Structurally novel highly potent proteasome inhibitors created by the structure-based hybridization of nonpeptidic belactosin derivatives and peptide boronates

Article abstract of DOI:10.1021/jm500045x

We previously developed highly potent proteasome inhibitor 1 (IC ₅₀ = 5.7 nM) and its nonpeptide derivative 2 (IC₅₀ = 29 nM) by systematic structure-activity relationship studies of the peptidic natural product belactosin A and subsequent rational topology-based scaffold hopping, respectively. Their cell growth inhibitory activities, however, were only moderate (IC₅₀ = 1.8 μM (1) and >10 μM (2)). We therefore planned to replace the unstable β-lactone warhead with a more stable boronic acid warhead. Importantly, belactosin derivatives bind mainly to the proteasome binding site, which is different from that occupied by known peptide boronate proteasome inhibitors such as bortezomib, suggesting that their hybridization might lead to the development of novel potent inhibitors. Here we describe design, synthesis, and biological activities of the newly developed potent hybrid proteasome inhibitors. Interestingly, these hybrids, unlike bortezomib, were highly selective for proteasomes and have long residence times despite having the same boronic acid warhead.

Full text of DOI:10.1021/jm500045x

Article
pubs.acs.org/jmc
Structurally Novel Highly Potent Proteasome Inhibitors Created by
the Structure-Based Hybridization of Nonpeptidic Belactosin
Derivatives and Peptide Boronates
Shuhei Kawamura,†,⊥ Yuka Unno,§,⊥ Akira Asai,§ Mitsuhiro Arisawa,∥ and Satoshi Shuto*,†,‡
‡
†Faculty of Pharmaceutical Sciences and Center for Research and Education on Drug Discovery, Hokkaido University, Kita-12,
Nishi-6, Kita-ku, Sapporo 060-0812, Japan
§Graduate School of Pharmaceutical Sciences, University of Shizuoka, Yada, Shizuoka 422-8526, Japan
∥Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
S
* Supporting Information
ABSTRACT: We previously developed highly potent proteasome
inhibitor 1 (IC50 = 5.7 nM) and its nonpeptide derivative 2 (IC50 =
29 nM) by systematic structure−activity relationship studies of the
peptidic natural product belactosin A and subsequent rational
topology-based scaffold hopping, respectively. Their cell growth
inhibitory activities, however, were only moderate (IC50 = 1.8 μM
(1) and >10 μM (2)). We therefore planned to replace the
unstable β-lactone warhead with a more stable boronic acid
warhead. Importantly, belactosin derivatives bind mainly to the
proteasome binding site, which is different from that occupied by known peptide boronate proteasome inhibitors such as
bortezomib, suggesting that their hybridization might lead to the development of novel potent inhibitors. Here we describe
design, synthesis, and biological activities of the newly developed potent hybrid proteasome inhibitors. Interestingly, these
hybrids, unlike bortezomib, were highly selective for proteasomes and have long residence times despite having the same boronic
acid warhead.
