Identification of peptide substrate and small molecule inhibitors of Testis-Specific Serine/Threonine Kinase1 (TSSK1) by the developed assays

Article abstract of DOI:10.1021/jm9002846

In this paper, a peptide substrate (Pep8) of TSSK1 is identified. Using Pep8 as a substrate, two homogeneous and efficient assays for TSSK1 inhibitors screening have been developed, including luminescent kinase assay and LC-MS-based high-throughput assay.

Full text of DOI:10.1021/jm9002846

J. Med. Chem. 2009, 52, 4419–4428 4419
DOI: 10.1021/jm9002846
Identification of Peptide Substrate and Small Molecule Inhibitors of Testis-Specific
Serine/Threonine Kinase1 (TSSK1) By the Developed Assays
Leilei Zhang, Yu Yan, Zijie Liu, Zeper Abliz, and Gang Liu*
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
Received March 6, 2009
In this paper, a peptide substrate (Pep8) of TSSK1 is identified. Using Pep8 as a substrate, two
homogeneous and efficient assays for TSSK1 inhibitors screening have been developed, including
luminescent kinase assay and LC-MS-based high-throughput assay. Two classes of compounds were
identified that are able to efficiently inhibit phosphorylation catalyzed by TSSK1.
1. Introduction
2. Results and Discussion
Protein phosphorylation plays a critical role in the regula-
tion of many cellular processes. Protein kinases are among the
largest family of proteins known, with 518 putative protein
kinase genes that were defined within the human genome.1
Because abnormal kinase activities are associated with a wide
range of diseases, including leukemias, tumors, vascular dis-
eases, diabetes mellitus, and immune/inflammatory disorders,
protein kinases have now become one of the most important
groups of drug targets in addition to the G protein-coupled
receptors.2 The testis-specific serine/threonine kinase (TSSK)
family is one with exclusive or predominant expression in the
testis and consists of at least five members.3-10 TSSKs were
very recently validated as potential contraceptive targets.11,12
The expression of TSSK1 was restricted to the last stages of
spermatid maturation. Recent studies demonstrated that
TSSK1 is localized in the cytoplasm of late spermatids and
in structures resembling residual bodies, suggesting that
TSSK1 probably participates in the reconstruction of cyto-
plasm during sperm tail maturation.4 Therefore, inhibition of
TSSK1 may provide male contraception. However, neither
peptide substrate nor small molecule inhibitors of TSSK1
havebeen reported until recently, withTSKS(65 kDaprotein)
being shown to be phosphorylated at its Ser281 by TSSK2,
which shared high sequence similarity with TSSK1.4,13
2.1. Identification of TSSK1 Peptide Substrates. Identify-
ing biologically relevant substances for protein kinases is a
critical step to understanding the function of those new
enzymes. In particular, an inexpensive substrate is necessary
for high-throughput screening (HTSa) of kinase inhibitors.
To search for a peptide substrate of TSSK1, two representa-
tive kinase substrate screening kits were selected and syn-
thesized in this article, one from AnaSpec Corporate
(10 peptides)14 and the other from Cell Signaling Technology
(76 peptides).15 Accordingly, a total of 86 peptides were
synthesized simultaneously, employing a “split-mix” com-
binatorial chemistry strategy and the standard Fmoc peptide
chemistry by using Rf tag in a Microkan.16 All synthesized
crude peptides were subsequently analyzed using a fast liquid
chromatographic-mass spectrometry (LC-MS) system to
determine the purities and the correct molecular weights. The
crude peptides were dissolved in dimethyl sulfoxide (DMSO)
at a concentration of 5 mM and further diluted to 2 mM
with 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris)-buffer
(Tris-HCl, pH = 7.4) as working solutions. The substrate
screening was carried out at a final TSSK1 concentration of
1.0 μg/well with addition of 2 μM adenosine 50-triphosphate
(ATP). The phosphorylation of peptides was measured by
detection of ATP remaining after certain reaction times
using the Kinase-Glo reagent.17 The substrate identification
was subsequently performed as two runs. In the first, the
synthetic crude peptides were used directly to obtain poten-
tial substrate peptides and eliminate the most inactive pep-
tides. The active hits in the first run were then resynthesi-
zed and purified by HPLC to reproduce those results in
the second run. Two peptides (Pep8 and Pep4) showed
strikingly less luminescence, indicating that Pep4 and Pep8
were phosphorylated in the course of the TSSK1 reaction
and the ATP was consumed. Dose curve studies demon-
strated that Pep8 is more sensitive to phosphorylation than
Pep4 (Figure 1a). Further studies demonstrated that
these two peptides were unable to be phosphorylated experi-
mentally by cyclin-dependent kinase 2 and 4 (data not
shown). Pep4 contains multiple phosphorylation sites
of threonine and serines, however, the specific one is un-
known. It was reported that a mouse TSKS phosphopeptide
*To whom correspondence should be addressed. Phone/Fax: +86 10
63167165. E-mail:gangliu27@yahoo.com.
a Abbreviations: ATP, adenosine 50-triphosphate; BSA, bovine serum
albumin; BOP, benzotriazole-1-yl-oxy-tris-(dimethylamino)-phospho-
nium hexafluorophosphate; DFDNB, 1,5-difluoro-2,4-dinitrobenzene;
DIC, N,N0-diisopropylcarbodiimide; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; DTT, dithiothreitol; ESI, electrospray
ionization; HBTU, O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-
hexafluorophosphate; HOBt, N-hydroxybenzotriazole; HPLC, high-
performance liquid chromatography; HTS, high-throughput screening;
IC50, concentration for 50% inhibition; LC-MS, Liquid chromatogra-
phy-mass spectrometry; Pep4, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-
Ser; Pep8, Ala-Ala-Leu-Val-Arg-Gln-Met-Ser-Val-Ala-Phe-Phe-Phe-
Lys; Pep8-P, Ala-Ala-Leu-Val-Arg-Gln-Met-Ser(HPO3)-Val-Ala-Phe-
Phe-Phe-Lys; Pep8-P(O2), Ala-Ala-Leu-Val-Arg-Gln-Met(O2)-Ser
(HPO3)-Val-Ala-Phe-Phe-Phe-Lys; RP, reversed phase; Rt, retention
time; RLU, relative light units; Tris, 2-amino-2-hydroxymethyl-pro-
pane-1,3-diol; TIC, total ion chromatography; TOF-MS, time-of-flight
mass spectrometry; TFA, trifluoroacetic acid; UHP, urea-hydrogen
peroxide.
r
2009 American Chemical Society
Published on Web 06/16/2009
pubs.acs.org/jmc

4420 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zhang et al.
