Evaluation of potential Myt1 kinase inhibitors by TR-FRET based binding assay

Article abstract of DOI:10.1016/j.ejmech.2012.06.007

In the human cell cycle, the Myt1 kinase is a crucial regulator of the G2/M transition. Because this membrane-associated kinase is hard to obtain and assay, there is a distinct lack of data so far. Here we report the derivatization of a glycoglycerolipid which was shown previously to be active in a Myt1 activity assay. These compounds were tested in a binding assay together with a set of common kinase inhibitors against a full-length Myt1 expressed in a human cell line. Dasatinib exhibited nanomolar affinity whereas broad coverage inhibitors such as sunitinib and staurosporine derivatives did not show any effect. We also carried out docking studies for the most potent compounds allowing further insights into the inhibitor interaction of this kinase. The glycoglycerolipids showed no significant effects in the binding assay, endorsing the idea of a mechanism of action distant from the active site.

Full text of DOI:10.1016/j.ejmech.2012.06.007

European Journal of Medicinal Chemistry 61 (2013) 41e48
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Evaluation of potential Myt1 kinase inhibitors by TR-FRET based binding assay
Alexander Rohe a, Christiane Göllner a, Kanin Wichapong a, Frank Erdmann a, Ghassab M.A. Al-Mazaideh a,
,
,
*
Wolfgang Sippl a b, Matthias Schmidt a
a Department of Medicinal Chemistry, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, W.-Langenbeck-Str.4, 06120 Halle, Germany
b Freiburg Institute for Advanced Studies (FRIAS), University Freiburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In the human cell cycle, the Myt1 kinase is a crucial regulator of the G2/M transition. Because this
membrane-associated kinase is hard to obtain and assay, there is a distinct lack of data so far. Here we
report the derivatization of a glycoglycerolipid which was shown previously to be active in a Myt1
activity assay. These compounds were tested in a binding assay together with a set of common kinase
inhibitors against a full-length Myt1 expressed in a human cell line. Dasatinib exhibited nanomolar
affinity whereas broad coverage inhibitors such as sunitinib and staurosporine derivatives did not show
any effect. We also carried out docking studies for the most potent compounds allowing further insights
into the inhibitor interaction of this kinase. The glycoglycerolipids showed no significant effects in the
binding assay, endorsing the idea of a mechanism of action distant from the active site.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 31 January 2012
Received in revised form
31 May 2012
Accepted 3 June 2012
Available online 12 June 2012
Keywords:
Protein kinase Myt1
Binding assay
Inhibitor
Organic synthesis
Docking
1. Introduction
as Thr14. Abrogation of the G2 checkpoint by selective inhibition of
Wee1 resulted in a sensitization of p53-deficient tumor cells to
The cell cycle includes two checkpoints which provide the cell
with opportunities to monitor genomic integrity. These check-
points can delay the cell cycle progress and allow cells to repair
damaged DNA in order to prevent it from being passed on to
daughter cells. Many cancer cell lines show deficient G1 checkpoint
mechanisms, thus relying on the G2 checkpoint far more than
normal cells [1,2]. For this reason, G2 checkpoint abrogation is
a promising concept to damage cancerous cells in a preferential
way [3]. The main feature influencing the decision to enter mitosis
is a complex composed of Cdk1 and cyclin B. Cdk1 exists in
a monomeric form and is inactive as a kinase. Association with
cyclin B in S- and G2-phase renders the heterodimeric kinase
active. Once activated, the Cdk-cyclin complex can phosphorylate
hundreds of substrate proteins, finally leading to mitosis [4,5].
Cdk1/CycB is regulated by various feedback mechanisms of which
inhibitory phosphorylations at Thr14 and Tyr15 by the Wee family
kinases are considered essential [6]. Wee1 phosphorylates Cdk1
specifically at Tyr15 whereas Myt1 is dual-specific for Tyr15 as well
DNA-damaging agents [7] and was also shown to have single-agent
anti-tumor activity against sarcoma cells [8]. Indeed, pushing the
cell cycle forward into mitosis might be a more effective strategy to
treat cancer than stagnation of the cell cycle, though the optimal
target, be it Wee1, Myt1 or another, is not yet clear [9]. Myt1
downregulation by RNA interference did not lead to a significant
augmentation of doxorubicin effects. However, inhibition by a small
molecule inhibitor is proposed to be a promising option because
Myt1 binds Cdk1, altering equilibria and, thus, affecting G2/M
transition [10]. In addition, selective Myt1 inhibitors could help
analyze contributions of the various components involved in the
mitotic entry network [6]. Wee1 as a soluble protein is well
investigated and has been established in many kinase panels for
tests of potential inhibitors (e.g. Bruyère et al. [11]). In contrast,
Myt1 as a membrane associated kinase [12] is much more difficult
to prepare and test.
To the best of our knowledge, full-length Myt1 has never before
been tested against a set of common kinase inhibitors. Using
a coupled Cdk1 e Wee kinase activity assay, Wang et al. reported
the G2 checkpoint abrogator PD0166285 to be a potent ATP-
competitive inhibitor [13]. This compound proved to have strong
nanomolar effects on Wee1 and Myt1 in vivo and in vitro, but the
inhibitor is three times more selective for Wee1 and shows,
moreover, inhibitory properties against many other tyrosine
kinases such as c-Src, epidermal growth factor receptor, and
Abbreviations: BCA, bicinchoninic acid; Cdk1, cyclin dependent kinase 1; CycB,
cyclin B; DMSO, dimethylsulfoxide; DTT, dithiothreitol; GGL, glycoglycerolipid;
HEK, human embryonic kidney; HR-MS, high resolution mass spectrometry; TR-
FRET, time-resolved fluorescence resonance energy transfer.
* Corresponding author. Tel.: þ49 345 55 25188; fax: þ49 345 55 27018.
