Identification of Novel, Potent, and Orally Available GCN2 Inhibitors with Type i Half Binding Mode

Article abstract of DOI:10.1021/acsmedchemlett.9b00400

General control nonderepressible 2 (GCN2) is a master regulator kinase of amino acid homeostasis and important for cancer survival in the tumor microenvironment under amino acid depletion. We initiated studies aiming at the discovery of novel GCN2 inhibit

Full text of DOI:10.1021/acsmedchemlett.9b00400

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Letter
Identification of novel, potent and orally available
GCN2 inhibitors with type I half binding mode
Jun Fujimoto, Osamu Kurasawa, Terufumi Takagi, Xin Liu, Hiroshi Banno, Takuto Kojima, Yasutomi
Asano, Akito Nakamura, Tadahiro Nambu, Akito Hata, Tsuyoshi Ishii, Tomoya Sameshima, Yasuyuki
Debori, Maki Miyamoto, Michael G Klein, Richard Tjhen, Bi-Ching Sang, Irena Levin, Scott Weston Lane,
Gyorgy P. Snell, Ke Li, Georgia Kefala, Isaac D. Hoffman, Steve C Ding, Douglas R Cary, and Ryo Mizojiri
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00400 • Publication Date (Web): 19 Sep 2019
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Identification of novel, potent and orally available GCN2 inhibitors
with type I half binding mode
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Jun Fujimoto,§,* Osamu Kurasawa,§,‡ Terufumi Takagi,§ Xin Liu,§ Hiroshi Banno,§,‡ Takuto
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Kojima,§ Yasutomi Asano,§,‡ Akito Nakamura,§ Tadahiro Nambu,§,‡ Akito Hata,§ Tsuyoshi Ishii,§
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Tomoya Sameshima,§ Yasuyuki Debori,§,‡ Maki Miyamoto,§ Michael G. Klein,∥ Richard Tjhen,∥
Bi-Ching Sang,∥ Irena Levin,∥ Scott Weston Lane,∥ Gyorgy P. Snell,∥ Ke Li,∥ Georgia Kefala,∥
Isaac D. Hoffman∥, Steve C. Ding,∥ Douglas R. Cary§,∇ and Ryo Mizojiri§
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§Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555,
Japan
∥Takeda California, Inc. 10410 Science Center Drive, San Diego, CA 92121, United States
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KEYWORDS. GCN2, kinase inhibitor, C-helix, SBDD, ALL, asparaginase
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ABSTRACT: General control nonderepressible 2 (GCN2) is a master regulator kinase of amino acid homeostasis and im-
portant for cancer survival in the tumor microenvironment under amino acid depletion. We initiated studies aiming the
discovery of novel GCN2 inhibitors as first-in-class antitumor agents, and conducted modification of the substructure of
sulfonamide derivatives with expected type I half binding on GCN2. Our synthetic strategy mainly corresponding to the
C-helix allosteric pocket of GCN2, led to significant enhancement in potency and good pharmacokinetic profile in mice. In
addition, compound 6d, which showed slow dissociation in binding on GCN2, demonstrated antiproliferative activity in
combination with asparagine-depleting agent asparaginase in an acute lymphoblastic leukemia (ALL) cell line, and also
displayed suppression of GCN2 pathway activation with asparaginase treatment in the ALL cell line and mouse xenograft
model.
