Structure-activity relationship study, target identification, and pharmacological characterization of a small molecular IL-12/23 inhibitor, APY0201

Article abstract of DOI:10.1016/j.bmc.2014.03.036

Interleukin-12 (IL-12) and IL-23 are proinflammatory cytokines and therapeutic targets for inflammatory and autoimmune diseases, including inflammatory bowel diseases, psoriasis, rheumatoid arthritis, and multiple sclerosis. We describe the discovery of APY0201, a unique small molecular IL-12/23 production inhibitor, from activated macrophages and monocytes, and demonstrate ameliorated inflammation in an experimental model of colitis. Through a chemical proteomics approach using a highly sensitive direct nanoflow LC-MS/MS system and bait compounds equipped with the FLAG epitope associated regulator of PIKfyve (ArPIKfyve) was detected. Further study identified its associated protein phosphoinositide kinase, FYVE finger-containing (PIKfyve), as the target protein of APY0201, which was characterized as a potent, highly selective, ATP-competitive PIKfyve inhibitor that interrupts the conversion of phosphatidylinositol 3-phosphate (PtdIns3P) to PtdIns(3,5)P₂. These results elucidate the function of PIKfyve kinase in the IL-12/23 production pathway and in IL-12/23-driven inflammatory disease pathologies to provide a compelling rationale for targeting PIKfyve kinase in inflammatory and autoimmune diseases.

Full text of DOI:10.1016/j.bmc.2014.03.036

Bioorganic & Medicinal Chemistry 22 (2014) 3021–3029
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship study, target identification, and
pharmacological characterization of a small molecular IL-12/23
inhibitor, APY0201
Nobuhiko Hayakawa a, Masatsugu Noguchi a, Sen Takeshita a, Agung Eviryanti a, Yukie Seki a,
Hikaru Nishio a, Ryohei Yokoyama a, Misato Noguchi a, Manami Shuto a, Yoichiro Shima a,
Kanna Kuribayashi a, Shunsuke Kageyama a, Hiroyuki Eda a, Manabu Suzuki a, Tomohisa Hatta b,
Shun-ichiro Iemura b, Tohru Natsume b, Itsuya Tanabe a, Ryusuke Nakagawa a, Makoto Shiozaki a,
a,
Kuniya Sakurai a, Masataka Shoji a, Ayatoshi Andou a, , Takashi Yamamoto
⇑
⇑
a Research Institute, Ajinomoto Pharmaceuticals Co. Ltd, 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa 210-8681, Japan
b Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku,
Tokyo 135-0064, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Interleukin-12 (IL-12) and IL-23 are proinflammatory cytokines and therapeutic targets for inflammatory
and autoimmune diseases, including inflammatory bowel diseases, psoriasis, rheumatoid arthritis, and
multiple sclerosis. We describe the discovery of APY0201, a unique small molecular IL-12/23 production
inhibitor, from activated macrophages and monocytes, and demonstrate ameliorated inflammation in an
experimental model of colitis. Through a chemical proteomics approach using a highly sensitive direct
nanoflow LC–MS/MS system and bait compounds equipped with the FLAG epitope associated regulator
of PIKfyve (ArPIKfyve) was detected. Further study identified its associated protein phosphoinositide
kinase, FYVE finger-containing (PIKfyve), as the target protein of APY0201, which was characterized as
a potent, highly selective, ATP-competitive PIKfyve inhibitor that interrupts the conversion of phospha-
tidylinositol 3-phosphate (PtdIns3P) to PtdIns(3,5)P2. These results elucidate the function of PIKfyve
kinase in the IL-12/23 production pathway and in IL-12/23-driven inflammatory disease pathologies to
provide a compelling rationale for targeting PIKfyve kinase in inflammatory and autoimmune diseases.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 6 February 2014
Revised 19 March 2014
Accepted 21 March 2014
Available online 13 April 2014
Keywords:
APY0201
IL-12/23
PIKfyve
Target identification
1. Introduction
inflammation.1 IL-12 induces interferon-gamma (IFN-
c) produc-
tion from NK and T cells, which contributes to phagocytic cell acti-
vation as well as inflammation and has an important role in
defense against infection and intracellular pathogens. IL-23, a more
recently discovered member of the IL-12 family, shares the p40
protein subunit with IL-12 and promotes Th1 response, but its
functions differ from those of IL-12.2 Furthermore, IL-23 is impor-
tant in late developmental functions of Th17 cells, which are essen-
tial for immune effector activity and pathogenicity.3,4
Interleukin-12 (IL-12, also called IL-12p70) is a heterodimeric
proinflammatory cytokine comprising two subunits, named p35
and p40, and is involved in type 1 helper T (Th1) cell-mediated
Abbreviations: ArPIKfyve, associated regulator of PIKfyve; DNLC, direct nanoflow
liquid chromatography; GSK3, glycogen synthase kinase 3; IBD, inflammatory
bowel disease; IFN-c, interferon-gamma; IL-12, interleukin-12; LPS, lipopolysac-
The predominant physiological producers of IL-12 and IL-23 are
macrophages and dendritic cells, followed by neutrophils.5 Previ-
ous studies have reported the involvement of the downstream
pathway of phosphoinositide 3-kinase (PI3K) in IL-12 production
from dendritic cells and monocytes.6,7 One downstream substrate
of PI3K is a Ser/Thr protein kinase mammalian target of rapamycin
(mTOR), which acts as a negative regulator of IL-12 production by
production of IL-10. Moreover, glycogen synthase kinase 3 (GSK3),
charide; mTOR, mammalian target of rapamycin; PBMC, peripheral blood mono-
nuclear cells; PI3K, phosphoinositide 3-kinase; PIKfyve, phosphoinositide kinase,
FYVE finger containing; PSL, prednisolone; PtdIns3P, phosphatidylinositol 3-phos-
phate; SAC, Staphylococcus aureus Cowan I strain; SAR, structure–activity relation-
ship; TG-PEC, thioglycollate-induced mouse peritoneal exudate cells; TNF-
necrosis factor-alpha.
a, tumor
⇑
Corresponding authors. Tel.: +81 44 210 5823; fax: +81 44 210 5875.
E-mail addresses: ayatoshi_andou@ajinomoto.com (A. Andou), takashia_
yamamoto@ajinomoto.com (T. Yamamoto).
http://dx.doi.org/10.1016/j.bmc.2014.03.036
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

3022
N. Hayakawa et al. / Bioorg. Med. Chem. 22 (2014) 3021–3029
another downstream target of PI3K pathways, positively regulates
IL-12 production through the IL-10-independent pathway.
Although the inflammatory effector functions of Th1 and Th17
are crucial for eliminating pathogens, overproduction of proinflam-
matory cytokines IL-12/23 leads to development of serious patho-
logical conditions in diverse inflammatory diseases, such as
Crohn’s disease,3,8 ulcerative colitis,3 psoriasis,9,10 and rheumatoid
arthritis.11 The therapeutic potential of IL-12/23 inhibition has
been validated in both clinical and preclinical studies. Previous
reports on mice deficient for IL-12p40 or IL-12p19 subunits have
proven the central role of IL-12/23 in various inflammatory states
in animal models related to colitis,12 multiple sclerosis,13 arthri-
tis,14 and psoriasis.15 Ustekinumab, which is an injectable mono-
clonal antibody that neutralizes IL-12p40, has been approved for
treating moderate to severe plaque psoriasis,9,15–17 and it has
shown effectiveness in clinical trials in patients with Crohn’s dis-
ease.18 Thus, an orally available small molecule capable of inhibit-
ing IL-12/23 production is strongly needed as another option for
patients.19–22
a
c
H
N
IL-12p70
IC50 = 8.4 nM
100
75
50
25
0
N
N
N
N
N
N
O
-9 -8 -7 -6 -5
APY0201 log[M]
b
6000
IL-12p70
IC50 = 8.4 nM
IL-12p40
100
75
50
25
0
4000
2000
0
IC50 = 99 nM
-9 -8 -7 -6 -5
APY0201 log[M]
-9 -8 -7 -6 -5
APY0201 log[M]
8000
6000
4000
2000
0
IL-12p40
In the course of phenotypic cell-based screening and directed
medicinal chemistry campaigns using the production level of IL-
12p70 as index, we generated APY0201 (Compound 1, Fig. 1a), which
possesses a pyrazolo[1,5-a]pyrimidine ring as its structural core.
