New Series of Potent Allosteric Inhibitors of Deoxyhypusine Synthase

Article abstract of DOI:10.1021/acsmedchemlett.0c00331

Deoxyhypusine synthase (DHPS) is the primary enzyme responsible for the hypusine modification and, thereby, activation of the eukaryotic translation initiation factor 5A (eIF5A), which is key in regulating the protein translation processes associated with tumor proliferation. Although DHPS inhibitors could be a promising therapeutic option for treating cancer, only a few studies reported druglike compounds with this inhibition property. Thus, in this work, we designed and synthesized a new chemical series possessing fused ring scaffolds designed from high-throughput screening hit compounds, discovering a 5,6-dihydrothieno[2,3-c]pyridine derivative (26d) with potent inhibitory activity; furthermore, the X-ray crystallographic analysis of the DHPS complex with 26d demonstrated a distinct allosteric binding mode compared to a previously reported inhibitor. These findings could be significantly useful in the functional analysis of conformational changes in DHPS as well as the structure-based design of allosteric inhibitors.

Full text of DOI:10.1021/acsmedchemlett.0c00331

pubs.acs.org/acsmedchemlett
Letter
New Series of Potent Allosteric Inhibitors of Deoxyhypusine
Synthase
Yuta Tanaka,* Osamu Kurasawa, Akihiro Yokota, Michael G. Klein, Bunnai Saito,
Shigemitsu Matsumoto, Masanori Okaniwa, Geza Ambrus-Aikelin, Noriko Uchiyama, Daisuke Morishita,
Hiromichi Kimura, and Shinichi Imamura
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ABSTRACT: Deoxyhypusine synthase (DHPS) is the primary enzyme responsible for the hypusine modification and, thereby,
activation of the eukaryotic translation initiation factor 5A (eIF5A), which is key in regulating the protein translation processes
associated with tumor proliferation. Although DHPS inhibitors could be a promising therapeutic option for treating cancer, only a
few studies reported druglike compounds with this inhibition property. Thus, in this work, we designed and synthesized a new
chemical series possessing fused ring scaffolds designed from high-throughput screening hit compounds, discovering a 5,6-
dihydrothieno[2,3-c]pyridine derivative (26d) with potent inhibitory activity; furthermore, the X-ray crystallographic analysis of the
DHPS complex with 26d demonstrated a distinct allosteric binding mode compared to a previously reported inhibitor. These
findings could be significantly useful in the functional analysis of conformational changes in DHPS as well as the structure-based
design of allosteric inhibitors.
KEYWORDS: Deoxyhypusine synthase (DHPS), eukaryotic translation initiation factor 5A (eIF5A),
nicotinamide adenine dinucleotide (NAD), spermidine, allosteric inhibitor
binds to DHPS similarly to spermidine11−13 and possesses
eoxyhypusine synthase (DHPS) is an enzyme that
ε
inhibitory activity; nonetheless, the potential application of 1 is
Dactivates via hypusine [N -(4-amino-2-hydroxybutyl)-
limited by its polyamine-like structure (Figure 1A).14−16 We
lysine] modification the eukaryotic translation initiation factor
recently reported a novel allosteric inhibitor (2) and its
cocrystal structure with DHPS, which revealed the dynamic
conformational change in the DHPS structure.17 However, its
application for the further biological evaluation of DHPS is still
limited due to the narrow structure−activity relationship
(SAR) of its related compounds, although the structural
analysis suggests that the allosteric pocket is druggable. Hence,
5A (eIF5A), which is a regulator of the protein translation
processes involved in tumor growth.1−5 This modification
involves two enzymatic steps. DHPS catalyzes the first rate-
limiting step, which is the cleavage of the polyamine
spermidine: the 4-aminobutyl moiety is transferred to the ε-
amino group of one specific lysine residue of the eIF5A
precursor to form a deoxyhypusine intermediate.6−8 In the
second step, deoxyhypusine hydroxylase (DOHH) converts
the deoxyhypusine-containing intermediate into hypusine-
containing mature eIF5A (Supporting Information). Blocking
the biological activity of eIF5A by inhibiting the DHPS-
mediated modification of its precursor could be an effective
strategy in cancer therapy.9,10 N1-Guanyl-1,7-diaminoheptane
(GC-7) (1), a substrate (spermidine) mimetic compound,
Received: June 16, 2020
Accepted: July 30, 2020
Published: July 30, 2020
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© XXXX American Chemical Society
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A

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more potent and druglike inhibitors are needed for detailed
biologic studies on the therapeutic potential of targeting the
DHPS-mediated hypusine synthesis. Here, we describe a new
series of potent DHPS inhibitors discovered through SAR
studies from hit compounds obtained via a high-throughput
screening (HTS). Among them, the X-ray crystallographic
analysis of a highly potent compound (26d, IC50 = 0.0092 μM)
in complex with DHPS demonstrates a distinct binding mode
compared to the previously reported 2.