principle among all active sites, which is nucleophilic attack of
the N-terminal Thr hydroxyl group, but it is the singularity of
each substrate binding channel that determines the chemical
nature of the specificity (S) pockets and accommodates the
ligand’s side chains (P sites) with respect to their amino acid
progression. Interestingly, translation of the immunoprotea-
some is induced by interferon-γ to be primarily expressed in
hematopoietic cells,11 whereas constitutive proteasome is
ubiquitously expressed in all kinds of cells. Thus, blocking
either the constitutive proteasome or immunoproteasome has a
major impact on specific biological potencies.3,6a
Belactosin A is a naturally occurring tripeptide metabolite
produced by Streptomyces sp., that comprises L-alanine, 3-(trans-
2-aminocyclopropyl)-L-alanine, and a chiral carboxy-β-lactone
moiety (Figure 1)12 and inhibits proteasome ChT-L activity13
by acylating the active site Thr residue via its strained β-lactone-
opening, as confirmed by X-ray crystallographic analysis of
belactosin derivatives in complex with proteasome.14 Impor-
tantly, belactosin A and its derivatives are the only known
proteasome inhibitors that bind to the primed substrate binding
site of the proteasome as well as the nonprimed binding
site14,15 so that it is an attractive lead compound for developing
INTRODUCTION
■
The ubiquitin−proteasome system is the major pathway for
systematic degradation of intracellular proteins1 and is involved
in many physiologically important cellular processes, including
signal transduction,2 the immune response,3 the unfolded
protein response,4 and cell cycle progression.5 The 20S
proteasome, the catalytic core particle of proteasome, is an
attractive target molecule for anticancer and antiautoimmune
drugs because proteasome inhibition causes cell cycle arrest and
induces apoptosis.6 In fact, bortezomib (Figure 1), a dipeptide
boronic acid proteasome inhibitor, is a currently available
prescription drug against various cancer types and represents
one of the best known blockbuster drugs.7
The eukaryotic 20S proteasome contains three active β
subunits β1, β2, and β5, which are responsible for the caspase-
like (C-L), trypsin-like (T-L), and chymotrypsin-like (ChT-L)
activities, respectively,8 whereas vertebrates possess an even
more elaborate version of proteasomes9 that harbor three
different types of the 20S proteasome: constitutive protea-
somes, immunoproteasomes, and thymoproteasomes. Each of
these proteasome types has its own set of active sites:10 the
constitutive proteasome incorporates subunits β1c, β2c, and
β5c, the immunoproteasome incorporates subunits β1i
(LMP2), β2i (MECL-1), and β5i (LMP7), and the
thymoproteasome incorporates subunits β1i, β2i, and β5t.
The mechanism of peptide bond cleavage follows a universal
Received: January 9, 2014
Published: February 13, 2014
© 2014 American Chemical Society
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Figure 1. Known proteasome inhibitors.
proteasome inhibitors that are unique from the currently
known inhibitors.
We performed systematic structure−activity relationship
(SAR) studies of belactosin A to develop a highly potent
proteasome inhibtor 1.14c,16 On the basis of the results of our
SAR and the binding mode studies,17 we identified a
remarkably simplified nonpeptide inhibitor 2 by the topology-
based scaffold hopping of 1.18 Although 2 had remarkably high
proteasome inhibitory activity (IC50 = 29 nM), its cell growth
inhibitory effect was insignificant (IC50 > 10 μM), perhaps due
to the instability of the β-lactone warhead under biological
condition.19 To develop stabilized analogues of belactosin A,
we previously designed and synthesized a series of belactosin
analogues in which the β-lactone moiety was replaced with a β-
lactam17 or more sterically hindered β-lactones19 as a warhead,
but this kind of modification significantly impaired proteasome
inhibitory activity (Figure 2).20 Therefore, we planned to
develop more potent cell growth inhibitors by replacing the β-
lactone warhead of nonpeptide inhibitor 2 with another
chemotype of stable warhead.
Peptide boronates are the most valuable class of proteasome
inhibitors as represented by the clinically useful drug
bortezomib and drug candidates CEP-1877021 and MLN-
970822 (Figure 1), which are currently undergoing clinical trials.
This class of inhibitors binds only to the nonprimed binding
site of proteasome23 and exhibits remarkable cell growth
inhibitory effects, in which the boronic acid moiety functions as
a very effective warhead.21,22,24 Therefore, we thought that
replacing the β-lactone moiety of the nonpeptide belactosin A
derivatives with a boronic acid warhead might allow us to
identify the structurally novel proteasome inhibitors with
potent cell growth inhibitory effects.
On the basis of the above-mentioned results and
considerations, we designed and synthesized a series of hybrids
of the nonpeptide belactosin A derivatives and peptide
boronates and investigated various biological properties of
these compounds such as their inhibitory effects on constitutive
20S proteasome subunits and immunoproteasome, cell growth
inhibitory effects, and the reversibility of their proteasome
inhibition. The most potent compound 4a identified in the
present study has inhibitory effect on ChT-L activity that is as
strong as or even stronger than that of bortezomib, and the
inhibitory effects against proteolytic enzymes are highly
selective to proteasome. Interestingly, 4a has a long residence
time compared to bortezomib despite having the same boronic
Figure 2. Belactosin A derivatives previously developed by us.
acid warhead as bortezomib. Here we report our results in
detail.
