://www.r-project.org/, v. 2.7.0).
18 For rate deter-
mination, kinetic data were fit using 3 met hods: a linear model
using least squares (“linear,” calculated using the lm function in
robustbase R package, v. 0.2-7), a linear model using only the
difference between the first and last time points (“delta”), and a
robust linear model using an iteratively re-weighted least squares
algorithm (“robust,” calcu lated using the lmrob function in robust
ba se R p ac ka ge , v. 0. 2-7).
19 For hit identification, the fina l 5 min of
data (6 data points in total including the 15 min time point) were
fit using the “linear” method. The fitted rates were the values
used in the calculations of all plate statistics. Only plates passing
the minimum Z ′ and Z -factor thresholds of 0.4 and 0.4, respec-
tively, were accepted. Initial screening hits were determined on
a plate-by-plate basis by identifying compounds with activities
that were simultaneously outliers from the negative control and
variable compound populations. The outlier cutoffs were calcu-
lated as t he upper fourth plus 1.5 times the fourth spread (the
upper fourth and fourt h spread are similar to the third quartile
and interquartile range, respectively), a robust statistic that cor-
responds to a P value of ∼0.005 for normal distr ibutions.
20 For
Z ′ calculations, 16 positive and 16 negative controls were used
unless other wise stated. Reaction rate was transformed from AU/
min to m M NADH/min using an ex tinction coeff icient of 6.349
AU/mM/cm for NADH and an approximate path length of 0.4 cm
for assays performed at 25 μL f inal volume in a 384-well plate.
Reaction rates for dose–response data were calculated as
descr ibed above. Rates were then normalized to DMSO and DFMO
controls and sigmoidal cur ves with variable slopes were fit using
Graphpad Prism 4.03. No constraints were used when fitt ing this
data.
Receiver Operating Characteristic Analysis
Receiver operating characteristic (ROC) analysis was per-
formed using custom R scripts (http://www.r-project.org/, v. 2.9.0)
and the R rocr package (v. 1.0.2). The ROC test set was composed
of 63 compounds that uniformly sample from the distribution of
observed primary screen activities (−7% to 103%). A compound
was considered a true positive if it inhibited ODC in a dose-depen-
dent fashion, had no effect in the PEPC-MDH assay system, and
showed a 2-fold increase in reaction r ate when screened with 300
nM ODC. The ROC curve was formed by plotting the true positive
rate vs. the false positive rate as a function of decreasing prima ry
screen activity. Conf idence intervals for the ROC area under the
cur ve (AUC) were calculated from 200 bootstrap simulations.
R E S U L T S
Optimization of ODC-PEPC-MDH-Linked Assay
The assay system was chosen in part because the linking sys-
tem was available from Ther mo Fisher Scientif ic, simplifying
quality control and large-scale reagent sourcing. This system was
designed for clinical detection of bicarbonate in bod ily f luids, and
is extremely sensitive to low levels of dissolved CO
2 , which is in
equilibrium with bicarbonate in aqueous solutions. Unfortunately,
this sensitivit y is a disadvantage in HTS applications. The back-
ground signal produced by atmospheric CO
2 reduces the assay
linear time and causes reagents placed on the screening deck
to rapidly degrade. However, if reagents are stored, dispensed,
incubated, and read under a nitrogen atmosphere, this liability is
largely mitigated. When the assay is performed under nitrogen,
the signal-to-noise ratio increases from 2.5 to 8.1 and t here is a
significant increase in reagent stability (see Fig. 1A ). For this rea-
son, all optimization and screening experiments were performed
under an inert nitrogen atmosphere as described.
The assay rate was linear with respect to ODC concentration
from 20 to ∼600 nM. A final concentration of 150 nM ODC was
chosen for the primary screen, based upon signal-to-noise ratio
and Z ′ score (>4 and >0.5, respectively, Fig. 1B ). The final PLP
concentration chosen was 60 μM, ∼300-fold over the literature
reported K
m ,
21 to minimize the probability of finding compounds
that interf er ed di rec tl y w it h P LP, o r comp et ed for th e PLP-bi nd ing
site. The assay buffer was not optimized, and was formulated to
match the commercial linking buffer in order to maintain perfor-
mance of the linking system. Several methods to remove dissolved
CO
2 from the buffer were tested including vacuum degassing and
nitrogen sparging. None offered significant reductions in back-
ground signal over using fresh MilliQ Sy nthesis grade water (data
not shown). To ensure that the assay system was performing com-
parably to other literature methods, the K
m for ornithine was mea-
sured. The va lues obtained (420 ± 22 μM, Fig. 1C ) were consistent
with liter ature values (370–50 0 μM).