APY0201 works differently from anti-IL-12/23 antibodies and acts
by inhibiting production of these proinflammatory cytokines with
characteristic selectivity over other cytokines, including tumor necro-
125
100
75
50
25
0
TNF-α
IC50 = 16 nM
IC50 > 10μM
-9 -8 -7 -6 -5
APY0201 log[M]
-9 -8 -7 -6 -5
APY0201 log[M]
sis factor-alpha (TNF-a). Despite its unique and interesting pharma-
40000
30000
20000
10000
0
cological profile, the target protein of APY0201 was unknown.
In this article, we describe our chemical proteomics approach
that uses a direct nanoflow liquid chromatography (DNLC) system
and bait compounds tagged with the FLAG peptide epitope23,24 to
identify phosphoinositide kinase, FYVE finger-containing (PIKfyve)
kinase as the APY0201-binding protein.25 DNLC is a unique flow
system equipped with a novel splitless nanoflow gradient elution
device that allows the use of a fritless high-resolution electrospray
interface column at an extremely slow flow rate (>50 nL/min). A
combination of this pressure-resistant (approximately 100 kg/
cm2) LC system with data-dependent collision-induced dissocia-
tion tandem mass spectrometry (MS/MS) on a Q-TOF hybrid mass
spectrometer plus automated data processing facilitated identifica-
tion of protein components in low femtomole amounts of a func-
tional multiprotein complex that was prepared from one dish of
cultured cells.
PIKfyve is a mammalian type III phosphatidylinositol phosphate
(PIP) lipid kinase that acts on PtdIns3P to generate phosphatidylin-
ositol 3,5-bisphosphate (PtdIns(3,5)P2). PIKfyve has an important
role in pleiotropic cell functions, such as intracellular compartment
morphology, membrane trafficking pathways, and endosomal
dynamics;26,27 inhibition of this kinase led to endosomal enlarge-
ment and cytoplasmic vacuolation due to impaired PtdIns(3,5)P2
synthesis.28,29 However, no report had indicated the immunological
function of PIKfyve concerning the regulation of IL-12/23 produc-
tion and therapeutic potential as an anti-inflammatory drug target
until we disclosed this PIKfyve-IL-12/23 pathway in the patent arti-
cle30 and conference papers31,32 in 2011 and 2012. In the present
work, we performed pharmacological evaluation of APY0201to
determine the effect of selective inhibition of PIKfyve kinase on
immunomodulatory activity and anti-inflammatory potential.
TNF-α
d
4000
3000
2000
1000
0
IC50 > 10μM
-9 -8 -7 -6 -5
APY0201 log[M]
*
Figure 1. APY0201 selectively inhibits IL-12/23 production. (a) Chemical structure
of APY0201. (b) Inhibition profiles of the cytokine production of APY0201 in mouse
TG-PEC. The cells were stimulated by 100 ng/mL IFN-
indicated concentrations of APY0201. Supernatants were tested for IL-12p70, IL-
12p40, and TNF- . The black columns show activated conditions in the presence of
the vehicle control. Data are expressed as means sds. in at least three experi-
ments. (c) Human PBMC from healthy volunteers (n = 3) were exposed to either the
control or to APY0201 and stimulated as described in (b) to evaluate cytokine
production. Black columns show activated conditions in the presence of the vehicle
control. Data were normalized to the detected cytokine levels for each volunteer in
activated hPBMC (139.2, 201.7, and 592.8 ng/mL for IL-12p70; 419.8, 555.2, and
c and 0.05% (v/v) SAC with the
a
902.1 ng/mL for IL-12p40; 13,226, 14,731, and 16,281 ng/mL for TNF-
shown as means sds. for three experiments. (d) Shown is the APY0201 inhibition
of IFN /SAC-stimulated IL-12p70 production in an ex vivo experiment using BALB/c
a). Data are
c
mice (n = 8). ELISA was used to measure the amount of IL-12p70 in the whole blood
drawn 30 min after the subjects received oral administration of APY0201. ⁄P < 0.005
compared with the respective vehicle-treated group.
lipopolysaccharide (LPS) or the Staphylococcus aureus Cowan I
strain (SAC).33 Using IFN-
c/SAC as a stimulant (Fig. 1b and c),
cell-based assays in thioglycollate-elicited mouse peritoneal mac-
rophages or human peripheral blood mononuclear cells (PBMC)
were performed to evaluate the production of IL-12p70, IL-
2. Results
2.1. APY0201 showed selective inhibition of IL-12/23 production
12p40, and TNF-
a to identify the inhibitory profile of APY0201
IFN-
c priming is considered to be critical for IL-12 production of
for cytokines. In stimulated thioglycollate-induced mouse perito-
murine macrophages and human monocytes by costimulation with
neal exudate cells (TG-PEC), APY0201 strongly inhibited IL-12p70

N. Hayakawa et al. / Bioorg. Med. Chem. 22 (2014) 3021–3029
3023
and IL-12p40 production, with IC50 values of 8.4 and 16 nM,
respectively. Potent inhibition for IL-12p70 production was also
observed in human PBMC at the same IC50 value as observed in
TG-PEC, which implied that the inhibitory activity of APY0201 on
IL-12p70 was similar in rodents and humans. APY0201 also inhib-
ited IL-12p40 at 99 nM in human PBMC. APY0201 showed signifi-
cant selectivity for the production of IL-12p70 and IL-12p40 over
IL-12p70 production, which implied that this pharmacophore is
crucial for the activity.
On the basis of the obtained structure–activity relationships
(SAR), 3 and 6 were selected as ‘warheads’ and incorporated into
positive and negative bait compounds, respectively. Two linkers,
C12- and PEG-based types that possessed different physicochemical
properties (Fig. 3), were used to connect the FLAG epitope. These
TNF-
a
, and this selectivity was maintained across species. To date,
four bait compounds, 7–10, were added into cell lysate prepared
from IFNc/SAC-stimulated TG-PEC to capture the interacting pro-
tein. Affinity purification with an anti-FLAG antibody was used to
purify the bait compound–protein complex. Proteins in the eluate
were digested with Lys-C endoproteinase, and DNLC–MS/MS was
used to directly analyze the resulting peptide fragments to identify
bait compound-associated proteins. Sixty-seven proteins were
some small molecules, such as phosphodiesterase 4 inhibitors,34
toll-like receptor 4 signaling inhibitor,35 and 14-3-3f modulators,19
have shown inhibition of IL-12/23 production, but none of these
compounds have a reported mode of action with the characteristic
selectivity observed for APY0201.
Mouse whole blood was drawn 30 min after oral administration
of APY0201; it was then incubated with a mixture of IFN
c
and SAC
found using this approach (Table
information).