To identify a new series of DHPS inhibitors, we conducted
an HTS via an enzyme assay. Several hit compounds
containing a 3-pyridyl group such as 3 and 4 (Figure 1B),
whose chemical structures differ from those of other reported
inhibitors (Figure 1A), showed inhibitory activities toward
DHPS.17−20 An initial SAR study revealed that the 3-pyridine
moiety is essential for their inhibitory activity (data not
shown). Based on the structural similarity among these
compounds, we presumed that a fused ring system with 3-
pyridine is a pharmacophore of their series for inhibiting
DHPS, as shown in Figure 1B; thus, we started chemical
modifications according to the designed molecules maintaining
the possible pharmacophore.
Figure 1. (A) Chemical structures of spermidine and reported DHPS
inhibitors. (B) Structures of HTS hits (3 and 4), and a possible
pharmacophore. The DHPS inhibitory activities were determined by
an enzyme assay. IC50 values and 95% confidence intervals were
calculated via the nonlinear regression analysis of the percent
inhibition data.
a
Scheme 1. Synthesis of Benzyl Derivatives 6a−d
Benzyl derivatives with designed fused rings 6a−d were
synthesized via benzylation of 5a−d as illustrated in Scheme 1.
The precursors 5a−d were prepared as described in the
Supporting Information.
Scheme 2 illustrates the synthesis of 5,6-dihydrothieno[2,3-
c]pyridine derivatives 9a−g from the intermediate 5d.
Alkylation of 5d with methyl 2-bromo-2-phenylacetate gave
the racemic ester 7a, which was successively reduced by
NaBH4 in the presence of CaCl2 to yield the alcohol 8a.
Conversion into the racemic amine 9a was performed via
introduction of phthalimide by Mitsunobu reaction and
subsequent treatment with hydrazine monohydrate at 70 °C
in EtOH. The optical resolution of 9a via chiral HPLC
a
Reagents and conditions: (a) benzyl bromide, NaH, DMF.
a
Scheme 2. Synthesis of Compounds 7a, 8a, and 9a−g
a
Reagents and conditions: (a) methyl 2-bromo-2-phenylacetate, potassium tert-butoxide, THF, 90%; (b) NaBH4, CaCl2, MeOH; (c) (i)
phthalimide, triphenylphosphine, diisopropyl azodicarboxylate, toluene; (ii) hydrazine monohydrate, EtOH, 70 °C; (d) 10a−b, potassium tert-
butoxide, THF; (e) K2CO3, MeOH, H2O, 80 °C, 62%; (f) 11, potassium tert-butoxide, DMF, 52%; (g) 2 M NaOH, MeOH, 87%.