E-mail address: Matthias.schmidt@pharmazie.uni-halle.de (M. Schmidt).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.007

42
A. Rohe et al. / European Journal of Medicinal Chemistry 61 (2013) 41e48
platelet-derived growth factor receptor [14,15]. A natural product
originally derived from marine algae was reported to have strong
Due to reasons of data compression we herein report data for final
products only.
inhibitory effects on Myt1 (IC50 0.12
m
g/ml) and, additionally,
Mass spectra (MS) were recorded off-line with nano-ESI (Prox-
eon emitters, Odense, Denmark) on a LTQ-Orbitrap XL (Thermo
Fisher Scientific, San Jose, USA). 1H NMR and 13C NMR spectra were
recorded on a Varian Gemini 2000 and a Varian Inova 500;
chemical shifts were referenced to residual solvent signals and
showed high selectivity over other kinases such as Akt and Chk1
[16]. Chemically, the isolated product is a glycoglycerolipid (GGL),
whose scaffold is totally different from that of conventional
inhibitors. We synthesized the respective GGL and performed
a systematic derivatization to exclude unspecific effects and to
investigate the actual mechanism.
In addition to the unknown inhibition pattern, Myt1 assay
development is hindered by a restrictive substrate acceptance [17].
Therefore, we used a TR-FRET based kinase binding assay [18] to
evaluate potential inhibitors.
reported in ppm (d). Chromatography was performed on silica gel
(Merck silica gel 60, 40e63 mesh) by MPLC. Therefore, columns
were prepared with a Cartridger C-670 (Büchi). Fractions were
sampled with a Fraction Collector C-660 (Büchi) by discontinuous
enhancement of polarity, for pressure we used a Pump Module C-
601 and a Pump Manager C-615 (Büchi). TLC was carried out on
silica gel plates (E. Merck 60 F254); spots were detected visually by
ultraviolet irradiation (254 nm) or by spray detection (solution of
0.5 g thymol and 5 ml conc. sulfuric acid in 100 ml ethanol) and
heated to 130 ꢀC for 10 min. All reagents were used as purchased
unless stated otherwise. Solvents were dried according to standard
procedures. All reactions were carried out under an atmosphere of
dry argon. All chemicals were purchased from Sigma-Aldrich
(Schnelldorf, Germany).
2. Chemistry
2.1. Organic synthesis
2.1.1. Strategy
A collection of GGLs based on the reported Myt1 inhibitor
was synthesized. Previously, we described the neosynthesis
of 1,2-dipalmitoyl-3-(N-palmitoyl-60-amino-60-deoxy-
a
-D
-gluco-
2.1.2. Characterization of final products
2.1.2.1. 1,2-Dipalmitoyl-3-(N-palmitoyl-60-amino-60-deoxy-
a-D-glu-
syl)-sn-glycerol (1a) in a multistep strategy starting from a-meth-
ylglucopyranoside [19]. Based on these results, we altered the
hexopyranose core from the glucoside to the epimeric mannoside
and galactoside with respect to the anomeric configuration.
Furthermore, using the glucoside as a starting point, the palmitoyl
chains were shortened to octanoyl chains. Synthesis route and
conditions are displayed in Scheme 1. Briefly, the starting material
was blocked in position 6 by tritylation of the primary hydroxyl
group. After benzylation of the remaining secondary hydroxyl
groups we removed the trityl residue to convert the 6-OH group
into an azide after activation with methanesulfonyl chloride.
Demethylation in position 1 led to a glycosyl donor which reacts
after activation with (S)-1,2-isopropylideneglycerol. After hydro-
lysis of the ketal, the characteristic hydrophobic chains can be
inserted by acylation. Reduction of the azido group yielded the
respective amine that was N-acetylated to realize the third acyl
chain. Finally, catalytic hydrogenation gave the aspired products.
Identities were confirmed by NMR spectroscopic analysis and high
resolution mass spectrometry (HR-MS).
cosyl)-sn-glycerol (1
(m, 1H, eNHeCOe), 5.19e5.23 (m, 1H, sn2), 4.78 (d, 1H, JH-1/H-
a
). 1H NMR: 500 MHz, CDCl3:
d
¼ 5.85e5.89
3
¼ 3.7 Hz, H-1), 4.36 (dd, 1H, 2Jsn1/sn1 ¼11.8 Hz, 3Jsn1/sn2 ¼ 4.0 Hz,
0
2
2
3
sn1), 4.11 (dd, 1H, Jsn1/sn1 ¼ 11.8 Hz, Jsn1 /sn2 ¼ 5.7 Hz, sn10),
0
0
2
3
0
3.97e4.03 (m, 1H, H-6), 3.77 (dd, 1H, Jsn3/sn3 ¼ 10.7 Hz, Jsn2/
¼ 4.4 Hz, sn3), 3.71 (t, 1H, 3J ¼ 9.4 Hz, H-3), 3.61 (dd, 1H, 2Jsn3/
sn3
sn30
3
¼ 10.7 Hz, Jsn3 /sn2 ¼ 6.0 Hz, sn30), 3.54e3.57 (m, 1H, H-5), 3.46
0
3
3
(dd, 1H, JH-1/H-2 ¼ 3.7 Hz, JH-2/H-3 ¼ 9.4 Hz, H-2), 3.08 (t, 1H,
3J ¼ 9.4 Hz, H-4), 3.00e3.04 (m, 1H, H-60), 2.27e2.30 (m, 4H, 2ꢁ
(eOeCOeCH2e)), 2.21e2.24 (m, 2H, eNHeCOeCH2e), 1.55e1.63
(m, 6H, 3ꢁ (eCOeCH2eCH2e(CH2)12eCH3)), 1.21e1.31 (m, 72H,
3ꢁ (eCH2eCH2e(CH2)12eCH3)), 0.86 (t, 9H, J ¼ 7.0 Hz, 3ꢁ (eCH3));
13C NMR: 100 MHz, CDCl3:
d
¼ 13.4, 23.1, 24.7, 25.3, 25.7,
28.5e29.8, 32.1, 33.7, 34.7, 36.9, 39.9, 62.2, 66.6, 69.8, 70.3,
71.3,72.6, 73.6, 76.8, 99.7, 172.8, 173.3, 175.4; MALDI TOF mass
spectrum: m/z 990 (M þ Naþ). HRFABMS m/z 968.8121 [M þ Hþ];
(calcd for C57H110NO10 968.8130).
A general illustration of the compounds synthesized is shown in
Table 1. Characterization of intermediate products for 1a is already
described in Ref. [19]. In terms of the other final products, charac-
terization of intermediate compounds was carried out analogously.
2.1.2.2. 1,2-Dipalmitoyl-3-(N-palmitoyl-60-amino-60-deoxy-
cosyl)-sn-glycerol (1
). 1H NMR: 500 MHz, CDCl3:
¼ 6.68e6.72
(m, 1H, eNHeCOe), 5.32e5.36 (m, 1H, sn2), 4.58 (dd, 1H, Jsn1/
b-D-glu-
b
d
2
Scheme 1. General scheme of organic synthesis for the glycoglycerolipids starting from the a-methylglycoside in a 13 step synthesis route.