21
Adaptation to cellular stress is one of the most fundamental 47 by 1a are filed in the patent as inhibitors of GCN2.12 Similar to
22 features of cancer cells.1 Dysregulation of the integrated stress 48 this series, we have reported 1b with potent enzymatic and
23 response (ISR) has been implicated with various types of dis- 49 cellular GCN2 inhibitory activities, and in vitro biology of
24 eases including cancer.2 Pharmacological modulation of the 50 GCN2 inhibition was explored using this agent.13 However,
25 ISR is expected to be a novel approach to treat cancer.3-5 Gen- 51 further animal testing was suspended due to its poor pharma-
26 eral control nonderepressible 2 (GCN2) is a serine/threonine- 52 cokinetic (PK) profile. Compound 2 (RAF-265) is a B-RAF
53 inhibitor and advanced into clinical trials for the treatment of
54 metastatic melanoma.14 This compound was reported to bind
55 to GCN215 with expected DFG-out binding mode (type II) that
56 is observed in binding to B-Raf.14 In 2015, a series of pyra-
57 zolones including 3 as protein kinase R-like endoplasmic re-
58 ticulum kinase (PERK) inhibitors has been disclosed, and
59 some of this derivatives showed GCN2 inhibitory activities as
60 an off-target effect.16 The representative compound was re-
61 ported to bind to inactive DFG-out conformation of PERK
27 protein kinase, one of eukaryotic initiation factor 2 kinase
28 that is a master regulator in the ISR.6 GCN2 is activated by
29 binding to uncharged transfer RNA (tRNA) and regulates
30 amino acid homeostasis.7-8 Several reports indicate that GCN2
31 is important for cancer survival in the tumor microenviron-
32 ment with amino acid depletion.9-11 However, development of
33 potent, selective and orally available inhibitors targeting the
34 GCN2 pathway has been limited, and the potential of GCN2
35 inhibitors as cancer therapeutic agents has not been reported.
36 Therefore we initiated investigation to devise a series of novel, 62 with packing an allosteric binding pocket close to C-helix by
37 potent and orally available GCN2 inhibitors endowed with 63 its 2-phenyl substituent of the pyrazolone (type II half). Most
38 high kinase selectivity as first-in-class antitumor agents. Dur- 64 recently, sulfonamides including 4 have been reported, which
39 ing in-house high-throughput screening campaign, there were 65 demonstrated binding effect not only on B-Raf as a primary
40 no promising compounds and most of compounds showed 66 target but on GCN2 and assumed to bind to B-Raf with the
41 weak to moderate GCN2 inhibitory activity with promiscuity 67 activation loop folded in (DFG-in) occupying an cavity adja-
68 cent to C-helix in their computational analyses (type I half).17
42 in terms of kinase selectivity. Thus we surveyed reported
69 Our in-house GCN2 enzymatic assay18 revealed that these
43 compounds possessing inhibitory activity or binding effects on
44 GCN2 for embarking our medicinal chemistry program. As
45 shown in Figure 1, four distinct chemical series have been
46 disclosed to date. Triazolopyrimidine derivatives represented
70 compounds 1−4 showed moderate to potent inhibitory activity
71 (IC50 values are shown in Figure 1), and among these with
72 characteristic differences in both chemical scaffolds and bind-
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ing modes, the expected type I half inhibitor 4 would be highly 46 provement of PK and/or physicochemical properties of a mol-
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attractive as our footholds. As disclosed in the supplementary
material of the manuscript, this agent possessed high selectivi-
ty for B-Raf, GCN2 and ZAK.17 On the other hand, other
chemical series were deprioritized on our program due to in-
sufficient target specificity and/or undesirable kinase inhibi-
tion regarding toxicity concerns in in-house assay. As far as
we know, kinase inhibitors with type I half binding mode are
rare compared to typical type I and II binders, which led us
47 ecule with maintaining potencies (Figure 2B).
10 one possible assumption that the induced conformational
11 change around C-helix with DFG-in folding are available
12 only in the limited kinases, and type I half inhibitor would
13 realize high target specificity. Therefore, we initiated our me-
14 dicinal chemistry efforts from compound 4 aiming type I half
15 GCN2 inhibitor with enhanced potency and excellent selec-
16 tivity over off-target kinases, especially in B-Raf. In addition,
17 improvement of PK profile and aqueous solubility of this se-
18 ries to conduct in vivo validation study is mentioned in this
19 report.
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49 Figure 2. Docking study of 4 with GCN2 homology model.
20 Figure 1. Reported compounds possessing GCN2 inhibitory
21 activities.
50 (A) Left; overview of modeling of 4/GCN2 homology model.
51 Right; overlay of a solvent accessible protein surface for the
52 binding site around C-helix of 4/GCN2 (purple mesh) and
53 vemurafenib/B-Raf (gray, PDB:3OG7). (B) Illustration of the
54 drug design which led to compounds 6a−g described in this
55 article.
