A universal, fully automated high throughput screening assay for pyrophosphate and phosphate release from enzymatic reactions

Article abstract of DOI:10.2174/138620710790218203

The malachite green assay is often used for measuring the presence of inorganic mono-phosphate concentrations. Some studies have adapted this assay for use in monitoring enzymatic reactions and have suggested its potential use in high throughput screening (HTS). With the increasing availability of laboratory automation, some studies are starting to explore the possibility of conducting limited, semi-automated versions of the assay. Here we report the optimization and complete adaptation of the malachite green assay to a fully automated, HTS platform that can be performed unattended with standard, commercially available, automated liquid-handling systems. The assay is universal for the majority of enzymes that release phosphate or pyrophosphate. Moreover, the assay is fully scalable from smaller drug screening efforts (～20,000 wells per day) to ultra-high throughput environments (～200,000 wells per day). The assay uses cost-effective, commercially available reagents, and can be used to perform automated IC₅₀ value and kinetic parameter determination. Finally, we demonstrate the utility of the assay via the initial, primary screening of 100,080 compounds against two target enzymes from Bacillus anthracis, O-succinylbenzoyl-CoA synthetase and nicotinate mononucleotide adenylyltransferase.

Full text of DOI:10.2174/138620710790218203

Combinatorial Chemistry & High Throughput Screening, 2010, 13, 27-38
27
A Universal, Fully Automated High Throughput Screening Assay for
Pyrophosphate and Phosphate Release from Enzymatic Reactions
Scott D. Pegan*, Yang Tian, Valerie Sershon and Andrew D. Mesecar*
Center for Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy, the
University of Illinois at Chicago, Chicago, IL 60607, USA
Abstract: The malachite green assay is often used for measuring the presence of inorganic mono-phosphate
concentrations. Some studies have adapted this assay for use in monitoring enzymatic reactions and have suggested its
potential use in high throughput screening (HTS). With the increasing availability of laboratory automation, some studies
are starting to explore the possibility of conducting limited, semi-automated versions of the assay. Here we report the
optimization and complete adaptation of the malachite green assay to a fully automated, HTS platform that can be
performed unattended with standard, commercially available, automated liquid-handling systems. The assay is universal
for the majority of enzymes that release phosphate or pyrophosphate. Moreover, the assay is fully scalable from smaller
drug screening efforts (~20,000 wells per day) to ultra-high throughput environments (~200,000 wells per day). The assay
uses cost-effective, commercially available reagents, and can be used to perform automated IC50 value and kinetic
parameter determination. Finally, we demonstrate the utility of the assay via the initial, primary screening of 100,080
compounds against two target enzymes from Bacillus anthracis, O-succinylbenzoyl-CoA synthetase and nicotinate
mononucleotide adenylyltransferase.
Keywords: Malachite green, high throughput screening, pyrophosphatase, assay techniques, phosphate.
INTRODUCTION
OSB-CoA synthetase first transfers the AMP protion of ATP
to O-succinylbenzoate (OSB) to form an OSB-AMP
intermediate and the first product of the reaction,
pyrophosphate (PPi; Fig. 1B). During the second step, CoA
reacts with OSB-AMP to form OSB-CoA and AMP [2]. The
kinetic mechanism for the OSB-synthetase reaction is an
ordered Bi Uni Uni Bi Iso ping-pong reaction with ATP
binding first followed by the binding of OSB. The release of
product occurs in the order of PPi followed by OSB-CoA and
then AMP [4].
Enzymes that release phosphate in the form of
pyrophosphate or monophosphate (ERPs) comprise one of
the most diverse sets of proteins with respect to the reactions
that they catalyze. ERP members exist as simple
pyrophosphatases, protein phosphatases, ATPases and
GTPases as well as adenyltransferase and synthetases. Not
surprisingly, many of these enzymes have been shown to
mediate essential pathways for sustaining bacterial life in
Bacillus subtillus making them attractive targets for drug
development [1]. To capitalize on the prevalence of these
enzymes in several critical cellular pathways, a HTS assay
that requires minimal adaptation between ERPs, yet allows
for large-scale HTS with standard laboratory resources, is
required. To probe the potential for developing such a
universal assay for screening ERPs against large compound
libraries, we employed two diverse pharmaceutical targets
from Bacillus anthracis, O-succinylbenzoyl-CoA synthetase
(OSB-CoA synthetase; EC 6.2.1.26) and nicotinate
mononucleotide adenyltransferase (NMAT; EC 2.7.7.1).
OSB-CoA synthetase and NMAT each catalyze critical
reactions essential to the growth and survival of B. anthracis
and other bacteria. Although these two reactions generate
entirely different products, both enzymes can be regarded as
nucleotidases (Fig. 1A).
Whereas OSB-CoA synthetase is critical for the electron
transport chain, NMAT serves as a key enzyme in the
bacterial synthesis of NAD(P) [1, 5]. NMAT’s role in the
pathway is the generation of nicotinate adenine dinucleotide
(NaAD) and PPi from nicotinate mono-nucleotide (NaMN)
and ATP (Fig. 1C). NMAT utilizes a random bi bi kinetic
mechanism with negative cooperatively associated with the
binding of substrates [6]. Recent reports have identified
NMAT and other enzymes in the pathway as undergoing
significant up-regulation during the critical growth phase of
Bacillus anthracis in macrophages, suggesting NMAT as a
potential drug target [5].
Currently, there are several assays for both enzymes that
could be potentially adapted to HTS (Table 1). Some of
these assays are enzyme specific, while others exploit the
production of PPi. OSB-CoA synthetase specific assays
include the detection of the product AMP or the detection of
DHNA-CoA formation that is generated via coupling of the
OSB-CoA reaction to the downstream reaction of DHNA-
CoA synthetase [4, 7, 8]. Options for NMAT specific assays
are more limited since AMP becomes fused to NaMN
through the reaction. However, a low-throughput method of
detecting NaAD formation via HPLC has been used to
measure the activity of NMAT [6, 9].
OSB-CoA synthetase catalyzes the fourth reaction in the
B. anthracis menaquinone biosynthetic pathway, which is
responsible for generating the key electron transport
molecule menaquinone (MK-8) [2, 3]. To form OSB-CoA,
*Address correspondence to these authors at the Center for Pharmaceutical
Biotechnology, University of Illinois at Chicago, 900 South Ashland
Avenue (M/C 870), Chicago, IL 60607-7173, USA; Tel. 312-996-1877;
Fax: 312-413-9303; E-mails: mesecar@uic.edu or pegan@uic.edu
1386-2073/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