RESULTS AND DISCUSSION
■
Design of Compounds. The two X-ray crystal structures
of a proteasome in complex with bortezomib23 or belactosin A
derivative 1 are superimposed in Figure 3,14c which clearly
shows that bortezomib binds to the nonprimed binding site
with its P2 side chain oriented toward the vacant primed
binding site and that 1 binds mainly to the primed binding site.
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Figure 3. Superposition of the X-ray crystal structures of bortezomib23 (gray tube; PDB code 2F16) and belactosin A derivative 114c (green tube;
PDB code 4J70) in complex with proteasome.
Figure 4. Design of hybrid inhibitors of nonpeptide belactosin A derivatives and peptide boronates.
Figure 5. Predicted binding mode of 4a by docking simulation.
Therefore, we assumed that elongation of the P2 side chain of
bortezomib toward the primed binding site would allow us to
develop novel hybrid inhibitors of peptide boronates and
belactosin A derivatives. As mentioned above, we previously
developed nonpeptide derivative 2 by topology-based scaffold
hopping of 1,18 and therefore we decided to take advantage of 2
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Table 1. Inhibitory Effects of Compounds 3a−5a, 3b−5b, and Bortezomib on Human 20S Proteasome and HCT116 Cell
Growth
a
Based on three experiments.
for designing hybrid compounds to reduce the number of
peptide bonds. Thus, we designed hybrid compounds 3a−5a
and 3b−5b, in which the moiety derived from the nonpeptidic
scaffold of 2 with two aromatic rings was ligated with the P2
side chain via an amide linkage (Figure 4).25,26 To search for
the precise three-dimensional positioning of the two aromatic
rings and the boronic acid warhead for effective proteasome
binding in these hybrid compounds, we designed compounds
with Asp (m = 1) or Glu (m = 2) P2 residues and different
lengths of alkyl linkers (n = 0−2).
To justify the molecular design described above, we
performed docking simulation of the designed hybrids 3a−5a
and 3b−5b by following the previously described docking
procedure.17 As an example, the predicted binding mode of 4a
is shown in Figure 5. In the simulated binding mode, the
moiety originating from the nonpeptidic belactosin A derivative
is accommodated in the primed binding site and the moiety
originating from the bortezomib is accommodated in the
nonprimed binding site, respectively, as we expected. We
therefore synthesized these newly designed hybrid molecules
and evaluated their biological effects.
Ac-nLPnLD-AMC, and Ac-RLR-AMC, respectively, and the
results are summarized in Table 1. All of these compounds
strongly inhibited proteasome ChT-L activity, with IC50 values
ranging from 2.7 to 5.7 nM, which are lower than that of
bortezomib (IC50 = 7.0 nM). The P2 Asp-type (m = 1)
compounds 3a−5a showed subunit selectivity similar to that of
bortezomib, with IC50 values ranging from 120 to 150 nM for
C-L and 1100 to 2700 nM for T-L activities. The inhibitory
potency of the P2 Glu-type (m = 2) compounds 3b−5b on T-L
activity (IC50 = 2400−3000 nM) was similar to that of the Asp-
type compounds, but these compounds were more potent
inhibitors of C-L activity (IC50 = 46−76 nM) than the Asp-type
compounds, suggesting that Glu was more favored than Asp as
the P2 residue to be accommodated in the binding site of the
β1 subunit. In these compounds, the linker length (n = 0−2)
had only a small impact on the proteasome inhibitory activities,
suggesting that the flexible nature of the linker moieties permits
the insertion of the two aromatic groups into primed binding
site regardless of their length.
We also evaluated cell growth inhibitory effects of the
compounds on HCT116 cells. As summarized in Table 1, all of
these compounds effectively inhibited cell growth. Interestingly,
the P2 Asp-type compounds 3a−5a were more potent cell
growth inhibitors (IC50 = 32−45 nM) than the P2 Glu-type
compounds 3b−5b (IC50 = 64−110 nM).
SAR Analysis. The inhibitory effects of synthesized
compounds 3a−5a and 3b−5b and bortezomib on the ChT-
L, C-L, and T-L activities of human 20S proteasomes were
measured using the chromophoric substrates Suc-LLVY-AMC,
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Table 2. Inhibitory Effects of Compounds 6a−9a on Human 20S Proteasome and HCT116 Cell Growth
a
Based on three experiments.