22 ,
23 T he relatively low orni-
thine concentration chosen for screening (625 μM, 1.5 × K
m ) was
selected to maximize the probability of finding or nith ine compet-
itive inhibitors. The k
cat determined under screening conditions
(0.71 s
−1 at ∼22°C) is ∼10-fold lower than the reported literature
adt.2009.0249.indd 178 4/19/2010 11:37:45 AM
DECARBOXYLASE ENZYME HTS
© MARY ANN LIEBERT, INC. • VOL . 8 NO. 2 • APRIL 2010 ASSAY and Drug Development Technologies 179
values.
21 This was determined to be a temperature effect, and it
was possible to replicate literat ure values by performing the assay
at 37°C. Due to limitations with our integrated plate reader, all
HTS was performed at room temperature (∼22°C).
Finally, the amount of linking enzyme mix (Infinity CO
2 ) was
optimized ( Fig. 1D ). While the signal remained linear over the
20-min assay window for both 60% and 45% Infinity CO
2 , 60%
was chosen in order to maximize the linear time available during
screening and reduce dependence on precise timing during the
process. The percentage was not increased past 60% due to the
fact that absorbance values were already approaching 1.2 at this
concentration. The assay system’s tolerance for DMSO was also
tested, and was shown to be up to 10% at optimized screening
conditions (data not shown).
Optimization of Secondary PEPC-MDH Assay
In light of the fact that this assay is dependent upon the activit y
of the 2 linking enzy mes as well as that of ODC, a secondar y assay
0
01020304050
Time (min)
60 70 80 90
0 0.0
0.00
0.25
0.50
0.75
1.00
1.25
AU340
2.5 5.0 7.5 10.0
Time (min)
Optimization of Infinity CO2 Percentage
12.5 15.0 17.5 20.0
0.0000
0.0025
0.0050
Rate (mM/min)
0.0075
5,000 10,000
Determination of Orn Km
Km = 420 ± 22 μM
kcat = 0.78 ± 0.02 s–1
15,000 20,000 25,000
Orn (μM)
0.00 0.0
0.1
0.2
0.3
0.4
0.5
0.6
Z′ Value
0.7
0.8
0.9
0.01
0.02
0.03
0.04
250 500 750 1,000 1,250 1,500
0.00
0.25
0.50
0.75
1.00
[ODC (nM)]
Optimization of ODC Levels
Reaction Rate (mM NADH/min)
Rate = 0.020 AU/min
Rate = 0.0162 AU/min
Rate = 0.008 AU/min
Rate = 0.002 AU/min
Effect of Nitrogen Atmosphere
A
CD
B
AU
F i g . 1 . O p t i m i z a t i o n o f a s s a y c o n d i t i o n s . ( A ) Increase in background signal under normal atmosphere. The signal seen here in green is
solely from atmospheric CO
2 (orange = nitrogen atmosphere, no ornithine; green = standard atmosphere, no ornithine; blue = nitrogen
atmosphere, 625 μM ornithine; red = standard atmosphere, 625 μM ornithine). Quadruplicate data were collected in a 384-well microplate
as described in the presence of the indicated concentrations of ornithine. All other reagent concentrations were identical to the optimized
conditions described in Materials and Methods. The nitrogen atmosphere was maintained as described with O
2 levels under 2.5%. ( B )
Optimization of enzyme levels. ▼ = Z ′ values, ■ = reac tion r ates. Dashed line indicates st andar d Z ′ cutoff value of 0.5. Da ta wer e collected
in a 384-well microplate using optimized assay conditions with varied fi nal ornithine decarboxylase (ODC) concentrations. Z ′ Values were
calculated using 8 positive (1 mM difl uoromethylornithine (DFMO)) and 8 negative (DMSO) controls. The signal window at 150 nM ODC is
∼5-fold. ( C ) Determination of ODC K m at 150 nM ODC and 60 μM PLP. Data were collected in a 384-well plate as described and fi t to the
Michaelis–Menten equation. ( D ) Optimization of Infi nity CO
2 percentage (■ = 60% Infi nity CO
2 , ▲ = 45% Infi nity CO
2 , ▼ = 30% Infi nity CO
2 ,
♦ = 15% Infi nity CO
2 , • = 7.5% Infi nity CO
2 ). Data were collected in 384-well plates as described at optimized assay conditions with varied
Infi nity CO
2 . Data points were taken every 15 s in quadruplicate. The plot of AU
340 vs. time at 60% Infi nity CO
2 represents a typical data set
under optimized assay conditions with a ∆AU
340 of ∼0.4 AU.