A. Rohe et al. / European Journal of Medicinal Chemistry 61 (2013) 41e48
43
Table 1
28.8e29.6, 31.9, 33.4, 34.5, 36.7, 39.8, 61.8, 66.1, 69.7, 70.2, 71.4, 72.5,
73.6, 76.7, 99.9, 172.5, 173.7, 175.2; MALDI TOF mass spectrum: m/z
990 (M þ Naþ). HRFABMS m/z 990.79492 [M þ Naþ]; (calcd for
C57H109NaNO10 990.7949).
Synthesized glycoglycerolipids. The sugar core was varied from glucose (1a, 1b) to
galactose (2a, 2b) and mannose (3a, 3b). Using the glucoside, octanoyl chains were
inserted instead of palmitoyl chains (4
a
).
5
R
2.1.2.5. 1,2-Dipalmitoyl-3-(N-palmitoyl-60-amino-60-deoxy-
ctosyl)-sn-glycerol (3
). 1H NMR: 500 MHz, CDCl3:
(m, 1H, eNHeCOe), 5.21e5.25 (m, 1H, sn2), 4.86 (d, 1H, JH-1/H-
a-D-gala-
4
NH
R
2
R
a
d
¼ 5.90e5.93
O
3
3
R
6
R
¼ 3.4 Hz, H-1), 4.30 (dd, 1H, 2Jsn1/sn1 ¼ 11.7 Hz, 3Jsn1/sn2 ¼ 3.4 Hz,
HO
0
2
1
R
2
3
sn1), 4.11 (dd, 1H, Jsn1/sn1 ¼ 11.7 Hz, Jsn1 /sn2 ¼ 5.9 Hz, sn10),
3.64e3,85 (m, 7H, H-2, H-3, H-4, H-5, H-6, sn3, sn30), 3.08e3.13 (m,
1H, H-60), 2.28e2.32 (m, 4H, 2ꢁ (eOeCOeCH2e)), 2.18 (t, 2H,
7
0
0
R
R1
R2
R3
R4
R5
Pa
P
P
P
R6
R7
DPGb
H
DPG
H
J
¼
7.3 Hz, eNHeCOeCH2e), 1.55e1.62 (m, 6H, 3ꢁ
1
1
2
2
3
3
4
a
b
a
b
a
b
a
OH
OH
H
H
H
OH
OH
H
OH
OH
OH
OH
H
H
H
H
H
OH
OH
H
H
DPG
H
DPG
H
DPG
H
(eCOeCH2eCH2e(CH2)12eCH3)), 1.21e1.30
(m,
72H,
3ꢁ
(eCH2eCH2e(CH2)12eCH3)), 0.86 (t, 9H, J ¼ 7.3 Hz, 3ꢁ (eCH3)); 13
C
NMR: 100 MHz, CDCl3:
d
¼ 13.5, 23.5, 24.6, 25.1, 25.6, 28.4e29.7,
H
32.0, 33.7, 34.7, 36.8, 39.6, 62.0, 66.3, 69.9, 70.7, 71.1, 72.4, 73.8,
OH
OH
OH
P
DPG
H
76.6, 99.7, 172.7, 173.3, 175.4; MALDI TOF mass spectrum: m/z 990
H
H
H
P
Naþ). HRFABMS m/z 968.8125 [M
þ
Hþ]; (calcd for
OH
Cc
DCGd
(M
þ
a
b
c
C57H110NO10 968.8130).
P: palmitoyl.
DPG: 1,2-dipalmitoyl-sn-glycerol.
C: capryloyl.
2.1.2.6. 1,2-Dipalmitoyl-3-(N-palmitoyl-60-amino-60-deoxy-
ctosyl)-sn-glycerol (3
). 1H NMR: 500 MHz, CDCl3:
(m, 1H, eNHeCOe), 5.25e5.30 (m, 1H, sn2), 4.35 (dd, 1H, Jsn1/
b-D-gala-
d
DCG: 1,2-dicapryloyl-sn-glycerol.
b
d
¼ 6.28e6.32
2
¼11.9 Hz, 3Jsn1/sn2 ¼ 3.4 Hz, sn1), 4.32 (d, 1H, 3JH-1/H-2 ¼ 7.6 Hz,
¼ 11.9 Hz, Jsn1/sn2 ¼ 2.1 Hz, sn1), 4.19e4.22 (m, 2H, H-1, sn10),
3
sn10
sn10
2
H-1), 3.99e4.11 (m, 2H, H-6, sn10), 3.74e3.83 (m, 2H, sn3, sn30),
3.57 (t, 1H, 3J ¼ 9.2 Hz, H-3), 3.35 (t, 1H, 3J ¼ 9.2 Hz, H-2), 3.22e3.27
(m, 1H, H-5), 3.13 (t, 1H, 3J ¼ 9.2 Hz, H-4), 3.04e3.09 (m, 1H, H-60),
2.23e2.33 (m, 6H, 3ꢁ (eOeCOeCH2e)), 1.50e1.63 (m, 6H, 3ꢁ
(eCOeCH2eCH2e(CH2)12eCH3)), 1.20e1.31 (m, 72H, 3ꢁ
3.80e3.86 (m, 3H, H-4, H-6, sn3), 3.73 (dd, 1H, Jsn3/sn3 ¼ 10.7 Hz,
0
3
3
0
0
Jsn2/sn3 ¼ 6.7 Hz, sn3 ), 3.57e3.61 (m, 1H, H-2), 3.54 (dd, 1H, JH-2/H-
3
¼ 9.2 Hz, JH-3/H-4 ¼ 2.1 Hz, H-3), 3.47e3.50 (m, 1H, H-5),
3
3.24e3.29 (m, 1H, H-60), 2.27e2.32 (m, 4H, 2ꢁ (eOeCOeCH2e)),
2.22 (t, 2H, J ¼ 7.3 Hz, eNHeCOeCH2e), 1.56e1.62 (m, 6H, 3ꢁ
(eCH2eCH2e(CH2)12eCH3)), 0.86 (t, 9H, J ¼ 6.7 Hz, 3ꢁ (eCH3)); 13
C
(eCOeCH2eCH2e(CH2)12eCH3)), 1.20e1.31
(m,
72H,
3ꢁ
NMR: 100 MHz, CDCl3:
d
¼ 13.1, 23.4, 24.6, 25.2, 25.8, 28.3e29.7,
(eCH2eCH2e(CH2)12eCH3)), 0.86 (t, 9H, J ¼ 7 Hz, 3ꢁ (eCH3)); 13C
32.2, 33.5, 34.8, 36.7, 40.0, 62.1, 66.3, 69.6, 70.1, 71.4, 72.4, 73.5,
76.7, 99.3, 172.9, 173.3, 175.1; MALDI TOF mass spectrum: m/z 990
(M þ Naþ). HRFABMS m/z 968.81242 [M þ Hþ]; (calcd for
C57H110NO10 968.8130).