28 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1
Pegan et al.
Fig. (1). ERPs that catalyze adenyltransferase reactions. (A) General adenyltransferase reaction. (B) OSB-CoA synthetase catalyzed reaction
(C) NMAT catalyzed reaction. Preferred inorganic phosphatae (Pi)-based detection assays. (D) Adenyltransferase-Luciferase based assay.
(E) 2-amino-6-mercapto-7-methyl purine (MESG)-based assay. (F) Malachite Green assay.
There are currently four predominant assays for
measuring PPi production that are used routinely (Fig. 1D-
F). The first is a luminescence assay that uses ATP sulfate
adenyltransferase to produce ATP from adenine 5'-
phosphosulfate and PPi. The ATP produced is directly
coupled to the luciferase reaction where it reacts with
luciferin to produce light (Fig. 1D). Although this assay has
been used to great success in DNA sequencing and utilized
in a few other applications for pyrophosphate detection, the
assay requires that ATP be produced continually and in low
concentrations for detection. In contrast, ERPs such as OSB-
synthetase and NMAT require that high concentrations of
ATP be present in their assays which would thereby interfere
with detection [10, 11].
A fluorescence-based method for detection of PPi also
recently became available. This assay relies on
monophosphate binding to a modified, coumarin labeled,
phosphate-binding protein purified from Escherichia coli
(PBP) [12]. With a Kd of 0.1 ꢀM for Pi, the PBP assay is a
sensitive assay; however, the required N-[2-(1-

Pyrophosphate and Phosphate Release from Enzymatic Reactions
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1 29
Table 1. Current Assays Available for NMAT and OSB-CoA Synthetase
Assay
Type
Enzyme
Specific
Fully Commercially
Available
Reagent Cost
(Each Well)
Reporting Abs
Range (nm)
Sensitivity
Complexity
Assay Type
Malachite
Green
low, one coupled
enzyme
No
No
Yes
Yes
No
1-30 μM Pi
>1μM Pi
<5 cents
>10 cents
<5 cents
623
380
392
End-point
Continuous
Continuous
medium, two
coupled enzyme
MESG
MenB
coupled
low, one coupled
enzyme
Yes
>10 μM OSB-CoA
MDCC-
PBP
medium, two
coupled enzyme
ex 430 /
em 450
No
No
>0.1 μM Pi
>15 cents
Continuous
NMAT
HPLC
Assay
low, no couple
enzyme required
Yes
Yes
Yes
>2 μM NaaD
> 2 μM Pi
>10 cents
>16 cents
NA
340
End-point
AMP
detection
high, three
coupled enzyme
No*
Continuous
*Assay can only be performed on reactions that produce AMP.
maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide
(MDCC)-labeled PBP is not commercially available and
approximately 50% less expensive than the alternative
assays (Table 1). Cost becomes more of a lead factor as HTS
campaigns are increasingly being performed against larger
libraries with replicates or multiple concentrations for
quantitative HTS (qHTS) [15].
requires synthesis via
a complex, multi-step process
involving protein purification of a mutated version of PBP,
modification with MDCC, and removal of indigenous
monophosphate using purine nucleoside phosphorylase and
7-methylguanosine (MEG) [12]. As a result, this assay is
unlikely to be adapted to HTS due to its complexity and
associated expense (Table 1).
The longer wavelength range for measuring activity also
favors the MG assay. A recent trend is to perform qHTS to
detect false positives and negatives by analysis of multiple
inhibitor concentrations on the assay [15]. However, this
increases enormously the number of assays needed to be
performed. An alternative method focuses on a reporting
wavelength that is minimally impacted by library
interference caused by absorbance or autofluorescence of a
compound. Data collected by NIH Chemical Genomics
Center (NCGC), a member of the NIH Molecular Libraries
Screening Centers Network (MLSCN), illustrated that from a
typical compound library of 58,450 compounds, 6,462
compounds exhibited emission and excitation wavelengths at
340 nm and a 450 nm (PubChem AID: 590). No compounds
were detected when the same library was screened using
excitation and emission wavelengths of 570 nm and 647 nm
(PubChem AID: 592). Since absorbance of light is a
requirement for excitation, MG, with its wavelength range
between 590 and 650 nm, would be expected to have
minimal interference from current compound libraries used
for HTS [16].
The remaining two assays for detection of PPi are
colorimetric. The amino-6-mercapto-7-methylpurine ribonucleo-
side (MESG) assay relies on the detection of a 2-amino-6-
mercapto-7-methylpurine product formed from the reaction
of MESG and free mono-inorganic phosphate (Pi) by purine-
nucleoside phosphorylase (PNP) [13]. In comparison to the
MESG assay, the malachite green assay (MG) capitalizes on
the strong absorption shifts created by Pi entering into a
malachite green, phosphomolydbate complex at low pH [14].
Although all the aforementioned assay methods are valid
and have the sensitivity required for inhibitor identification,
we weighed the cost, commercial availability, complexity,
and wavelength range for our final selection criteria. In
addition, we sought an assay that could be used universally
for any enzyme that releases PPi or Pi. For each of these
criteria, the malachite green assay for Pi detection showed
the most potential. Unlike most of the other assays, MG can
be adapted for ERPs that release pyrophosphatase by
requiring only one coupling enzyme to be added, inorganic
pyrophosphatase (IPP), to cleave PPi to monophosphate Pi
(Fig. 1E, F). This minimizes the number of enzymes that
would have to be counter-screened to differentiate any false
positive hits from potential inhibitors identified during
screening. Furthermore, the assay requires no rare or
commercially unavailable substrates and no proprietary
enzymes. Cost analysis, based on the most expensive
reagents required for each assay, puts the MG assay at
A recent study suggests that the MG assay could have the
potential to be adapted to a 384-well assay format [17]. In
another study, it was shown that a standalone, multi-
dispensing system could be used to semi-automate a
phosphatase assay for HTS and demonstrated feasibility
against a small library of 3,280 compounds [18]. Although
this partially-automated method was successful, it still
required substantial human intervention to track reaction
times and to transfer plates between devices. As with cost,
these human tasks can become daunting as substantially