B
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a
Scheme 3. Synthesis of Compounds 22a−b and 26a−d
a
Reagents and conditions: (a) bromine, AcOH, 61%; (b) 3-pyridineboronic acid, Pd(dppf)Cl2·CH2Cl2, Cs2CO3, DME, H2O, 80 °C, 72%; (c)
trifluoromethanesulfonic anhydride, pyridine; (d) (i) (E)-1-ethoxyethene-2-boronic acid pinacol ester, Pd(dppf)Cl2·CH2Cl2, Cs2CO3, DME, H2O,
80 °C; (ii) 12 M HCl, THF, 30% (two steps); (e) 19a−b, 2-picoline borane, MeOH, AcOH; (f) NaOMe, MeOH; (g) 4 M HCl in EtOAc; (h)
23a−d, 2-picoline borane, MeOH, AcOH; (j) (i) phthalimide, triphenylphosphine, diisopropyl azodicarboxylate, toluene; (ii) hydrazine
monohydrate, EtOH, 70 °C.
afforded both its enantiomers: the S- (9b) and the R-form
(9c). The absolute configuration of each enantiomer was
determined through single-crystal X-ray analysis (Supporting
Information). Using the same procedures for 9a, compounds
9d−e were synthesized from 5d and the corresponding
bromides 10a−b, which were prepared as described in the
Supporting Information. The Boc group of 9e was removed in
the presence of K2CO3 at 80 °C in MeOH and H2O to obtain
the indole derivative 9f. By treating 5d with the bromide 11,
we formed the benzoyl derivative 12, which was hydrolyzed to
yield the alcohol 13, which was then converted into the amine
9g similarly to 9a.
Scheme 3 describes an alternative method to obtain 5,6-
dihydrothieno[2,3-c]pyridine derivatives. By using 3-hydrox-
ythiophene 14 as a starting material, triflate 17 was synthesized
via bromination at the 4-position, Suzuki coupling, and
successive triflation of the resulting phenol 16. The
introduction of an ethyl vinyl ether via Suzuki coupling
followed by hydrolysis under an acidic condition yielded the
aldehyde 18. The 5,6-dihydrothieno[2,3-c]pyridine scaffold
was constructed via the reductive amination of 18 with the
racemic amines 19a−b and the subsequent intramolecular
cyclization in the presence of NaOMe in MeOH, producing
the cyclized compounds 21a−b. The removal of the Boc group
yielded the racemates 22a−b. In a similar way, the alcohols
25a−d were synthesized through the cyclization of the amines
24a−d, which were prepared from 18 and the commercially
available R-form or racemic amines 23a−d. The application of
the same reactions shown in Scheme 2 to alcohols 25a−d gave
the amines 26a−d.
To verify the potentials of each bicyclic scaffold having the
3-pyridyl moiety, the derivatives with a benzyl group derived
from the hit compound 4 were evaluated in the enzyme assay
(Table 1). The conversion of the thieno[3,2-d]pyrimidine ring
in 4 to isoquinolone 6a improved the inhibitory activity (IC50
= 8.7 μM). Dihydroisoquinolone 6b also showed a comparable
potency to 6a, indicating that the left ring of the bicyclic
scaffold is not necessarily aromatic and the nitrogen atom on
the 1-position of the thieno[3,2-d]pyrimidine (4) weakens the
potency. A significant loss of potency was observed by
changing the position of the 3-pyridine moiety (6c, IC50
>
100 μM), indicating that the orientation of the substituents
from the scaffold is a key factor. Furthermore, the evaluation of
scaffolds such as fused the 6,5- (6d) and 5,6-membered (27)
bicyclic rings revealed the strong inhibitory activity of 5,6-
dihydrothieno[2,3-c]pyridine 6d (IC50 = 6.3 μM).
C
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Table 1. SAR for Fused Ring Scaffolds 6a−d and 27
Table 2. SAR for Substituents of 5,6-Dihydrothieno[2,3-
c]pyridine-7(4H)-one Derivatives
a
IC50 values were calculated via the nonlinear regression analysis of
the percent inhibition data. 95% confidence intervals are shown in the
Supporting Information.