56 Scheme 1. General synthetic procedurea
57 aReagents and conditions: (a) 8, pyridine; (b) 10, Pd(II) catalyst, Cs2CO3,
58 DMSO; (c) additional reactions such as reduction and substitution.
22
There have been no co-crystal structures of type I half in-
23 hibitor with GCN2 reported. In order to develop our synthetic
24 strategy by structure-based drug design (SBDD) approach, we
25 constructed a GCN2 homology model by combining the re-
26 ported co-crystal structure of vemurafenib (type I half B-Raf
27 inhibitor)19/B-Raf with those of type II GCN2 inhibitor 520
28 /GCN2 that was obtained and analyzed by Takeda (PDB:
29 6N3L). Utilizing these ligand-protein complex structures, a
30 docking model of 4 with GCN2 was successfully developed
31 (left image of Figure 2A.), which indicated a type I half bind-
32 ing mode of 4 to GCN2 and was informative to design new
33 analogues. In this model, the pyridin-3-yl sulfonamide moiety
34 of 4 is located in a lipophilic cavity around C-helix of GCN2,
35 and relatively larger space exists around this moiety, com-
36 pared to vemurafenib/B-Raf (purple vs. gray in the right image
37 of Figure 2A). Therefore, incorporation of a substituent on this
38 right-hand aromatic portion fleshing out the allosteric pocket
39 was put in a main strategic focus of our synthetic efforts to
40 enhance GCN2 inhibitory activity through gaining additional
41 interaction with the enzyme and selectivity against overall
42 kinases, especially in B-Raf. In addition, optimization of p-
43 fluorophenyl 1H-pyrazolo[3,4-b]pyridine portion as a hinge
44 binding part, which would extend to a solvent region, was
45 planned with expectation that this could contribute to im-
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Scheme 1 outlined the general synthetic procedure for sul-
60 fonamides 6. Condensation of substituted aromatic sulfonyl
61 chlorides 8 with 3-ethynyl-2,4-difluoroaniline (7),17 yielded
62 intermediates 9. Then Sonogashira coupling reaction with
63 halogenated heteroaromatic reagents (10) and following treat-
64 ments such as reduction, deprotection and substitution reac-
65 tions were conducted to provide the target compounds 6. As
66 an alternative, the intermediate 11 was synthesized by So-
67 nogashira coupling reaction of 7 and 10, and compounds 6
68 were obtained by sulfonamidation of 11 with 8. The detail
69 procedures are described in Supporting Information.
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Table 1. Biological activities and solubilities of 4 and 6a−g 22 the substituent on the pyridine (ring B) would contribute on
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8
23 additional lipophilic interaction with GCN2. According to this
24 result, contribution of substituents on the ring B was exhaust-
25 ively investigated, which revealed that installment of a small
26 and lipophilic substituent such as methyl, methoxy and halo-
27 gen at ortho or meta-position resulted in enhancement of po-
28 tency (data not shown). Particularly, compound 6b possessing
29 ortho, meta-dichloro phenyl ring exhibited substantial en-
30 hancement of both enzymatic and cellular GCN2 inhibitory
31 activities (IC50 = 3.7 and 190 nM, respectively) probably due
32 to gaining further lipophilic interaction with GCN2. The aque-
33 ous solubilities of these analogues were still insufficient, how-
34 ever, the introduction of a hydroxymethyl group into the meta-
35 position of the ring B (6c) found effective to make a molecule
36 more water-soluble. Notably, 6c showed a slight increase of
37 potencies from 6b, which suggested potential hydrogen bond-
38 ing between the hydroxymethyl group and GCN2. Regarding
39 the hinge-binding moiety, replacement of pyrazolopyridine
40 with similar bivalent 2-aminopyrimidine (6d) was investigated
41 and resulted in potent cellular activity with nano-molar level
42 of IC50 value (9.3 nM) with expectedly improvement of aque-
43 ous solubility through decrease of the number of aromatic
44 rings. Instead of ortho, meta-disubstituted phenyl group of the
45 ring B, introduction of ortho, meta-disubstituted pyridine was
46 permitted regarding potency (6e). Finally, optimization of the
47 substituent on pyrimidine-2-amine moiety, which is assumed
48 to extend to solvent region in our docking model, was con-
49 ducted for adjustment of physicochemical properties, which
50 led to 6f and 6g with maintaining potent GCN2 inhibitory
51 activities and improved aqueous solubilities.