30 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1
Pegan et al.
larger libraries and replication become prevalent in modern
HTS and qHTS campaigns. Finally, another major concern
of the MG assay has been raised regarding the use of
concentrated sulfuric acid during the development step since
hydrolysis of ATP could occur over time thereby degrading
the quality of the results for HTS studies [19].
a Molecular Devices SpectraMax 384 variable wavelength
spectrometer using transparent Falcon 96-well assay plates.
384 Format Assay Miniaturization and Optimization for
NMAT and OSB-CoA Synthetase HTS
Experiments to optimize release of PPi for OSB-CoA
synthetase and NMAT reactions were conducted in 384-well
plates by varying enzyme concentrations and reaction
incubation time. For OSB-CoA synthetase reactions, a
mixture of 8 μM OSB, 16 μM ATP, 128 μM CoA, and 1 mM
MgCl2 was used to assay activity over a 15 min interval.
Similar NMAT reactions were conducted using a mixture of
15 ꢀM NaMN, 124.9 ꢀM ATP, and1 mM MgCl2 over a 14.5
min interval. Both enzyme reactions were quenched by the
addition of the malachite green development solution. The
effect of 2.5% DMSO and 1.5 μM BSA on enzyme activity
were also determined with assays measured using the
Molecular Devices SpectraMax 384 variable wavelength
To address the issues and concerns described above, we
have developed a fully automated version of the assay MG
assay in a 384-well format capable of screening over 10,000
compounds, in duplicate in a 10-h work day using a
relatively simple liquid-handling robot. Moreover, we have
applied this assay to the pyrophophatases OSB-CoA
synthetase and NMAT, screening 100,080 compounds
against each target in an automated HTS format. Finally, we
explore the practical nature of implementing the MG assay
on small-scale HTS platforms as well as data quality and
throughput.
ꢀ
MATERIALS AND METHODS
Sources of Materials
spectrometer. All reactions were carried out at 23 C and
included 0.15 U/mL of the coupling enzyme, IPP.
The product of the reaction, NaAD, was used as a control
inhibitor at a single concentration of 28.6 ꢀM for the purpose
of assay validation. A concentration range of 15 to 1000 μM
of NaAD was used to determine the IC50 values for both the
MG and MESG assays. The conditions used for the IC50
value determination of NaAD for the MG assay were
otherwise unchanged from HTS assay conditions. For the
MESG assay, the conditions were the same as MG
conditions except that 0.5 U of PNP and 300 ꢀM of MESG
were added. The activity of NMAT in the MESG assay was
monitored continuously over time at an absorbance
wavelength of 360 nm as previously described [4, 6]. All
IC50 curves were fit to the equation %I = 100% / (1 +
(Ki/[NaAD])) using non-linear regression and the Enzyme
Kinetics Module in the program SigmaPlot (Systat)... The
percent inhibition values (%I) were calculated using
[(sample)-(negative control)]/[([positive control]-[negative
control] X 100%.
Dimethyl sulfoxide (DMSO), bovine serum albumen
(BSA), nicotinate adenine mononucleotide (NaMN),
inorganic pyrophosphatase (IPP) from S. cerevisiae,
malachite green, nicotinate adenine dinucleotide (NaAD),
ATP, sodium phosphate, ammonium molybdate, magnesium,
Tween 20, sulfuric acid and CoA were all purchased from
Sigma-Aldrich. O-Succinylbenzoate (OSB) was synthesized
according to previously described methods [20].
Transparent, 384-well plates (Matrix ScreenMates) were
used for all HTS and assay optimization. All compound
libraries were sourced from Chembridge. His-tagged NMAT
and OSB-CoA synthetase proteins were prepared using
established protocols [4, 6].
Malachite Green Dye Preparation
The malachite green dye mixture was prepared in 2 L
batches in accordance with previously described methods
[21]. In short, 300 mL of 36 N sulfuric acid stock was mixed
with 1.5 L of water and then cooled down to room
temperature. Malachite green powder (2.2 g) was then added
and mixed by stirring with a magnetic stir bar and stir plate.
Prior to use, 10 mL of the malachite green dye was mixed
with 2.5 mL of 7.5% ammonium molybdate and 0.2 mL 11%
(v/v) Tween 20. The final mixture, defined as the malachite
green development solution was added in a 1:4 ratio to
reactions and incubated for 15 min prior to being read at
either 623 nm or 612 nm.
Compound Library Source and Composition
Compounds were sourced from a two libraries acquired
from ChemBridge. Library 1 (Diver Set/CNS Set) contained
50,080 compounds, and Library 2 (Novacore) contained
50,000 compounds. Compounds in both libraries obey
Lipinski rules but differ in their diversity [22]. Compounds
in Library 1 were selected to maximize diversity, whereas
compounds in Library 2 were centered on derivatives of 47
different precursor templates. The 384-well daughter plates
used in the study were comprised of 320 compounds from
four 96 well mother plates that contained 80 compounds
each at 10 mM in DMSO, resulting in 157 daughter plates.
The final daughter plate layout contained 16 positive control
wells and 16 negative control wells
Absorbance Spectra and Pi Standard Curves
Absorbance spectra and Pi standard curves were
generated by addition of various amounts of Pi to 200 μL of
either NMAT HTS reaction conditions (30 mM Tris pH 7.5
250 mM NaCl, 1 mM MgCl2, 15 ꢀM NaMN, and 124.9 ꢀM
ATP) or OSB-CoA synthetase HTS reaction conditions (50
mM Tris HCl 7.5, 1 mM MgCl2, 8 μM OSB, 16 μM ATP,
128 μM CoA and 100 mM NaCl). Absorbance spectra were
measured using a Varian Cary 50 Bio variable-wavelength
spectrometer using 1 mL cuvettes with 1.0 cm pathlengths.
In contrast, standard Pi measurements were performed using
Automated Screening Procedure
Fully automated, high throughput screens of 100K
compounds against OSB-CoA synthetase and NMAT were
performed in duplicate using a Tecan Freedom Evo 200
liquid handling station. The robotics platform is equipped
with a mounted 3X3 Temo 96 tip pipetting head, fixed-tip,

Pyrophosphate and Phosphate Release from Enzymatic Reactions
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1 31
Fig. (2). Schematics for the Robotics Deck Layout, Assay Flow and Plate-Set Scheduling. (A) TECAN Freedom Evo deck layout of reagents
and robotic components for OSB-CoA synthetase and NMAT. Boxes represent locations of substrates (yellow), library daughter plates
(olive), NMAT/OSB-CoA synthetase (brown), DMSO (green), ethanol (teal), blotting paper (white), 384-pin tool (lavender), tips (dark
orange), negative control buffer (magenta), and assay plates (blue). Deck locations that are visited multiple types of reagent are colored
proportionately according those reagents where as locations visited by different plates of the same type are annotated with (X). (B) Assay
flow chart. Plate sets are shown as their respective replicates (A, B) with wells colored to corresponded to well location and reagent added.
(C) Boxes are shaded according to platform components being utilized. Orange boxes indicate a Temo 3X3 in a 96 head-dispense mode and
lavender boxes represent the Temo 3X3 when fitted with a 384-pin tool. Tan boxes represents a TeShake, blue boxes an Abgene 300 plate
sealer, and magenta boxes the LiHa arm. The red line and green shaded box represents a GeniosPro plate reader measuring absorbance
values at 612 nm during the NMAT enzyme HTS runs. All boxes are scaled according to time, with the largest box equating to 10.5 min.
Numbers correspond to; (1) the addition of a negative control, (2) substrate addition, (3) compound addition, (4) library daughter plate
sealing, (5) enzyme addition, (6) 2 min shake, (7) 3 min reaction incubation, (8) malachite green development solution addition, and (9) a
10.5 min shake for development. In-line assay detection (10) and red lines denote items that pertain only to the NMAT HTS screen.
liquid-handling arm (Liha Arm), plate transfer robot,
automated plate sealer (Abgene ALPS-300), and a Tecan
orbital shaker (Te-shake) (Fig. 2A). All compounds were
assayed in duplicate by simultaneously processing two
separate assay plates (Fig. 2B). To achieve simultaneous
operation of all of the main robotic elements, the Temo, Te-
shake, and Liha operations were synchronized in a manner to
allow for up to three plate sets to be processed in different
phases of the assay at the same time. The precise timing
scheme for each step illustrated in Fig (2B) is presented in
Fig. (2C).