NMR: 100 MHz, CDCl3:
d
¼ 13.8, 23.2, 24.7, 25.3, 25.7, 28.5e29.8,
32.2, 32.7, 34.3, 36.9, 39.9, 62.2, 66.6, 69.8, 70.3, 71.3,72.6, 73.6,
76.8, 99.6, 172.5, 173.4, 175.1; MALDI TOF mass spectrum: m/z 990
(M þ Naþ). HRFABMS m/z 990.79577 [M þ Naþ]; (calcd for
C57H109NaNO10 990.7949).
2.1.2.3. 1,2-Dipalmitoyl-3-(N-palmitoyl-60-amino-60-deoxy-
nosyl)-sn-glycerol (2
). 1H NMR: 500 MHz, CDCl3:
a-D-man-
¼ 6.04e6.07
a
d
2.1.2.7. 1,2-Diocteoyl-3-(N-octeoyl-60-amino-60-deoxy-
sn-glycerol (4
). 1H NMR: 500 MHz, CDCl3:
eCH2eNHeCOe), 5.19e5.23 (m, 1H, sn2), 4.78 (d, 1H, JH-1/H-
a-D-glucosyl)-
¼ 5.88e5.93 (m, 1H,
(m, 1H, eNHeCOe), 5.15e5.18 (m, 1H, sn2), 4.77 (s, 1H, H-1), 4.30
a
d
(dd, 1H, 2Jsn1/sn1 ¼11.7 Hz, Jsn1/sn2 ¼ 3.9 Hz, sn1), 4.10 (dd, 1H, 2Jsn1/
3
3
0
¼ 11.7 Hz, 3Jsn1 /sn2 ¼ 5.9 Hz, sn10), 4.01e4.06 (m, 1H, H-6), 3.95
¼ 4 Hz, H-1), 4.36 (dd, 1H, 2Jsn1/sn1 ¼11.9 Hz, Jsn1/sn2 ¼ 4 Hz, sn1),
3
sn10
2
0
0
(d, 1H, 3J ¼ 2.6 Hz, H-2), 3.84 (dd, 1H, JH-2/H-3 ¼ 2.9 Hz, JH-3/H-
4.11 (dd, 1H, 2Jsn1/sn1 ¼ 11.9 Hz, 3Jsn1 /sn2 ¼ 5.9 Hz, sn10), 3.99 (ddd,
3
3
0
0
¼ 9.3 Hz, H-3), 3.73 (dd, 1H, 2Jsn3/sn3 ¼ 10.7 Hz, 3Jsn2/sn3 ¼ 4.9 Hz,
1H, JH-5/H-6 ¼ 2.6 Hz, JeNHe/H-6 ¼ 8.2 Hz, JH-6/H-6 ¼ 15 Hz, H-6),
3
3
2
0
0
4
2
3
2
3
sn3), 3.56 (dd, 1H, Jsn3/sn3 ¼ 10.7 Hz, Jsn2/sn3 ¼ 5.4 Hz, sn30),
3.50e3.53 (m, 1H, H-5), 3.43 (t, 1H, 3J ¼ 9.3 Hz, H-4), 2.98e3.03 (m,
1H, H-60), 2.22e2.30 (m, 6H, 3ꢁ (eCOeCH2e)),1.55e1.64 (m, 6H, 3ꢁ
3.77 (dd, 1H, Jsn3/sn3 ¼ 11 Hz, Jsn2/sn3 ¼ 4.8 Hz, sn3), 3.71 (t, 1H,
0
0
3J ¼ 9.5 Hz, H-3), 3.61 (dd, 1H, 2Jsn3/sn3 ¼ 11 Hz, Jsn2/sn3 ¼ 5.9 Hz,
3
0
0
3
3
3
sn30), 3.56 (ddd, 1H, JH-4/H-5 ¼ 9.5 Hz, JH-5/H-6 ¼ 2.6 Hz, JH-5/H-
0
(eCOeCH2eCH2e(CH2)12eCH3)), 1.21e1.30
(m,
72H,
3ꢁ
¼ 2.9 Hz, H-5), 3.46 (dd, 1H, 3JH-1/H-2 ¼ 4.0 Hz, 3JH-2/H-3 ¼ 9.5 Hz, H-
6
(eCH2eCH2e(CH2)12eCH3)), 0.86 (t, 9H, J ¼ 6.8 Hz, 3ꢁ (eCH3)); 13
C
2), 3.08 (t, 1H, 3J ¼ 9.5 Hz, H-4), 3.03 (ddd, 1H, JH-5/H-6 ¼ 2.9 Hz,
3
0
3
2
JeNHe/H-6 ¼ 4.4 Hz, JH-6/H-6 ¼ 15 Hz, H-60), 2.60 (bs, 3H, 3ꢁ
0
0
NMR: 100 MHz, CDCl3:
d
¼ 13.3, 23.2, 24.8, 25.2, 25.7, 28.5e29.7,
32.3, 33.6, 34.9, 36.4, 39.7, 62.0, 66.4, 69.7, 70.0, 71.1,72.5, 73.5,
76.8, 99.6, 172.5, 173.7, 175.2; MALDI TOF mass spectrum: m/z 990
(M þ Naþ). HRFABMS m/z 990.7900 [M þ Naþ]; (calcd for
C57H109NaNO10 990.7949).