Furthermore, we evaluated proteasome and cell growth
inhibitory activities of 3a′, which is a boronic acid congener of
3a. As shown in Table 1, the inhibitory activities of 3a′ (ChT-L
activity, IC50 = 9.1 nM; HCT116 cell growth, IC50 = 80 nM)
were comparable to those of 3a, suggesting that the pinanediol
ester works as a promoiety and does not have significant effect
on the activities, in accord with the results reported
previously.26
On the basis of the above-mentioned results, we next
investigated the SAR at the P3 site with compounds 6a−9a,
and the results are summarized in Table 2. The inhibitory
potency on ChT-L activity was not improved by the P3
modification, however, introduction of large substituents
effectively improved selectivity for ChT-L activity. Particularly,
compound 9a with the biaryl (3-phenyl-pyridin-2-yl) P3
substituent had very weak inhibitory potency on C-L activity
(IC50 = 1000 nM) despite its remarkable inhibitory potency on
ChT-L activity (IC50 = 7.5 nM). Because CEP-18770, with the
same biaryl P3 substituent as 9a, strongly inhibits C-L activity
with an IC50 value of around 70 nM,21 the binding to the
primed site in these hybrid compounds might differentiate
recognition of the P3 substituents.
evaluated the biological activities of compounds 10a and 11a, in
which the moiety was entirely removed (10a) or one of the
aromatic rings was removed (11a), and the results are
summarized in Table 3. Compared with 10a without the
primed site-binding moiety, compounds 11a with one aromatic
ring and 4a with two aromatic rings exhibited 10- and 20-times
greater inhibitory effects on ChT-L activity, respectively,
demonstrating that the moiety ligated into the P2 side chain
largely contributes to increase their inhibitory potency.
Compound 10a had almost no effect on cell growth (IC50
=
2500 nM), which likely reflects its lowered proteasome
inhibitory activity and membrane permeability. Although 11a
had proteasome inhibitory effects comparable or even higher
than those of 7a and 8a, it had only moderate cell growth
inhibitory effects (IC50 = 130 nM), which might be due to
differences in the membrane permeability, biological stability,
and/or binding reversibility, as described below.
Thus, we rationally developed a novel class of proteasome
inhibitors with hybrid structures of nonpeptidic belactosin A
derivatives and peptide boronates, having proteasome inhib-
itory effects that were comparable or even higher than those of
bortezomib. In addition, these compounds effectively inhibited
HCT116 cell growth.
Finally, we investigated the impact of the moiety derived
from nonpeptidic belactosin A derivatives ligated into the P2
side chain on their biological activities. For this purpose, we
Inhibitory Effects on β5i of Immunoproteasome. The
therapeutic benefits of selective inhibition of immunoprotea-
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Table 3. Inhibitory Effects of Compounds 10a and 11a on Human 20S Proteasome and HCT116 Cell Growth
a
Based on three experiments.
somes were recently demonstrated, especially for autoimmune
diseases.3 On the other hand, inhibition of both constitutive
proteasomes and immunoproteasomes is desirable for potent
cell growth inhibitory activity.27 Thus, we investigated the
inhibitory activity of the newly identified hybrid inhibitors 4a
and 11a on β5i of immunoproteasomes. The results are shown
in Figure 6, compared with the results of bortezomib, PR-957,
and lactacystin, which were shown to be nonselective, β5i
selective, and β5 selective inhibitors, respectively, inconsistent
with the previous report.3,6a,28 The compound 4a and 11a were
shown to be slightly selective for β5i compared to the
nonselective inhibitor bortezomib, while they have the same
boronic acid warhead. The structures originating from the
nonpeptidic belactosin derivatives might differentiate the
selectivity between β5 and β5i subunits.