GCN2a
Solubilityd
IC50 (nM)
Enzymeb
Cellc
3300
compd.
Ring A
Ring B
(μg/mL)
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720
4
<0.14
0.25
<0.08
3.0
(430 - 1200) (1800 - 6000)
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(33 - 58)
2300
6a
6b
6c
6d
(1400 - 3700)
3.7
190
(2.7 - 5.1)
(130 -270)
1.3
24
(18 - 32)
(0.8 - 2.1)
1.8
9.3
(6.7 - 13)
6.3
(1.2 - 2.8)
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To assess whether our compounds suppress GCN2-
53 mediated eIF2α phosphorylation, the effects of 4 and 6c−e on
54 eIF2 phosphorylation were evaluated in U2OS cells during
55 amino acid starvation (Table S1). Consistent with the cellular
56 activities (suppression of ATF4) shown in Table 1, 6c−e
57 showed inhibition of eIF2α phosphorylation. Our further anal-
58 ysis revealed that GCN2 inhibitory activities in the enzymatic
59 assay of our sulfonamide derivatives showed a good correla-
60 tion with those in the cellular assays whereas a set of PERK
61 selective inhibitors prepared in house did not result in a clear
62 correlation (Figure S3 and S4). These results proved the va-
63 lidity of our GCN2 cellular assays.
6e
2.4
20
(13 - 30)
1.4
(AcOH salt)
(1.9 - 3.1)
4.4
14
(8.5 - 22)
6f
4.0
(3.4 - 5.7)
3.9
8.2
(5.7 - 12)
6g
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(2.8 - 5.4)
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4
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aValues in parentheses indicate 95% confidence interval. bInhibition of
human GCN2 enzymatic activity. cSuppression of ATF4 induction in
U2OS cells with amino acid-free medium. dKinetic solubility was meas-
ured at pH6.8.
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The biological activities and aqueous solubilities of sulfon-
amide derivatives are summarized in Table 1. The reported
compound 4 showed moderate GCN2 inhibitory activities
confirmed by in-house enzymatic and cellular assays (IC50
=
10 720 and 3300 nM, respectively),18 which should be enhanced
11 to render a chemical tool to probe in vivo pharmacological
12 effects of GCN2 inhibition. In addition, the aqueous solubility
13 of 4 is low (<0.14 g/ml at pH6.8), which would result from
14 the large number of the aromatic rings composing the mole-
15 cule. According to the synthetic strategy mentioned in Figure
16 2, we quickly checked the removal of para-fluorophenyl por-
17 tion from the hinge-binding pyrazolopyridine (ring A) does
18 not affect GCN2 inhibitory activity (data not shown), and the
19 introduction of meta-bromo atom into the right hand pyridine
20 ring (6a) demonstrated a >16-fold increase of potency in bio-
21 chemical assay (IC50 = 43 nM) from 4, which suggested that
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Table 2. Pharmacokinetic parameters and kinetics data of
4 and 6d−g
55 these results, we conclude that 6d possesses unique slow-
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56 binding profile in GCN2 inhibition with some cellular PERK
57 inhibitory activity and 6e is a potent GCN2 inhibitor endowed
58 with high kinase selectivity. Both compounds were selected
59 for further pharmacological studies. The studies using 6e have
60 been reported,13 and the biological tests for 6d are described
61 in the latter part of this article.
PKa
b
Cmax
CLc
AUCpod MRTpoe
GCN2 dilution assayh
F f (%) fupg
compd. (ng/mL) (mg/h/kg) (ng/mL*h)
(h)
1.8
1.7
3.2
1.9
1.6
T1/2 (min.)