(eOH)), 2.27e2.31 (m, 4H, 2ꢁ (eCOeCH2e(CH2)5eCH3)), 2.21e2.24
(m, 2H, eNHeCOeCH2e(CH2)5eCH3), 1.56e1.64 (m, 6H, 3ꢁ
(eCH2eCH2e(CH2)4eCH3)),
1.22e1.30
(m,
24H,
3ꢁ
(e(CH2)2e(CH2)4eCH3), 0.84e0.87 (m, 9H, 3ꢁ (eCH3)); 13C NMR:
100 MHz, CDCl3:
d
¼ 14.0, 22.6, 24.8, 24.9, 25.6, 28.9e29.7, 31.6,
2.1.2.4. 1,2-Dipalmitoyl-3-(N-palmitoyl-60-amino-60-deoxy-
nosyl)-sn-glycerol (2
). 1H NMR: 500 MHz, CDCl3:
1H, eNHeCOe), 5.30e5.34 (m, 1H, sn2), 4.55 (dd, 1H, Jsn1/
b-
D
-man-
¼ 6.69e6.73 (m,
34.1, 34.2, 36.4, 39.8, 62.1, 66.9, 69.9, 70.1, 71.1, 72.4, 73.1, 76.7, 99.4,
173.2, 173.4, 175.7; MALDI TOF mass spectrum: m/z 655 (M þ Naþ).
HRFABMS m/z 654.41877 [M þ Naþ]; (calcd for C33H61NaNO10
654.4193).
b
d
2
¼12.1 Hz, 3Jsn1/sn2 ¼ 2.9 Hz, sn1), 4.50 (s, 1H, H-1), 3.96e4.07 (m,
sn10
3H, H-2, H-6, sn10), 3.76e3.84 (m, 2H, sn3, sn30), 3.59 (dd, 1H, 3JH-2/H-
¼ 2.6 Hz, JH-3/H-4 ¼ 9.5 Hz, H-3), 3.48 (t, 1H, 3J ¼ 9.5 Hz, H-4),
2.2. Computational chemistry: ligand preparation and molecular
docking study
3
3
3.12e3.18 (m, 2H, H-5, H-60), 2.24e2.32 (m, 6H, 3ꢁ (eCOeCH2e)),
1.55e1.64 (m, 6H, 3ꢁ (eCOeCH2eCH2e(CH2)12eCH3)), 1.20e1.31
(m, 72H, 3ꢁ (eCH2eCH2e(CH2)12eCH3)), 0.86 (t, 9H, J ¼ 7 Hz, 3ꢁ
All ligands were constructed using MOE2010.10 [20] and energy
minimized using the MMFF94 force field with a convergence
(eCH3)); 13C NMR: 100 MHz, CDCl3:
d
¼ 13.8, 23.3, 24.5, 25.6, 25.9,

44
A. Rohe et al. / European Journal of Medicinal Chemistry 61 (2013) 41e48
criteria of 0.01 kcal/mol. The protonation state of the ligand was
assigned at pH 7.1 using the protonated-3D module implemented
in MOE2010.10. Dasatinib and PD0166285 were docked into the
binding pocket of the X-ray structure of a truncated Myt1 (PDB code
3P1A, active form of Myt1) using GOLD version 5.0 [21,22].
According to our previous studies [23,24], GOLD is able to correctly
predict the binding mode of Wee1 kinase inhibitors. Moreover, we
were able to dock the pyridopyrimidine derivatives, which have the
same scaffold as PD0166285, into the binding pocket of Wee1
kinase [23]. Since Wee1 and Myt1 show a high structural similarity,
we used the same protocol as applied for Wee1 kinase docking for
Myt1 kinase docking. We defined Cys190 located at the hinge
region as center of the binding site with a radius of 20 Å. Beside free
docking we tested also the influence of protein hydrogen bond
constraints to the hinge region residues Glu188 and Cys190
(backbone heteroatoms). Water molecules found at the binding
pocket of Myt1 were also applied for docking using ‘toggle’ mode in
GOLD.
dasatinib (7) and tyrphostin AG 1478 (6). However, binding of the
latter was too weak to determine IC50 values due to limitations of
the assay system (see Discussion). Dasatinib bound to Myt1 with an
IC50 of about 63 nM (Fig. 1).
The positive control PD0166285 had an IC50 of 7.1 nM in this
assay. The chemical structures of compounds which affected Myt1
are shown in Fig. 2. The scaffold of PD0166285 and tyrphostin AG
1478 show a high degree of similarity.
The potent inhibitors were subjected to further in silico studies
to investigate their potential interaction at the kinase binding site.
3.2. Docking studies
3.2.1. Examination of the Myt1 ATP binding pocket
We first analyzed the X-ray structure of Myt1 (PDB Code 3P1A)
and Wee1 (PDB code 1X8B). Even though the sequence identity
between Wee1 and Myt1 is rather low (35.3%) [17], the overall 3D-
structures, especially the binding pocket, are quite similar as shown
in Fig. 3.
Root mean square deviation (RMSD) between these two struc-
tures is 1.73 Å (backbone atoms). Therefore, it is reasonable to use
the same protocol for Myt1 docking as for Wee1 docking. As dis-
cussed previously [24], the X-ray structures of Wee1-inhibitor
complexes reveal the interaction between inhibitors and the resi-
dues Glu377 and Cys379 located at the hinge region. It is well
known from other kinases that the residues at the hinge region play
an important role for interacting with ATP-competitive inhibitors
or ATP. Thus, the key residues where inhibitors and ATP bind are
Glu188, Cys190 and the gatekeeper residue of Myt1 (Thr187) as
displayed in Fig. 3.
3. Results
3.1. Set of common kinase inhibitors
Because common kinase inhibitors have not been tested on
human Myt1 kinase so far, we used known pan-kinase inhibitors
such as staurosporine as well as more selective tyrosine kinase
inhibitors which have been approved and marketed such as lapa-
tinib and imatinib to obtain a first inhibition profile.
All compounds were tested three times at two different
concentrations. Any substance binding >50% at 10
mM was sub-
jected to further testing and IC50 values were determined. Quan-
titative data is expressed as mean ꢂ standard error. Curves were
fitted to the data by GraphPad Prism 5.01 (San Diego, CA) using
sigmoidal dose response with a variable slope. PD0166285 (5) was
used as a positive control. Compounds and respective effects at
a concentration as indicated are displayed in Table 2.
Most of the tested compounds did not affect Myt1. The kinase
was insensitive even towards highly promiscuous inhibitors such as
staurosporine and bisindolylmaleimide I. The effects of erlotinib
and gefitinib were negligible. Major effects could be determined for
3.2.2. Molecular docking
In our previous work [23], we were able to dock the pyr-
idopyrimidine derivatives into the binding pocket of Wee1 kinase.
