Identification, structure modification, and characterization of potential small-molecule SGK3 inhibitors with novel scaffolds

Article abstract of DOI:10.1038/s41401-018-0087-6

The serum and glucocorticoid-regulated kinase (SGK) family has been implicated in the regulation of many cellular processes downstream of the PI3K pathway. It plays a crucial role in PI3K-mediated tumorigenesis, making it a potential therapeutic target for cancer. SGK family consists of three isoforms (SGK1, SGK2, and SGK3), which have high sequence homology in the kinase domain and similar substrate specificity with the AKT family. In order to identify novel compounds capable of inhibiting SGK3 activity, a high-throughput screening campaign against 50,400 small molecules was conducted using a fluorescence-based kinase assay that has a Z' factor above 0.5. It identified 15 hits (including nitrogen-containing aromatic, flavone, hydrazone, and naphthalene derivatives) with IC₅₀ values in the low micromolar to sub-micromolar range. Four compounds with a similar scaffold (i.e., a hydrazone core) were selected for structural modification and 18 derivatives were synthesized. Molecular modeling was then used to investigate the structure-activity relationship (SAR) and potential protein–ligand interactions. As a result, a series of SGK inhibitors that are active against both SGK1 and SGK3 were developed and important functional groups that control their inhibitory activity identified.

Full text of DOI:10.1038/s41401-018-0087-6

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ARTICLE
Identification, structure modification, and characterization of
potential small-molecule SGK3 inhibitors with novel scaffolds
Grace Qun Gong1,2,3, Ke Wang4, Xin-Chuan Dai1, Yan Zhou1, Rajesh Basnet1,5, Yi Chen1, De-Hua Yang1, Woo-Jeong Lee2,
Christina Maree Buchanan2,3, Jack Urquhart Flanagan3,6, Peter Robin Shepherd2,3, Ying Chen4 and Ming-Wei Wang1,4,5
The serum and glucocorticoid-regulated kinase (SGK) family has been implicated in the regulation of many cellular processes
downstream of the PI3K pathway. It plays a crucial role in PI3K-mediated tumorigenesis, making it a potential therapeutic target for
cancer. SGK family consists of three isoforms (SGK1, SGK2, and SGK3), which have high sequence homology in the kinase domain
and similar substrate specificity with the AKT family. In order to identify novel compounds capable of inhibiting SGK3 activity, a
high-throughput screening campaign against 50,400 small molecules was conducted using a fluorescence-based kinase assay that
has a Z' factor above 0.5. It identified 15 hits (including nitrogen-containing aromatic, flavone, hydrazone, and naphthalene
derivatives) with IC50 values in the low micromolar to sub-micromolar range. Four compounds with a similar scaffold (i.e., a
hydrazone core) were selected for structural modification and 18 derivatives were synthesized. Molecular modeling was then used
to investigate the structure-activity relationship (SAR) and potential protein–ligand interactions. As a result, a series of SGK
inhibitors that are active against both SGK1 and SGK3 were developed and important functional groups that control their inhibitory
activity identified.
Keywords: serum and glucocorticoid-regulated kinase; SGK3; high-throughput screening; inhibitors; molecular modeling
Acta Pharmacologica Sinica (2018) 0:1–11; https://doi.org/10.1038/s41401-018-0087-6
INTRODUCTION
phosphorylated at the threonine residue by PDK1, and mTORC2 is
proposed to be responsible for phosphorylating the serine residue
[1]. Phosphorylation at both sites is required for full activation of
SGK [2].
The serum and glucocorticoid-regulated kinase (SGK) family
consists of serine/threonine kinases that act as effectors down-
stream of the phosphatidylinositol 3-kinase (PI3K) pathway [1].
SGKs are involved in the regulation of many cellular components
including ion channels, enzymes, transporters and transcription
factors, thereby affecting a wide range of biological processes
including cell proliferation, survival, stress, and transportation of
ions and nutrients [2].
This kinase family has three highly homologous isoforms: SGK1,
SGK2, and SGK3 encoded by the SGK1, SGK2, and SGK3 genes,
respectively [3, 4]. SGKs have three domains, an N-terminal
variable domain, a catalytic domain and a C-terminal hydrophobic
domain [2, 5, 6]. The catalytic domain and C-terminal hydrophobic
domain can also be collectively referred to as the kinase domain.
SGK shares a similar domain structure and has high sequence
identity in the kinase domain with the AKT family of proteins;
therefore, they also have similar substrate specificities with AKT [2,
3, 7]. The SGK family is regulated by phosphorylation downstream
of the PI3K pathway and has two phosphorylation motifs [8]:
a threonine residue in the activation loop of the catalytic
domain (T320 in SGK3), and a serine residue in the hydrophobic
motif of the C-terminal domain (S486 in SGK3) [2, 3]. SGK is
The role of the PI3K/AKT signaling pathway in cancer
development has been well known for many decades, however
in recent years, studies have shown that a subset of tumors is
dependent on PI3K and PDK1 but not AKT signaling [9–11].