2 and the Supplementary
to induce cytokine production (Fig. 1d) for examining the ex vivo
response of IL-12p70 production. Oral APY0201 at a 30 mg/kg dose
showed significant reduction of IL-12p70 production (78% inhibi-
tion relative to that of the vehicle control), which implied that
the inhibitory potential of APY0201 against IL-12 was confirmed
in the animal experiment.
Previously, we experienced that many of these detected pro-
teins were not the true binding protein of the target compound,
probably because some proteins interact with bait compounds
non-specifically or bind to parts other than the critical ‘warhead’
moiety. Thus, only the case with ‘Found’ for both positive control
bait compounds (7 and 9) was considered to be a ‘true positive’,
and only the case with ‘Negative’ for both negative controls (8
and 10) was considered to be a ‘true negative’. Consequently, only
the VAC14 homolog, also known as the associated regulator of PIK-
fyve (ArPIKfyve),36 was found with both positive bait compounds
but not with both negative compounds.
2.2. APY0201 bound to the protein complex incorporating the
ArPIKFyve
The unique cytokine selectivity profile and oral efficacy of
APY0201 led us to investigate the mode of action of this com-
pound. To explore the APY0201 binding protein, we adopted a
chemical proteomics approach that included a comprehensive
analysis of compound–protein binding by affinity-based isolation
of the complex (Fig. 2).
2.3. APY0201 was identified as an ATP-competitive inhibitor of
PIKfyve kinase
To identify the binding proteins of our IL-12/23 production
inhibitors, substitution of a pyridine moiety connected to the pyr-
azolo[1,5-a]pyrimidine ring of APY0201 was investigated to design
bait compounds tagged with the FLAG epitope at the appropriate
position (Table 1). A simple phenyl group was tolerated as replace-
ment for the pyridine ring (2) in terms of inhibition of IL-12p70
ArPIKfyve is a scaffold protein that exists as a trimolecular
complex with at least two proteins, PIKfyve and Sac domain-
containing phosphatase 3 (Sac3), and predominantly localizes in
late endosomes and lysosomes of mammalian cells.29 This homo-
log physically associates with PIKfyve and Sac3 on the basis of
co-immunoprecipitation assays with endogenous or overexpressed
proteins;27 thus, the obtained result could be considered to mean
that APY0201 binds to the protein complex incorporating the
ArPIKfyve. This protein complex has a significant role in the bio-
synthesis and turnover of the signaling lipid PtdIns(3,5)P2, which
is a low abundance phosphoinositol that was reported to be an
essential mediator of critical endocytic membrane homeostasis.37
production as well as of selectivity over TNF-
showed 82% inhibition for the expression level of IL-12b mRNA
on mTG-PEC cells stimulated with SAC/IFN in our preliminary
a. The compound 2
c
experiment. Further introduction of piperadine on the phenyl
group in compound 2 (3) showed activity equipotent to that of 2.
Further acylation of the secondary nitrogen atom in the piperidine
ring in 3 (4 and 5) showed that the activity and selectivity of
APY0201 was maintained, which indicated the possibility of teth-
ering at this position through the acylpiperidine structure.
Replacement of the m-tosylhydrazone of APY0201 with a simple
chlorine atom (6) resulted in dramatic loss of inhibition against
PIKfyve is stimulated by
a regulatory complex containing
ArPIKfyve and Sac3, whereas PtdIns(3,5)P2 degradation is cata-
lyzed, at least in part, by the phosphatase activity of Sac3.
In 2008, Jefferies reported the PIKfyve inhibitor YM201636
(Fig. 4a) that blocked PtdIns(3,5)P2 production and reversibly
impaired endosomal trafficking in NIH3T3 cells, which mimicked
the effect induced by depleting PIKfyve with short interfering
RNA (siRNA).38 This compound was found through an investigation
Prey
to determine ATP-competitive PI3K p110a inhibitors possessing
(Cell lysate)
critical morpholine in their structures.39 The morpholine ring is a
frequently used substructure of several PI3K inhibitors that allows
formation of a hydrogen bond between a morpholino oxygen atom
and an NH proton of the ‘hinge’ region in the ATP-binding site of
the targeted kinase.40 YM201636 has a morpholino-pyrimidine
scaffold that is common to APY0201. These two findings, that is,
the possibility of a PIKfyve–ArPIKfyve–Sac3 complex acting as a
binding protein of APY0201 and the structural overlap between
YM201636 and APY0201, suggested the hypothesis that APY0201
would block the function of PIKfyve and PIKfyve is the key regulat-
ing enzyme for IL-12/23 production. The inhibitory activity of
Bait
compound
TG-PEC
anti-FLAG Ab
Beads
Binding protein
identification
DNLC-MS/MS
Figure 2. Shown is the schematic strategy for a chemical proteomics approach to
target identification using highly sensitive DNLC–MS/MS system and bait
compound equipped with the FLAG peptide epitope. Cell lysate was prepared from
mouse TG-PEC cells incubated with a mixture of IFN and SAC as stimulants. Bait
compound was added to capture the interacting protein complex. The bait
compound–protein complex was purified by immunoaffinity chromatography with
the anti-FLAG antibody. Proteins in the column eluate were digested with Lys-C
endoproteinase, and DNLC–MS/MS was used to analyze the obtained mixture of
peptides to identify the binding proteins of inhibitors of IL-12/23 production.
a
a
c
YM201636 for the production of IL-12p70 in IFN
TG-PEC was tested and showed negligible influence on TNF-
duction up to 10 M (details are provided in the Supplementary
c/SAC-stimulated
a
pro-
l

3024
N. Hayakawa et al. / Bioorg. Med. Chem. 22 (2014) 3021–3029
Table 1
Structure–activity relationship (SAR) study on APY0201 (1)
R2
N
R1
N
N
N
O
Compound
R1
R2
IL-12p70 (IC50 nM)
8.4 2.5
TNF-a (IC50 nM)
H
N
N
N
APY0201 (1)
>10
>10
>10
>10
lM
H
N
N
N
N
N
2
3
4
23 0.81
21 8.0
4.8 2.7
lM
lM
lM
H
N
HN
N
H
N
N
N
O
H
N
N
N
5
6
18 0.68
>2000
>10
>10
l
M
M
O
Cl
HN
N
l
Modifications were made at the para position of the pyridine ring in APY0201 to identify the appropriate position for tethering the FLAG peptide epitope. Compound 3 was
introduced into bait compounds as the positive control, and 6 was used as the negative control.
C
Linker
12
Warhead
N
FLAG peptide
H
N
O
O
O
O
H
N
N
N
N
N
N
N Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
H
N
N
O
O
N
O
7
8
H
N
Cl
N
N Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
H
N
N
N
O
PEG-based Linker
Warhead
N
FLAG peptide
H
N
O
H
H
N
N Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
O
O
O
O
O
N
O
O
O
O
O
O
N
N
N
N
O
O
O
O
N
O
9
O
H
H
N
Cl
N
N Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
O
N
N
N
N
N
O
10
Figure 3. Shown are the bait compounds for target identification. The compounds were designed to have a ‘warhead’ moiety and the FLAG peptide epitope connected with
two different types of linkers. A lipophilic C12 linker was used for compounds 7 and 8, and a hydrophilic PEG-based linker was introduced in compounds 9 and 10. Bait
compounds 7 and 9 were used as positive controls, and 8 and 10 were designed as negative controls.
information). This inhibitory profile for cytokine production was
similar to the one for APY0201.