adt.2009.0249.indd 179 4/19/2010 11:37:45 AM
SMITHSON ET AL.
180 ASSAY and Drug Development Technologies APRIL 2010
to test the effects of hits on the linking enzymes was designed.
Titration of exogenous sodium bicarbonate into the reaction mix-
ture showed a K
mapp of 0.47 ± 0.04 mM, which is consistent with
repor ted literature values for the K
m of PEPC from E. coli (0.1–0.3
mM)
24 ,
25 ( Fig. 2A ). A bicarbonate concentration of 1.5 × K
mapp
(0.75 mM) was chosen for testing inhibitors. A final concentration
of 60% Infinity CO
2 was used in order to mimic the primary assay
screening conditions. At these reagent concentrations, a linear
time of 7 min was observed with a ∆AU of ∼0.8 ( Fig. 2B ). A signal
window of 26 was observed and a Z ′ of 0.75 was calculated using
no bicarbonate reagent as the positive control.
Testing of Known Inhibitors and Selection of Positive
Controls
The only know n ODC inhibitor available in large enough quan-
tities for use as a HTS control compound is DFMO. At optimized
assay conditions in the ODC-PEPC-MDH-linked system, DFMO
exhibited an IC
50 of 200 ± 40 μM, which is consistent with the
literature K
iapp value of 160 μM ( Fig. 3A ).
26 No effect on the IC
50
value was seen when the amounts of link ing enzyme present were
changed. Additionally, it was possible to make 1 M stock solutions
of DFMO in DMSO that were stable for several months at room
temperature. Stock solutions at this concentration allowed effi-
cient pin transfer of DFMO into the control wells in nanoliter vol-
umes and permitted th is compound to be used as positive control
in screening at a final concentration of 1 mM. Inhibitors of the
linking enzymes were ineffective under nor mal screening condi-
tions ( Fig. 3B and 3C ). Neither baicalein, a PEPC inhibitor, nor
isoquinoline, a MDH inhibitor, had any effect at concentrations
up to 1 mM under optimized assay conditions, well past their
reported K
i values of 0.79 and 200 μM, respectively.
27 ,
28 However,
if the percentage of Infinity CO
2 was reduc ed , inh ib it ion by b aica-
le in beca me app ar en t, wi th IC
50 values approaching literature val-
ues. Isoquinoline was not observed to have any inhibitor y effects
under any conditions tested; t his is unsurprising given its poor
potency.
In the PEPC-MDH secondar y assay system, DFMO did not
have any effect below 10 mM concentrations. This inhibition is
the result of a pH decrease in the assay solution due to the fact
that the hydrochloride salt of DFMO was u sed to make inhibitor
stock solutions. Baicalein, the PEPC inhibitor, was not obser ved
to have any effect on the secondary assay at norma l screening
levels of Infinity CO
2 . However, as in the case of the ODC-PEPC-
MDH system, when linking enzyme levels were dropped, inhibi-
tion by baicalein was detected. Isoquinoline had no effect on the
2 enzyme system under any conditions tested.
Optimization of the High-Throughput Assay
Before perfor ming a ny screening, the stability of the assay
was tested using the fully automated system. Location-dependent
effects were measured across a microplate prepared using the
automated assay system. The only significant effect seen was a
slight increase in high signal for the outside columns ( Fig. 4A and
4B ). This did not affect Z ′ values significantly due to t he fact that
negative control wells were placed on both interior and edge col-
umns (8 negative controls in columns 1 and 2, and 8 in columns
13 and 14). In order to
maximize the amount
of data gathered across
a wide range of variable
compounds, a 20-min
kinetic read with time
points every minute was
used during the initial
screening efforts.