Among these downstream effectors, SGK isoforms are proposed to
be important for tumorigenesis, especially given the structural and
substrate similarity between SGK and AKT [2]. In a screen to
identify PIK3CA mutant tumors with minimal activation of AKT,
SGK3 was identified as a PDK1 substrate that mediated increased
cell viability [12]. It was shown that forced expression of SGK3 can
increase cell growth and anchorage independent growth in
hepatocellular carcinoma [13]. SGK3 is also somehow linked with
estrogen-dependent breast cancer [14] as well as androgen-
mediated prostate cancer [15]. In addition, SGK1 is also implicated
in many cancers [16–18], and is involved in cell growth and
survival regulation [19–21]. SGK activation is thus proposed to be a
mechanism of resistance to AKT inhibitors, as breast cancer cell
lines with high expression levels of SGK1 were resistant to AKT
inhibition [17]. Altogether, these demonstrate a role of the SGK
1The National Center for Drug Screening and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai
201203, China; 2Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand; 3The Maurice Wilkins Centre, Auckland, New Zealand;
4School of Pharmacy, Fudan University, Shanghai 201203, China; 5University of Chinese Academy of Sciences, Beijing 100049, China and 6Auckland Cancer Society Research
Centre, Auckland, New Zealand
Correspondence: Ying Chen (yingchen71@fudan.edu.cn) or Ming-Wei Wang (mwwang@simm.ac.cn)
These authors contributed equally: Grace Qun Gong, Ke Wang, Xin-Chuan Dai.
Received: 11 April 2018 Accepted: 10 June 2018
©
CPS and SIMM 2018

Identification of potential small-molecule SGK3 inhibitors
GQ Gong et al.
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family in PI3K-mediated tumorigenesis, making it a potential
therapeutic target for cancer.
protein S6 (pSer235/236) peptide were purchased from PerkinEl-
mer (Boston, MA, USA). MgCl2, EGTA, EDTA, Tris, and Tween-20
were bought from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). Dimethyl sulfoxide (DMSO) and dithiothreitol
(DTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The
SGK3 recombinant human protein used for the HTS campaign was
procured from Life Technologies (Carlsbad, CA, USA), and the
assay plates (ProxiPlate-384 F Plus) were acquired from PerkinEl-
mer. Staurosporine was purchased from Selleck Chemicals
(Houston, TX, USA). HTRF KinEASE STK S3 kit was the product of
Cisbio (Codelet, France). SGK1, SGK3, and AKT1 used for
concentration-response studies of modified compounds were
bought from Carna Bioscience (Kobe, Japan). Cell lines were
obtained from Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences (Shanghai, China). CCK-8 solution was
procured from Dojindo Laboratories (Kumamoto, Japan). Cell Lytic
M was acquired from Sigma-Aldrich. BCA kit was purchased from
Beyotime (Shanghai, China). Denaturing buffer was bought from
New Cell and Molecular Biotech Co, Ltd. (Suzhou, China).
Nitrocellulose membranes (0.45 μm) were obtained from Merck
Millipore (Darmstadt, Germany). Antibodies were procured from
Cell signaling Technology (Beverly, MA, USA).
Given the role of SGK in cancer signaling, a number of SGK1
inhibitors have been discovered (Fig. 1). The crystal structures of 1
(PDB: 3HDM) and 2 (PDB: 3HDN) in complex with SGK1 showed
that the azaindole nitrogen of these two inhibitors interact with
the linker forming donor-acceptor interactions with Ile 179 and
Asp 177 [22]. In both 1 and 2, the carboxylic acid group makes an
electrostatic interaction with the catalytic Lys 127. This interaction
is important, as analogs lacking the carboxylic group had
significantly reduced potency. GSK650394, similar in structure to
1 and 2 was discovered and characterized by Sherck et al. [23]. It is
not selective between the SGK isoforms, but it is selective for SGK1
over AKT, and is able to inhibit androgen-mediated prostate
cancer cell (LNCaP) growth.
Compounds with a different scaffold were also reported by
Merck [24]. The para-phenol group in 3 is proposed to interact
with the linker, and the carbonyl to interact with Lys 127 [25].
Halland et al. [25] identified 7 via virtual screening, which was
proposed to form hydrogen bond interactions with Ile 179 and
Asp 177 using its aminoindazole scaffold. Further optimization of
7 led to the discovery of 14g and 14n that were approximately
50-fold and 75-fold selective for SGK1 over SGK3, respectively, but
not selective for SGK1 over SGK2. EMD638683, another SGK1
inhibitor similar to those unmasked by Merck, was able to lower
blood pressure in mice with hyperglycemia and salt excess [26]. It
is not selective between the SGK isoforms, but selective for SGK
over other kinases including AKT.
An in silico study by Ortuso et al. [27] identified SI113, which is
active against SGK1. It orients the OH group towards the linker,
donating a hydrogen bond to Asp 177, accepting a hydrogen bond
from Ile 179 and making π-stacking interaction with the lysine side
chain. It was able to decrease the growth of RKO colon carcinoma,
MCF-7 breast carcinoma, and A-172 glioblastoma cells [28].