Figure 1. (a) Pep8 (AALVRQMSVAFFFK) was able to be phosphorylated at lower concentrations of TSSK1 in a dose-dependent manner
than Pep4 (PLSRTLSVSS). (b) Sequence alignment of the TSKS phosphopeptide with Pep4 and Pep8. Asterisks indicate the phosphorylation
positions. Question marks represent the plausible phosphorylation sites. RLU = relative light units.
(HGLSPATPIQGCSGPPGS*PEEPPR) by TSSK2 was
identified from immunoprecipitated complexes of TSKS
and its antibody by IMAC-LC-MS/MS method.13 Analy-
sis of sequence alignment mouse TSKS phosphopeptide with
Pep4 and Pep8 did not reveal the conserved residues, in-
dicating that Pep4 and Pep8 are novel substrates of TSSK1
(Figure 1b).
are typically colorless, not fluorescent or luminescent, and
not radioactive, traditional kinase assays usually require
some modification of the substrates for compatibility with
the means of detection, including fluorescent tagging, iso-
topic labeling, or enzyme coupling. Recent efforts have
recognized that mass spectrometric techniques are useful
for measuring enzyme activities and enzyme inhibitor screen-
ing as well as for studying enzyme kinetics.40,41 LC-MS
techniques can detect the reaction products directly and
quantitatively, avoiding modification of substrates or sec-
ondary enzymatic reactions that are irrelevant to the target
enzyme reaction. Thus, an LC-MS-based assay could be
applied to any enzyme reaction system providing that the
substrates and products are distinguishable by molecular
weight and retention behavior on the basis of reasonable
ionization efficiency.
2.3.1. Synthesis of Pep8-P and Pep8-P(O2). To develop an
LC-MS-based assay, phosphorylated product (Pep8-P) and
an internal standard (Pep8-P(O2)) were required, particu-
larly to optimize the LC separation conditions for elimina-
tion of overlap between Pep8 and Pep8-P. There are
currently two strategies for the synthesis of phosphopeptide.
The building block approach uses protected phosphoamino
acids and the global phosphorylation method involves post-
synthetic phosphorylation of unprotected hydroxyl groups
on the solid support. Owing to the commercial availability of
suitably protected Fmoc-Ser(PO(OBzl)-OH)-OH, the Pep8-
P was directly coupled to peptide on solid-phase using O-
benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-
phosphate (HBTU) and N-hydroxybenzotriazole (HOBt) as
activator and additive, respectively. Deprotection of the
side chain and cleavage of Pep8-P from the resin were
achieved simultaneously by treatment of the resin with a
cocktail solution of trifluoroacetic acid (TFA)/H2O/thioa-
nisole (v/v/v = 95:2.5:2.5). After purification of Pep8-P by
semipreparative HPLC, direct confirmation was indicated
by an increase in the molecular weight of Pep8 of 80 Da,
corresponding to the addition of one phosphate group.
Because the Pep8-P contains a methionine in sequence,
the sulfone analogue of Pep8-P at Met, with its physical and
chemical properties close to Pep8-P, was chosen herein to be
the internal standard. Many methods of preparing sulfones
from sulfides have been reported. Among them, urea-
hydrogen peroxide (UHP) oxidation is a simple and safe
method.21 UHP, an adduct of hydrogen peroxide and urea,
is a mild, inexpensive, stable, and relatively safe oxidant.
2.2. Identification of Small Molecule Nonpeptide Inhibitors
with a Luminescent Kinase Assay. To further identify the
nonpeptide inhibitors of TSSK1, the kinase reaction condi-
tions were optimized with respect to the amount of ATP,
kinase, and final peptide substrate concentration. Accord-
ingly, serial 2-fold dilutions of ATP resulted in a linear
correlation with the amount of ATP depleted (Figure 2a),
therefore, the middle concentration of 1.5 μM of ATP and
excess Pep8 (20 μM) were used for selection of the optimal
TSSK1 concentration (Figure 2b). Recordings during the
optimization of the TSSK1 concentration resulted in the
largest change in luminescence in wells that were missing
Pep8 compared with the reaction wells in which the kinase
reaction were completed in the presence of Pep8, where the
concentration of TSSK1 was 0.5 μg/well. In addition, the
amount of ATP depleted also increased as Pep8 increased
(Figure 2c). Obvious luminescence changes were observed
when Pep8 was at 10 μM. In all, the final conditions for HTS
of TSSK1 inhibitor were defined as 1.5 μM of ATP, 0.5 μg/
well of TSSK1, and 10 μM of Pep8.
Compounds with 14 different chemical skeletons
(Figure 3a) were in stock in our laboratory as 50 mM
solutions. These compounds were assembled through
a scaffold-directed program, aimed at developing benzo-
fused privileged structures with druglike properties.18-29
A total of 640 single pure compounds were screened with
initial concentration of 20 μM (see Supporting Information).
Among them, eight compounds showed reproducible
inhibitory activity (>40%) and two of them, 5-hydroxy-
8-methyl-4-propyl-7,8-dihydropyrano[3,2-g]chromene-2,6-
dione (7) and 1,10-(biphenyl-4,40-diyl)bis(2,2-dihydroxyetha-
none) (8), had measured IC50 values of 38.7 μM and 43.2 μM,
respectively (Figure 3b).
2.3. Development and Validation of Liquid Chromatogra-
phy-Mass Spectrometry (LC-MS) Based Assay. Conven-
tional kinase assays include fluorescence analysis,30-33
radioactive methods,34,35 luminescence (bioluminescence)
assays,36,37 and colorimetric detection of a quantifiable sub-
strate or product.38,39 Because natural substrates or products

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4421
Here, UHP was used for the first time to oxidize peptide,
and Pep8-P was conveniently and quantitatively converted
into its sulfone analogue (Pep8-P(O2)) (Scheme 1).
2.3.2. LC-MS-Based Assay Development. Theoretically,
either peptide substrate or its phosphorylated product
can be used for kinase activity evaluation by mass spec-
trometry analysis. However, primary experiments indi-
cated that the signal from the Pep8-P was more facilita-
ted to ionization than Pep8 (see Supporting Information).