NTi
7.0
NTi
>1440
381
4
45
801
313
48
93
6d
6e
6f
6g
1201
2469
1372
124
2588
13389
3578
163
79.9
63.5
0.03
0.04
62 X-ray co-crystal structures of 6d and 6e with GCN2 were
63 successfully resolved, which validated our SBDD and revealed
64 that both bind to GCN2 as DFG-in binding mode while bury-
65 ing the allosteric pocket adjacent to C-helix (Figure 3). The
66 compounds sit in the ATP-binding site of the kinase domain,
67 and two-pairs of hydrogen bonds were formed by 2-
68 aminopyrimidine portion of ligands with the backbone of
69 hinge residue Cys805. The oxygen atom of the sulfonamide
70 moiety forms a hydrogen bond with the backbone amide
71 Phe867 from the conserved DFG-motif. In addition, the inter-
72 action between the deprotonated nitrogen atom and Asp866
73 was assumed, which has been reported in the case of other
74 type I half B-Raf inhibitors including vemurafenib.24 The
75 right-hand rings of compounds pack between gatekeeper resi-
76 due Met802 and other hydrophobic residues (Leu640 and
77 Val637) around C-helix. Both co-crystal structures showed
78 almost identical alignment, but it is noteworthy that the dichlo-
79 ro hydroxymethyl phenyl ring of 6d is stabilized via the hy-
80 drogen bond formed with the backbone carbonyl oxygen of
81 Leu640 of C-helix, which is the clear contrast between the
82 binding feature of 6d and 6e. As mentioned in Table 2, com-
83 pound 6d demonstrated slower dissociation profile, and this
84 additional hydrogen bond with Leu640 of GCN2 could be a
85 key factor resulting in its characteristic binding kinetics.
150
835
51.9 <0.01
13.5 <0.01
236
>720
9
3
4
5
6
7
aCassette dosing test in C57BL/6J mice (1.0 mg/kg, po and 0.1 mg/kg, iv,
c
0−8 h). bCmax: maximum concentration. CL: clearance. dAUCpo: area
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e
under the curve (po). MRTpo: mean residence time (po). fbioavailability.
gFraction unbound in plasma. hAssay protocol is shown in Supporting
Information. iNot tested.
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The PK and binding kinetics of 6d−g were further investigat-
ed (Table 2). Compared to 4, favorable PK profiles of 6d−f in
10 mice were observed. Since the microsomal stabilities and cel-
11 lular permeabilities of 4 and 6d−f are high (data not shown),
12 the improvement of aqueous solubility in 6d−f (Table 1)
13 would be projected to their higher bioavailabilities than 4. In
14 addition, profiling of these compounds regarding target resi-
15 dence time was conducted, and it was noteworthy that 6d and
16 6g possessing a hydroxymethyl group at the meta-position of
17 the right-hand phenyl ring (ring B) exhibited much slower
18 dissociation properties (T1/2 = >1440 and >720 min, respec-
19 tively) compared to 6e and 6f, which possesses pyridine with-
20 out a hydroxymethyl group as the right-hand ring (T1/2 = 381
21 and 236 min, respectively).
22 With respect to target specificity, 6e and 6f were screened
23 against a panel of 468 kinases in a binding assay to determine
24 its selectivity (KINOMEscanTM from DiscoveRx Corp.).21 At a
25 concentration of 1000 nM, both did not bind to any of the 468
26 kinases tested with >99.5% binding affinity (<0.5% control),
27 except GCN2 (Figure S5). Unlike 4, these compounds did not
28 show any binding affinities to B-Raf at the same concentration
29 (100% control). The exquisite selectivity profile verified our
30 drug design aiming type I half GCN2 inhibitor.
31
In order to further examine selectivity, 6d and 6e were in-
32 vestigated regarding inhibitory activities against eIF2 kinase
33 PERK, which was not included in the above panel. As illus-
34 trated in Table S2, both compounds showed potent PERK
35 inhibitory activity in enzymatic assay (6d: IC50 = 0.26 nM and
36 6e: IC50 = 3.8 nM). The ATP concentration used in PERK
37 enzyme assay (4 M; 4 × Km concentration) was extremely
86 Figure 3. The superposition of X-ray co-crystal structures of
87 6d (purple) and 6e (green) with GCN2. Key amino acid resi-
88 dues are depicted as sticks (gray) and hydrogen bonds are rep-
89 resented as dotted yellow lines.