The docking results showed a conserved binding, namely an H-
bond interaction between the NH and N atom of the inhibitor’s
pyrimidine ring (the main scaffold) with the Cys379 residue. For the
Myt1 kinase, the docking also showed the same binding mode, as
displayed in Fig. 4A. The H-bond interactions were found between
the NH atom and the pyrimidine ring of the inhibitor with Cys190.
The benzene ring with the chloro substituent at the ortho position
is located at the hydrophobic pocket. There is also another inter-
action between the benzene ring of the inhibitor with Pro192 and
Leu116, as shown in Fig. 4B.
Table 2
Test of common kinase inhibitors for effects on human full-length Myt1 kinase.
Binding of the respective compound is reported in [relative % tracer displaced].
Dasatinib is a well-known kinase inhibitor, and several X-ray
structures of kinases (active conformation) complexed with dasa-
tinib are available in the Protein Data Bank; for example PDB code
2GQG (Abl kinase), 3G5D (c-Src kinase), and 3K54 (Btk kinase).
Compound
Displacement
Displacement
IC50 [nM]
n.t.b
[%] at 5
n.d.a
m
M
[%] at 10
mM
Bisindolyl-
maleimide I
Dasatinib
Erlotinib
Gefitinib
HA-1077
Imatinib
n.d.
95.5 ꢂ 0.3
7.05 ꢂ 1.65
n.d.
97.1 ꢂ 0.3
63.0 ꢂ 1.1
8.15 ꢂ 0.65
6.85 ꢂ 2.35
n.d.
n.t.
n.t.
n.t.
n.d.
n.d.
n.d.
n.t.
K252a
n.d.
n.d.
n.t.
Lapatinib
PD0166285
Midostaurin
SB 203580
Staurosporine
Sunitinib
Tyrphostin
AG 1478
n.d.
n.t.
n.d.
n.d.
n.t.
n.d.
24.8 ꢂ 0.7
n.d.
n.t.
n.d.
n.d.
n.t.
n.d.
39.5 ꢂ 1.5
n.t.
7.2 ꢂ 1.1
n.t.
n.t.
n.d. up to 10 mM
n.t.
n.t.
U0126
Vatalanib
7.55 ꢂ 0.65
n.d.
n.d.
n.t.
n.t.
n.d.
a
n.d.: no displacement (<5%) at the specified assay concentration.
n.t.: not tested.
Fig. 1. Binding of dasatinib to Myt1. Data expressed as mean ꢂ standard error and
reported in [relative % tracer displaced]. The IC50 was determined 63.0 ꢂ 1.1 nM.
b

A. Rohe et al. / European Journal of Medicinal Chemistry 61 (2013) 41e48
45
Fig. 2. Chemical structures of the compounds affecting Myt1 in a significant manner: 5 (PD0166285), 6 (Tyrphostin AG 1478) and 7 (Dasatinib).
Superimposition of these X-ray structures (Fig. 5) reveals that
dasatinib not only forms H-bonds with residues at the hinge region
but also with the gatekeeper residue.
Our molecular docking results for dasatinib and Myt1 (PDB code
3P1A, active conformation) as displayed in Fig. 6A also yielded the
same binding mode as for the other protein kinases. H-bond
interactions were found between NH and the N atom of the thiazole
ring with Cys190, and between the NH atom with Thr186 (gate-
keeper residue for Myt1). The additional interaction between
dasatinib and Pro192 were also found as shown in Fig. 6B.
4. Discussion
The restrictive substrate acceptance of the human Myt1 kinase
led us to use a binding assay instead of an activity assay. In general,
the use of binding assays in drug discovery is not necessarily
accompanied by disadvantages. It has been shown that data gained
by a kinase binding assay may provide a better correlation to
cellular effects than a conventional enzyme activity assay. More-
over, the identification of hits targeting inactive kinase conforma-
tions is strongly facilitated compared to activity assays [18,25].
Even the largest available kinase panels contain only a fraction
of all 518 kinases of the human kinome, and often the kinase
domain only instead of the full-length protein. In addition, the
panels are often highly biased by containing mostly kinases that
can be easily purified and assayed. Additionally, as these kinases are
typically purified from non-mammalian cells, they do not contain
their naturally occurring post-translational modifications [26]. We
provide data for a difficult to obtain membrane-associated kinase
which is, to our best knowledge, not commercially available as an
enzymatically active protein. This kinase is of special interest
because it is involved in pathways of major importance for devel-
opment and survival of many cancer cells.
3.3. Biological evaluation of the glycoglycerolipids
The GGLs were tested three times in the kinase binding assay at
3
mg/ml and 30
mg/ml. Table 3 summarizes the results.
No effects on Myt1 could be determined. Only 2
a
showed
a negligible displacement of the kinase tracer at 30
mg/ml.
Staurosporine was found not to bind to Myt1. This result
supports a recent study with the Myt1 kinase domain which was
expressed in E. coli [27] and is, furthermore, cross-validated by the
fact that the staurosporine derivatives midostaurin and K252a did
not show any effect in concentrations up to 10
mM either. Also other
compounds which usually inhibit a broad range of kinases (e.g.
sunitinib) did not affect Myt1. Because assay development requires
positive controls, i.e. known inhibitors, these unusual inhibition
properties may be jointly responsible for the lack of data so far.
Higher concentrations of negative compounds could not be tested
because autofluorescent compounds can serve as FRET acceptors
via a non-specific diffusion enhanced FRET mechanism and there-
fore interfere by increased background signal and narrowed assay
window [18,28]. According to our experience, the presence of
compounds containing aromatic systems, as are present in most
kinase inhibitors, is therefore limited to about 10 mM.
Myt1 was expressed in a human cell line, which means any
posttranslational modifications are approximately as in the actual
human target cells. The tested kinase preparation showed catalytic
activity towards Thr14 and Tyr15 of Cdk1 [17]. Additionally, due to
the fact that the epitope tag is present in the kinase of interest only,
the assay system used is insensitive to contaminating kinases.