To efficiently separate Pep8-P from other reaction compo-
nents, several commercially available columns were inves-
tigated. Among them, a Kromasil C18 (50 mm ꢀ 4.6 mm)
column appeared to have the best efficacy for dealing
with desalt procedures in a relatively short gradient running
time. A formic acid/acetonitrile-based mobile phase at pH
2.9 was selected to achieve good HPLC performance as
well as good volatility and appropriate pH for positive ion
detection.
The sensitivity and accuracy of the mass spectrometry can
be greatly compromised by the presence of a buffer. There-
fore, a switching valve on the Agilent LC/MSD TOF was
successfully combined with efficient HPLC separation to
completely remove salt contaminants from the sample prior
to analysis of the mass spectrometry. Basically, a proper
HPLC gradient could elute all salts in a short time under
polar conditions and these could be switched into the
waste. The optimized HPLC conditions ensured that inor-
ganic components were completely eluted from the column,
but the substrate and phosphorylated substrate were still
retained in the column. In fact, a total of 2.5 min of gradi-
ent flow was switched into waste in this study to guaran-
tee at least 90 sample analyses without observing any
contamination.
As discussed above, Pep8-P and Pep8 were separated
efficiently under optimal HPLC conditions. For obtaining
good repeatability as well as good chromatographic peak
shape, an optimal fragmentor voltage was systemically
investigated. Because peptides are easily ionized to multiply
charged ions and show multiple charge distributions with
different fragmentor voltages, the resulting mass spectra of
Pep8-P showed prominent peaks at m/z 847.9 and m/z 565.3,
which correspond to the +2 and +3 charge states of
phosphorylated Pep8, respectively. Studies indicated that
the lowest fragmentor voltage (150 V) resulted in a promi-
nent +3 charged ion at m/z 565.3 ([M + 3H]3+). In con-
trast, the highest fragmentor voltage (250 V) gave mainly
+2 charged ions at m/z 847.9 ([M + 2H]2+). Optimally, at
230 V, m/z 847.9 was prominent and both good repeat-
ability and chromatographic peak shape were observed
simultaneously.
LC-MS and a one-point normalization factor were used
to determine the Km values of two substrates.42 In this
study, Pep8-P(O2), the sulfone analogue of Pep8-P, was
the internal standard for Km determination. Pep8-P and
Pep8-P(O2)’s TIC peaks were completely separated (see
Supporting Information). Substrate time courses for ATP
and Pep8 (Figure 4), respectively, were recorded in dupli-
cate at varied substrate concentrations, while other sub-
strate Pep8 or ATP was fixed at a saturated concentration.
A kinetic plot was generated by plotting the initial reaction
velocity against the different substrate concentrations. The
calculated Km values for ATP and Pep8 were 50.5 and 25.2
μM, respectively. In this article, therefore, we set ATP and
Pep8 substrates at 25 μM and 10 μM for LC-MS-based
Figure 2. Optimization of the luminescent kinase assay for TSSK1.
(a) Correlation of RLU readings and ATP concentrations (r2
=
0.997). Serial 2-fold dilutions of ATP were made in a 96-well plate in
50 μL of kinase reaction buffer (40 mM Tris-HCl, 20 mM MgCl2,
and 0.1 mg/mL bovine serum albumin (BSA)). (b) Luminescence
changes in the presence of TSSK1 after 2-fold serial dilutions in
wells missing the Pep8 and in wells with completed kinase reactions.
The amounts of ATP and Pep8 were 1.5 μM and 20 μM, respec-
tively. (c) ATP depletion increased as Pep8 increased, in reactions
where ATP and TSSK1 were 1.5 μM and 0.5 μg/well, respectively.
The byproducts of UHP oxidation are TFA and urea
(Scheme 1), which are easily washed away with water.
Therefore, UHP is particularly useful forpeptide sulfonation.

4422 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zhang et al.
Figure 3. Identification of TSSK1 inhibitors. (a) The selected compound skeletons. (b) Eight compounds showed reproducible inhibitory
activity (>40%).
Scheme 1. Oxidation of Pep8-P to Sulfonated Pep8-P(O2) at
the Methionine Residuea
To determine the optimal TSSK1 concentration and
incubation time, the kinase reaction was studied system-
atically at 150 ng/well, 75 ng/well, 37.5 ng/well, 18.75 ng/
well, and 0 ng/well of TSSK1, respectively. The Pep8 phos-
phorylation level was determined within 180 min. A linear
rate of product formation was observed within 120 min for
75 ng TSSK1 and within 60 min for 150 ng TSSK1 (Figure 5).
Although phosphorylation of Pep8 also occurred linearly
within the total testing time period for 18.75 and 37.5 ng of
TSSK1, Pep8-P level was low enough that the results may be
beyond the limitation of mass detection sensitivity. Hence, a
kinase amount of 75 ng and 90 min incubation time were
optimal for high-throughput screening (HTS) of chemical
libraries.
a Pep1
(HPO3)-Val-Ala-Phe-Phe-Phe-Lys-CONH2.
= NH2-Ala-Ala-Leu-Val-Arg-Gln-CO, Pep2 = -Ser
To qualify the method, the linearity of the method was
further investigated over a concentration range of 33.8-
676.8 ng of Pep8-P. The product peak area versus Pep8-P
concentration was plotted to generate a standard curve by
high-throughput identification of TSSK1 small molecule
inhibitors.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4423
Figure 4. The Kms of ATP and Pep8 were determined by LC-MS measurement. (a) Pep8 phosphorylation was time-dependent linear as the
ATP concentration varied from 3.12 to 200 μM in the presence of 0.5 μg/well TSSK1 and 30 μM Pep8. (b) Michaelis-Menten plot for ATP. The
K
m value of ATP was determined as 50.5 μM. (c) Pep8-P was linearly occurred in time-dependent manner as the Pep8 concentration varied from
0 to 40 μM in the presence of 0.5 μg/well TSSK1 and 200 μM ATP. (d) Michaelis-Menten plot for Pep8. The Km value of Pep8 is obtained as
25.2 μM. The Km values are calculated by Systat Sigmaplot 10.0.
injections (Figure 6b). In another experiment, 89 TSSK1
reactions were carried out simultaneously under optimal
conditions and analyzed by LC-MS. The precision evalu-
ated by repeatedly measuring the peak areas of Pep8-P was
10.6% (Figure 6c). All of these results indicate that this LC-
MS-based assay of TSSK1 reaction developed was highly
reproducible.