kinase in a nearly intact cellular context with endogenous binding
partners and post-translational modifications. The system using
cellular lysate was suitable for evaluation of the interaction
between compounds and PIKfyve, which works as a fully func-
tional kinase only in the trimolecular complex with ArPIKfyve
and Sac3.29 Native kinase profiling revealed that APY0201 inhib-
ited 96.1% and 99.6% of PIKfyve–probe binding at 30 and 300 nM,
respectively, and other than PIKfyve, only the kinases ITPK1 and
LOK consistently displayed >50% inhibition when treated with
Thus, we next used in situ native kinase profiling with ATP-
competitive biotinylated ATP acyl-phosphate probes for 24 lipid
kinases and 83 protein kinases to evaluate the binding affinities
of APY0201 in the ATP binding pocket of a panel of kinases, includ-
ing PIKfyve, in a lysate of Jurkat cells (Fig. 4c, more details are pro-
vided in the Supplementary information).41,42 This assay platform
is a powerful tool for studying the behavior of any lipid and protein

N. Hayakawa et al. / Bioorg. Med. Chem. 22 (2014) 3021–3029
3025
Table 2
FLAG-tagged bait compounds 7–10 and DNLC–MS/MS analysis were used to identify proteins in immunoprecipitates prepared with the anti-FLAG from activated mouse TG-PEC
cells
Gene symbol
Protein name
Bait compound
C12 Linker PEG Linker
10
Score
7
8
9
VAC14
JAK1
ArPIKFyve/VAC14
Janus kinase 1
Annexin A2 or pseudogene 2
Alpha 2 actin or beta actin or actin beta-like 2 or Found
cardiac muscle alpha actin 1 or actin gamma
1 propeptide or actin gamma 2
Found
—
Found
—
4
3
3
3
Found Found Found
Found
—
—
—
ANXA2|ANXA2P2
ACTA2|ACTB|ACTBL2|ACTC1|ACTG1|ACTG2
—
—
—
—
ARPC5
FECH
Actin-related protein 2/3 complex subunit 5
Ferrochelatase
Histone family
Found
Found
Found
—
—
—
—
—
—
—
—
—
3
3
3
H3F3A|H3F3B|HIST1H3B|HIST1H3D|HIST1H3F
|HIST1H3I|HIST2H3D|HIST2H3PS2|HIST3H3|LOC347376|LOC440093
MTHFD1L
Methylenetetrahydrofolate dehydrogenase
(NADP+ dependent) 1-like
Found
—
—
—
3
MYO1F
NQO2
CPT1A
PHB
SGPL1
VDAC1
VDAC1|VDAC3|VDAC4P
Myosin IF
Found
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3
3
3
3
3
3
3
NAD(P)H dehydrogenase, quinone 2
Carnitine palmitoyltransferase 1A liver
Prohibitin
Sphingosine-1-phosphate lyase 1
Voltage-dependent anion channel 1
Voltage-dependent anion channel 1 or 3 or 4
pseudogene
Found
Found
Found
Found
Found
Found
—
—
VDAC2
VDAC3
Voltage-dependent anion channel 2
Voltage-dependent anion channel 3
—
—
—
—
Found
Found
—
—
3
3
The analysis was repeated four times, and the observed peaks for at least two different analyses were denoted as ‘Found’. The symbol ‘—’ indicates that the corresponding
peaks were not observed or observed only once in the four repeated analyses. One point was added in case of ‘Found’ for positive controls, and ‘—’ was recorded for negative
controls; the cumulative points for each protein are shown in the ‘Score’ column. Only proteins with more than three points are shown. The full list of identified proteins is
provided in the Supplementary information.
300 nM of APY0201. No significant inhibitions of APY0201 were
observed for PI3Ks, mitogen-activated protein kinases, GSK3b, or
mTOR, all of which were reported to have a role in LPS-induced IL-
12 production from dendritic cells or monocytes.6,7 APY0201 was
remarkably selective for several other members of the lipid kinases,
including the PI4K and PI5K families. The calculated Gini coefficients
were 0.860 and 0.753 for 30 and 300 nM APY0201, respectively.43
This native kinase profiling assay was also performed for compound
3 to prove its selective and ATP-competitive PIKfyve inhibition as
well (83.9% inhibition at 300 nM), whereas compound 6 showed
negligible inhibition of the binding between PIKfyve and biotinylated
ATP acyl-phosphate probe (6.8% inhibition at 300 nM). Furthermore,
APY0201 was submitted to Cerep (Seattle, WA) for evaluation by a
to a decrease in the expression of IL-12p40, which is an integral
component unit of both IL-12 and IL-23.30–32 After we disclosed
the preliminary experimental data using PIKfyve-siRNA in the pat-
ent article30 and conference papers31,32 in 2011 and 2012, the
researchers at Novartis confirmed that blocking the phosphotrans-
ferase activity of PIKfyve leads to selective inhibition of IL-12/IL-
23p40 by regulating mRNA expression in 2013.44 The researchers
in Astellas followed our results.45
On the basis of the aforementioned promising profile, APY0201
was further characterized through pharmacokinetic (PK) studies
conducted in mice. The PK parameters obtained after intravenous
administration at 3 or 10 mg/kg and oral dosing at 30 mg/kg are
shown in Figure 6. For intravenous administration, the PK param-
eters of initial drug concentration (C0) and area under the curve to
time infinity (AUCinf) showed good linearity between the two
receptor binding assay at 10 lM, with a panel of 137 G protein-
coupled receptors (GPCRs), enzymes, ion channels, and transporters,
and it showed negligible inhibitions relative to the IC50 value in the
PIKfyve kinase assay. These results clearly suggested that APY0201 is
a potent, highly selective, ATP-competitive PIKfyve kinase inhibitor
that interrupts phosphorylation of PtdIns3P to yield PtdIns(3,5)P2.
As reported,44 Apilimod (Fig. 4d) is a selective PIKfyve kinase
inhibitor, but our data showed its inhibitory activity on some GPCRs
(>85% inhibition at A3, alpha2C, M3, KOP, 5-HT1A, 5-HT1B, 5-HT2D,
and 5-HT2B in Diversity profile assays in Cerep, Table S5 in the
Supplementary information). Due to the unique pyrazolo[1,5-a]
pyrimidine structure, APY0201 had superior selectivity over
Apilimod for these GPCRs, leading to favorable safety profile
(Tables S3–S5 in the Supplementary information).
doses, and APY0201 showed low total body clearance (CLtot
=
1.0 L/h/kg) (Fig. 6a). After oral administration, heart and portal
plasma concentrations were determined to assess the first-pass
effect (Fig. 6b). The maximum APY0201 concentration (Cmax) value
in the heart was 7.2 lM, and the plasma concentration was 3.5 lM
at 6 h. The apparent fraction absorbed and availability at the liver
were estimated to be 68% and 76% of the dose, respectively, which
indicated a good bioavailability of 52% (Table 3). Because good bio-
availability with oral administration was obtained, APY0201 was
examined for its oral therapeutic efficacy for once-daily dosing at
30 mg/kg.
Consistent with these results, the morpholine moiety in APY0201
has a crucial interaction with the hinge region of the kinase domain
through a hydrogen bond between the morpholino oxygen and
Leu1940 in the PIKfyve homology model (Fig. 5a and b).