38 lower than those in GCN2 enzyme assay (190 M; Km con-
39 centration) and cellular ATP levels (a few mM). Kinase inhibi-
40 tory activities of ATP competitive compounds decrease in
41 ATP concentration-dependent manner,22 which would result in
42 difficulty in the interpretation of selectivity. Therefore, cellu-
43 lar PERK assay was conducted by induction of CHOP expres-
44 sion in U2OS cells during thapsigargin-mediated ER stress in
45 order to examine selectivity against PERK under physiological
46 condition. As a result, 6d and 6e showed moderate to weak
47 PERK inhibitory activities in cells (IC50 = 230 and 9000 nM,
48 respectively, Table S2). Selectivity for GCN2 against PERK
49 under physiological condition was evaluated from inhibitory
50 activities of PERK in cells and that of GCN2 in cellular ATF4
51 assay, and was calculated to be 25-fold (6d) and 450-fold (6e),
52 respectively (Table S2). We also confirmed that the cellular
53 PERK inhibition of both compounds was weak in comparison
54 with the reference PERK inhibitor 1223 (Figure S6). Based on
90
To examine the impact of GCN2 inhibition on cancer cell
91 proliferation, acute lymphoblastic leukemia (ALL) CCRF-
92 CEM cells were treated with 6d in the presence of asparagine-
93 depleting agent asparaginase. CCRF-CEM cells are insensitive
94 to asparaginase, because asparaginase treatment induces as-
95 paragine synthetase through GCN2/ATF4 pathway activa-
96 tion.13 As shown in Figure S7, treatment with 6d greatly sensi-
97 tized CCRF-CEM cells to asparaginase. We also examined
98 whether the antiproliferative activity of 6d was mediated by
99 GCN2 inhibition. Importantly, the moderate antiproliferative
100 effects achieved by combining asparaginase and 6d treatment
101 were observed in GCN2-wildtype (WT) mouse embryonic
102 fibroblast (MEF) cells, but not in GCN2-knockout (KO) MEF
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(Figure S8). These results suggest that our GCN2 inhibitors 49 selectivity has been separately reported from our team.13 The
could be useful to treat cancer in combination with asparagi- 50 compound 6d with slow dissociation binding profile demon-
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nase.
51 strated suppression of GCN2 pathway activation with aspara-
52 ginase treatment in the CCRF-CEM cells and mouse xenograft
53 model. Notably, 6d displayed more potent in vivo target inhi-
54 bition than 6e. Further chemical optimization of this com-
55 pound will be warranted to achieve the exquisite target speci-
4
5
6
The GCN2 inhibition of 6d in asparaginase-treated CCRF-
CEM cells was further investigated. CCRF-CEM cells were
treated with 6d and asparaginase, and phosphorylated GCN2
7
8
(p-GCN2), phosphorylated eIF2 (p-eIF2) and ATF4 were 56 ficity like 6e. Our findings validate the potential of GCN2
measured by western blot analysis. As shown in Figure S9, 6d
demonstrated suppression on p-GCN2, p-eIF2 and ATF4
57 inhibition as a new approach for treating cancer.
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10 activated by asparaginase, which is consistent with our previ-
11 ous result using 6e.13 We also confirmed that ATF4 induction
12 observed in asparaginase-treated GCN2-WT MEF cells is not
13 occurred in GCN2-KO MEF cells, and clearly GCN2-
14 dependent.13 Other group convincingly demonstrated in
15 mammalian cells that amino acid starvation-mediated eIF2
16 phosphorylation and ATF4/CHOP expression depend on
17 GCN2.25 These results support that suppression on GCN2
18 pathway activation observed in Figure S9 results from GCN2
19 inhibition by 6d.
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59 ASSOCIATED CONTENT
60 Supporting Information Available
61 Data regarding characterization of compounds in this letter, de-
62 tailed experimental information of the biological activity assays,
63 and other crystallographic experiments are available free of
64 charge on the ACS Publications website at DOI: XXX.