To assess the high-throughput screening, we also esti-
mated the effect of added DMSO on the TSSK1 activity
prior to the evaluation of inhibitors. The results indi-
cated that DMSO less than 2% did not affect both the
analysis and enzymic reaction. To valuate the Z0-factor,
a total of 48 mass spectra were acquired, of which 24 spectra
were collected in the presence of TSSK1 (75 ng in the assay
buffer) and 24 were not (see Supporting Information).
Calculations based on these data provided a Z0 score of
0.836, indicating that the developed mass-based assay was
excellent.
2.4. High-Throughput Screening of Single Compound or
Mixture Compound Libraries by the Developed LC-MS-
Based Assay. 2.4.1. Screening Biphenyl Compounds. The
LC-MS-based assay was then used to screen small mole-
cule inhibitors of TSSK1. In above luminescent kinase
assay, compound 8 was identified, which belongs to biphenyl
compound scaffold. Twenty analogues in our stock library
were further screened as TSSK1 potential inhibitors seek-
ing more potent compound. Fortunately, one of these,
sodium 2,20-(biphenyl-4,40-diyl)bis(1-hydroxy-2-oxoethane-
sulfonate (9), which is the adduct of 8 and sodium hydro-
gen sulfite, was found to be more potent.43 Its IC50 value
Figure 5. Phosphorylation level of Pep8 as time changes of incuba-
tion and variable TSSK1 doses.
linear regression. The obtained high correlation coefficient
(0.99) demonstrates that this method was highly suitable
(Figure 6a).
The major advantage of the LC-MS-based assay was that
it could be automated. Because many factors influence MS
analysis, the reproducibility and stability of this method were
determined in the following manner. When Pep8-P was
dissolved in a buffer system (acetonitrile:kinase buffer=
50:50 (v/v)), the obtained RSD was 7.34% for 91 continuous

4424 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zhang et al.
Figure 6. (a) LC-MS peak area counts for Pep8-P was cor-
relatively linear with Pep8-P concentration (r2 > 0.99). (b) Re-
peatability of Pep8-P in 50% ACN-kinase buffer for 91 con-
tinuous injections. (c) Repeatability of Pep8-P peak area in 89
TSSK1 catalyzed reactions. The optimal reaction conditions were:
40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 2 mM Dithio-
threitol (DTT), 0.1 mg/mL BSA, 25 μM ATP, 75 ng TSSK1, and
10 μM Pep8. These results were obtained by LC-MS analysis
system (the analysis conditions are included in 4Experimental
Section).
Figure 7. Inhibitory curves of compound 9 to TSSK1 were illu-
strated by developed LC-MS-based assay with or without in-
ternal standard (Pep8-(O2)) (a,b) and a luminescent kinase assay
(c). X axis represents the log 10 value of the compound 9 con-
centration, while Y axis is the peak area of Pep8-p for (a), the
concentration of Pep8-p for (b), and the inhibitory rate for (c).
All of the IC50 values were plotted and calculated by the software
Origin 7.5.
was calculated by three different methods developed in
this study to give 19.1 μM for LC-MS-based assay with
internal standard (Pep8(O2)-P), 14.4 μM for LC-MS-
based assay without internal standard, and 24.8 μM for
a luminescent kinase assay, respectively (Figure 7a,b,c).
The smaller range in the results confirms the validity of

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4425
Figure 8. Novel 1,2,7-trialky-1H-imidazo[4,5-g]quinoxalin-6-ones that inhibited TSSK1 activity.
Scheme 2. Synthesis of 1,2,7-trialky-1H-imidazo[4,5-g]quinox-
alin-6-one
the developed assays.
2.4.2. Screening a chemical library of 1,2,7-trialky-1H-
imidazo[4,5-g]quinoxalin-6-one. Privileged structures have
been successfully exploited across and within different target
families and promises to be an effective approach to the
discovery and optimization of novel bioactive molecules.
Both benzimidazole and quinoxalin-2-one are important
pharmacophores and privileged structures in medicinal
chemistry encompassing a diverse range of biological activ-
ities. The incorporation of two privileged pharmacophores
may provide additional opportunities to discover novel lead
compounds using one synthetic process. A chemical library
of 1,2,7-trialky-1H-imidazo[4,5-g]quinoxalin-6-one was de-
signed and synthesized by our laboratory to integrate benzi-
midazole and quinoxalin-2-one privileged structures into
one molecule.26 This library was composed of five mixed
analogues per well, and the total purity of five compounds/
well is >80% by HPLC analysis (UV wavelength at 254 nm).
In the primary screening, 370 wells consisting of 1850
compounds were assayed at a total estimated concentration
of 20 μM of five compounds in each well. Every mixture well
was measured twice in parallel. A total of eight wells with
greater than 50% repetitive inhibition of TSSK1 activity
were selected for secondary screening. Each individual com-
pound in these eight positive wells was then resynthesized
as outlined in Scheme 2. Two fluorine groups on the star-
ting material 1,5-difluoro-2,4-dinitrobenzene (DFDNB)
(10) were subsequently and quantitatively displaced by alkyl
primary amines and amino acid methyl or ethyl esters in the
presence of N-ethyldiisopropylamine (DIPEA).30 Subse-
quently, the quantitative reduction of two aromatic nitro
groups of 11 to produce 12 was achieved in high yield using
HCOONH4 and Pd/C. In the presence of 5% acetic acid in
dioxane, treatment of 12 with aldehydes at 77 °C gave high
yields of 13. All of 40 compounds were finally characterized
by 1H NMR and high-resolution MS after recrystallization
or purification by semipreparative HPLC until the purity
was greater than 95% (see Supporting Information).
These 40 individual pure compounds were further tested for
inhibitory activity against TSSK1. Three compounds showed
positive activity (Figure 8). Herein, false-positive wells were
thought to arise from impurities in the combinatorial library.
Nevertheless, it was shown that mixture library synthesis and
mixture screening could greatly improve the efficiency of lead
compound discovery by a LC-MS-based assay.
3. Conclusion
In this study, a peptide substrate of TSSK1 was identified
for the first time. Furthermore, a luminescent kinase assay
using Kinase-Glo reagent and a highly efficient LC-MS-
based assay were developed for high-throughput identifica-
tion of novel TSSK1 inhibitors. Two classes of compounds

4426 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zhang et al.