2.5. APY0201 ameliorates inflammation in an experimental
model of colitis
To determine the impact of PIFKfyve inhibition on inflamma-
tory responses in vivo, we evaluated APY0201 in a mouse model
of inflammatory bowel disease (IBD) induced by the adoptive
transfer of IL-10 knockout (KO) CD4+ T cells.46,47 In this model,
splenocytes and mesenteric lymph node cells were collected from
diseased IL-10 deficient mice, and CD4+ T cells were purified up to
2.4. PIKfyve controls biosynthesis of IL-12 p40 by regulating
mRNA expression
On the basis of the experimental results described above, we
hypothesized that depletion of PIKfyve kinase activity would lead

3026
N. Hayakawa et al. / Bioorg. Med. Chem. 22 (2014) 3021–3029
a
b
IC50 = 5.2 nM
O
N
N
H
100
75
50
25
0
N
N
O
H2N
N
N
O
-9
-8
-7
APY0201 log[M]
c
DGKB
DGKG
DGKZ
DGKI
>90%
>75%
DGKA
DGKD
DGKH
DGKE
DGKQ
DGKK
IHPK3
IHPK1
IHPK2
IPMK
TRRAP
ITPKC
>50%
PI4K2B
PI4K2A
>35%
ITPKA
ITPKB
PIKfyve
PI4KAP2
PI4KA
PI4KB
PIK3C3
AGK
CERK
CERKL
PIK3CG
ATM
ATR
SPHK1
SPHK2
PIK3CD
PIK3CB PIK3CA
PIK3C2G
ITPK1
PRKDC
SMG1
mTOR
PIK3C2B
PIK3C2A
PIP5KL1
PIP4K2C
PIP5K1B
PIP4K2A
PIP4K2B
PIP5K1A
PIP5K1C
© 2012 Carna Biosciences, Inc.
d
H
N
N
O
N
N
N
N
O
Figure 4. (a) Chemical structure of YM201636. The key morpholino-pyrimidine
scaffold is highlighted in red. (b) Cell-free functional assay of full-length PIKfyve
kinase. APY0201 inhibited the conversion of PtdIns3P to [33P]PtdIns(3,5)P2 in the
presence of [33P]ATP. Data are presented as mean sd from triplicate experiments.
(c) Lipid (left) and protein (right) kinase selectivity profile for APY0201 shown on
the human lipid and protein kinome dendrogram. An in situ native kinase profiling
approach (KiNativ profile) using an ATP-competitive probe and Jurkat cells lysate
was used to determine the selectivity for 24 lipid kinases and 83 protein kinases.
The lipid kinases evaluated for APY0201 in KiNativ profiling are highlighted in red,
and the circles represent the percent inhibitions of APY0201 at 300 nM. The lipid
kinase dendrogram was generated on the basis of the alignment of the full-length
kinases and represented in radial view. The protein kinase dendrogram was adapted
with permission from Carna Biosciences, Inc. (d) chemical structure of Apilimod.
Figure 5. (a) Pictorial representation of the PIKfyve PIPK domain homology model.
The amino acid residues in the ATP binding site are explicitly shown. The conserved
lysine reacted with an ATP-acylphosphate probe in the KiNativ profiling assay is
highlighted in brown. (b) The complex with APY0201 (green) in the ATP-binding
site. Critical contact was observed in the morpholino oxygen of APY0201 with
Leu1940 (2.83 Å) in the ‘hinge’ region of the kinase.
significantly efficacious in a mouse model of colitis induced by
adoptive transfer of IL-10 KO CD4+ T cells without showing any
sign of adverse effects.
95%, which were transferred into female SCID mice to induce spon-
taneous colitis within 3 weeks. The severity of colitis was assessed
by wet colon weight. Stool consistency was scored to evaluate the
therapeutic effect of APY0201 on colitis. The animals were sacri-
ficed on day 21, and colon weights were measured. An increase
in the colon weight ratio was observed in the vehicle control group
for colitis compared with that in the normal control group. Daily
administration of APY0201 significantly reduced increases in colon
weight in a dose-dependent manner (Fig. 7a; 19.7%, 25.4%, and
73.3% reduction at 3, 10, and 30 mg/kg, respectively), and the effect
of APY0201 at 30 mg/kg was equivalent to that of prednisolone
(PSL) at 15 mg/kg B.I.D. (81.0% reduction, Fig. 7b). Examination of
the stool consistency showed that the vehicle control group exhib-
ited severe diarrhea on the day of the sacrifice, while APY0201 sig-
nificantly prevented development of diarrhea in a dose-dependent
manner (Fig. 7c and d). Twice a day administration of PSL amelio-
rated inflammation in this mouse model of colitis. However, treat-
ment with PSL led to a significant decrease in body weight after
chronic administration relative to that of the vehicle or normal
control groups, although the drug relieved diarrhea and reduced
colon weight (please see the Supplementary information). Con-
versely, administration of APY0201 caused no difference in body
weight relative to that of the normal control group. These results
clearly demonstrated that APY0201 was orally available and
3. Discussion
We identified the novel anti-inflammatory drug target PIKfyve
kinase that exists as a complex with ArPIKfyve and Sac3 and local-
izes primarily in late endosomes/lysosomes. In our chemical pro-
teomics approach using compound 3 as a ‘warhead’ in a positive
control bait compound, only ArPIKfyve was found to be a true posi-
tive, and direct interaction of compound 3 with PIKfyve was not
observed (Table 2). However, the in situ native kinase profiling
assay using an ATP acyl-phosphate probe demonstrated APY0201
to be a selective, ATP-competitive PIKfyve kinase inhibitor, and
the cell-free kinase functional assay using full-length PIKfyve
enzymes showed that APY0201 extensively reduced the kinase
activity of the PIKfyve enzyme. These data suggested that
APY0201 directly interacted with PIKfyve kinase at the ATP-bind-
ing site, which led to suppression of the conversion of PtdIns3P
to PtdIns(3,5)P2 to suppress the production of IL-12/23. Therefore,
the results implied that our target binding approach captured the
trimolecular protein complex of PIKfyve–ArPIKfyve–Sac3; how-
ever, only ArPIKfyve was detected among the three proteins, prob-
ably because of the sensitivity limit of the DNLC–MS/MS system or

N. Hayakawa et al. / Bioorg. Med. Chem. 22 (2014) 3021–3029
3027
600
500
400
300
200
100
0
a
b
a
b
100
10
1
600
500
400
300
200
100
0
3 mg/kg IV
10 mg/kg IV
***
***
0.1
0
1
2
3
Time (h)
30 mg/kg PO (systemic)
30 mg/kg PO (portal)
100
10
1
3 mg/kg QD
10 mg/kg QD
30 mg/kg QD
Normal
Vehicle
c
4
3
2
1
0
0
1 2 3 4 5 6 7
Time (h)
Figure 6. Plasma concentration (mean sd) after (a) intravenous and (b) oral
administration of APY0201. Female BALB/c mice (n = 3) were dosed intravenously at
10 mg/5 mL/kg in 50% PEG 400/50% water titrated with 1 N HCl at 3 or 10 mg/kg
and orally as a crystalline suspension in 0.5% methyl cellulose at 30 mg/10 mL/kg.
After oral administration, the heart and portal plasma concentrations were
separately determined to assess the apparent fraction absorbed.