Together with the affirmation of PD0166285 as a tightly binding
compound, our results appear valid. Previously, Myt1 was
Fig. 3. Comparison of the overall structure of Wee1 (PDB Code 1X8B) and Myt1 (PDB
Code 3P1A). The structure of Wee1 and Myt1 are shown as green and orange ribbon,
respectively. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

46
A. Rohe et al. / European Journal of Medicinal Chemistry 61 (2013) 41e48
Fig. 4. (A) GOLD docking solution of compound PD0166285 (magenta stick) in the binding pocket of Myt1 (B) Schematic representation of the interactions between compound
PD0166285 with the residues at the binding pocket of Myt1. H-bonds are displayed as dashed lines. (For interpretation of the references to color in this figure legend, the reader
referred to the web version of this article.)
suggested as a target of dasatinib using an MS-driven proteomics
approach [29]. However, this approach used a chemically altered,
immobilized dasatinib-derivative. Taking into account that former
comparable studies did not reveal Myt1 to be a target of dasatinib
[30], affirmation in a more specific way was needed. In our in vitro
test system, we found dasatinib to affect Myt1 strongly, with an IC50
value of about 63 nM, leading to the conclusion that Myt1 may
indeed be a target of dasatinib. However, the affinity of dasatinib
towards its reported primary targets, Abl and Src, lying in the
subnanomolar range [31], is much higher compared to Myt1. Future
work has to reveal whether Myt1 can be confirmed as a substantial
target also in cellular systems.
According to their binding modes, both potent inhibitors exploit
a hydrophobic pocket beyond the gatekeeper residue and not only
the regions targeted by ATP, favored by the small threonine residue
in this position. About 18% of all protein kinases contain a Thr-
gatekeeper. As this residue is not very bulky, it provides the
possibility to target the backpocket in a more specific way [32].
Therefore, systematic structure activity relationship studies are
Type III kinase inhibitors are compounds that bind exclusively to
sites other than the ATP-binding site. Due to steric reasons caused by
three fatty acid chains in the GGLs, a mechanism of action outside
the ATP-binding site is likely, making it Type III instead of ATP-
competitive. Even though most type III inhibitors do not directly
bind to the ATP site, the majority must either alter the active site in
a way that displaces the tracer or bind close to the active site. The
GGLs did not displace the tracer, although a maximum concentra-
tion up to 250-fold the reported IC50 was used. Considering the
potency of 1a in an activity assay, the conclusion can be drawn that
the effect of this class is unfolded distant from the active site. So far,
there is only one known example of a type III inhibitor which could
not be detected in the binding assay but in an activity assay. This
compound inhibited p38a depending on the substrate used [33,34].
The chemical structure of the GGLs together with the cellular
localization of Myt1, membrane-bound at the endoplasmic retic-
ulum, give rise to another thesis. All tested GGLs are very hydro-
phobic and therefore practically insoluble in water. Not until
solutizer (30% DMSO) or tenside were added could the compound
be dissolved. Therefore, these compounds are likely to interact with
more hydrophobic structures such as bilayers (membranes) and
micelles. As can be seen in Fig. 7, the membrane association motif
within the Myt1 protein sequence shows spatial proximity to both
the kinase domain and the Cdk1 interaction site [35].
needed to realize
selectivity.
a compound providing both affinity and
Because an interaction of the GGLs with the kinase domain was
not observed in our study, GGL effects on the substrate (Cdk1)
recognition of Myt1 might be responsible for their reported strong
inhibitory potential. The lone available crystal structure of Myt1
resolves the kinase domain only, which precludes further structural
studies.
From this point of view it is not surprising that the derivatized
GGLs also did not show a significant effect on Myt1, though these
results increase the validity of the study. However, a retest of these
compounds in an activity-based assay in future work is desirable to
investigate structure activity relationships and to learn about the
actual mechanism of action.
5. Conclusion
A first inhibitor profile for full-length human Myt1 was provided
and showed that Myt1 is quite hard to affect but, if so, the
compounds are far from being selective. The glycoglycerolipids as
potentially selective inhibitors are a second class of described
compounds which show a discrepancy between activity assay and
Fig. 5. Comparison of the binding mode of dasatinib (white stick) in the binding
pocket of different kinases (Abl: orange-2GQG, c-Src: magenta-3G5D, and Btk: cyan-
3K54). (For interpretation of the references to color in this figure legend, the reader
referred to the web version of this article.)

A. Rohe et al. / European Journal of Medicinal Chemistry 61 (2013) 41e48
47
Fig. 6. (A) GOLD docking solution of dasatinib (magenta stick) in the ATP-binding pocket of Myt1. (B) Schematic representation of the interactions between dasatinib and the
residues at the ATP-binding pocket of Myt1. (For interpretation of the references to color in this figure legend, the reader referred to the web version of this article.)
Table 3
was supplemented with DTT (2 mM), frozen in ethanol/dry ice and
Binding assay results of synthesized glycoglycerolipids for effect on human Myt1.
stored at ꢃ80 ꢀC. The purification was controlled by coomassie
stained SDS-PAGE analysis of all fractions. Determination of protein
concentrations was conducted via BCA-assay as described previ-
ously using BSA as protein standard [36]. Identity of Myt1 was
additionally proven via western blotting experiments (antibody
#4282; Cell Signaling).
Compound
Displacement [%] at 3
mg/ml
Displacement [%] at 30 mg/ml
1
1
2
2
3
3
4
a
b
a
b
a
b
a
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
7.53 ꢂ 0.04
n.d.
n.d.
n.d.
n.d.
6.2. Binding assay
n.d.: no displacement (<5%) at the specified assay concentration.
Tracer and Eu-labeled antibody used in this study were from
Invitrogen (Madison, WI, USA). Binding assays were performed in
384-well low volume plates (Optiplate #6007290, Perkin Elmer) at
room temperature in sterile filtered kinase buffer consisting of
50 mM HEPES (pH 7.5), 0.01% Brij-35, 10 mM MgCl2, and 1 mM
EGTA. For all experiments with a kinase inhibitor, a dilution series
of inhibitor was first prepared in 100% DMSO at 100-fold the
desired final assay concentration. From this master dilution series,
inhibitors were diluted 33.3-fold into kinase buffer, for an inter-
mediate concentration of inhibitor 3-fold of that to be used in the
binding assay. This fact emphasizes that they do not target the ATP-
site and also shows that this entire class should be evaluated in
future activity assays. The discovery of selective Myt1 inhibitors
will be of major importance to assess druggability of Myt1 and its
actual role in cells. Binding mode insights provided here may help
to develop such compounds by computer-aided drug design.