(biphenyl compounds and 1,2,7-trialky-1H-imidazo[4,5-g]
quinoxalin-6-ones) were identified as being able to efficiently
inhibit TSSK1-catalyzed phosphorylation. The LC-MS-
based assay proved to be more sensitive than the luminescent
method. For instance, the amount of kinase required for the
LC-MS-based assay was 75 ng, but 500 ng of TSSK1 was
required for the luminescent method when optimal kinase
reaction conditions were reached for TSSK1-catalyzed phos-
phorylation. In addition, the LC-MS-based assay directly
detects the native substrate, whereas the luminescent method
indirectly detects ATP consumption. The former is compati-
ble with mixture analysis and significantly enhances the
screening throughput.
depending on the particular sequence. In cases of insufficient
coupling, repeated coupling was performed until the reaction
was complete. The N-terminus was acetylated using 15% acetic
anhydride in DCM for 30 min. Several difficult sequences were
selected as controls during the course of the coupling reaction
and were monitored using the Kaiser test. Crude peptides were
cleaved using a one-step TFA cleavage method with different
cleavage conditions (according to peptide synthesis manuals,
such as Nova Biochem). The TFA cleavage mixture was added
to a tube containing a peptide resin, and the mixture was shaken
for 2 h at room temperature. Upon completion of the cleavage
reaction, the “peptide + resin” mixture was filtered and the tube
was rinsed with 2-3 mL of cleavage mixture. The solution was
treated with ice-cooled methyl tert-butyl ether/petroleum ether
(3:1) to precipitate the peptide. The crude peptides were washed
six times with ice-cooled methyl tert-butyl ether/petroleum ether
(3:1). Crude peptides were redissolved in 30-60% CH3CN-
H2O (v/v) and analyzed by LC/MS, indicating that the purities
were from 30% to 95% under UV 214 nm wavelength.
4.4. Synthesis of Pep8 and Pep8-P. Pep8 and Pep8-P, which
correspond with the sequences of Ala-Ala-Leu-Val-Arg-Gln-
Met-Ser-Val-Ala-Phe-Phe-Phe-Lys and Ala-Ala-Leu-Val-Arg-
Gln-Met-Ser(HPO3)-Val-Ala-Phe-Phe-Phe-Lys, respectively,
were manually synthesized according to standard Fmoc solid-
phase strategy.44 Noticeably, the coupling procedure of Fmoc-
Ser(PO(OBzl)-OH)-OH for synthesis of Pep8-P involved the
appropriate Fmoc-amino acids (5 equiv), HOBt (5 equiv), and
coupling reagent (HBTU, 5 equiv) dissolved in DMF containing
15 equiv of DIPEA. Double coupling was performed until the
reaction was complete. The N-terminus was acetylated using
15% acetic anhydride in DCM for 30 min. The side chain benzyl
group was removed using TFA/TIS/water (95:2.5:2.5) for 3 h.
Peptides were lyophilized and purified by HPLC until >97%
under UV 214 nm wavelength.
4.5. Synthesis of Internal Standard (Pep8-P(O2)). Freshly
prepared UHP (0.07 mmol) was dissolved in acetonitrile
(300 μL), and trifluoroacetic anhydride (50 μL) was added
slowly with stirring to achieve full dissolution. UHP-trifluor-
oacetic anhydride oxidate was then prepared. Pep8-P (12 mg)
was dissolved in acetonitrile (2 mL), and UHP-trifluoroacetic
anhydride oxidate (70 μL) was added with stirring at room
temperature. After completion of oxidation, the reaction mix-
ture was subjected to HPLC analysis, and the mixture was
filtered with a 0.45 μM membrane and subjected to desalting
with RP-HPLC until the purity was >97% under UV 214 nm
wavelength.
4.6. Km Determination. Stock solutions of Pep8-P (1 mM) and
Pep8-P(O2) (0.7 mM) were prepared in kinase buffer. The mixed
solution of Pep8-P(O2) (7 μM) and Pep8-P (10 μM) was then
prepared in 50% acetonitrile-kinase buffer (v/v). This mixture
was analyzed by the developed LC-MS analysis system. Thirty
samples were run sequentially with an injection volume of 15 μL.
The normalization factor was determined from eq 1, shown
below. APep8-P was the peak area of Pep8-P and AIS was the peak
area of the internal standard. This ratio was multiplied by the
respective concentrations of internal standard and Pep8-P in
order to obtain R, the normalization factor. The average R value
was used for subsequent calculations of product concentration.
4. Experimental Section
4.1. Materials. ATP disodium salt, BSA, DTT, (MgCl2
6
3
H2O), and Tris were purchased from Amresco (Solon, OH). All
Kinase-Glo reagents were obtained from Promega (Milano,
Italy). Purified TSSK1 was a gift from Shanghai Genomics
(Shanghai, China). Acetonitrile and water were HPLC gradient
grade. Formic acid was obtained from Fluka (Vienna, Austria).
All amino acids used for peptide synthesis and coupling reagents
(HBTU, BOP, and DIC) and HOBt were purchased from GL
Biochem Ltd. (Shanghai, China). Rink amide resin (loading
0.44 mmol/g, 1% DVB, 100-200 mesh) was purchased from
Hecheng, Co. (Tianjin, China). All amino acid methyl or ethyl
esters were purchased from Chem-Impex International, Inc.
(Wood Dale, IL). All alkyl primary amines and aldehydes were
purchased from Acros Organics (Geel, Belgium). All organic
solvents were redistilled after a proper drying program.
4.2. LC-MS Analysis System. Liquid chromatography/elec-
trospray/time-of-flight mass spectrometry in positive ionization
mode was used to separate and identify Pep8 and Pep8-P. The
analytes were separated using an HPLC (series 1100, Agilent
Technologies, Palo Alto, CA) equipped with a 50 mm ꢀ 4.6 mm
reversed-phase C18 analytical Kromasil column (Beijing Ana-
lytical Instrument Institute, China) with 5 μm particle diameter.