6
8
12 14 16 18 20
Days after transfer
Table 3
(c) Pharmacokinetic, blood-to-plasma concentration ratio (Rb), and plasma protein
binding (PPB) data for APY0201
Normal
Vehicle
3 mg/kg QD
10 mg/kg QD
30 mg/kg QD
d
Intravenous dose (mg/kg)
C0 M)
V1 (L/kg)
AUCinf
CLtot (L/h/kg)
Rb
3
10
27.6
0.9
12.2
0.8
20
19
18
(l
6.2
1.2
2.9
1.0
1.1
92
(l
g h/mL)
PPB (%)
Oral
Dose (mg/kg)
30
30
Heart
7.2
12.3
68
Portal
16.3
16.8
Cmax
AUClim
Fa,app (%)
Fh (%)
(
l
M)
(l
g h/mL)
0
2
4
6
8
10 12 14 16 18 20
76
Days after transfer
BA,lim (%)
52
Figure 7. APY0201,
a selective inhibitor of PIKfyve and inhibitor of IL-12/23
Rb and PPB were measured at 25 lM.
production, ameliorates colon inflammation in an IL-10À/À cell transfer colitis
model. APY0201 or PSL was administered orally to SCID mice with experimental
colitis induced by spleen/mesenteric lymph node cells from diseased IL-10À/À mice
(n = 8). Data are presented as means s.ds. (a) APY0201 was administered at 3, 10,
and 30 mg/kg daily, and colon weight was measured at 21 days. ⁄⁄⁄P < 0.0001
Dunnett’s test (vs vehicle). (b) 15 mg/kg PSL was administered twice a day, and
colon weight was measured at 21 days. ⁄⁄⁄P < 0.0001 Dunnett’s test (vs vehicle). (c)
Stool consistency was measured at days 12, 14, 16, 18, and 20. The vehicle control
group exhibited diarrhea. APY0201 administration prevented diarrhea in a dose-
dependent manner. (d) Body weight was measured once every 2 days, and no
difference was observed in body weight after APY00201-treatment relative to that
in the normal control group.
the amount of protein in the lysate. In the PIKfyve homology
model, APY0201 interacts with the ‘hinge’ region of the ATP-bind-
ing site in the kinase domain with its pyridine ring directed toward
the outer solvent space (Fig. 5a and b). Thus, tethering the FLAG
epitope at the 4-position of the pyridine ring was an appropriate
approach for designing bait compounds that may interact with
the ATP-binding site of PIKfyve kinase.
The relationship between PIKfyve inhibition and IL-12/23 pro-
duction inhibition was investigated. Vacuolation is a cellular event
strongly associated with decreased levels of PtdIns(3,5)P2,38 which
was produced from PtdIns3P only as a result of PIKfyve kinase
activity. Thus, vacuolation could be a functional biomarker of cel-
lular PIKfyve inhibition and of the reduction of PtdIns(3,5)P2 in
the cell. The EC50 values of cellular vacuolation after addition of
compounds were consistent with their IC50 values for IL-12p70
production, which suggested that these two phenotypic cellular
events were simultaneously induced by exposure to a PIKfyve
inhibitor. It is reported that depletion of PIKfyve resulted in
inhibition of IL-12/23 production at the transcriptional level and
provided indications of decreasing cellular PtdIns(3,5)P2.44 There-
fore, it is strongly suggested that PIKfyve inhibition and cellular
PtdIns(3,5)P2 reduction are closely linked to inhibition of IL-12/
23 production from activated macrophages.
Recently, Sbrissa et al. reported that the PIKfyve inhibitor
YM201636 reduced not only PtdIns(3,5)P2, but also PtdIns5P in
various cell lines.48 Our results implied that IL-12/23 production
inhibition by APY0201 from activated macrophages is through

3028
N. Hayakawa et al. / Bioorg. Med. Chem. 22 (2014) 3021–3029
PIKfyve inhibition. However, we could not yet elucidate the whole
mechanism of observed IL-12/23-inhibitory activity that could
include reduction of PtdIns(3,5)P2, PtdIns3P declination, or other
unknown pathways.
its pharmacological profile in vitro and in vivo, the strategy for tar-
get identification, and the biological characterization of PIKfyve
kinase as an anti-inflammatory drug target were presented. The
results from this investigation should be useful in drug discovery
targeted at novel agents for treating autoimmune and inflamma-
tory diseases. Our findings provide a new understanding of the
function of PIKfyve kinase in the IL-12/23 production pathway
and IL-12/23–driven inflammatory disease pathologies. In addi-
tion, these findings support the development of a selective PIKfyve
kinase inhibitor as a therapeutic modality for autoimmune disor-
ders, such as IBD.
Previous reports have stated that a critical phenotype was
observed in mice with a null mutation in ArPIKfyve or PIKfyve.
The ArPIKfyve-deficient mice showed early postnatal lethality,
had 50% of the wild-type PtdIns(3,5)P2 and PtdIns5P levels, and
exhibited massive neurodegeneration with vacuolated cells in
multiple regions of the brain.49 Nearly all PIKfyve homozygous
KO mutant mice died at an early stage of embryonic development,
with significant reduction of cellular PtdIns(3,5)P2;50 however,
mice with haploid PIKfyve gene deletion developed normally, with
no defects in gross abnormalities, growth rate, and size, although
they showed only approximately 60–65% of the wild-type lipid lev-
els.50 Thus, it is noteworthy that administration of APY0201 at the
efficacious dose did not induce signs of adverse effects, and no
swollen cells were observed in mice treated with 30 mg/kg that
were sacrificed 1 day after the final administration (data not
shown). These results suggested that a small molecular PIKfyve
inhibitor reversed the kinase activity because it was eliminated from
the tested animals. Although a detailed elucidation of the mecha-
nism underlying modulation of IL-12/23 production by PIKfyve inhi-
bition is still being sought, the observed therapeutic margin for
APY0201 showed promise for clinical development of the com-
pound as a PIKfyve-selective small molecular inhibitor in IL-12/
23–related inflammatory and autoimmune diseases, such as IBD.
APY0201 is an orally-available PIKfyve kinase inhibitor that is
selective for all tested kinases, GPCRs, ion channels, and enzymes,
and it is a powerful tool for understanding the role of PIKfyve and
PtdIns(3,5)P2 in immunological and inflammatory responses. The
administration of APY0201 can be used to assess PIKfyve function
in immune cells and animals with normal architecture of the PIK-
fyve–ArPIKfyve–Sac3 complex. Exposure to APY0201 in vitro
blocked production of IL-12/23, which highlighted the effect of
selective inhibition of this kinase. The data presented here indicate
a unique role of PIKfyve kinase in cytokine production. Because
PIKfyve inhibition blocks production of IL-12 and IL-23 from acti-
vated macrophages, APY0201 may control the cytokine modula-
tion function of these cell types and reduce the pathological
proinflammatory cytokines IL-12/23 with negligible influence on
4. Experimental procedures
4.1. Chemical synthesis
See the Supplementary information for synthetic procedures
and characterization data.
4.2. Cell isolation
Mouse TG-PEC cells were collected from female BALB/c mice
(6 weeks) as described in the Supplementary information. Human
PBMC were isolated from the peripheral blood of healthy volun-
teers, as described in the Supplementary information.
4.3. Mouse whole blood ex vivo assay
The vehicle (0.5% methyl cellulose) and tested compound were
orally administered to female BALB/c mice (6 weeks). After 30 min,
the mice were anesthetized, and blood samples were collected by
cardiac puncture. The detailed procedures for the preprocessing
and analysis are described in the Supplementary information.
4.4. Cell stimulations
Mouse TG-PEC or human PBMC were incubated with the tested
compound in the presence of 100 ng/mL mouse or human IFN-
c
and 0.05%w/v SAC, as described in the Supplementary information.
4.5. Cytokine measurements
other cytokines, including TNF-a. Moreover, the therapeutic
impact of PIKfyve inhibition in a mouse model of IBD included
reduction in inflammation without affecting body weight.
IL-12p70, IL-12p40, and TNF-
ELISA, as described in the Supplementary information.
a levels were determined by
In the in vitro assay using mouse whole blood for IL-12p70-
inhibitory activity, APY0201 demonstrated potent inhibitory activ-
ity against IL-12p70 production with the IC50 value of 880 nM. At
4.6. Murine IL-10À/À cell transfer colitis
the therapeutic dose (30 mg/kg daily), the mean Cmax was 7.2 lM,
Colitis was induced in female SCID mice (n = 8) by adoptive
transfer of spleen and mesenteric lymph node cells from diseased
IL-10À/À mice, as described previously.46,47 The tested compound
was administered from day 0 to mice with experimental colitis.