Primary Screen
and Optimization
of Kinetic Fitting
Parameters
For the initial proof-
of-concept screen, the
bioactive collection
at St. Jude Children’s
Research Hospital (8,832
data points for ∼3,600
0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
AB
123 4
Rate (mM NADH/min)
5
[HCO3(mM)]
6789 0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
51015
Time (min)
Rate = 0.087 AU/min
Rate = 0.002 AU/min
AU
20 25
Km = 0.47 ± 0.04 mM
F i g . 2 . Optimization of PEPC-MDH assay system. Data were collected under nitrogen as described in Materials
and Methods. All data were collected under nitrogen in 384-well microplates. ( A ) Determination of the K m appar-
ent with respect to bicarbonate. The value obtained is consistent with literature values.
24,25 Data were fi t to the
Michaelis–Menten equation. ( B ) Determination of assay linear time (■ = 0.75 mM HCO
3 , ▲ = no HCO
3 ). Under
optimized assay conditions, the bicarbonate signal was linear for ∼7 m i n .
adt.2009.0249.indd 180 4/19/2010 11:37:46 AM
DECARBOXYLASE ENZYME HTS
© MARY ANN LIEBERT, INC. • VOL . 8 NO. 2 • APRIL 2010 ASSAY and Drug Development Technologies 181
unique molecules in 23 384-well plates) was selected for testing.
Absolute replicate number for each compound varied from 1 to 7
depending on vendor library composition and plating. The com-
pounds were screened at 10 μM against the ODC-PEPC-MDH sys-
tem using a 20-min kinetic read. Eight posit ive controls (1 mM
DFMO) and 8 negative controls (DMSO) were placed in colum ns 1,
2 and 13, 14 to help detect possible edge effects. The scatter plot
for percent activ ity across the library is shown in Figure 4C . The
screening r un took 8.7 h to complete using our automated system.
We tested 3 different met hods for determining the assay rates
( Fig. 5 ). Fitting using the later portion of the data (time points
10–20) improved Z ′. The “delta” model performed similarly to the
“linear” model on average, albeit with slightly higher variance.
The “robust” model performed the best when all time points were
included, but fai led to converge without at least 15 time points.
The “ linea r” me thod using ti me points fr om 15 t o 20 min wa s c ho -
sen to calculate endpoint values as a compromise between assay
run time and Z ′.
0.1
–25
0
25
50
75
100
ABC
% Inhibition
0
25
50
75
100
D
% Inhibition
0
25
50
75
100
E
% Inhibition
0
25
50
75
100
F
% Inhibition
–25
–25
0
25
50
75
100
% Inhibition
–25
0
25
50
75
100
% Inhibition
1 10 100
[Inhibitor (μM)]
DFMO
ODC Inhibitor, Ki = 160 μM*
ODC-PEPC-MDH
DFMO
PEPC-MDH
Baicalein
PEPC-MDH
Isoquinoline
PEPC-MDH
Baicalein
PEPC Inhibitor, Ki = 0.79 μM**
ODC-PEPC-MDH
Isoquinoline
MDH Inhibitor, Ki = 200 μM***
ODC-PEPC-MDH
1,000 10,000
0.1 1 10 100
[Inhibitor (μM)]
1,000 10,000 0.10.01 1 10 100
[Inhibitor (μM)]
1,000 10,000 0.10.01 1 10 100
[Inhibitor (μM)]
1,000 10,000100,000
0.1 1 10 100
[Inhibitor (μM)]
1,000 0.1 1 10 100
[Inhibitor (μM)]
1,000 10,000
F i g . 3 . Performance of known inhibitors in the ODC-PEPC-MDH and PEPC-MDH assays (■ = 60% Infi nity CO
2 , ▲ = 45% Infi nity CO
2 , ▼ =
30% Infi nity CO
2 , ♦ = 15% Infi nity CO
2 ). Data were collected under optimized screening conditions as described unless otherwise noted.