Inhibitory Effects on Other Proteases. Proteasome
inhibitors with a boronic acid moiety as the active warhead
generally suffer from their promiscuous inhibitory activity on
various serine proteases because of the high reactivity of the
warhead with hydroxyl groups.29 In fact, peripheral neuropathy
Figure 6. The binding activity of 4a and 11a to β5 and β5i subunits
investigated by active-site ELISA.3 The activity to β5i (Δ) subunits of
due to bortezomib treatment, which is the major dose-limiting
toxicity of bortezomib, is thought to be caused by its off-target
activity on serine proteases.29b Thus, the inhibitory activity of
the newly identified hybrid inhibitors 4a and 11a on cathepsin
A and cathepsin G (serine proteases) and cathepsin B (cysteine
proteas) were examined. As summarized in Table 4, bortezomib
showed inhibitory activity on cathepsin A and cathepsin G,
while carfilzomib, with an epoxyketone warhead, did not inhibit
these serine proteases as reported previously by Arastu-Kapur
et al.29b The carfilzomib, however, inhibited cathepsin B,
●
immunoproteasome and β5 ( ) subunits of the constitutive
proteasome were normalized to values derived from DMSO-treated
controls. The known proteasome inhibitors, bortezomib, PR-957, and
lactacystin, were used as controls. The IC50 values of these compounds
were as follows: 4a, IC50 = 140 nM (β5), 31 nM (β5i); 11a, IC50 = 280
nM (β5), 59 nM (β5i); bortezomib, IC50 = 140 nM (β5), 78 nM
(β5i); PR-957, IC50 > 1.0 μM (β5), 450 nM (β5i); lactacystin, IC50
500 nM (β5), > 3.0 μM (β5i).
=
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Table 4. Inhibitory Effects of 4a, 11a, Bortezomib, and
Carfilzomib on Cathepsin A, B, and G
a
IC50 [μM]
compd
4a
cathepsin A
cathepsin B
cathepsin G
>30
>30
9.2
>30
>30
>30
11
>30
>30
3.1
11a
Figure 8. Immunoblot analysis of p53 and cleaved PARP in HCT116
cells treated with DMSO (D), 4a, 11a, or bortezomib (B).
bortezomib
carfilzomib
>30
>30
a
Based on three experiments.
cycle arrest and caused an accumulation of p53, resulting in
HCT116 cell apoptosis similar to bortezomib.
probably because of the high reactivity of its epoxyketone
warhead with the thiol group. In contrast, compounds 4a and
11a exhibited no inhibitory activity on any of these proteases.
Their large P2 moiety comprised of the nonpeptidic scaffold
originating from the belactosin derivative might effectively
improve the selectivity for proteasome. Thus, these hybrid
inhibitors seem to be promising candidates for the development
of anticancer drugs without off-target toxicities due to the
inhibition of proteases other than proteasome.
Mechanism of Cell Growth Inhibition. To clarify the
mechanism of cell growth inhibition, we examined the cell cycle
distribution of HCT116 cells treated with compounds 4a and
11a. As shown in Figure 7, 4a and 11a inhibited cell cycle
progression at the G2/M stage, similar to bortezomib and the
belactosin derivative 1.14c
Reversibility of Proteasome Inhibition. Although
peptide boronate proteasome inhibitors bind covalently to
proteasome, the reaction between the boronic acid warhead
and the proteasome Thr hydroxyl group is reversible. The
dissociation half-life of two peptide boronates, MLN9708 and
bortezomib, from β5 is reported as 18 and 110 min,
respectively.22 Because these two inhibitors have the same
boronic acid warhead, the difference between their dissociation
half-lives seems to arise from their structures other than the
warhead.
To investigate the impact of the moiety originating from the
nonpeptidic belactosin A derivatives on the dissociation half-
life, we preincubated proteasome with 4a or 11a, and the ChT-
L activity was measured before and after dialysis, in comparison
with bortezomib and carfilzomib as controls for a reversible and
an irreversible inhibitor, respectively.22,28,32 As shown in Figure
9, the inhibitory activity of compound 11a and bortezomib on
Figure 9. ChT-L activity of the human 20S proteasome treated with
4a, 11a, bortezomib, or carfilzomib before and after wash-out.