To date, the number of SGK inhibitors identified is limited, and
none are selective for the SGK3 isoform. Therefore, we conducted
a high-throughput screening (HTS) campaign against the SGK3
isoform in an attempt to identify selective inhibitors and/or novel
scaffolds that can be optimized to produce potent inhibitors.
Kinase assay
Two protocols were used for the kinase assay. Protocol A was used
for the HTS and concentration-response studies of the hit
compounds. Compounds (1 μL/well) dissolved in DMSO were
diluted in 20 μL/well kinase buffer (50 mmol/L Tris, pH 7.5, 1
mmol/L EGTA, 10 mmol/L MgCl2, 2 mmol/L DTT, 0.01% Tween-20),
and 2 μL/well of the diluent was added to the detector plate. The
wells were incubated with SGK3 protein (0.25 ng/well), ULight-
rpS6 peptide (50 nmol/L) and ATP (10 μmol/L) for 1 h at room
temperature with a total reaction volume of 10 μL/well. Eu-anti-
phospho-rpS6 (2 nmol/L) in 12 mmol/L EDTA (10 μL) was added
into the plate and incubated for another hour at room
temperature. The fluorescence at 665 and 620 nm was read using
the EnVision Plate Reader (PerkinElmer), and the experiment was
carried out in duplicates. The fluorescence signal was calculated as
665 nm/620 nm.
Protocol B was used for concentration-response studies of the
modified compounds. Compounds (1 μL/well) dissolved in DMSO
were diluted in 40 μL/well kinase buffer (provided by HTRF
KinEASE STK S3 kit and supplemented with 10 mmol/L MgCl2 and
1 mmol/L DTT), and 4 μL/well of the diluent was added to the
detector plate. The wells were incubated with SGK1 (1.2 ng/well),
MATERIALS AND METHODS
Reagents
LANCE Ultra Europium-anti-phospho-40S ribosomal protein S6
(Ser235/236) and LANCE Ultra ULight™-phospho-40S ribosomal
Fig. 1 Structures of published SGK inhibitors
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Identification of potential small-molecule SGK3 inhibitors
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SGK3 (0.08 ng/well), or AKT1 (0.12 ng/well), STK substrate 3-biotin
(1 μmol/L), and ATP (10 μmol/L) for 30 min at room temperature
with a total reaction volume of 10 μL/well. STK antibody-cryptate
(5 μL) and 5 μL streptavidin-XL665 (62.5 nmol/L) in detection
buffer were added to the plate and incubated for another hour
at room temperature. The fluorescence at 665 and 620 nm was
read using the EnVision Plate Reader (PerkinElmer), and the
experiment was carried out in duplicates. The fluorescence signal
was calculated as 665 nm/620 nm.
electrophoresis (6 and 8% gel) and transferred onto nitrocellulose
(0.45 μm) membranes. The membranes were incubated with 5%
(w/v) non-fat dried milk dissolved in TBS-T (1 mol/L Tris-HCl, 0.15
mol/L NaCl, 0.05% (v/v) Tween-20, pH 7.4) for 1 h at room
temperature. The membranes were then washed three times, 10
min each and incubated with primary antibody (1:1000) overnight
at 4 °C in 5% w/v BSA dissolved in TBS-T. The membranes were
again washed three times, 10 min each, and then incubated with
anti-rabbit IgG HRP-linked antibody (1:5000) in 5% (w/v) non-fat
dried milk dissolved in TBS-T for 1 h at room temperature.
Following three-time wash (10 min each), they were reacted with
SuperSignal® West Dura substrate in a ChemiDocTM MP Imaging
System (ThermoFisher Scientific, Waltham, MA, USA).
HTS campaign
The compound library used for the screening of SGK3 inhibitors
consisted of 50,400 pure synthetic and natural compounds.
Samples dissolved in 100% DMSO were applied to the primary
screening, with an average final concentration of 20 μmol/L for
each compound. In each 384-well assay plate, 24 wells were used
as positive controls (2 μmol/L staurosporine), 24 wells as negative
controls (no compounds), and 16 wells as blank controls
(no protein or compounds). Compounds showing greater than
50% inhibition relative to negative controls were considered as
“hits”.
Molecular modeling
Homology modeling of SGK3. The amino-acid sequence of human
SGK3 was downloaded from the NCBI database (accession code:
Q96BR1), and sequence similarity search was performed using the
NCBI BLAST server. Three crystal structures of the human SGK1
kinase domain (PDB: 2R5T, 3HDM and 3HDN) were selected as the
template to construct SGK3. Sequence alignment of SGK1 and
SGK3 was carried out using Discovery Studio [30]. Twenty models
were generated, and the model with the lowest Discrete
Optimized Protein Energy score was submitted to 200 steps of
energy minimization with steepest descent. The quality of the
model was evaluated using the Profiles-3D protocol in Discovery
Studio, and a Ramachandran plot was also generated.