Mobile phase A was acetonitrile with 0.1% formic acid, and
mobile phase B consisted of water with 0.1% formic acid. The
HPLC conditions were as follows: 5% A for 1.5 min, followed
by a gradient to 25% A in 0.1 min, then a linear gradient
progressing from 25% A to 60% A in 4.9 min, after which a
gradient increased A to 95% in 1 min, followed by a stop time
of 7.5 min and a post time of 1.5 min 5% A. The flow rate was
0.8 mL/min, and 60 μL of sample was injected. The HPLC
system was connected to a time-of-flight mass spectrometer
(MSD-TOF, Agilent Technologies)equipped with an electrospray
interface under the following operating parameters: capillary
2500 V, nebulizer 30 psig, drying gas 10 L/min, gas temperature
300 °C, fragmentor 230 V, skimmer 50 V. The mass axis was
calibrated using the mixture provided by the manufacturer over
the m/z 50-3200 range. A second orthogonal sprayer with a
reference solution was used as a continuous calibration using
the reference masses 121.0509 and 922.0098 m/z. Spectra were
acquired over the m/z 50-3000 range at a scan rate of 1 scan/s.
4.3. Peptide Library Synthesis. The peptide library containing
86 peptides was synthesized using a Fmoc-based synthesis
protocol and mix-and-split approach using the IRORI sorting
system. Rink amide resin (28 mg) was put into every MicroKan
using a resin-packing device. At the same time, an Rf tag was put
in and MicroKans were covered by a parallel capping device.
The tag was coded before the first coupling and decoded
before the next coupling step. The resin-bound Fmoc-protected
peptide was treated twice with 20% piperidine in DMF for
15 min for deprotection. The coupling procedure involved the
appropriate Fmoc-amino acids (3 equiv), HOBt (3 equiv), and
coupling reagent (BOP or DIC, 3 equiv) dissolved in DMF.
Coupling reactions were allowed to proceed for different times,
ðAPep8-PÞ½internal stdꢁ
R ¼
ðeq1Þ
ðAISÞ½Pep8-Pꢁ
Determination of Km for two substrates (ATP concentrations
ranged from 3.13 to 200 μM and Pep 8 concentrations ranged
from 0 to 40 μM) was performed using the LC-MS assay under
conditions of saturation of the other substrates as follows:
[ATP] = 200 μM, [Pep8] = 30 μM. Data were collected in
duplicate and averaged. Other reaction components were 75 ng
TSSK1, 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/mL
BSA, and 2 mM DTT. Kinase reactions with 50 μL volume were
incubated at 37 °C. After incubation for different periods, the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4427
reactions were quenched with 50 μL of acetonitrile con-
taining 7 μM internal standard (Pep8-P(O2)), then centrifuged
(15000g ꢀ 10 min) and subjected to HPLC-MS analysis. The
Km value was determined by performing a nonlinear least-
squares best fit to the Michaelis-Menten equation using Systat
Sigmaplot 10.0.
pure compounds, LC-MS analysis of the kinase reaction
mixture and the mixture solution including internal standard
and Pep8-P, HPLC and MS analysis of Pep8, Pep8-P, and Pep8-
P(O2), table listing peptide library for TSSK1 peptide sub-
strate screening, Z0 factor determination, the structure, and
their physical data of single compound with 1,2,7-trialky-1H-
imidazo[4,5-g]quinoxalin-6-one. This material is available free
of charge via the Internet at http://pubs.acs.org.
4.7. Luminescent Kinase Assay. All TSSK1 reactions were
performed in a kinase buffer (40 mM Tris-HCl buffer, 20 mM
MgCl2, 0.1 mg/mL BSA, 2 mM DTT). To obtain the best
performance when using Kinase-Glo reagent, optimal kinase
conditions were established. TSSK1 was resuspended in 25 μL
of kinase buffer. The kinase reaction was performed by adding
25 μL of a mixture containing ATP and peptide substrate (Pep8)
in kinase buffer. The final reaction conditions were 0.5 μg/well
TSSK1, 10 μM Pep8, and 1.5 μM ATP. The reaction was
incubated for 2 h at 37 °C, and then an equal volume of
Kinase-Glo reagent (50 μL) was added. As a control, the kinase
reaction was performed with the same samples in the absence of
peptide substrate (no pep8) and without TSSK1. Meanwhile,
staurosporine was chosen as a positive control at the concentra-
tion of 100 μM. Samples were then incubated for 10 min at room
temperature, and the luminescence developed was recorded by
a luminometer and expressed as RLU.
References
(1) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam,
S. The protein kinase complement of the human genome. Science
2002, 298, 1912–1934.
(2) Cohen, P. Protein kinases;the major drug targets of the twenty-
first century?. Nat. Rev. Drug Discovery 2002, 1, 309–315.
(3) Bielke, W.; Blaschke, R. J.; Miescher, G. C.; Zurcher, G.; Andres,
A. C.; Ziemiecki, A. Characterization of a novel murine testis-
specific serine/threonine kinase. Gene 1994, 139, 235–239.
(4) Kueng, P.; Nikolova, Z.; Djonov, V.; Hemphill, A.; Rohrbach, V.;
Boehlen, D.; Zuercher, G.; Andres, A. C.; Ziemiecki, A. A Novel
Family of Serine/Threonine Kinases Participating in Spermiogen-
esis. J. Cell Biol. 1997, 139, 1851–1859.
(5) Zuercher, G.; Rohrbach, V.; Andres, A. C.; Ziemiecki, A. A novel
member of the testis specific serine kinase family, tssk-3, expressed
in the Leydig cells of sexually mature mice. Mech. Dev. 2000, 93,
175–177.
(6) Visconti, P. E.; Hao, Z.; Purdon, M. A.; Stein, P.; Balsara, B. R.;
Testa, J. R.; Herr, J. C.; Moss, S. B.; Kopf, G. S. Cloning and
chromosomal localization of a gene encoding a novel serine/
threonine kinase belonging to the subfamily of testis-specific
kinases. Genomics 2001, 77, 163–170.
(7) Bucko-Justyna, M.; Lipinski, L.; Burgering, B. M.; Trzeciak, L.
Characterization of testis-specific serine-threonine kinase 3 and its
activation by phosphoinositide-dependent kinase-1-dependent
signalling. FEBS J. 2005, 272, 6310–6323.
(8) Chen, X.; Lin, G.; Wei, Y.; Hexige, S.; Niu, Y.; Liu, L.; Yang, C.;
Yu, L. TSSK5, a novel member of the testis-specific serine/threo-
nine kinase family, phosphorylates CREB at Ser-133, and stimu-
lates the CRE/CREB responsive pathway. Biochem. Biophys. Res.