32 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1
Pegan et al.
Overall, OSB-CoA synthetase and NMAT HTS assays
followed the same nine step process which required only
three major liquid addition steps to each well (Fig. 2B). Step
1 involved adding 17.5 ꢀL of each enzyme’s respective
enzymatic buffer solution, containing no enzyme, to the 16
negative control wells per plate. Step 2, a 52.5 ꢀL mixture of
desired substrates was then added. Subsequently, during step
3, 0.2 ꢀL of 10 mM compound stocks in DMSO were
dispensed via two passes of a V&P Scientific 100 nL 384-
pin tool. Daughter plates were sealed immediately after use
by an ALP-300 (Abgene) plate sealer during step 4.
RESULTS AND DISCUSSION
Optimization of Malachite Green Reagent for OSB-CoA
Synthetase and NMAT Reaction Conditions
Traditional advantages for using a colorimetric assay
such as MG are its ease of measurement, simplicity of
instrumentation, and potentially high signal-to-noise ratio.
Added to these, the MG assay in particular detects within a
wavelength range that minimizes false positives or negatives.
The basis for the colorimetric detection of inorganic
phosphate via MG is tied to the absorbance of the dye-
phosphomolybdate complex formed under conditions of low
pH during the development stage [16]. The low pH condition
also serves as a suitable method for quenching most
enzymatic reactions, as most enzymes, including NMAT and
OSB-CoA synthetase, are inactive in the 0-3 pH range.
However, these conditions also have the tendency to
precipitate reaction components, including the enzyme itself
[23]. As a result, various detergents and acids have been
employed to reduce development time while keeping
reaction components soluble [16]. For our studies, we
utilized sulfuric acid and Tween 20 as our acid and
detergent, a combination based on a previously described
immunoassay [21].
To initiate enzymatic reactions in step 5, 17.5 ꢀL of
solution containing either 100 nM OSB-CoA synthetase or
50 nM NMAT was added for a final concentration of 25 nM
and 12.5 nM. Plates were shaken at 1600 rpm for 2 min (step
ꢀ
6) and allowed to incubate at 23 C for an additional 3 min
(step 7) prior to being quenched by 17.5 μL malachite green
development solution (step 8). In step 9, each plate set was
then shaken at 1600 rpm for 10.5 min to allow for
development.
For detection of enzyme activity in step 10, we decided
to evaluate the logistical and timing benefits of using a deck-
accessible plate reader versus one located in separate
location of the lab, i.e. not attached to the robot deck, since it
is anticipated that different labs will have one or the other
setup. We therefore conducted the NMAT screen using the
deck-accessible plate reader and the OSB-CoA synthetase
assays with the offline detector. The absorbance values for
the NMAT plate sets were read immediately after
In order to determine the optimal wavelength range of
signal detection for assays containing components for OSB-
CoA synthetase and NMAT reactions, the malachite green
reagent was added to solutions containing the respective
enzyme reagents, include ATP, at previously determined Km
values, with and without 8 μM inorganic phosphate [4, 6].
An absorbance spectrum of the phosphate containing NMAT
and OSB-CoA synthetase samples, after baseline correction
against water, produced three peaks at 444 nm, 590 nm, and
630 nm, within the 370 to 800 nm spectral region (Fig. 3A).
For phosphate-free samples, a 444 nm peak was present with
almost the same intensity as the phosphate containing
samples. Although a peak at 620 nm was also observed, its
intensity was minimal in comparison to the phosphate
containing spectra in the same range. As a result, we
determined the optimum detection range to be between 590
and 630 nm for MG. This range minimized interference from
substrates, compound libraries, and protein.
development by
a
robot accessible GENios Pro
monochromatic filter spectrometer at 612 nm (step 10). In
contrast, plates for the OSB-CoA synthetase assay were
stored on the deck for up to the length of 6 plate sets (2.25 h)
prior to being transported manually to a Molecular Devices
SpectraMax 384 variable wavelength spectrometer and read
at 623 nm. Final concentrations for the OSB-CoA synthetase
HTS were 28.5 μM compound, 7.75 μM OSB, 15.5 μM
ATP, 124 μM CoA, 50 mM Tris HCl 7.5, 1 mM MgCl2, 100
mM NaCl, 1.5 μM BSA, and 0.15 U/mL IPP. For NMAT
HTS, final concentrations were 28.5 μM compound, 15ꢀM
NaMN, 124.9 ꢀM ATP, and 1 mM MgCl2.
Data from all screens were processed using a custom
program script based on Perl language that was developed by
the authors. This program provided matching of sample
wells to compound ID numbers and raw absorption data as
well as calculation of percent inhibition of each sample well
and its replicate. Mean inhibition and duplicate plate set Z'-
factor (ZDPS) values were also provided by the program using
Eq. (1)-(2).
Miniaturization of the Malachite Green Assay for 384-
Well Formats
For the purpose of adapting the MG assay for use in 384-
well format, a standard curve at 623 nm was generated from
multiple concentrations of Pi in wells containing a total
reagent volume of 87.5 uL (Fig. 3B). Robust linear
relationships were observed between 0-20 μM Pi for buffers
containing reagents for OSB-CoA synthetase and NMAT
reactions. Based on the linear relationship observed, we
focused on conditions that produced 7.5 μM of
pyrophosphate and therefore 15 μM Pi after adding excess
inorganic pyrophosphatase. In order to achieve 15 μM Pi
generation for OSB-CoA synthetase and NMAT assays, the
primary substrates were fixed at 7.75 μM OSB and 15 μM
NaMN, respectively. All other reagents involved in the
reaction had their concentrations fixed at their respective Km
values to provide for an assay that was sensitive to all forms
of inhibition, also known as a balanced assay (Table 2A, B)
Abssample-Absμ ꢀ
(
)
% Inhibition =
Eq. (1)
Absμ + -Absμ ꢀ
(
)
3ꢁ c+ +3ꢁ cꢀ
(
)
ZDPS = 1ꢀ
Eq. (2)
| μ
c
+
+μ
c
ꢀ
|
Both the ZDPS-factor and mean inhibition calculations
used standard deviations (ꢁ) and means (μ) that were based
on the 32 control wells, either positive or negative, sourced
from both replicate plates.