The mice were sacrificed for assessing inflammation 21 days after
cell transfer. The severity of colitis was assessed according to wet
colon weight. Scoring of stool consistency was performed once in
2 days (0: normal beaded stool, 2: soft stool, 4: diarrhea). The
detailed experimental procedure is provided in the Supplementary
information.
which is sufficient to inhibit IL-12p70 production in whole blood
almost completely, and the estimated plasma concentration at
12 h is still close to 880 nM, which indicated that an appropriate
amount of APY0201 was maintained to inhibit IL-12p70 production
for nearly a half day (Fig. 6b). Thus, the concentration of APY0201 at
this experimental dose could be consistent with the observed ther-
apeutic effect in the IL-10À/À cell transfer colitis model.
In summary, a chemical proteomics approach using a highly
sensitive DNLC–MS/MS system and bait compounds equipped with
the FLAG epitope was used in this investigation, and further study
demonstrated that the associated protein PIKfyve kinase was the
possible target protein of APY0201, which is a selective and potent
IL-12/23 production inhibitor. The data by researchers at Novartis
supported these discussions.44 Characterization showed that
APY0201 was a potent, highly selective, ATP-competitive PIKfyve
kinase inhibitor that strongly inhibited IL-12/23 production
in vitro and ameliorated inflammation in an experimental model
of colitis. The structure–activity relationship study on APY0201,
4.7. PK profile
Female BALB/c mice (n = 3) were used. For intravenous admin-
istration, the tested compound was dissolved in 80% PEG 400/
20% water (3 or 10 mg/mL/kg) for a dose of 3 or 10 mg/kg, respec-
tively. For oral administration, the tested compound was sus-
pended in 0.5% methyl cellulose (30 mg/5 mL/kg). Blood samples
were collected at designated time points by cardiac puncture

N. Hayakawa et al. / Bioorg. Med. Chem. 22 (2014) 3021–3029
3029
14. McIntyre, K. W.; Shuster, D. J.; Gillooly, K. M.; Warrier, R. R.; Connaughton, S. E.;
Hall, L. B.; Arp, L. H.; Gately, M. K.; Magram, J. Eur. J. Immunol. 1996, 26, 2933.
15. Gottlieb, A.; Menter, A.; Mendelsohn, A.; Shen, Y. K.; Li, S.; Guzzo, C.; Fretzin, S.;
Kunynetz, R.; Kavanaugh, A. Lancet 2009, 373, 633.
(systemic) or from the portal vein (portal) under anesthesia. The
detailed procedures for preprocessing, analysis, and calculation of
the PK parameters are described in the Supplementary information.
16. Leonardi, C. L.; Kimball, A. B.; Papp, K. A.; Yeilding, N.; Guzzo, C.; Wang, Y.; Li,
S.; Dooley, L. T.; Gordon, K. B. Lancet 2008, 371, 1665.
4.8. IC50 determination
17. Papp, K. A.; Langley, R. G.; Lebwohl, M.; Krueger, G. G.; Szapary, P.; Yeilding, N.;
Guzzo, C.; Hsu, M. C.; Wang, Y.; Li, S.; Dooley, L. T.; Reich, K. Lancet 2008, 371, 1675.
18. Sandborn, W. J.; Feagan, B. G.; Fedorak, R. N.; Scherl, E.; Fleisher, M. R.; Katz, S.;
Johanns, J.; Blank, M.; Rutgeerts, P. Gastroenterology 2008, 135, 1130.
19. Boyce, J. P.; Brown, M. E.; Chin, W.; Fitzner, J. N.; Paxton, R. J.; Shen, M.; Stevens,
T.; Wolfson, M. F.; Wright, C. D. Bioconjug. Chem. 2008, 19, 1775.
20. Brown, M. E.; Fitzner, J. N.; Stevens, T.; Chin, W.; Wright, C. D.; Boyce, J. P.
Bioorg. Med. Chem. 2008, 16, 8760.
21. Nazarian, R.; Weinberg, J. M. Curr. Opin. Investig. Drugs 2009, 10, 1236.
22. Wada, Y.; Lu, R.; Zhou, D.; Chu, J.; Przewloka, T.; Zhang, S.; Li, L.; Wu, Y.; Qin, J.;
Balasubramanyam, V.; Barsoum, J.; Ono, M. Blood 2007, 109, 1156.
23. Nakagawa, T.; Shirane, M.; Iemura, S.; Natsume, T.; Nakayama, K. I. Genes Cells
2007, 12, 709.
GraphPad Prism version 5 (GraphPad Software) was used to
determine IC50s.
4.9. Statistical analysis
SAS version 6.12 software (SAS Institute, Cary, NC, USA) and
Dunnett’s method were used to perform statistical analysis, as out-
lined in the Supplementary information.
4.10. Target identification
24. Natsume, T.; Yamauchi, Y.; Nakayama, H.; Shinkawa, T.; Yanagida, M.;
Takahashi, N.; Isobe, T. Anal. Chem. 2002, 74, 4725.
25. Shisheva, A.; Sbrissa, D.; Ikonomov, O. Mol. Cell. Biol. 1999, 19, 623.
26. Ho, C. Y.; Alghamdi, T. A.; Botelho, R. J. Traffic 2012, 13, 1.
27. Shisheva, A. Cell Biol. Int. 2008, 32, 591.
Bait compounds equipped with FLAG peptide epitopes (7–10)
were incubated with the cell lysate prepared from IFNc/SAC-stim-
28. Ikonomov, O. C.; Sbrissa, D.; Shisheva, A. Am. J. Physiol. Cell Physiol. 2006, 291, C393.
29. Sbrissa, D.; Ikonomov, O. C.; Fu, Z.; Ijuin, T.; Gruenberg, J.; Takenawa, T.;
Shisheva, A. J. Biol. Chem. 2007, 282, 23878.
30. Takeshita, S.; Yamamoto, T.; Andou, A.; Okutsu, T.; Evyrianti, A.; Fukuchi, N.;
Kageyama, S. 2011. Patent WO2011081171.
31. Yamamoto, T.; Andou, A.; Hayakawa, N.; Noguchi, M.; Takeshita, S.; Yokoyama,
R.; Eviryanti, A.; Seki, Y.; Nishio, H.; Shuto, M.; Noguchi, M.; Shima, Y.;
Kuribayashi, K.; Kageyama, S.; Eda, H.; Iemura, S.; Natsume, T.; Nakagawa, R.;
Shiozaki, M.; Sakurai, K.; Shoji, M. National Meeting of the American Chemical
Society: San Diego, CA, 2012; MEDI13.
32. Andou, A.; Yamamoto, T.; Eviryanti, A.; Seki, Y.; Nishio, H.; Shuto, M.; Noguchi,
M.; Shima, Y.; Takeshita, S.; Yokoyama, R.; Hayakawa, N.; Noguchi, M.;
Kageyama, S.; Eda, H.; Shiozaki, M.; Nakagawa, R.; Sakurai, K.; Shoji, M.
Gastroenterology 2012, 142.
ulated mouse TG-PEC. The bait compound–protein complex was
immunoprecipitated with a bead-bound anti-FLAG antibody, and
the bait compound-associated proteins were then digested by
Lys-C endoproteinase. A DNLC–MS/MS system was used to analyze
the resulting peptides, as described previously.23,24 The analysis
was repeated four times, and the observed peaks for at least two
different analyses were denoted as ‘Found’ in Table 2 and in
Table S2 in the Supplementary information. The detailed procedure
of the experiment is found in the Supplementary information.