Commun. 2005, 333, 742–749.
(9) Hao, Z.; Jha, K. N.; Kim, Y. H.; Vemuganti, S.; Westbrook, V. A.;
Chertihin, O.; Markgraf, K.; Flickinger, C. J.; Coppola, M.; Herr,
J. C.; Visconti, P. E. Expression analysis of the human testis-
specific serine/threonine kinase (TSSK) homologues. A TSSK
member is present in the equatorial segment of human sperm.
Mol. Hum. Reprod. 2004, 10, 433–444.
(10) Spiridonov, N. A.; Wong, L.; Zerfas, P. M.; Starost, M. F.; Pack, S.
D.; Paweletz, C. P.; Johnson, G. R. Identification and character-
ization of SSTK, a serine/threonine protein kinase essential for
male fertility. Mol. Cell. Biol. 2005, 25, 4250–4256.
(11) Xu, B.; Hao, Z.; Jha, K. N.; Digilio, L.; Urekar, C.; Kim, Y. H.;
Pulido, S.; Flickinger, C. J.; Herr, J. C. Validation of a testis specific
serine/threonine kinase [TSSK] family and the substrate of TSSK1
& 2, TSKS, as contraceptive targets. Soc. Reprod. Fertil. Suppl.
2007, 63, 87–101.
(12) Herr, J. C.; Xu, B.; Hao, Z. Validation of TSSK family members
and TSKS as male contraceptive targets. U.S. Patent US 2008/
0274117 A1, 2008.
(13) Xu, B.; Hao, Z.; Jha, K. N.; Zhang, Z.; Urekar, C.; Digilio, L.;
Pulido, S.; Strauss, J. F.; Flickinger, C. J.; Herr, J. C. Targeted
deletion of Tssk1 and 2 causes male infertility due to haploinsuffi-
ciency. Dev. Biol. 2008, 319, 211–222.
(14) AnaSpec; www.anaspec.com, accessed October 6, 2005.
(15) Cell Signaling Technology; www.cellsignal.com, accessed October
6, 2005.
(16) Hu, H.; Li, L.; Kao, R. Y.; Kou, B.; Wang, Z.; Zhang, L.; Zhang,
H.; Hao, Z.; Tsui, W. H.; Ni, A.; Cui, L.; Fan, B.; Guo, F.; Rao, S.;
Jiang, C.; Li, Q.; Sun, M.; He, W.; Liu, G. Screening and identifica-
tion of linear B-cell epitopes and entry-blocking peptide of severe
acute respiratory syndrome (SARS)-associated coronavirus
using synthetic overlapping peptide library. J. Comb. Chem.
2005, 7, 648–656.
(17) Promega; www.promega.com, accessed September 18, 2007.
(18) Zhao, H. Y.; Liu, G. Solution-phase parallel synthesis of diverse
1,5-benzodiazepin-2-ones. J. Comb. Chem. 2007, 9, 1164–1176.
(19) Li, L.; Wang, Z. G.; Chen, Y. Y.; Yuan, Y. Y.; Wang, G. X.; Liu, G.
Design and synthesis of novel tricycles based on 4H-benzo[1,4]
thiazin-3-one and 1,1-dioxo-1,4-dihydro-2H-1λ6-benzo[1,4]thia-
zin-3-one. J. Comb. Chem. 2007, 9, 959–972.
4.8. LC-MS-Based Assay. The LC-MS-based TSSK1 assay,
an end-point assay that measures Pep8-P production, was per-
formed in a 0.5 mL Eppendorf tube with a reaction volume of 50
μL. All TSSK1 reactions were performed in a kinase buffer (40 mM
Tris-HCl buffer, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM DTT).
TSSK1 was resuspended in 25 μL of kinase buffer. The kinase
reaction was performed by adding 25 μL of a mixture containing
ATP and peptide substrate (Pep8) in kinase buffer. The final
reaction conditions were 75 ng/well TSSK1, 10 μM Pep8, and
25 μM ATP. The reaction was incubated for 90 min at 37 °C.
Enzyme reactions were quenched by addition of acetonitrile
(50 μL) and centrifuged (15000g ꢀ 10 min). A 60 μL volume of
quenched reaction mixture was subjected to HPLC-MS analysis.
4.9. Preparation of Individual 1,2,7-Trialkyl-1H-imidazo[4,5-
g]quinoxalin-6-one Compounds. To a stirred solution of DFDNB
(204 mg, 1.0 mmol) in THF (20 mL), DIPEA (4.0 mmol), and
NH2CH(R1)COOMe HCl (1.0 mmol) were added sequentially.
3
The reactions were then continued for 3 h at room temperature
with gentle stirring. Continuously, exactly 1.0 mmol of alkyl
primary amine was added and allowed to stand for an additional
18 h. The solvent was removed under reduced pressure to give
compound 11, which was dissolved in dichloromethane, washed
with water, and dried. Compound 11 was then dissolved in
a mixed solvent of THF (10 mL) and EtOH (10 mL), and 10%
Pd/C was added, followed by immediate addition of ammonium
formate (1.26 g). The suspensions were stirred continuously
at room temperature. When hydrogenation was complete, the
Pd/C was filtered and the solvent removed to give crude
compound 12. After purification, parallel synthesis was applied
to obtain compound 13. To each reaction tube in a 96-well H+P
parallel synthesizer, 0.16 mmol of compound 12 and 0.32 mmol
of aldehyde were added and dissolved in 6 mL dioxane, to which
300 μL of acetic acid was added. The suspensions were stirred at
77 °C for 8 h, after which the solvent was removed to give the
crude product. All reactions were monitored by LC-MS. All
the products were recrystallized or purified by preparative RP-
HPLC and were further characterized by LC/MS and 1H NMR.
Acknowledgment. This study was supported by the National
High Technology Research and Development Program of China
(National 863 Program, no. 2006AA020501). We thank Dr. Shu
Fang and Sun Xiaoqing (Shanghai Genomics, Inc.) for their nice
collaboration on TSSK1 expression and purification as well as
the assistance of TSSK1 peptide substrate identification.
Supporting Information Available: Protein sequence and
purification methods of TSSK1, screening results of 640 single

4428 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zhang et al.
(20) Zhao, H. Y.; Liu, G. Solution-phase parallel synthesis of 2,3-
dihydro-1,5-benzothiazepin-4(5H)-ones. J. Comb. Chem. 2007, 9,
756–772.