Pyrophosphate and Phosphate Release from Enzymatic Reactions
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1 33
Fig. (3). Malachite green assay optimization for OSB-CoA synthetase and NMAT. (A) Spectral scans of OSB-CoA synthetase and NMAT
under the conditions of the assay with and without 8 ꢀM Pi. NMAT conditions are indicated with 8 ꢀM Pi (gray; dashed) and without 8 ꢀM
Pi (gray; solid). In contrast, OSB-CoA synthetase is depicted as 8 ꢀM Pi (black; solid) and without 8 ꢀM Pi (black; dashed). (B) Standard Pi
curve for malachite green assay carried out in NMAT reaction conditions (red) and OSB-CoA synthetase reaction conditions (blue). NMAT
and OSB-CoA synthetase had slopes of 0.0522 ± 0.0004 (dark red) and 0.0502 ± 0.0018 (blue) respectively. (C) Reaction progress curves at
varying NMAT concentrations; 12.5 nM (ꢀ), 25 nM (ꢁ), and 50 nM (ꢁ) are shown in gray, or at varying OSB-CoA synthetase
concentrations; 25 nM (ꢀ), 50 nM (ꢁ), and 100 nM (ꢁ) are shown in black. (D) The IC50 values of NaAD towards NMAT was determined
using the MG (black) and the MESG (grey) assays. Data were fit as described in Materials and Methods, and the resulting IC50 values were
65.5 ± 5.3 μM for the MG assay and 36.3 ± 4.9 μM for the MESG assay.
[4, 6]. OSB-CoA synthetase and NMAT concentrations were
chosen by varying enzyme concentration and monitoring Pi
generation over time. OSB-CoA synthetase and NMAT
concentrations of 100 nM and 12.5 nM were selected based
on the amount of Pi generation over a 5-min time interval
(Fig. 3C). These concentrations proved to be optimal for the
automation strategy for reasons described below.
To validate MG’s ability to detect potential inhibitors, we
chose to capitalize on the inhibition of NMAT by its product
NaAD [9]. Validation of an assay normally requires that a
proven inhibitor be tested to ensure that a decrease in signal
is tied only to the enzyme of interest. In cases where no such
inhibitors are known, the product of a reaction can in some
incidences serve as an inhibitor. To demonstrate this
possibility, we utilized our understanding of the NMAT
product inhibition reaction kinetics and used NaAD which is
a competitive product inhibitor [6]. We determined the IC50
value for NaAD by both the MG endpoint assay and the HTS
assay under conditions used in the full HTS screens. The
response of NMAT to increasing concentrations of NaAD
resulted in an IC50 value of 65.5 ± 5.3 μM as determined by
the MG assay which is comparable to the IC50 of 36.3 ± 4.9
μM determined from continuous MESG assay under the
same HTS conditions (Fig. 3D). Inclusion of 28.6 μM NaAD
in sample wells reduced the activity of NMAT by
approximately 66% on average. This value falls within the
range that would be expected if the concentration of the
inhibitor is near its IC50 value.
To prevent promiscuous inhibitors from producing false
positive hits in the assays and to stabilize OSB-CoA
synthetase and NMAT for up to the 3 hrs needed for an
automated HTS run, bovine serum albumin (BSA) was
added to the assay mixture to a final concentration of 1.5 uM
[24]. Since DMSO was expected to be present at
concentrations of up to 2.5% as a result of all library
compounds being dissolved in 100% DMSO, the effects of
both BSA and DMSO on the activities of OSB-CoA
synthetase, NMAT, and the coupling enzyme IPP, were
tested. No reduction in catalytic activity was observed by the
addition of either BSA or DMSO (Table 2C) [25].

34 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1
Pegan et al.
Table 2. OSB-CoA Synthetase and NMAT Km Substrate
Correlations with DMSO and BSA Control Data
Coulter, and Panasonic allows for screening 24,576
compounds in duplicate, 58,982 per 24 h period (Fig. 2A). In
addition to allowing for relatively high throughput capacity,
the automation scheme allows for the entire assay to be
performed on one robot deck without any manual
manipulations or off deck storage of plates, which has been
typical of other similar HTS assays [18, 19]. As a result, a
substantial increase in reproducibility and time savings were
achieved. It would also be possible to run the assays
continuously over a 24 h time period assuming that the
particular enzyme under study by the investigators is stable
over extended time periods. In our case, we found that
NMAT and OSB-CoA synthetase were most stable for up to
10 h on the robot deck, and we found it valuable to perform
real-time evaluation of Z-factors to ensure the best data
quality for our enzymes.
A: OSB-CoA Synthetase Substrate Apparent Km Valuesa
[ATP]
OSB Km (ꢀM)
CoA Km (ꢀM)
[OSB]
4 μM
7.0 ± 0.6
68.7 ± 6.5
2 μM
8 μM
12.8 ± 0.97
132 ± 13
4 μM
16 μM
8.8 ± 0.7
149 ± 12
8 μM
32 μM
8.0 ± 0.8
189 ± 19
16 μM
ATP Km (ꢀM)
CoA Km (ꢀM)
38.6 ± 6.3
94.0 ± 18
35.9 ± 4.7
126 ± 18
19.3 ± 3.8
231 ± 18
28.1 ± 3.2
240 ± 27
[CoA]
32 μM
64 μM
128 μM
256 μM
OSB Km (ꢀM) 0.74 ± 0.19 0.62 ± 0.13
ATP Km (ꢀM) 1.4 ± 0.4 2.4 ± 0.4
1.4 ± 0.2
4.4 ± 0.4
1.5 ± 0.2
3.2 ± 0.5
The implementation of the automation scheme also
demanded an additional consideration in terms of reaction
times and library compound concentrations. To fully utilize
the time-saving overlapping method and eliminate dwell
time, the reactions were limited to 5 min. This time interval
required that the enzyme concentrations be 100 nM for OSB-
CoA synthetase and 12.5 nM for NMAT in order to achieve
a maximum signal-to-noise ratio (Fig. 3C). A concentration
of 28.5 μM was chosen for the library compounds since this
concentration could be readily achieved by two transfers
from the 10 mM stock concentrations of the compounds
using a 100 nL capacity pin tool within the optimal time
frame. This delivery volume also limited the final DMSO
concentration to 0.28% which is well below the 2.5% DMSO
which is tolerable for both enzymes (Table 2C).
B: NMAT Substrate Apparent Km Valuesa
[ATP]
10 ꢀM
11 ± 1
25 ꢀM
28 ± 4
300 ꢀM
15 ± 2
1 mM
21 ± 1
NaMN Km (ꢀM)
[NaMN]
10 ꢀM
119 ± 20
25 ꢀM
93 ± 15
300 ꢀM
107 ± 13
1 mM
ATP Km (ꢀM)
142 ± 28
C: DMSO and BSA Effects on Percent Activityb
Enzyme
2.5% DMSO BSA (1.5 uM)
Both
NMAT
OSB-CoA synthetase
IPP
93 ± 3
98 ± 3
106 ± 2
98 ± 3
99 ± 2
97 ± 6
101 ± 3
102 ± 1
103 ± 1
aAll Km determinations were derived from previous kinetic studies of B. anthracis'
OSB synthetase and NMAT [4, 6]. bPercent Activity calculated using the formula,
[A623 with additives]/ [A623 without additives] X 100.
Beyond the expediency of using 28.5 μM as the final
compound concentration in the assay, this concentration was
deemed reasonable for the detection of inhibitors with Ki
values of below 50 μM. Since the concentrations of the three
substrates (OSB, ATP and CoA) used in the assay were
within 0.5 to 4 times their Km values, the detection of
inhibitors that could act via any mode of inhibition, i.e.
noncompetitive, competitive or uncompetitive or mixed, is
expected [26]. Since it is unclear at this time as to which
mode of inhibition will eventually lead to the best antibiotic
against OSB-CoA synthetase, probing for an inhibitor with
any mode of action was deemed the best strategy in the
initial screening campaign.
Implementation of an Automated HTS Malachite Green
Assay
The implementation of automated high throughput
screens on large compound libraries requires that at least two
criteria be met, high throughput and reproducibility. To
achieve both of these criteria, we chose to develop a robotic
scheme that would allow all compounds to be tested in
duplicate (Fig. 2). Our scheme provides for the simultaneous
operations of the liquid handling, plate, and 96 multi-
dispensing robot arms of a TECAN Freedom Evo 200. As a
result, the processing of multiple 384-well assay plates can
be performed simultaneously. The final scheme using a
robotics platform that can accommodate reagents and
consumables for eight plates that can be screened in
duplicate during one 2.5 h time period allowed for a
throughput of over 10,000 compounds, 24,576 wells total, in
a typical ten hour work day.
Large Scale Screening and Data Processing of a 100K
Compound Library
To explore the practicality of using MG for large
libraries, OSB-CoA synthetase and NMAT were each
screened separately against two libraries comprising
approximately 100K commercially available compounds.
Positive controls for each of the assays ranged from 0.65 to
1.0 absorbance units at 623 nm for OSB-CoA synthetase,
and at 612 nm for NMAT. Negative controls for OSB-CoA
synthetase ranged from 0.21 to 0.40 absorbance units, and
negative controls for NMAT ranged from 0.17 to 0.19
absorbance units (Fig. 4A, B).
The throughput was limited in our case only by the lack
of on deck storage of reagents and plates. Depending on the
assay platform in different labs, we anticipate that the
strategy outlined in Fig. (2) would be directly scalable
pending the liquid handler in use. Although a basic TECAN
Freedom Evo 200 was used here, addition of a deck
accessible carousel for plate storage and a dedicated reagent
dispenser with a large reservoir for this deck, or other similar
liquid handlers to include those from CyBio, Beckman
Z'-factors for the assays were calculated using a modified
version of the traditional Z'-factor calculation by aggregating
all 32 positive controls and 32 negative controls of both