4.11. PIKfyve homology modeling and docking study
33. Ma, X.; Chow, J. M.; Gri, G.; Carra, G.; Gerosa, F.; Wolf, S. F.; Dzialo, R.;
Trinchieri, G. J. Exp. Med. 1996, 183, 147.
34. Claveau, D.; Chen, S. L.; O’Keefe, S.; Zaller, D. M.; Styhler, A.; Liu, S.; Huang, Z.;
Nicholson, D. W.; Mancini, J. A. J. Pharmacol. Exp. Ther. 2004, 310, 752.
35. Ii, M.; Matsunaga, N.; Hazeki, K.; Nakamura, K.; Takashima, K.; Seya, T.; Hazeki,
O.; Kitazaki, T.; Iizawa, Y. Mol. Pharmacol. 2006, 69, 1288.
36. Sbrissa, D.; Ikonomov, O. C.; Strakova, J.; Dondapati, R.; Mlak, K.; Deeb, R.;
Silver, R.; Shisheva, A. Mol. Cell Biol. 2004, 24, 10437.
37. Ikonomov, O. C.; Sbrissa, D.; Fenner, H.; Shisheva, A. J. Biol. Chem. 2009, 284,
35794.
The known crystal structure of PIP4KIIb (PI4K2B, PDB: 1BO1)51
was used as a template with the Molecular Operating Environment
(MOE) software52 to produce a homology model of PIKfyve kinase.
PIP4KIIb had the closest sequence identity to PIKfyve kinase. The
docking function present in the MOE software package was used to
dock APY0201 into the obtained homology model of PIKfyve. The
detailed procedure is provided in the Supplementary information.
38. Jefferies, H. B.; Cooke, F. T.; Jat, P.; Boucheron, C.; Koizumi, T.; Hayakawa, M.;
Kaizawa, H.; Ohishi, T.; Workman, P.; Waterfield, M. D.; Parker, P. J. EMBO Rep.
2008, 9, 164.
Acknowledgments
39. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Yamano, M.;
Okada, M.; Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Workman, P.; Waterfield, M.
D.; Parker, P. Bioorg. Med. Chem. Lett. 2007, 17, 2438.
40. Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann,
M. P.; Williams, R. L. Mol. Cell 2000, 6, 909.
41. Patricelli, M. P.; Nomanbhoy, T. K.; Wu, J.; Brown, H.; Zhou, D.; Zhang, J.;
Jagannathan, S.; Aban, A.; Okerberg, E.; Herring, C.; Nordin, B.; Weissig, H.;
Yang, Q.; Lee, J. D.; Gray, N. S.; Kozarich, J. W. Chem. Biol. 2011, 18, 699.
42. Patricelli, M. P.; Szardenings, A. K.; Liyanage, M.; Nomanbhoy, T. K.; Wu, M.; Weissig,
H.; Aban, A.; Chun, D.; Tanner, S.; Kozarich, J. W. Biochemistry 2007, 46, 350.
43. Graczyk, P. P. J. Med. Chem. 2007, 50, 5773.
44. Cai, X.; Xu, Y.; Cheung, A. K.; Tomlinson, R. C.; Alcázar-Román, A.; Murphy, L.;
Billich, A.; Zhang, B.; Feng, Y.; Klumpp, M.; Rondeau, J. M.; Fazal, A. N.; Wilson,
C. J.; Myer, V.; Joberty, G.; Bouwmeester, T.; Labow, M. A.; Finan, P. M.; Porter, J.
A.; Ploegh, H. L.; Baird, D.; De Camilli, P.; Tallarico, J. A.; Huang, Q. Chem. Biol.
2013, 20, 912.
We thank Dr. A. Okano, Dr. A. Mitsui, Dr. T. Okutsu, Dr. T. Yos-
hida (Ajinomoto Pharmaceuticals), Dr. N. Yamada, Y. Manabe (Aji-
nomoto), and Prof. Kazuo Shinya (AIST) for their valuable advice
and discussion.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.036.
References and notes
45. Nakamura, N.; Yuri, M.; Hiramoto, M.; Terajima, M.; Kaneko, Y.; Hattor, K.;
Yokota, H. In HUPO 12th Annual World Congress; POS-01-169, Ed.: Yokohama,
Japan, 2013.
46. Andou, A.; Hisamatsu, T.; Okamoto, S.; Chinen, H.; Kamada, N.; Kobayashi, T.;
Hashimoto, M.; Okutsu, T.; Shimbo, K.; Takeda, T.; Matsumoto, H.; Sato, A.;
Ohtsu, H.; Suzuki, M.; Hibi, T. Gastroenterology 2009, 136, 564.
47. Ikenoue, Y.; Tagami, T.; Murata, M. Int. Immunopharmacol. 2005, 5, 993.
48. Sbrissa, D.; Ikonomov, O. C.; Filios, C.; Delvecchio, K.; Shisheva, A. Am. J. Physiol.
Cell Physiol. 2012, 303, C436.
49. Zhang, Y.; Zolov, S. N.; Chow, C. Y.; Slutsky, S. G.; Richardson, S. C.; Piper, R. C.;
Yang, B.; Nau, J. J.; Westrick, R. J.; Morrison, S. J.; Meisler, M. H.; Weisman, L. S.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17518.
50. Ikonomov, O. C.; Sbrissa, D.; Delvecchio, K.; Xie, Y.; Jin, J. P.; Rappolee, D.;
Shisheva, A. J. Biol. Chem. 2011, 286, 13404.
1. Trinchieri, G. Immunol. Today 1993, 14, 335.
2. Boniface, K.; Blom, B.; Liu, Y. J.; de waal Malefyt, R. Immunol. Rev. 2008, 226,
132.
3. Morrison, P. J.; Ballantyne, S. J.; Kullberg, M. C. Immunology 2011, 133, 397.
4. D’Elios, M. M.; Del Prete, G.; Amedei, A. Expert Opin. Ther. Targets 2010, 14, 759.
5. Trinchieri, G. Int. Rev. Immunol. 1998, 16, 365.
6. Martin, M.; Rehani, K.; Jope, R. S.; Michalek, S. M. Nat. Immunol. 2005, 6, 777.
7. Ohtani, M.; Nagai, S.; Kondo, S.; Mizuno, S.; Nakamura, K.; Tanabe, M.;
Takeuchi, T.; Matsuda, S.; Koyasu, S. Blood 2008, 112, 635.
8. Peluso, I.; Pallone, F.; Monteleone, G. World J. Gastroenterol. 2006, 12, 5606.
9. Croxtall, J. D. Drugs 2011, 71, 1733.
10. Torti, D. C.; Feldman, S. R. J. Am. Acad. Dermatol. 2007, 57, 1059.
11. Annunziato, F.; Cosmi, L.; Liotta, F.; Maggi, E.; Romagnani, S. Nat. Rev.
Rheumatol. 2009, 5, 325.
51. Rao, V. D.; Misra, S.; Boronenkov, I. V.; Anderson, R. A.; Hurley, J. H. Cell 1998,
94, 829.
52. MOE2009.10.; Chemical Computing Group.
12. Neurath, M. F. Nat. Med. 2007, 13, 26.
13. Cua, D. J.; Sherlock, J.; Chen, Y.; Murphy, C. A.; Joyce, B.; Seymour, B.; Lucian, L.;
To, W.; Kwan, S.; Churakova, T.; Zurawski, S.; Wiekowski, M.; Lira, S. A.;
Gorman, D.; Kastelein, R. A.; Sedgwick, J. D. Nature 2003, 421, 744.