Figure 7. Cell cycle profiles of HCT116 cells treated with DMSO (a),
100 nM of bortezomib (b), 4a (c), or 11a (d). After 24 h, the cells
were harvested and stained with propidium iodide. The DNA content
of each sample was analyzed by flow cytometry.
the ChT-L activity was significantly attenuated by wash-out,
while compound 4a retained strong inhibitory activity even
after wash-out like carfilzomib, which has an epoxyketone
warhead. These findings suggest that the interaction of the two
aromatic groups in 4a effectively prolongs its residence time,
whereas the interaction by the one aromatic group in 11a does
not. Because the drug-target residence time is an important
factor for in vivo efficacy and target selectivity of ligands,33 4a
might be an attractive lead for developing anticancer drugs with
prolonged in vivo efficacy and reduced off-target toxicity.
Furthermore, we investigated the proteasome inhibitory
effects of these compounds in a cellular system by immunoblot
analysis. Figure 8 shows the clear accumulation of a tumor
suppressor gene product p53, which is a well-known
proteasome target,30 in HCT116 cells treated with 4a and
11a. In addition, cleavage of poly(ADP-ribose) polymerase
(PARP), a prominent marker of apoptosis,31 was observed in
the treated cells, indicating activation of the apoptotic pathway
in the cells.
CHEMISTRY
■
Synthesis of target compounds 3a−11a and 3b−5b is shown in
Scheme 1. The pinanediol ester of leucine boronic acid 1234
was condensed with Boc-Asp(OBn)-OH or Boc-Glu(OBn)-
OH by the mixed anhydride method to yield 13a or 13b,
These results suggest that proteasome inhibition by the
newly identified hybrid inhibitors 4a or 11a induced G2/M cell
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a
Scheme 1. Synthesis of Target Compounds 3a−11a and 3b−5b
a
Reagents and conditions: (a) Boc-Asp(OBn)-OH, PivCl, Et3N, CH2Cl2, 0 °C to rt; (b) Boc-Glu(OBn)-OH, PivCl, Et3N, CH2Cl2, 0 °C to rt; (c)
TFA, CH2Cl2, 0 °C to rt; (d) Ac2O, Et3N, CH2Cl2, 0 °C, 68% for 14a (3 steps from 12), 84% for 14b (3 steps from 12); (e) Pd/C, H2, THF; (f) 15,
EDC, HOAt, DIEA, CH2Cl2, −18 °C to rt, 68% for 3a (2 steps from 14a), 78% for 3b (2 steps from 14b); (g) 16, EDC, HOAt, DIEA, CH2Cl2, −18
°C to rt, 77% for 4a (2 steps from 14a), 84% for 4b (2 steps from 14b); (h) 17, EDC, HOAt, DIEA, CH2Cl2, −18 °C to rt, 71% for 5a (2 steps from
14a), 75% for 5b (2 steps from 14b); (i) R-Cl, Et3N, CH2Cl2, 0 °C, 49% for 6a (R = 2,5-dichlorobenzoyl, 5 steps from 12), 53% for 7a (R = benzoyl,
5 steps from 12); (j) R-OH, PivCl, Et3N, CH2Cl2, 0 °C to rt, 47% for 8a (R = pyrazine-2-carbonyl, 5 steps from 12), 54% for 9a (R = 6-
phenylpyridine-2-carbonyl, 5 steps from 12); (k) CH3NH2·HCl, EDC, HOAt, DIEA, CH2Cl2, 0 °C to rt, 2 steps 18% from 14a; (l) 4-
(benzyloxy)butan-1-amine·HCl, EDC, HOAt, DIEA, CH2Cl2, −18 °C to rt, 2 steps 76% from 14a.
respectively. The Boc group of 13a and 13b was removed by
treatment with TFA/CH2Cl2 and following acetylation of the
resulting amino group with Ac2O afforded 14a and 14b,
respectively. The Bn group of 14a and 14b was removed by
hydrogenolysis, and subsequent condensation with 15−17 gave
target compounds 3a−5a and 3b−5b, respectively.
or acyl chlorides gave target compounds 6a−9a. The Bn group
of 14a was removed, and following condensation with CH3NH2
or 4-(benzyloxy)butan-1-amine gave target compounds 10a or
11a, respectively.