Pyrophosphate and Phosphate Release from Enzymatic Reactions
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1 35
Fig. (4). High throughput screening (HTS) quality factors for OSB-CoA synthetase and NMAT for library 1 and library 2. (A) OSB-CoA
synthetase positive mean (ꢀ) and negative mean (ꢁ) calculated using all the control samples from both plate A and plate B with error bars for
standard deviation. Black lines represent the means of positive and negative controls calculated across each library. (B) NMAT positive
mean (ꢀ) and negative mean (ꢀ) calculated using all the control samples from both plate A and plate B with error bars for standard
deviation. Black lines represent the means of positive and negative controls calculated across each library. (C) ZDPS-factors calculated from
control samples from plate A and plate B of an individual assay plate set. OSB-CoA synthetase ZDPS-factors in (ꢁ) and NMAT (ꢁ). Lines
represent the mean ZDPS calculated from each library for the NMAT MG (black) and OSB-CoA synthetase MG (grey) assays.
plates instead of treating each plate independently. This
allowed for generation of a “duplicate plate set Z'-factor”
(ZDPS). The ZDPS-factor, in comparison to the single plate Z'-
factor calculations, does not treat each plate independently
but as one plate. As a result, the quality between the
replicates is captured in the ZDPS-factor value. All plate sets
for NMAT and OSB-synthetase possessed a ZDPS-factor
value greater then 0.5, the minimum benchmark for the
assays to be considered "excellent assay" [27]. The average
ZDPS-factor of the entire screen of 100,080 compounds for
the NMAT MG assay was 0.86±0.02 and for the OSB-
synthetase MG assay was 0.71±0.09 (Fig 4c).
The coefficient of variance’s (Cv) taken from a typical
384-well plate were consistent or better than previous
manual or partially automated HTS applications of MG
(Table 3) [18]. However, the signal-to-noise ratios between
the NMAT and OSB-CoA synthetase assays differed
significantly. Whereas the NMAT assay had a signal-to-
noise ratio of 23.2, the value for OSB-CoA synthetase was
only 5.3 when controls were compared from a typical plate.
This difference is believed to result partially from OSB-CoA
synthetase having a higher absorbance value for the negative
control resulting from background absorbance of the reaction
components. The OSB-CoA synthetase assay also differed in
terms of detection procedures from those of the NMAT