Based on the above-mentioned SAR results, we further
explored the substituents on the left side of the 5,6-
dihydrothieno[2,3-c]pyridine scaffold (Table 2). Since the hit
compound 3 bearing a hydroxymethyl group at the benzyl
position showed inhibitory activity, we expected that the
introduction of a polar functional group at the corresponding
benzylic position of 6d would be tolerable. Indeed,
introduction of the hydroxymethyl group (8a) significantly
enhanced the potency although a methyl ester (7a) was
unfavorable, indicating the necessity of a hydrogen donor for
the inhibitory activity. The conversion of the primary alcohol
to an amine further improved the potency, and the racemate
amine 9a exhibited a strong IC50 value of 0.018 μM. Moreover,
we found that R- form 9c is a eutomer after separation of each
enantiomer of 9a. On the other hand, neither the extension of
the alkyl chain (9g) nor the conformational restriction of the
amine moiety via cyclization (22a) increased the potency
compared to 9a.
a
b
Refer to Table 1. Racemate.
evaluate the inhibitory potency of compounds and the
competitiveness against NAD or spermidine at each Km or
higher concentrations although there is a limitation to analyze
precise kinetic profiles due to 120 min incubation time needed
in this assay. To analyze the inhibition mode of the newly
discovered chemical series, the potent compound 9c was
evaluated under the high NAD concentration (Table 3); its
DHPS inhibition at 250 μM NAD was 29-fold lower than at 14
μM NAD, indicating a NAD competitive profile similar to that
of 2.17 The X-ray cocrystal analysis of the new chemical series
revealed its binding mode and a mechanism of the NAD
competition (vide infra).
In a previous work, the allosteric inhibitor 2 showed
nicotinamide adenine dinucleotide (NAD), a coenzyme of
DHPS, competitive profile as a result of dramatic conforma-
tional changes of DHPS induced by its binding.17 We
employed the radiometric assay using [3H]-spermidine to
To further improve the inhibition potency and identify a
promising tool molecule, we successively converted the
benzene ring on the left part into other hydrophobic
substituents (Table 3). We used a DHPS concentration of
D
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Table 3. Conversion of Benzene Ring to Other
Hydrophobic Substituents
a
b
Refer to Table 1. The metabolism clearance of each compound was
c
examined by using mice liver microsomes. Determined by using
Daylight software. Racemate. Not determined.
d
e
a
Table 4. Pharmacokinetic Parameters of 26d in Mice
b
b
c
c
c
d
Vdss
CLtotal
Cmax (ng/
AUC0−8h
MRT
(h)
F
(mL/kg)
(mL/h/kg)
mL)
(ng·h/mL)
(%)
8491
8965
4.8
13.1
2.46
11.7
a
Figure 2. Complex crystal structure of compound 26d with DHPS
(PDB ID 6WL6). All images were prepared using PyMOL.22 (A)
Tetramer with each monomer indicated by a different color in cartoon
model. Each 26d molecule is represented as a sphere. (B) 26d bound
allosteric site. 26d is shown as a stick model in salmon. The protein is
colored by chain and shown as a cartoon, while the two residues,
Gly282 and Lys287, are shown as stick models. The black dashed
circle indicates a loop structure that was conformationally changed
from an α-helix in GC-7 bound structure (PDB ID 6P4V). NAD and
GC-7 are shown in surface representation by superposition of the
complex (PDB ID 6P4V) in pink and deep blue, respectively.
Compound 2 is shown by the superposition of the complex (PDB ID
6PGR), which is shown as a stick model in magenta. (C) Schematic
diagrams of 26d (PDB ID 6WL6) and 2 (PDB ID 6PGR) bound
conformations in one dimer of a DHPS tetramer.
Cassette dosing. Male Institute of Cancer Research mice (n = 3).
b
c
d
0.1 mg/kg, iv. 1 mg/kg, po. Bioavailability.
0.024 μM in the enzyme assay (Supporting Information).