All of these target compounds were purified by reverse-phase
Target compounds 6a−11a were synthesized similarly. The
Bn group of 13a was removed, and subsequent condensation
with 16 afforded 18a. The Boc group of 18a was removed, and
subsequent condensation with corresponding carboxylic acids
column chromatography because pinanediol esters of these
peptide boronic acids are unstable under usual silica gel column
chromatography conditions.35
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cytokine production and attenuates progression of experimental
arthritis. Nature Med. 2009, 15, 781−787.
CONCLUSIONS
■
We successfully developed a highly potent novel chemotype of
proteasome inhibitors using a rational hybridization strategy
with nonpeptide belactosin derivatives and peptide boronates.
These new inhibitors exhibited high cell-growth inhibitory
activity due to their proteasome inhibitory activity. These
inhibitors have the same boronic acid active warhead as
bortezomib, but unlike bortezomib, the hybrids are highly
selective for proteasomes without inhibiting serine proteases
and have long residence times compared to bortezomib. Thus,
these newly identified proteasome inhibitors are promising
candidates for the development of anticancer drugs without the
off-target toxicities that are problematic in bortezomib
treatment.
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ASSOCIATED CONTENT
* Supporting Information
Experimental details of synthesis, biological evaluations,
computational simulations, and combustion analysis data for
target compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
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S
AUTHOR INFORMATION
Corresponding Author
*Phone/fax: +81-11-706-3769. E-mail: shu@pharm.hokudai.ac.
■
jp.
Author Contributions
(8) Groll, M.; Ditzel, L.; Lowe, J.; Stock, D.; Bochtler, M.; Bartunik,
H. D.; Huber, R. Structure of 20S proteasome from yeast at 2.4A
resolution. Nature 1997, 386, 463−471.
⊥S.K. and Y.U. contributed equally.
Notes
The authors declare no competing financial interest.
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fulfill their role. Curr. Opin. Struct. Biol. 2003, 13, 665−673.
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Crystal Structures Reveal Differences in Substrate and Inhibitor
Specificity. Cell 2012, 148, 727−738. (b) Murata, S.; Sasaki, K.;
Kishimoto, T.; Niwa, S.-i.; Hayashi, H.; Takahama, Y.; Tanaka, K.
Regulation of CD8+ T Cell Development by Thymus-Specific
Proteasomes. Science 2007, 316, 1349−1353.
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J. B.; Micklem, D. R.; Ruurs, P.; Sylvain, C.; Lu, Y.; Shenk, K. D.;
Bennett, M. K. Carfilzomib can induce tumor cell death through
selective inhibition of the chymotrypsin-like activity of the proteasome.
Blood 2009, 114, 3439−3447.
(12) Asai, A.; Hasegawa, A.; Ochiai, K.; Yamashita, Y.; Mizukami, T.;
Belactosin, A. a novel antitumor antibiotic acting on cyclin/CDK
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(14) (a) Groll, M.; Larionov, O. V.; Huber, R.; De Meijere, A.
Inhibitor-binding mode of homobelactosin C to proteasomes: new
insights into class I MHC ligand generation. Proc. Natl. Acad. Sci. U. S.
A. 2006, 103, 4576−4579. (b) Korotkov, V. S.; Ludwig, A.; Larionov,
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(15) The binding sites of proteasome which bind to substrate P
residues (from the N-terminal to the cleavage site) and P′ residues
ACKNOWLEDGMENTS
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This investigation was supported by Grant-in-Aids for Scientific
Research (21390028) from the Japan Society for the
Promotion of Science and for Creation of Innovation Centers
for Advanced Interdisciplinary Research Areas Program from
Ministry of Education, Culture, Sports, Science and Technol-
ogy, Japan.
ABBREVIATIONS USED
■
AMC, aminomethylcoumarin; Boc, t-butoxycarbonyl; C-L,
caspase-like; ChT-L, chymotrypsin-like; DIEA, diisopropyle-
thylamine; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide; HOAt, 1-hydroxy-7-azabenzotriazole; LMP2,
large multifunctional peptidase 2; LMP7, large multifunctional
peptidase 7; MECL-1, multicatalytic endopeptidase complex-
like 1; Piv, pivaloyl; Suc, succinyl; T-L, trypsin-like
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