36 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1
Pegan et al.
assay which also may have contributed to negative and
positive fluctuations in control samples.
intrinsically lower negative control values, the NMAT assay
generated more consistent control and ZDPS-factor values.
Table 3. Typical* Plate Controls
Finally, the results from both assays have so far allowed
for numerous 'hit' compounds to be identified with IC50
values ranging from approximately 1 to 100 ꢀM depending
on the percent inhibition cut-off values selected (data not
shown). For example, using an automated IC50 determination
and the MG assay, a typical hit in the OSB-CoA synthetase
screen with 33.1 % mean inhibition between the duplicates,
resulted in an IC50 of 36.7 ± 4.6 (Fig. 5C). For NMAT, a
primary hit with a 56.1 % mean inhibition between the
duplicates resulted in an IC50 of 20.0 ± 3.3 (Fig. 5D).
Although the primary mode of inhibition (competitive, non-
competitive, un-competitive or mixed) for these compounds
is currently unknown, these preliminary data indicate that a
good range of inhibitory potencies can be detected.
OSB-CoA
Synthetase
Plate A
OSB-CoA
Synthetase
Plate B
NMAT NMAT
Plate A Plate B
Mean Signal**
Signal CV
0.82
3%
0.82
3%
0.81
5%
0.82
3%
Mean
Background**
0.17
0.17
0.35
0.36
Background CV
Signal/Noise
1%
1%
3%
5.3
2%
5.3
23.2
23.2
*Plate Set 72 from Library 1.
** Absorbance at 623 nm for OSB-CoA synthetase and 612 nm for NMAT.
CONCLUSIONS
Although the OSB-CoA synthetase assay was automated
up to step 9, we evaluated the logistical and timing issues
surrounding off-line detection, a scenario that exists in many
labs. The absorbance detection relied on manually
transporting the plates to the microplate plate reader and then
manually feeding the plates, one-by-one, into the reader. We
found such an operation problematic for a number of
reasons. First, it was difficult logistically to continue to
remove plates from the robot deck and then transport them to
the microplate reader though it was in the same room.
Second, having to utilize a person full-time to perform this
manual operation removes them from more practical
exercises such as having them evaluate the quality of the
data in real time. Third, due to the impracticality of having
each duplicated set of plates measured exactly at the same
post-quenching time on our robotics platform, some OSB-
CoA synthetase plates were left unmeasured for up to 2.25 h,
the length of an OSB-CoA HTS run, before having the
absorbance measured at 623 nm. Thus, precise timing of
measurements using an offline detector is impractical
compared to on-deck detection.
A large number of current laboratories that perform HTS
for drug discovery and molecular probe development do not
have the resources necessary for the advanced robotics
required for ultra-HTS or qHTS of large libraries. Here we
demonstrate a scalable alternative to this approach by
screening two ERPs from B. anthracis using only a single
commercially available liquid handling robot prevalent in the
HTS community. Using this assay, small scale robotics
platforms such as the one used in these studies can readily
screen 20K to 30K wells per 10 to 12 h work day.
Importantly, the assay should be scaleable to most robotics
systems, including commercial systems such as the
kalypsys® screening systems, that are capable of screening
up to 500K wells per day.
The subsequent benefits of utilizing this assay strategy
are far reaching. As shown, the introduction of even one
manual step, such as reading the plates for OSB-CoA
synthetase, could introduce unnecessary fluctuations and
increase time commitments of the researcher. When the
manual step is removed, no detrimental effects on the Cv’s,
signal-to-noise ratio, or Z'-factors are observed, and the
values either exceeded or were comparable to previous
studies. Furthermore, automating the reading of assay plates
proved to be an effective method of dealing with the issue of
ATP hydrolysis at low pH conditions. Not only were no
manual steps required while carrying out the assay for
NMAT or OSB-CoA synthetase, but once established, no
reprogramming was required to adapt the assay between
NMAT, OSB-CoA synthetase. Consequently, reprogram-
ming is assumed to be not required to screen other
pyrophosphatases or phosphatases in general. In total, this
method allowed for a throughput of 10,000 compounds in
duplicate per 10 h period, and a total of 100,080 compounds
were screened in total for each enzyme. Since many
enzymatic pathways, including those that NMAT and OSB-
CoA synthetase belong to, normally contain not one but
several ERPs, this universal HTS assay could potentially
allow for the rapid screening of entire pathways or multiple
single enzymatic reactions, at facilities having small-scale
liquid handling robotics. In addition, this assay is capable of
automating the measurement of IC50 values for hit validation
and secondary assays, as well as for the determination of
kinetic parameters in multi-substrate enzymatic reactions as
Other issues also arose with offline versus online
measurements. As observed with previous assays, the low
pH conditions of development can result in the hydrolysis of
nucleotides over long time periods, thus affecting both the
negative and positive control values [18, 19, 28]. The
hydrolysis results not only in a variance in the control
absorbance values but also in a variance in ZDPS-factor
calculations per each plate set for OSB-CoA synthetase (Fig.
4c; Table 3). In comparison, the NMAT assay included
detecting absorbance through the use of an on-deck
spectrometer that could be accessed by automation (Fig. 2B,
C). Thus, each plate set was read in the same time frame of
the previous plate set. This time saving technique removed
the impact of acid-promoted ATP hydrolysis during the
NMAT screen and in return eliminated the fluctuations seen
in the OSB-CoA synthetase assay. Moreover, an increase in
reproducibility was observed with the NMAT MG assay
over the OSB-CoA synthetase assay. By comparing each
sample’s relationship with its duplicate through a replicate
plot, the NMAT sample wells had minimal dispersion from
the ideal relationship in comparison to the OSB-CoA
synthetase sample wells (Fig. 5A, B). In addition, with the

Pyrophosphate and Phosphate Release from Enzymatic Reactions
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1 37
Fig. (5). HTS assay results for OSB-CoA synthetase and NMAT compound library screens. Replicate plots for the percent inhibition
resulting from assay sets, i.e. plate A versus plate B, are shown in (A) for the 100,080 compounds screened against OSB-CoA synthetase,
and (B) for the 100,080 compounds screened against NMAT. The black line in each panel represents an ideal replicate relationship with a
slope of 1. Sample wells with large, negative, percent inhibition values, 40 for OSB-CoA synthetase and 12 for NMAT, that can result from
machine pippeting errors or compound interference, were omitted to provide greater clarity for comparison of the remaining samples
between NMAT and OSB-CoA synthetase. The IC50 values for two 'hit' compounds identified from the primary screen were determined for
OSB-CoA synthetase (C) and NMAT (D).
we have demonstrated for NMAT and OSB-CoA synthetase
[4, 6]. The HTS assay for ERPs described here should
therefore provide the research community with a scalable
efficient screening assay for multiple robotics platforms as
well as provide an opportunity for assay design to move
beyond an enzyme target based HTS to one that is pathway
or organism oriented.
REFERENCES
[1]
Kobayashi, K.; Ehrlich, S.D.; Albertini, A.; Amati, G.; Andersen,
K.K.; Arnaud, M.; Asai, K.; Ashikaga, S.; Aymerich, S.; Bessieres,
P.; Boland, F.; Brignell, S.C.; Bron, S.; Bunai, K.; Chapuis, J.;
Christiansen, L.C.; Danchin, A.; Débarbouille, M.; Dervyn, E.;
Deuerling, E.; Devine, K.; Devine, S.K.; Dreesen, O.; Errington, J.;
Fillinger, S.; Foster, S.J.; Fujita, Y.; Galizzi, A.; Gardan, R.;
Eschevins, C.; Fukushima, T.; Haga, K.; Harwood, C.R.; Hecker,
M.; Hosoya, D.; Hullo, M.F.; Kakeshita, H.; Karamata, D.;
Kasahara, Y.; Kawamura, F.; Koga, K.; Koski, P.; Kuwana, R.;
Imamura, D.; Ishimaru, M.; Ishikawa, S.; Ishio, I.; Le Coq, D.;
Masson, A.; Mauël, C.; Meima, R.; Mellado, R.P.; Moir, A.;
Moriya, S.; Nagakawa, E.; Nanamiya, H.; Nakai, S.; Nygaard, P.;
Ogura, M.; Ohanan, T.; O'Reilly, M.; O'Rourke, M.; Pragai, Z.;
Pooley, H.M.; Rapoport, G.; Rawlins, J.P.; Rivas, L.A.; Rivolta, C.;
Sadaie, A.; Sadaie, Y.; Sarvas, M.; Sato, T.; Saxild, H.H.; Scanlan,
E.; Schumann, W.; Seegers, J.F.; Sekiguchi, J.; Sekowska, A.;
Séror, S.J.; Simon, M.; Stragier, P.; Studer, R.; Takamatsu, H.;
Tanaka, T.; Takeuchi, M.; Thomaides, H.B.; Vagner, V.; van Dijl,
J.M.; Watabe, K.; Wipat, A.; Yamamoto, H.; Yamamoto, M.;
Yamamoto, Y.; Yamane, K.; Yata, K.; Yoshida, K.; Yoshikawa,
H.; Zuber, U.; Ogasawara, N. Ogasawara, essential Bacillus subtilis
genes. Proc. Natl. Acad. Sci. USA, 2003, 100(8), 4678-4683.
ACKNOWLEDGEMENTS
This work was funded by a grant from NIH/NIAID (U19
AI056575). We would like to thank Dick Mitchell, Systat for
use of a beta version of SigmaPlot HTS macro, Institute for
Tuberculosis Research for use of their chemical library, and
Dr. David Crich’s lab at University of Illinois Chicago for
synthesizing the OSB used in the study.