Since the tight-binding21 possibly caused an underestimation
of the IC50 value of a highly potent compound, we analyzed the
inhibitory activities also under high NAD concentrations to
obtain weakened IC50 values for the SAR analysis of the NAD
competitive inhibitors. The conversion of the benzene ring
(9c) into bicyclic rings such as benzothiophene (9d) and
indole (9f) decreased the inhibitory activity, while the
compound with a cyclohexane substituent (26a) exhibited
improved potency under high NAD concentration (IC50 = 0.17
μM). This result encouraged us to pursue other alkyl
substituents. Although n-hexane derivative 22b also showed
potent activity, its metabolic stability in mice liver microsome
was diminished, probably due to high lipophilicity. Therefore,
we examined shorter and/or branched alkyl chains (26b−d) to
reduce the lipophilicity, resulting in the discovery of the
isobutyl derivative 26d, which exhibited the most potent
inhibitory activity among the 5,6-dihydrothieno[2,3-c]pyridine
derivatives (IC50= 0.069 μM under high NAD condition). In
addition, 26d showed orally available pharmacokinetic profiles
in mice (Table 4). Further investigations are needed to verify
E
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surprise, this Lys287 is overlapped with the spermidine binding
site in the DHPS/2 complex structure (Figure 2B, C). Overall,
these results indicate the versatility of the allosteric site as a
druggable pocket, which can be utilized for exploring DHPS
inhibitors.
Additionally, this complex structure well explained the
results of the SAR studies (Figure 3). Regarding the 3-pyridyl
moiety, a hydrogen bonding was observed between a nitrogen
atom of the pyridine ring and a side chain of Asp262 and/or a
main chain of Ser233 via a water molecule, confirming that it is
required for the potency. Furthermore, the carbonyl oxygen on
the bicyclic scaffold formed a hydrogen bond with Lys287. The
primary amine of 26d also formed a hydrogen bond with
Asp243 (side chain) and Asp238 (main chain), resulting in
high affinity in the pocket and strong inhibitory activity. The
alkyl side chain occupies a hydrophobic pocket surrounded by
Phe100, Ala270, Met278, and Val286; however, there might be
other residues surrounding the alkyl side chain because the
288−298 and 316−334 residues are disordered in the 26d
complex. As described in Table 1, the orientation of
substituents on the fused ring scaffolds is important for the
potency, which is reasonable to maintain or stabilize the
interactions between inhibitor and DHPS.
In summary, we discovered a new class of potent allosteric
DHPS inhibitors via SAR studies by initially exploring bicyclic
scaffolds suggested by the pharmacophore of HTS hit
compounds. These compounds were characterized as NAD
competitive inhibitors, and the X-ray cocrystal analysis
revealed that the potent inhibitor 26d binds to the allosteric
pocket in a newly identified binding mode, distinct from that of
the previously reported inhibitor 2. The identification of the
novel binding mode with the compound possessing improved
enzymatic inhibitory activity and favorable PK properties
suggests the promise of using the allosteric site as a druggable
pocket, and these results would pave the road for the structure-
based design for further lead optimization of allosteric
inhibitors targeting DHPS.
Figure 3. (A) Allosteric binding site of 26d. DHPS is shown at the
surface (PDB ID 6WL6). 26d and a part of the surrounding residues
and water molecules are represented as sticks and spheres,
respectively. The colors are the same as in Figure 2B. The dashed
lines indicate the hydrogen bonds between 26d and the protein/water
molecules. (B) Schematic diagram of interactions in stabilizing 26d at
the allosteric site.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00331.
Figure of the DHPS-eIF5A pathway and chemical
reaction of DHPS, 95% confidence intervals of enzyme
IC50, synthetic experimental details and characterization
data, single-crystal X-ray structures of 9b and 9c, assay
protocols, and cocrystal data of 9c and 26d (PDF)
the therapeutic potentials of the inhibitor, such as confirmation
of effects in in vivo tumor xenograft models as well as in eIF5A-
amplified cancer cells. However, we believe that the identified
chemical series can be a good lead molecule.