Compounds were allowed to equilibrate with enzymes for 20 min prior to addition of substrate. Percent inhibition is defi ned as (1 − v i / v o ) ×
100. At Infi nity, CO
2 percentages lower than 45% reaction rates were calculated from the fi rst 10 min of the reaction rather than the normal
20 min used at higher percentages due to limitations in assay linear time (see Fig. 1D ). For PEPC-MDH assay results, rates were determined
from the fi rst 5 min (see Fig. 2B ). ( A ) Performance of difl uoromethylornithine (DFMO), a known ornithine decarboxylase (ODC) inhibitor, in
the ODC-PEPC-MDH assay with varying amount s of linking enzymes. Note that linking enzyme concentrations do not effect the per formance
of DFMO. ( B ) Performance of baicalein, a known inhibitor of PEPC, at several different fi nal linking mix concentrations in the ODC-PEPC-MDH
assay. At normal screening conditions (60% Infi nity CO
2 ) no inhibition is seen, even from this submicromolar inhibitor. However, upon dilu-
tion of the linking mix, inhibition becomes apparent . ( C ) Perfor mance of isoquinoline, a known inhibitor of MDH in the ODC-PEPC-MDH assay
at several Infi nity CO
2 percentages. No inhibition was seen in any condition tested. Reaction rates at Infi nity CO
2 concentrations lower than
45% were calculated from the fi rst 10 min of data due to limitations in assay linear time (see Fig. 1D ). ( D ) Performance of DFMO in the PEPC-
MDH assay system at varying levels of Infi nity CO
2 . ( E ) Performance of baicalein in the PEPC-MDH assay system at varying levels of Infi nity
CO
2 . As with the ODC-PEPC-MDH system, a dependency on the amount of linking enzymes present greatly effects the amount of inhibition
seen. ( F ) Performance of isoquinoline in the PEPC-MDH assay. No inhibition was observed under any conditions tested. *Literature value, 26
**Literature value,
27 ***Literature value. 28
adt.2009.0249.indd 181 4/19/2010 11:37:46 AM
SMITHSON ET AL.
182 ASSAY and Drug Development Technologies APRIL 2010
The overall data quality for t he 3-enzyme assay was satisfac-
tory, with an average Z ′ value of 0.68 across all plates. The robust
outlier cutoff yielded 84 pri mary hits, of which 52 were unique.
Internal duplication of compounds afforded an assessment of
assay reproducibility. Active repli-
cates were detected 86% of the time
with an average activity CV of 16%,
and 97% of the time with an average
act ivity C V of on ly 3% when restrict-
ing replicates to those from the same
vendor.
Secondary Testing
To further characterize the hits,
aliquots of each unique sample were
cherry-picked and subjected to full
dose–response st udies consisting of
10 points in a 1:3 dilution series (top
= 100 μM). Thirty t hree of the initial
52 unique hits displayed a full dose
response while 15 displayed partial
dose responses in the ODC-PEPC-
MDH system—a 92% confirmation
rate. None of the samples signif i-
cantly affected the PEPC-MDH sys-
tem at concentrations up to 100 μM
at 60% Infinity CO
2 under optimized
assay conditions. To further confirm
that inhibition of ODC was the rate-
limiting step in the reaction cascade,
the samples were re-screened at 300
nM ODC, and the rates at the IC
50 were
compared to those determined at 150
nM ODC. A ll samples displayed the
predicted 2-fold increase at the higher
enzyme concentration, confirming
that inhibition of ODC was the rate-
limiting step. These compounds were
designated as true positives for the
ROC analysis.
ROC Analysis
ROC analysis provides an assess-
ment of the discriminatory power of
an assay that is nonparametric (ie,
does not assume Gaussian distribu-
tion) and that is independent of both
the number of true positives and the
threshold for classifying hits. The ROC AUC is the probabi lity
that the assay will ra nk a randomly chosen true positive a head
of a randomly chosen true negative. A random assay—one that
cannot discriminate true positives from tr ue negatives—has an
0
0.0000
0.0009
0.0019
Reaction Rate (mM NADH/min)
0.0029
0.0039
0.0049
A
C
0.0000
0.0009
0.0019
Reaction Rate (mM NADH/min)
0.0029
0.0039
0.0049
B
64 128 192
Well Number by Row
0
0
50
100
% Activity
150
2,000 4,000
Compounds in Order Screened
Scatterplot of High Throughput Data
6,000 8,000
256 320 384 0 64 128 192
Well Number by Column
256 320 384
F i g . 4 . ODC-PEPC-MDH-linked plate geographic effects and high-throughput data scatter plot.