65 Accession Codes
66 X-ray co-crystal structural data have been deposited in the Protein
67 Data Bank with accession codes 6N3L, 6N3N, 6N3O.
20 Finally, 6d was evaluated in CCRF-CEM xenograft model.
21 In the previous report, 6e suppresses GCN2 pathway activa-
22 tion to the basal level following pre-treatment with asparagi-
23 nase when orally dosed at 10 mg/kg to the same mice model.13
24 The xenograft mice were pretreated with 1,000 U/kg asparagi-
25 nase for 24 h and then 6d was orally administered at dosed of
26 0.3, 1 and 3 mg/kg, respectively. The effects of 6d on GCN2
27 pathway was assessed by autophosphorylation of GCN2 and
28 ATF4 levels 8 h after treatment of the inhibitor. As shown in
29 Figure 4, treatment with 6d at 3 mg/kg suppressed both self-
30 phosphorylation of GCN2 and the downstream effector ATF4
31 to the basal level following pre-treatment with asparaginase,
32 and this phenomenon is also observed with highly selective
33 compound 6e.13 This result suggested that 6d functionally
34 inhibits GCN2 and the downstream effector in vivo, even
35 though the involvement of PERK inhibition with 6d is not
36 completely excluded. Interestingly, the minimum effective
37 dose of 6d (3 mg/kg) was lower than 6e (10 mg/kg)13 despite
38 its shorter duration of drug concentration than 6e (Table 2).
39 We hypothesize that the slower dissociation binding profile of
40 6d might contribute to the long-lasting pharmacological effect.
68 AUTHOR INFORMATION
69 Corresponding Author
70 *Email: jun.fujimoto@takeda.com.
71 Present Addresses
72 ‡ Axcelead Drug Discovery Partners, Inc.: 26-1, Muraoka-
73 Higashi 2-chome, Fujisawa, Kanagawa, Japan.
74 ∇PeptiDream, Inc.: 3-25-23, Tonomachi, Kawasaki-ku, Kawasa-
75 ki, Kanagawa, Japan.
76 Author Contributions
77 The manuscript was written through contributions of all authors.
78 All authors have given approval to the final version of the manu-
79 script.
80 Notes
81 The authors declare no competing financial interest.
82 ACKNOWLEDGMENT
83 We acknowledge Shunsuke Ebara, Yuka Hasegawa and Ya-
84 suko Tsuchiya for biological assay. Yoshihiko Hirozane and
85 Ikuo Miyahisa provided data regarding kinase selectivity. Jun-
86 pei Takeda and Katsuhiko Yamamoto provided physicochemi-
87 cal data of the compounds. Sachiko Shiotani and Katsunori
88 Takashima led proper project management. The staff of the
89 Berkeley Center for Structural Biology supported beamline
90 5.0.3 at the Advanced Light Source. The Advanced Light
91 Source is supported by the Director, Office of Science, Office
92 of Basic Energy Sciences, of the U.S. Department of Energy.
93 This research used resources of the Advanced Photon Source,
94 a U.S. Department of Energy (DOE) Office of Science User
95 Facility operated for the DOE Office of Science by Argonne
96 National Laboratory.
41 Figure 4. Suppression of GCN2 pathway activation in CCRF-
42 CEM xenograft tumor at 8 h after oral administration of 6d
43 (0.3, 1 and 3 mg/kg, qd) with pre-treatment of asparaginase
44 (1000 U/kg, qd, ip).
97 ABBREVIATIONS
98 GCN2, general control nonderepressible 2; ALL, acute lym-
99 phocytic leukemia; ISR, integrated stress response; tRNA,
100 transfer RNA; PERK, protein kinase R-like endoplasmic retic-
101 ulum kinase; SBDD, structure-based drug design; PK, phar-
102 macokinetics; ATF4, activating transcription factor 4; p-
103 GCN2, phosphorylated GCN2; PD, pharmacodynamics;
104 Cs2CO3, cesium carbonate; DMSO, dimethyl sulfoxide.
45
In summary, potent and orally available GCN2 inhibitors as
46 potentially first-in-class antitumor agents were successfully
47 identified through our strategy aiming type I half inhibitors.
48 Detailed biological evaluation of 6e endowed with high kinase
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