38 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 1
Pegan et al.
[2]
Bryant, R.W.; Bentley, R. Menaquinone biosynthesis: conversion
of o-succinylbenzoic acid to 1, 4-dihydroxy-2-naphthoic acid and
menaquinones by Escherichia coli extracts. Biochemistry, 1976,
15(22), 4791-4796.
Kwon, O., Bhattacharyya, D.K.; Meganathan, R. Menaquinone
(vitamin K-2) biosynthesis: overexpression, purification, and
screening: a titration-based approach that efficiently identifies
biological activities in large chemical libraries. Proc. Natl. Acad.
Sci. USA, 2006, 103(31), 11473-11478.
Van Veldhoven, P.P.; Mannaerts, G.P. Inorganic and organic
phosphate measurements in the nanomolar range. Anal. Biochem.,
1987, 161(1), 45-48.
Bernal, C., Palacin, C.; Boronat, A.; Imperial, S. A colorimetric
assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-
erythritol 4-phosphate synthase activity. Anal. Biochem., 2005,
337(1), 55-61.
Rowlands, M.G., Newbatt, Y.M.; Prodromou, C.; Pearl, L.H.;
Workman, P. Aherne, W. High throughput screening assay for
inhibitors of heat-shock protein 90 ATPase activity. Anal.
Biochem., 2004, 327(2), 176-183.
Mok, M.T.S.; Edwards, M.R. Critical sources of error in
colorimetric assay for UDP-N-acetylglucosamine pyrophosphory-
lase. Anal. Biochem., 2005, 343(2), 341-343.
Leete, E.; Bodem, G.B. Biosynthesis of shihunine in Dendrobium
pierardii. J. Am. Chem. Soc., 1976, 98(20), 6321-6325.
Baykov, A.A.; Evtushenko, O.A.; Avaeva, S.M. A malachite green
procedure for orthophosphate determination and its use in alkaline
phosphatase-based enzyme immunoassay. Anal. Biochem., 1988,
171(2), 266-270.
Lipinski, C.A., Lombardo, F.; Dominy, B.W.; Feeney, P.J.
Experimental and computational approaches to estimate solubility
and permeability in drug discovery and development settings. Adv.
Drug Deliv. Rev., 2001, 46(1-3), 3-26.
Geladopoulos, T.P.; Sotiroudis, T.G.; Evangelopoulos, A.E. A
malachite green colorimetric assay for protein phosphatase activity.
Anal. Biochem., 1991, 192(1), 112-116.
[16]
[17]
[3]
properties of o-succinylbenzoyl-coenzyme
a
synthetase from
Escherichia coli. J. Bacteriol., 1996, 178(23), 6778-6781.
Tian, Y.; Mesecar, A.D. Bacillus anthracis o-Succinylbenzoyl-CoA
synthetase: reaction kinetics and a novel inhibitor mimicking its
reaction intermediate. Biochemistry, 2008, 47, 12434-12447.
Bergman, N.H.; Anderson, E.C.; Swenson, E.E.; Janes, B.K.;
Fisher, N.; Niemeyer, M.M.; Miyoshi, A.D.; Hanna, P.C.
Transcriptional profiling of Bacillus anthracis during infection of
host macrophages. Infect. Immun., 2007, 75(7), 3434-3444.
Sershon, S.C.; Santarsiero, B.D.; Mesecar, A.D. Kinetic and
structural evidence of negative cooperativity in substrate binding to
nicotinate mononucleotide adenylyltransferase (NMAT) from
Bacillus anthracis. J. Mol. Biol., 2008, 385, 867-888.
Bhattacharyya, D.K.; Kwon, O.; Meganathan, R. Vitamin K2
(menaquinone) biosynthesis in Escherichia coli: evidence for the
presence of an essential histidine residue in o-succinylbenzoyl
coenzyme A synthetase. J. Bacteriol., 1997, 179(19), 6061-6065.
Zheng, R.; Blanchard, J.S. Steady-state and pre-steady-state kinetic
analysis of Mycobacterium tuberculosis pantothenate synthetase.
Biochemistry, 2001, 40(43), 12904-12912.
Sershon, S.C.; Santarsiero, B.D.; Mesecar, A.D. Kinetic and
structural evidence of negative cooperativity in substrate binding to
nicotinate mononucleotide adenylyltransferase (NMAT) from
Bacillus anthracis. J. Mol. Biol., 2009, 385(3), 867-888.
Ahmadian, A.; Ehn, M.; Hober, S. Pyrosequencing: history,
biochemistry and future. Clin. Chim. Acta., 2006, 363(1-2), 83-94.
Nyren, P.; Lundin, A. Enzymatic method for continuous
monitoring of inorganic pyrophosphate synthesis. Anal. Biochem.,
1985, 151(2), 504-509.
[4]
[5]
[18]
[19]
[6]
[7]
[20]
[21]
[8]
[9]
[22]
[23]
[24]
[10]
[11]
McGovern, S.L.; Caselli, E.; Grigorieff, N.; Shoichet, B.K. A
common mechanism underlying promiscuous inhibitors from
virtual and high throughput screening. J. Med. Chem., 2002, 45(8),
1712-1722.
Garzon, J.; DeFelipe, J.; Rodriguez, J.R.; DeAntonio, I.; Garcia-
Espana, A.; Sanchez-Blazquez, P. Transport of CSF antibodies to
Galpha subunits across neural membranes requires binding to the
target protein and protein kinase C activity. Brain Res. Mol. Brain
Res., 1999, 65(2), 151-166.
[25]
[12]
[13]
Pais, J.E.; Bowers, K.E.; Stoddard, A.K.; Fierke, C.A. A
continuous fluorescent assay for protein prenyltransferases
measuring diphosphate release. Anal. Biochem., 2005, 345(2), 302-
311.
Webb, M.R. A continuous spectrophotometric assay for inorganic
phosphate and for measuring phosphate release kinetics in
biological systems. Proc. Natl. Acad. Sci. USA, 1992, 89(11),
4884-4887.
Itaya, K.; Ui, M. A new micromethod for the colorimetric
determination of inorganic phosphate. Clin. Chim. Acta., 1966,
14(3), 361-366.
[26]
[27]
Copeland, R.A. Mechanistic considerations in high throughput
screening. Anal. Biochem., 2003, 320(1), 1-12.
Zhang, J.H.; Chung, T.D.; Oldenburg, K.R. A simple statistical
parameter for use in evaluation and validation of high throughput
screening assays. J. Biomol. Screen., 1999, 4(2), 67-73.
Quan, A.; Robinson, P.J. Rapid purification of native dynamin I
and colorimetric GTPase assay. Methods Enzymol., 2005, 404, 556-
569.
[14]
[15]
[28]
Inglese, J., Auld, D.S.; Jadhav, A.; Johnson, R.L.; Simeonov, A.;
Yasgar, A.; Zheng, W.; Austin, C.P. Quantitative high throughput
Received: October 22, 2008
Revised: January 21, 2009
Accepted: March 27, 2009