Assays were performed in 384-well plates as described and rates were calculated from the fi nal
6 data points from a 20-min observation. ■ = High signal (DMSO), ▲ = mid signal (200 μM
difl uoromethylornithine (DFMO)), ▼ = low signal (1 mM DFMO). ( A ) Column effects resulting in
a slight increase in signal on the outer columns. ( B ) Data from 3A arranged by row, showing that
signal across rows is constant. The signal window for the assay is also apparent in these fi gures
and is ∼4.5. ( C ) Data from the small-scale high-throughput assay of the bioactive compound col-
lection at St. Jude Children’s Research Hospital. Green circles = positive control (1 mM DFMO),
red circles = negative control (DMSO), blue circles = hit compound, black circles = inactive
compound. Magenta line = 99th percentile cutoff, orange line = 95th percentile cutoff. Percent
activity was calculated by normalizing kinetic reaction rates (20-min data collection with the
fi nal 6 data points fi t as described in Materials and Methods) to positive and negative controls.
The 8,832 total data points were collected from ∼3,600 unique compounds. Absolute replicate
number for each compound varied from 1 to 7, depending on vendor library composition and
plating. The assay took 8.7 h to complete using our automation system.
adt.2009.0249.indd 182 4/19/2010 11:37:47 AM
DECARBOXYLASE ENZYME HTS
© MARY ANN LIEBERT, INC. • VOL . 8 NO. 2 • APRIL 2010 ASSAY and Drug Development Technologies 183
AUC = 0.5. The ROC AUC for the ODC assay is 0.90 ( Fig. 6 , 0.82–
0.97, 95% confidence inter val). Setting the cutoff at >30% in the
primary screen would retu rn >70% of all t rue positives, while
yielding ∼10% of all true negatives.
Hit Compounds
Due to t he nature of the compound librar y, this proof-of-concept
screen was not expected to yield many well-behaved inhibitors w ith
drug-like scaffolds. Indeed, a la rge number of quinone-containing
compound s were identi fied, a long with se veral compou nd series con-
taining Michael acceptors and other reactive groups. Though these
compounds are not likely to be good lead candidates, they do inhibit
the enzyme and were useful in the validation of the assay proto-
col. ODC has an active site cysteine residue and is known to require
large amounts of reducing agent to be active in vitro . Therefore, it
would be expected to be sensitive to potential alkylators as well as
oxidizing compounds. ODC has historically been a difficult target
to inhibit, and it is likely that screening a larger compound library
wil l be necessary to find well-behaved inhibitors.
D I S C U S S I O N
This study represents the first effort to develop an assay suit-
able for screening decarbox ylases in a high-throughput manner.
Alt hough it is a linked enzy me assay, the lac k of susceptibility to
known inhibitors of the li nking enzymes suggests that relatively
few false positive hits will be identified due to inhibition of the
other enzymes in the mix. Baicalein, a known inhibitor of PEPC
with a K
i of 0.79 μM , did not affect the assay appreciably until the
linking enzyme mix had been diluted 2-fold and even then only
inh ibited the reaction by 50%. Isoquinoline, a know n inhibitor
of MDH with a literature K
i of 200 μM, did not i nhibit the assay
at any tested condition. Furthermore, none of the hits identified
at 10 μM in the primary screen affected the dual enzyme link-
ing system at concentrations up to 100 μM. Although the exact
concentrations of the link ing enzymes in the mi xture are not dis-
closed by the manufacturer, these observations suggest that high
concentrations of both PEPC and M DH are present, and that the
assay is extremely resistant to inhibitors of the link ing enzymes
under optimized conditions (60% Infinity CO
2 , 600 μM Orn, 60
μM PLP, and 0.01% Triton-X).