Concentration-response study
Concentration-response study was performed essentially the same
as described above, except that test compounds were hand-
picked (average stock concentration: 10 mmol/L) and diluted (1:5)
seven times to give a total of eight concentrations. The final
compound concentration ranged from 100 μmol/L to 1.28 nmol/L.
Each compound was tested in duplicates and the concentration-
response curves and IC50 values were generated with GraphPad
Prism 5 (GraphPad, San Diego, CA, USA) using log (inhibitor) vs.
response equation in nonlinear regression analysis.
Ligand and protein preparation. All ligands were created with the
two-dimensional Sketcher module in Maestro 10.1. They were
prepared using the LigPrep option of Schrodinger [31]. Ligand
minimization was done using the MMFFs force field, and possible
ionization states at pH 7.0 2.0 were generated using Epik. All
possible stereoisomers were generated and a maximum of 32
conformers were obtained. The human SGK1 kinase domain
(3HDM) was prepared first using Discovery Studio to clean the
protein and add missing side chains and residues using CHARMm
minimization, with all parameters kept as default [30]. The protein
was then prepared using the protein preparation wizard tool
implemented in Maestro 10.1 [31]. Water molecules were deleted
and protein ionization states were generated at pH 7.0 3.0, and
minimized with the OPLS_2005 force field with all parameters
kept as default.
Cell culture and maintenance
LNCaP prostate cancer cells were maintained in RPMI1640
medium (ATCC modification) containing 10% fetal bovine serum
and 100 units/mL penicillin/streptomycin. T47D breast cancer cells
were maintained in RPMI1640 medium supplemented with 10%
fetal bovine serum and 100 units/mL penicillin/streptomycin. The
cells were incubated at 37 °C in a humidified atmosphere
containing 5% CO2.
Cell viability assessment
LNCaP and T47D cells were seeded at 50,000 cells/well in 96-well
plates. The plate was incubated for 42 h in a humidified incubator
at 37 °C and 5% CO2. Compounds dissolved in DMSO at a
concentration between 25 μmol/L and 0.39 μmol/L were added,
with a final DMSO concentration of 0.5%. DMSO without inhibitor
was used as the negative control. The plate was then incubated
for 24 h at 37 °C, and 10 μL of CCK-8 solution were added to each
well. The plate was further incubated for 4 h at 37 °C and
absorbance was measured at 450 nm using the EnSpire Multi-
mode Plate Reader (PerkinElmer). The data were then graphed
and analyzed with GraphPad Prism 5.
Molecular docking. Two docking methods were employed in this
study. The first used GLIDE implemented in Maestro 10.1
(Schrodinger) [32]. The receptor grid was defined as a 20 Å×
20 Å× 20 Å box centroid of the crystalized ligand. Hydrogen bond
constraints were set at the backbone amide nitrogen of Ile 179
and carbonyl of Asp 177 in SGK1, and the backbone amide
nitrogen of Val 243 and carbonyl of Asp 241 in SGK3. Docking was
performed with the XP (Extra Precision) protocol and 20 poses at
most were kept per ligand. All other settings were kept as default.
The second method employed was the Induced Fit Docking
(IFD) protocol using the OPLS_2005 force field in Maestro 10.1
(Schrodinger) [33]. The docking cavity for SGK1 was centered on
the ligand in the crystal structure, and the docking cavity for SGK3
was centered on the ligand of 1 docked into SGK3 using GLIDE.
The size of the box was generated automatically. Different
combinations of hydrogen bond constraints were used in the
docking, with constraints set at the backbone amide nitrogen and
carbonyl of Ile 179, the backbone carbonyl of Asp 177 and the side
chain of Lys 127 in SGK1. Constraints were set at the backbone
amide nitrogen of Val 243 and the carbonyl of Asp 241 in SGK3.
Docking was carried out using SP (Standard Precision). Residues
within 10 Å of ligand poses were refined, and structures within
30 kcal/mol of the best structure were re-docked. All other settings
were kept as default.