(33) Kupcho, K.; Somberg, R.; Bulleit, B.; Goueli, S. A homogeneous,
nonradioactive high-throughput fluorogenic protein kinase assay.
Anal. Biochem. 2003, 317, 210–217.
(21) Wang, Z. G.; Yuan, Y. Y.; Chen, Y. Y.; Sun, G. C.; Wu, X. H.;
Zhang, S. M.; Han, C. Y.; Wang, G. X.; Li, L.; Liu, G. Parallel
solution-phase synthesis of 4H-Benzo[1,4]thiazin-3-one and 1,1-
(34) Tritsch, D.; Hemmerlin, A.; Rohmer, M.; Bach, T. J. A sensitive
radiometric assay to measure D-xylulose kinase activity. J. Bio-
chem. Biophys. Methods 2004, 58, 75–83.
(35) Park, Y. W.; Cummings, R. T.; Wu, L.; Zheng, S.; Cameron, P. M.;
Woods, A.; Zaller, D. M.; Marcy, A. I.; Hermes, J. D. Homo-
geneous proximity tyrosine kinase assays: scintillation proximity
assay versus homogeneous time-resolved fluorescence. Anal. Bio-
chem. 1999, 269, 94–104.
(36) Tagliati, F.; Bottoni, A.; Bosetti, A.; Zatelli, M. C.; degli Uberti, E.
C. Utilization of luminescent technology to develop a kinase
assay: Cdk4 as a model system. J. Pharm. Biomed. Anal. 2005,
39, 811–814.
(37) Baki, A.; Bielik, A.; Molnar, L.; Szendrei, G.; Keseru, G. M.
A High throughput luminescent assay for glycogen synthase
kinase-3β inhibitors. Assay Drug Dev. Technol. 2007, 5, 75–83.
(38) Chapman, E.; Wong, C. H. A pH sensitive colorometric assay for
the high-throughput screening of enzyme inhibitors and substrates:
a case study using kinase. Bioorg. Med. Chem. 2002, 10, 551–555.
dioxo-1,4-dihydro-2H-1λ6-benzo[1,4]thiazin-3-one
derivatives
from 1,5-difluoro-2,4-dinitrobenzene. J. Comb. Chem. 2007, 9,
652–660.
(22) Yuan, Y. Y.; Liu, G.; Li, L.; Wang, Z. G.; Wang, L. Synthesis
of diverse benzo[1,4]oxazin-3-one-based compounds using 1,5-
difluoro-2,4-dinitrobenzene. J. Comb. Chem. 2007, 9, 158–170.
(23) Yang, T. M.; Liu, G. Solution-phase parallel synthesis of 3,5,6-
substituted indolin-2-ones. J. Comb. Chem. 2007, 9, 86–95.
(24) Liu, G.; Li, L.; Kou, B. B.; Zhang, S. D.; Zhang, L.; Yuan, Y. Y.;
Ma, T.; Shang, Y.; Li, Y. C. Benzofused tricycles based on
2-quinoxalinol. J. Comb. Chem. 2007, 9, 70–78.
(25) Kou, B. B.; Zhang, F.; Yang, T. M.; Liu, G. Simultaneous solid-
phase synthesis of quinoxalinone and benzimidazole scaffold
libraries. J. Comb. Chem. 2006, 8, 841–847.
(26) Zhang, J.; Zhang, L.; Zhang, S. D.; Liu, G. Solution-phase parallel
synthesis of
ꢀ
a
1,2,7-trialkyl-1H-imidazo[4,5-g]quinoxalin-6-ol
(39) Wang, Z.; Levy, R.; Fernig, D. G.; Brust, M. Kinase-catalyzed
library scaffold. J. Comb. Chem. 2005, 7, 657–664.
(27) Li, L.; Liu, G. Multistep parallel synthesis of substituted 5-amino-
benzimidazoles in solution phase. J. Comb. Chem. 2004, 6, 811–821.
(28) Zhang, L.; Liu, G. Parallel approach for solution-phase synthesis
of 2-quinoxalinol analogues and their inhibition of LPS-induced
TNF-R release on mouse macrophages in vitro. J. Comb. Chem.
2004, 6, 431–436.
(29) Wu, X. H.; Liu, G. Solution-phase reductive cyclization of
2-quinoxalinol analogs: systematic study of parallel synthesis..
Mol. Diversity 2004, 8, 165–174.
(30) Jia, Y.; Quinn, C. M.; Gagnon, A. I.; Talanian, R. Homogeneous
time-resolved fluorescence and its applications for kinase assays in
drug discovery. Anal. Biochem. 2006, 356, 273–281.
(31) Seethala, R.; Menzel, R. A homogeneous, fluorescence polari-
zation assay for src-family tyrosine kinases. Anal. Biochem. 1997,
253, 210–218.
modification of gold nanoparticles: a new approach to colorimetric
kinase activity screening. J. Am. Chem. Soc. 2006, 128, 2214–2215.
(40) Greis, K. D. Mass spectrometry for enzyme assays and inhibitor
screening: an emerging application in pharmaceutical research.
Mass Spectrom. Rev. 2007, 26, 324–339.
(41) de Boer, A. R.; Lingeman, H.; Niessen, W. M. A.; Irth, H. Mass
spectrometry-based biochemical assays for enzyme inhibitor
screening. Trends Anal. Chem 2007, 26, 867–883.
(42) Ge, X.; Sirich, T. L.; Beyer, M. K.; Desaire, H.; Leary, J. A.
A strategy for the determination of enzyme kinetics using electro-
spray ionization with an ion trap mass spectrometer. Anal. Chem.
2001, 73, 5078–5082.
(43) Wu, Y. L.; Wang, L.; Jiang, X. J.; Huang, L. Studies on the
antiviral compounds III. The synthesis of aromatic R-glyoxals.
Acta Pharm. Sin. 1965, 12, 254–266.
(44) Liu, G.; Lam, K. S. “One-bead one-compound” combinatorial
library method. In Combinatorial Chemistry, Practical Approach;
Fenniri, H., Ed.; Oxford University Press: NewYork, 2000; pp 33-49.
(32) Seethala, R.; Menzel, R. A fluorescence polarization competition
immunoassay for tyrosine kinases. Anal. Biochem. 1998, 255, 257–262.