6. Experimental protocols
6.1. Kinase expression and purification
assay (containing 3% DMSO). To 5
ml of each concentration of
inhibitor to be tested, appropriate assay reagents (15 nM Myt1,
10 nM tracer, 2 nM Eu-labeled anti-His-tag antibody) were added to
bring the final volume to 15 ml and the final DMSO concentration to
1%. In general, all assays were performed with 3 replicates of each
inhibitor concentration. Assay plates were read using a Perkin
Elmer EnVision plate reader together with standard Eu-based TR-
FRET settings with 340 nm excitation wavelength and emission
monitored at 615 nm (donor) and 665 nm (acceptor). Emission
In this study, full-length Myt1 was expressed in the human cell
line HEK293. Cells were cultured in Dulbecco’s modified eagle
medium (DMEM), supplemented with 10% FCS and GlutaMAX at
37 ꢀC in a humidified incubator. Transfection was carried out with
pcDNA3.1-His-Myt1 expression plasmid using lipofectamine 2000
(Invitrogen) according to recommendations of the supplier. Cells
were harvested and resuspended in lysis buffer containing 25 mM
TriseHCl pH 7.8,150 mM NaCl, 10 mM MgCl2, 10% glycerol,1% Triton
X-100 and Complete EDTA-free protease inhibitor cocktail (Roche,
Basel, Switzerland). Cells were finally lysed by shear stress using
a 25 g needle and the lysate shaken on ice for 1 h. After centrifu-
gation at 10 000 g for 15 min, the supernatant was loaded onto
a HIS-Select column (Sigma) pre-equilibrated with buffer A (50 mM
TriseHCl pH 7.8; 150 mM NaCl; 3 mM MgCl2; 20% glycerol; 0.5%
Triton X-100). The column was washed with buffer B (buffer
A þ 5 mM Imidazole) and eluted with buffer C (buffer A þ 250 mM
Imidazole). All Steps were carried out at 4 ꢀC and the final eluate
intensities were measured over a 100 ms window following a 60 ms
postexcitation delay. A ratio of raw acceptor/donor was calculated
and normalized relative to wells containing fully bound or fully
competed tracer (results reported in relative percent tracer dis-
placed). PD0166285 was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA), Vatalanib and Erlotinib were from Chem-
ieTek (Indianapolis, IN, USA), all other kinase inhibitors were ob-
tained from LC Laboratories (Woburn, MA, USA). Sunitinib was
a kind gift from Pfizer (Groton, CT, USA). Curve fitting and data
analysis were carried out using GraphPad Prism software (Graph-
Pad Software, Inc., La Jolla, CA, USA).
Author contribution
Alexander Rohe: In vitro tests, kinase expression and purifica-
tion, preparation of manuscript. Christiane Göllner: Organic
synthesis, preparation of manuscript. Kanin Wichapong: Molecular
modeling, preparation of the manuscript. Frank Erdmann: Kinase
Fig. 7. Illustration of the full-length Myt1 sequence and respective function of selected
regions. The membrane association motif provides spatial proximity to the kinase
domain as well as the Cdk1 interaction site, rendering both functional regions possible
sites of action for the glycoglycerolipids.

48
A. Rohe et al. / European Journal of Medicinal Chemistry 61 (2013) 41e48
of PD 166285, a new nanomolar potent and broadly active protein tyrosine
kinase inhibitor, J. Pharmacol. Exp. Ther. 283 (1997) 1433e1444.
[16] B.-N. Zhou, S. Tang, R.K. Johnson, M.P. Mattern, J.S. Lazo, E.R. Sharlow,
K. Harichd, D.G.I. Kingston, New glycolipid inhibitors of Myt1 kinase, Tetra-
hedron 61 (2005) 883e887.
expression and purification. Ghassab M. A. Al-Mazaideh: Organic
synthesis. Wolfgang Sippl: Supervision, research planning, prepa-
ration of manuscript. Matthias Schmidt: Organic synthesis, super-
vision, research planning, preparation of manuscript.
[17] A. Rohe, F. Erdmann, C. Bäßler, K. Wichapong, W. Sippl, M. Schmidt, In vitro
and in silico studies on substrate recognition and acceptance of human
Acknowledgment
PKMYT1,
a Cdk1 inhibitory kinase, Bioorg. Med. Chem. Lett. 22 (2012)
1219e1223.
[18] C.S. Lebakken, S.M. Riddle, U. Singh, W.J. Frazee, H.C. Eliason, Y. Gao,
L.J. Reichling, B.D. Marks, K.W. Vogel, Development and applications of
a broad-coverage, TR-FRET-based kinase binding assay platform, J. Biomol.
Screen. 14 (2009) 924e935.
[19] C. Göllner, C. Philipp, B. Dobner, W. Sippl, M. Schmidt, First total synthesis of
1,2-dipalmitoyl-3-(N-palmitoyl-60-amino-60-deoxy-alpha-D-glucosyl)-sn-
glycerol e a glycoglycerolipid of a marine alga with a high inhibitor activity
against human Myt1-kinase, Carbohydr. Res. 344 (2009) 1628e1631.
[20] MOE 2010.10, Chemical Computing Group, Inc., Montreal, Canada, 2010.
[21] G. Jones, P. Willett, R.C. Glen, Molecular recognition of receptor-sites using
a genetic algorithm with a description of desolvation, J. Mol. Biol. 245 (1995)
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This work was supported by research funds from “Kultusmi-
nisterium des Landes Sachsen-Anhalt”. We thank Pfizer for
providing sunitinib. We thank Prof. Manfred Jung from the Albert-
Ludwigs-University Freiburg i. Br. for instrumentational support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.06.
007. These data include MOL files and InChiKeys of the most
important compounds described in this article.
[22] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and valida-
tion of a genetic algorithm for flexible docking, J. Mol. Biol. 267 (1997)
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[23] K. Wichapong, M. Lawson, S. Pianwanit, S. Kokpol, W. Sippl, Postprocessing of
protein-ligand docking poses using linear response MM-PB/SA: application to
Wee1 kinase inhibitors, J. Chem. Inf. Model. 50 (2010) 1574e1588.
[24] K. Wichapong, M. Lindner, S. Pianwanit, S. Kokpol, W. Sippl, Receptor-based
3D-QSAR studies of checkpoint Wee1 kinase inhibitors, Eur. J. Med. Chem. 44
(2009) 1383e1395.
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