To unravel the binding mode of the newly discovered
compounds, we defined the X-ray crystal structure of the
DHPS/26d complex via soaking experiments with a 2.15 Å
resolution (Figure 2). The results revealed a tetramer of this
binary complex similar to the previously reported structure of
the DHPS/2 complex;17 26d was bound to the allosteric
pocket generated via an α-helix unfolding, which was induced
by the inhibitor binding. Despite the fact that the binding site
of 26d partially overlaps that of 2, their binding modes turned
out to be quite different. In contrast to 2, Gly282 is located
near a NAD binding site but neither flips nor seems to
interrupt the NAD binding. This implies a different mechanism
of competitive inhibition of 26d to NAD compared to 2.
Instead of Gly282, upon the 26d binding, Lys287 moves to
another NAD binding site and hinders its pocket. To our
Accession Codes
The atom coordinates and structure factors for the DHPS/9c
and DHPS/26d complexes have been deposited in the Protein
Data Bank with the accession codes 6WKZ, and 6WL6,
respectively.
AUTHOR INFORMATION
■
Corresponding Author
Yuta Tanaka − Pharmaceutical Research Division, Takeda
Pharmaceutical Company Limited, Fujisawa, Kanagawa 251-
8555, Japan; orcid.org/0000-0002-6110-7990;
Phone: +81-466-32-1164; Email: yuta.tanaka@takeda.com
F
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Letter
Authors
NAD, nicotinamide adenine dinucleotide; SAR, structure−
activity relationship
Osamu Kurasawa − Pharmaceutical Research Division, Takeda
Pharmaceutical Company Limited, Fujisawa, Kanagawa 251-
8555, Japan
Akihiro Yokota − Pharmaceutical Research Division, Takeda
Pharmaceutical Company Limited, Fujisawa, Kanagawa 251-
8555, Japan
Michael G. Klein − Department of Structural Biology, Takeda
California, San Diego, California 92121, United States
Bunnai Saito − Pharmaceutical Research Division, Takeda
Pharmaceutical Company Limited, Fujisawa, Kanagawa 251-
8555, Japan
Shigemitsu Matsumoto − Pharmaceutical Research Division,
Takeda Pharmaceutical Company Limited, Fujisawa,
Kanagawa 251-8555, Japan
Masanori Okaniwa − Pharmaceutical Research Division,
Takeda Pharmaceutical Company Limited, Fujisawa,
Kanagawa 251-8555, Japan
Geza Ambrus-Aikelin − Department of Structural Biology,
Takeda California, San Diego, California 92121, United States
Noriko Uchiyama − Pharmaceutical Research Division, Takeda
Pharmaceutical Company Limited, Fujisawa, Kanagawa 251-
8555, Japan
Daisuke Morishita − Pharmaceutical Research Division, Takeda
Pharmaceutical Company Limited, Fujisawa, Kanagawa 251-
8555, Japan
Hiromichi Kimura − Pharmaceutical Research Division,
Takeda Pharmaceutical Company Limited, Fujisawa,
Kanagawa 251-8555, Japan
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Complete contact information is available at:
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Author Contributions
All authors contributed equally and have given approval to the
final version of the manuscript.
Notes
The authors declare no competing financial interest.
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synthase. J. Med. Chem. 2020, 63, 3215−3226.
ACKNOWLEDGMENTS
■
We thank all members of the DHPS Program Team at Takeda
for their contribution. We thank Katsuaki Oda for assistance
with chiral separation and Mitsuyoshi Nishitani for assistance
with single-crystal X-ray study along with deposition of data to
the Cambridge Crystallographic Data Centre. We also thank
Weston Lane and Gyorgy Snell for diffraction data collection
and Jaya Bhatnagar for assistance with crystallization. The
GM/CA@APS has been funded in whole or in part with
federal funds from the National Cancer Institute (ACB-12002)
and the National Institute of General Medical Sciences (AGM-
12006). This research used resources of the Advanced Photon
Source, a U.S. Department of Energy (DOE) Office of Science
User Facility operated for the DOE Office of Science by
Argonne National Laboratory under Contract No. DE-AC02-
06CH11357.
ABBREVIATIONS
■
DHPS, deoxyhypusine synthase; eIF5A, eukaryotic translation
initiation factor 5A; DOHH, deoxyhypusine hydroxylase;
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