Western blot analysis
Western blot analysis was carried out as previously described with
minor modifications [29]. T47D cells were seeded in 12-well tissue
culture plates at 4.2 × 105 cells/well, and after a day they were
serum starved overnight. The cells were treated with DMSO only,
10 μmol/L or 20 μmol/L of test compounds for 60 min with a final
DMSO concentration of 0.5%, and then stimulated with 100 nmol/
L of insulin. They were lysed with Cell Lytic M containing protease
inhibitor and phosphatase inhibitor cocktail on ice. The concen-
tration of protein was determined using the BCA kit according to
the manufacturer’s instructions. The samples were incubated with
denaturing buffer at 37 °C for 10 min. Protein samples
were separated by sodium dodecyl sulfate polyacrylamide gel
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Data analysis
RESULTS
Assay validation
Z' factor was evaluated using 48 positive control wells, 48 negative
control wells and 32 blank control wells before the HTS campaign:
Firstly, protein titration was performed to determine the optimal
concentration of SGK3 for the HTS assay, and the EC80 (0.25 ng/
well) was selected (Fig. 2a). An incubation time course was
evaluated, the signal started to plateau after 1 h as shown in
Fig. 2b, therefore 1 h was selected as the incubation time. ATP
titration was performed to determine the optimal ATP concen-
tration (Fig. 2c), and the Km of ATP was 15.88 μmol/L. DMSO is
the most commonly employed solvent to dissolve organic
compounds in HTS, and DMSO did not affect the assay
performance at concentrations up to 2.5% (Fig. 2d). Under
these optimized conditions, the IC50 value of the inhibitor
control, staurosporine, was 0.14 μmol/L, comparable to those
reported in the literature [34–36] (Fig. 2e). The final concentra-
tions of ULight-rpS6 peptide and Eu-anti-phospho-rpS6 were 50
nmol/L and 2 nmol/L, respectively, according to the manufac-
turer’s instructions.
3 ´ ðSDPC þ SDNCÞ
Z0 ¼ 1 À
jMPC À MNCj
MPC means the mean value of positive control wells. SDPC
means the standard deviation of positive control wells. MNC
means the mean value of negative control wells. SDNC means the
standard deviation of negative control wells.
Inhibition (%) was calculated using the following equation:
ꢀ
ꢁ
ST À SB
Inhibitionð%Þ ¼ 1 À
´ 100%
SPC À SB
ST means the signal of each compound–test well. SB means the
mean signal of blank control wells. SPC means the mean signal of
positive control wells.
Fig. 2 Optimization for the HTS assay. a Kinase activities at protein concentrations ranging from 100 ng/mL to 12.5 ng/mL were measured
(mean SEM, n = 3). b Kinase activities at different incubation times (0 min to 120 min) were measured (mean SEM, n = 3). c ATP titrations
from 1 mmol/L to 12.8 nmol/L (mean SEM, n = 3). d DMSO tolerance test, with DMSO concentrations ranging from 0.5 to 5% (mean SEM,
n = 3). e Concentration-response curve of staurosporine on SGK3 activity was generated with optimal conditions, from which IC50 value was
calculated (mean SEM, n = 3)
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Table 1. High-throughput screening campaign against SGK3
identified 15 hits with IC50 values below 25 μmol/L
Fig. 3 Z′ factor of the SGK3 assay was determined at optimized
conditions. Forty-eight replicates of signal (1% DMSO, black circle)
and background (2 μmol/L staurosporine, empty circle) were
investigated
Fig. 4 HTS campaign against SGK3 of 50,400 small-molecule
compounds
Assay performance
Both signals evoked by SGK3 alone and in the presence of an
inhibitor control (2 μmol/L staurosporine) were studied (Fig. 3). The
coefficient of variation (CV) value was 13.9%. The Z' factor for the
assay was 0.56 with an S/B ratio of 14.0. These characteristics
indicate that the system is of high quality and well-suited to
HTS [37].
Identification of potential SGK3 inhibitors
The results of the primary screening of SGK3 inhibitors are
presented in Fig. 4. 50,400 compounds were screened for
potential inhibition on SGK3, and 203 hits (0.40%) that showed
inhibitory activity higher than 50% were selected for confirmation.
In the secondary screening, 51 compounds demonstrated
consistent inhibition compared with the primary screening and
showed concentration-dependency at the two concentrations
used (20 μmol/L and 4 μmol/L). Among them, 15 synthetic
compounds, namely, nitrogen-containing aromatic, flavone,
hydrazone, and naphthalene derivatives, displayed IC50 values
below 25 μmol/L (Table 1). One scaffold containing a hydrazone
core of these hits was assessed as the starting point for medicinal
chemistry efforts.
Structure modification
Compounds WNN1027-H007, WNN1029-B004, WNN1042-B003,
and WNN1113-B006 were selected for further studies due to their
potency and the consistent activity across the different analogs.
Based on the hydrazone core structure, we introduced different
substituent groups into the aromatic ring in an attempt to obtain
SGK3 inhibitors with improved potency. The synthesis of target
compounds (18a–r) is illustrated in Scheme 1. 4-(N-acetylamino)
acetophenone (6a) was prepared from commercially available 4-
aminoacetophenone (5) and acetic anhydride at room tempera-
ture. In the presence of sulfuric and nitric acids, 1-(4-N-
acetylamino-3-nitrophenyl) ethanone (8a) and 3-nitro-4-
methoxyacetophenone (8b) were synthesized via the nitration
of 6a and 6b, respectively. Then, 8a was deacetylated in 6 N HCl to
produce 3-nitro-4-aminoacephenone (9), and reduction of 8b with
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Scheme 1 Reagents and conditions: (a) Ac2O, then H2O, room temperature (rt); (b) H2SO4, HNO3, rt; (c) 6 N HCl, reflux; (d) 2,4-difluorobenzene-
1-sulfonyl chloride, pyridine, rt, 1 h; (e) SnCl2·2H2O, DMF, rt, 24 h; (f) Ac2O, fuming HNO3, –30 °C; (g) BrCH2CO2Me or BrCH2CO2Et, K2CO3, DMF, rt;
(h) H2NNH2·H2O, EtOH, 90 °C; (i) various ketones or aldehydes, EtOH, HAc, rt, or reflux
SnCl2·2H2O gave 1-(3-amino-4-methoxyphenyl) ethanone (10).