However, the know n ODC inhibitor, DFMO, exhibited an IC
50
value of 206 ± 40 μM af ter 20 min of preincubation, which is
consistent with the literature K
iapp of 160 μM. ROC analysis, based
on a definition of true positive that requires ODC specificity,
quantitatively demonstrates the high discriminatory power of
the assay. Moreover, the high degree of agreement found between
1
–1
–0.8
–0.6
–0.4
–0.2
0
0.2
0.4
Z′
0.6
0.8
Start (min)
End (min)
111 555
Non-Robust Linear Curve Fitting 2 Point Delta Values Robust Linear Curve Fitting
10 10 15
5 10 15 20 10 15 20 15 20 20 5 10 15 20 10 15 20 15 20 20 5 10 15 20 10 15 20 15 20 20
1111 5551010151111 5 55101015
F i g . 5 . Effect of var ying curve fi tting methods on average Z ′ values for scaling screen. ▶ = Median, ◄ = mean, solid line = standard Z ′ cutoff
of 0.5. Data were collected as described in Materials and Methods under optimized assay conditions. Time points were taken ever y minute
for 20 min. Note that the fi rst 5 to 10 min are poorly behaved and that eliminating them dramatically increases the average Z ′ for the run.
This is primarily a result of a decrease in scatter rather than an increase in signal, since the average rates calculated were consistent for all
methods tested.
adt.2009.0249.indd 183 4/19/2010 11:37:49 AM
SMITHSON ET AL.
184 ASSAY and Drug Development Technologies APRIL 2010
replicate samples in the library f urt her underscores the robust-
ness of the assay system.
While the HTS assay requires a CO
2 -free atmosphere in order
to stabilize the reagents, other engineer ing controls may allevi-
ate this need. For example, sealed reagent bottles equipped with
nitrogen lines feeding bulk liquid dispensers would allow large
amounts of the Inf init y CO
2 reagent to be loaded into automated
systems without degrading. It shou ld be noted that performing
the assay under nor mal atmosphere decreases linear t ime from
∼60 to 40 min, meaning that timing in the screening run must
be monitore d close ly. T he effect of mov ing to norm al atmospheric
conditions for screening is primarily an increase in background
signal. This decreases the average Z ′ values observed from ∼0.7
to ∼0.5, a typical lower limit for high-th roughput assay systems.
Additionally, since t his assay is based on absorbance values, it
cannot be used in an endpoint fashion due to absorbance inter-
ference from the test compounds. To compensate for t his, it is
necessary to use a kinetic read. However, throughput was max-
imized by using a short kinetic read as it minimized movement
of the plate, which is often rate limiting in high-t hroughput
screens on integrated systems. The throughput for t his assay is
∼10 plates per hou r using ou r automated systems, when using a
5-min kinetic read.
The relatively high prima ry hit rate of 1.4% ref lects both the
liberal primary hit cutoff in and the nature of the bioactive collec-
tion. It is likely that the h it rate would decline signif icantly for a
more diverse compound libra ry, particular ly one curated to r emove
electrophiles and redox-active compounds. The ident ification of
active scaffold series known to be redox-active compounds
and/or nonspecif ic alkylators highlights the need for second-
ary assays that will address both of these liabilities in any
true HTS-effort aimed at discovering novel inhibitors of ODC.
However, since none of these compounds were found to effect
the linking enzymes, this sensitiv ity is due to the specific
decarboxylase screened, not the assay met hod itself.
In conclusion, this study describes a quick and accurate
method to screen ODC and other decarbox ylases in high
throughput. T he system is robust, generates hits with the
desired biochemical effects, and is superior to ex isting assays
that rely on either radiolabeled substrate or HPLC methods.
A C K N O W L E D G M E N T S
This work was supported by the American Lebanese
Syrian Associated Charities (ALSAC) and St. Jude Children’s
Research Hospital (SJCRH). A portion of this research was
performed while on appoint ment as a U.S. Department of
Homeland Secur ity (DHS) Fellow under the DHS Scholarship
and Fellowship Program, a program administered by the Oak
Ridge Institute for Science and Education (ORISE) for DHS
through an interagency agreement with the U.S. Department of
Energ y. ORISE is managed by Oak Ridge Associated Universities
under DOE contract number DE-AC05-00OR22750. A ll opinions
expressed in the article are the author’s and do not necessari ly
ref lect the policies and views of DHS, DOE, or ORISE. Additional
funding provided by National Institutes of Health grants (R01
AI34432) (to M.A .P.), and the Welch Foundation grant I-1257 (to
M.A.P.).
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Address correspondence to:
Dr. R. Kiplin Guy
Department of Chemical Biology and Therapeutics
St. Jude Children’s Research Hospital
262 Danny Thomas Place
Memphis, TN 38105
E-mail: kip.guy@stjude.org
adt.2009.0249.indd 185 4/19/2010 11:37:51 AM