Compounds 10 and 5 were treated with 2,4-difluorobenzene-1-
sulfonyl chloride to provide intermediates 11 and 12. Moreover,
pyrrole (13) was reacted with fuming nitric acid to form
3-nitropyrrole (15). Compounds 15, 19, and 22a–c were subse-
quently condensed with methyl or ethyl bromoacetate in the
presence of potassium carbonate (K2CO3) to produce intermedi-
ates 16, 20a, and 23a–c. These five compounds and the
commercially available 20b were then treated with hydrazine
hydrate to obtain 17, 21a–b and 24a–c, which were further
condensed with various aromatic aldehydes or ketones as well as
9, 10, 11, and 12, using acetic acid as a catalyst in EtOH, to provide
eighteen target compounds (18a–r; see Supplementary
Information).
IC50 values of the modified compounds are shown in Table 2.
Compound 18d has an IC50 of 1.12 μmol/L against SGK3, and
when the OH group was replaced with a pyridine in 18f, potency
of the compound decreased by approximately 3-fold. A similar
IC50 value for 18d and 18e suggests that addition of a methyl
group to 18d has minimal effects on the potency. In addition, the
presence of a second OH group in the ortho-position as in 18j did
not alter the potency compared with 18e. The results imply that
the OH group in the para-position is critical for inhibitory activity:
when it was replaced by a methyl (18g) or removed completely
(18i), activities of the compounds were greatly compromised.
Moreover, 18h was inactive despite having an OH group in the
ortho-position, further highlighting the importance of the para-OH
group.
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having two OH groups. Substituting NO2 and Cl groups off the
benzene as observed with 18a, 18k, and 18l did not have
significant effects on potency. The data also show that these
compounds in general are not selective for SGK3 over SGK1, but
are selective for SGK over AKT (Table 2).
Table 2. IC50 values of potential inhibitors of SGK3, SGK1, and AKT
Molecular modeling
The hits were active against both SGK1 and SGK3; hence they
were first docked into the SGK1 crystal structure (PDB: 3HDM)
using GLIDE. Based on the crystal structure, 1 makes two
hydrogen bond interactions with Asp 177 and Ile 179 at the
linker (Fig. 5), therefore, hydrogen bond constraints were set at
the backbone carbonyl of Asp 177 and amide of Ile 179, although
only one constraint needs to be satisfied. The results demonstrate
that the OH group of 18d was predicted to make a hydrogen
bond donor interaction with the backbone carbonyl of Asp 177
and an acceptor interaction with the amide nitrogen of Ile 179
(Fig. 6a). Compound 18f has a pyridine instead of an OH group at
the same position, and this compound is predicted to only make a
hydrogen bond interaction with Ile 179 (Fig. 6b).
To give more flexibility to the active site during docking, the
Induced Fit Docking (IFD) protocol within Maestro 10.1 was used.
Hydrogen bond constraints were initially added at the backbone
amide nitrogen of Ile 179 and the backbone carbonyl of Asp 177.
The higher scoring binding modes of 18d oriented the OH group
towards the linker, making a donor hydrogen bond with Asp 177
and an acceptor hydrogen bond with Ile 179, similar to the
binding mode observed with GLIDE (Fig. 6c). The model also
suggests that the carbonyl group can interact with the catalytic
lysine. When the OH group was replaced by a pyridine (18f), the
carbonyl group instead of the pyridine was preferentially oriented
towards the linker (Fig. 6d).
The docked models suggest that the ligands may also interact
with the catalytic lysine, therefore, additional hydrogen bond
constraints were added at the side chain of Lys 127. The higher
scoring poses continue to orient the OH group towards the linker;
however, a second type of binding mode was also identified. In
this binding mode the carbonyl group interacts with the linker,
potentially making three hydrogen bond interactions with Asp
177 and Ile 179, and the OH group was predicted to interact with
the catalytic lysine. Docking results of 18j and 18d suggest that
the OH group in the para-position may be responsible for
interacting with the lysine side chain (Fig. 6e, f). The limitation of
this second type of binding mode is that it is nearly always scored
lower than those that oriented the OH group towards the linker.
Both types of binding modes identified above are possible.
Therefore, in order to investigate which binding mode is more
likely, we designed two compounds 18m and 18n. Compound
18m has a sulfonamide group that can potentially make an
electrostatic interaction with the catalytic lysine (Fig. 7a), and this
has been demonstrated in other kinases [38]. Compound 18n, on
the other hand, is incapable of making electrostatic interactions
with the catalytic lysine. The results show that both compounds
did not have very high potency, with <50% inhibition at 100 μmol/
L. Docking results suggest that the sulfonamide in the para-
position may be better suited than the meta-position (Fig. 7b).
Therefore 18q and its analogs 18o, 18p, and 18r were synthesized
and tested, and were shown to be inactive. Altogether, these
suggest that the first binding mode shown in Fig. 6a–c may be
more likely than binding mode two.
3D structure of the SGK3 model. Currently, no crystal structure of
the kinase domain of SGK3 is available in the Protein Databank
(PDB), therefore, homology modeling was used to generate a
model of the SGK3 kinase domain. Sequence alignment indicated
that the sequence identity in the kinase domain is approximately
74% between SGK1 and SGK3. Three crystal structures of SGK1 are
available from PDB, two in complex with inhibitors (PDB: 3HDM
When the NH group of 18d was replaced by S (18b), potency
decreased by approximately 20-fold, indicating that the NH group
is important. This is further supported by 18c, where potency was
lost when the NH group was removed, despite the compound
Acta Pharmacologica Sinica (2018) 0:1 – 11

Identification of potential small-molecule SGK3 inhibitors
GQ Gong et al.
8
and 3HDN) and one in complex with AMP-PNP, an ATP mimetic
(PDB: 2R5T). These structures were selected as templates to
construct the SGK3 model using Discovery Studio. The Ramachan-
dran plot showed that 97.7% of the residues in the SGK3 model
(Fig. 8a) were either in the most favorable regions or in the
additionally allowed regions, and only seven residues were
located in the disallowed region (Fig. 8b). This implies that the
overall conformations of the main chains and side chains are
reasonable. Verification of the model using the Profiles-3D
protocol showed that the verified score was 101.03 (expected
high score was 135.595 and the expected low score was 61.0176).
This protocol assesses the compatibility of the 3D structure of the
protein model with the sequence of residues it contains. The
verified score is higher than the expected low score and closer to
the expected high score indicating that the model is of good
quality.
The hits were also docked into the SGK3 model built by
homology modeling, and the results were similar to those
observed in SGK1, with the OH groups of the compounds
preferentially oriented towards the linker (Fig. 9). Interestingly,
binding mode 2 where the carbonyl is predicted to interact with
the linker and the OH group to interact with the catalytic lysine
was not observed in SGK3.
Fig. 5 Crystal structure of the human SGK1 kinase domain in
complex with 1 (PDB: 3HDM)
Fig. 6 Compounds docked into the SGK1 active site. a Predicted binding mode of 18d using GLIDE with hydrogen bond constraints set at
D177 and I179. b Predicted binding mode of 18f docked using GLIDE with hydrogen bond constraints set at D177 and I179. c Predicted
binding mode of 18d using IFD with hydrogen bond constraints added at D177 and I179. d Predicted binding mode of 18f using IFD with
hydrogen bond constraints added at D177 and I179. e Predicted binding mode of 18j using IFD with hydrogen bond constraints added at
K127, D177, and I179. f Predicted binding mode of 18d using IFD with hydrogen bond constraints added at K127, D177, and I179
Acta Pharmacologica Sinica (2018) 0:1 – 11

Identification of potential small-molecule SGK3 inhibitors
GQ Gong et al.
9
Fig. 7 Compounds docked into the SGK1 active site using IFD with hydrogen bond constraints added at D177 and I179. a Predicted binding
mode of 18m. b Predicted binding mode of 18q
Fig. 8 Homology model of the SGK3 kinase domain. a Homology model of SGK3 kinase domain in cartoon representation. The N-terminal
lobe is colored in cyan and the C-terminal lobe is colored in magenta. b Ramachandran plot calculated for the SGK3 kinase domain using
Discovery Studio
Effects of compounds in vitro
100 nmol/L of insulin. It was observed that phosphorylation of
SGK3, NDRG1, and TSC2 were unaffected (data not shown).
These cell-based experimental data indicate that the inhibitory
activity of the compounds under investigation may not be
strong enough for this inhibitory activity to be translated from
biochemical assays to cellular assays. Cells are more complex
than protein, and compounds must cross the lipid membrane to
be active within the cell, which may, therefore, reduce the
concentration of compounds at the target site and contribute to
the lack of inhibition in LNCaP and T47D cells.
The hit compounds were tested in LNCaP and T47D cells for
their effects on cell viability at a concentration between 0.39
μmol/L and 25 μmol/L using the CCK-8 assay. The results show
that following 24 h of exposure, 18b, 18d, 18e, 18 f, and 18k
had minimal effects on cell growth and viability (data not
shown). The inhibitors were also investigated for effects
downstream of SGK3, and western blot analysis was conducted
in T47D cells. The cells were inhibited for 60 min at 10 and
20 μmol/L of a given test compound, and then stimulated with
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Identification of potential small-molecule SGK3 inhibitors
GQ Gong et al.
10
Fig. 9 Predicted binding modes of compounds in SGK3. a Compound 18d docked into SGK3 using GLIDE, with hydrogen bond constraints
added at D241 and V243. b Compound 18a docked into SGK3 using the IFD protocol, with hydrogen bond constraints added at D241 and
V243
DISCUSSION
and 75-fold more selective for SGK1 over SGK3, respectively, but
were not selective for SGK1 over SGK2.
SGK is a family of serine/threonine kinases involved in the
regulation of many cellular processes including cell growth and
survival downstream of PI3K, and is considered a second AKT in
cell signaling [1, 2, 8]. Through the identification of tumors that are
independent on AKT but dependent on PDK1 signaling, the SGK
family of proteins has become a new therapeutic target for
anticancer drug discovery [9–11]. SGK and AKT have >50%
sequence identity in the kinase domain, and hence share similar
substrate specificity [2, 3]. Similar to AKT, SGK is also implicated in
many cancers including breast and prostate cancer [13–18]. To
date, only a limited number of inhibitors for the SGK family have
been identified. Therefore, there is an urgent need for the
discovery and development of new SGK inhibitors.
In this study, we present a HTS campaign in attempt to identify
new compounds that are active against the SGK family of proteins.
To apply the fluorescence-based assay to HTS, many factors were
considered and optimized to maximize the S/B ratio. As shown in
Fig. 2, the signal was affected by protein amount, incubation time,
and ATP concentration. In addition, DMSO tolerance was also
examined because it may limit the maximum concentration in
which a compound could be tested. The results show that at
concentrations of up to 2.5%, DMSO did not affect the assay
performance. In addition to the S/B ratio, the Z' factor and CV from
the validation experiments demonstrated that this assay is well-
suited for HTS [37].
Initial docking studies in SGK1 with hydrogen bond constraints
set at the backbone amide nitrogen of Ile 179 and the carbonyl of
Asp 177 (PDB: 3HDM) oriented the OH group towards the linker.
Docking with the IFD protocol using hydrogen bond constraints at
Ile 179 and Asp 177 also preferentially oriented the OH group
towards the linker, agreeing with the binding mode suggested by
Halland et al. [25] of a similar compound. In addition, we have also
identified a second binding mode where the carbonyl group was
oriented towards the linker, and the OH group was oriented
towards the catalytic lysine (Fig. 6e, f). However, compounds
designed based on this binding mode have low potency,
indicating that the first predicted binding mode in agreement
with those identified by Halland et al. [25] might be more likely.
Furthermore, docking of the compounds into our model of SGK3
demonstrate that all the compounds were oriented in the first
binding mode.
In summary, we have developed and validated a fluorescence-
based assay to directly measure the kinase activity of SGK3. We
used this assay to screen compounds in a 384-well plate format
and identified hit compounds that were then modified and
characterized in biochemical and cellular assays, resulting in the
discovery of low micromolar inhibitors of SGK. SAR and molecular
modeling studies were then carried out to better understand
ligand–protein interactions that control the potency.
Our HTS campaign identified fifteen hit compounds that had
IC50 values in the low micromolar to sub-micromolar range. Of
these, four compounds were selected for structural modification
because they have the same hydrazone core, which may be the
key scaffold to display bioactivity. These compounds are also
structurally similar to those discovered by Merck [24]. This
indicates that our HTS method is capable of identifying hits that
resemble those known to be active against SGKs. We used a
combination of SAR studies and molecular modeling to identify
functional groups that may be important for inhibiting SGK
activity. Our modified compounds were active against both SGK3
and SGK1, indicating that they are non-selective between the
isoforms. Due to the high sequence homology (74% between
SGK1 and SGK3) of the SGK kinase domain, selectivity may be
difficult to achieve by designing ATP-competitive inhibitors
binding to the active site. Based on the currently available data,
majority of the known SGK inhibitors do not appear to be selective
between the isoforms [22–27], with the exception of 14g and 14n
identified by Halland et al. [25] that were approximately 50-fold
ACKNOWLEDGEMENTS
We would like to thank Prof. Wei Fu for providing the facilities and advice for the
molecular modeling, and Ji Wu for technical support during the HTS. This work was
partially supported by grants from the Ministry of Science and Technology of China
(2014DFG32200), Shanghai Science and Technology Development Fund
(15DZ2291600) and the Thousand Talents Program in China. The funders had no
role in study design, data collection and analysis, decision to publish, or manuscript
preparation.
AUTHOR CONTRIBUTIONS
PRS, YC, and M-WW designed research; GQG, KW, XCD, YZ, W-JL, CMB, RB, and YiC
performed experiments; GQG, KW, XCD, JUF, W-JL, CMB, YiC, DHY, PRS, YC, and M-
WW analyzed data; GQG, XCD, KW, and M-WW wrote the paper.
ADDITIONAL INFORMATION
Competing interests: The authors declare no competing interests.
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Identification of potential small-molecule SGK3 inhibitors
GQ Gong et al.
11
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