Potent proteasome inhibitors derived from the unnatural cis-cyclopropane isomer of belactosin a: Synthesis, biological activity, and mode of action

Article abstract of DOI:10.1021/jm4002296

The natural product belactosin A (1) with a trans-cyclopropane structure is a useful prototype compound for developing potent proteasome (core particle, CP) inhibitors. To date, 1 and its analogues are the only CP ligands that bind to both the nonprimed S1 pocket as well as the primed substrate binding channel; however, these molecules harbor a high IC₅₀ value of more than 1 μM. We have performed structure-activity relationship studies, thereby elucidating unnatural cis-cyclopropane derivatives of 1 that exhibit high potency to primarily block the chymotrypsin-like active site of the human constitutive (cCP) and immunoproteasome (iCP). The most active compound 3e reversibly inhibits cCP and iCP similarly with an IC₅₀ of 5.7 nM. X-ray crystallographic analysis of the yeast proteasome in complex with 3e revealed that the ligand is accommodated predominantly into the primed substrate binding channel and covalently binds to the active site threonine residue via its β-lactone ring-opening.

Full text of DOI:10.1021/jm4002296

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Article
Potent Proteasome Inhibitors Derived from the Unnatural Cis-Cyclopropane
Isomer of Belactosin A: Synthesis, Biological Activity, and Mode of Action
Satoshi Shuto, Shuhei Kawamura, Yuka Unnno, Anja List, Takuma
Sasaki, Motohiro Tanaka, Mitsuhiro Arisawa, Akira Asai, and Michael Groll
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4002296 • Publication Date (Web): 02 Apr 2013
Downloaded from http://pubs.acs.org on April 16, 2013
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Potent Proteasome Inhibitors Derived from the Unnatural CisꢀCyclopropane Isomer of
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Belactosin A: Synthesis, Biological Activity, and Mode of Action
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Shuhei Kawamura,a,# Yuka Unno,b,# Anja List,c Motohiro Tanaka,d Takuma Sasaki,d Mitsuhiro Arisawa,a Akira Asai,b
Michael Groll,c and Satoshi Shuto*,a.
aFaculty of Pharmaceutical Sciences, Hokkaido University, Kitaꢀ12, Nishiꢀ6, Kitaꢀku, Sapporo 060ꢀ0812, Japan
bGraduate School of Pharmaceutical Sciences, University of Shizuoka, Yada, Shizuoka 422ꢀ8526, Japan
cCenter for Integrated Protein Science at the Department of Chemistry, Chair of Biochemistry, Technische Universität
München, Lichtenbergstrasse 4, 85747 Garching, Germany
dSchool of Pharmacy, Aichi Gakuin University, 1ꢀ100 Kusumotoꢀcho, Chikusaꢀku, Nagoya 464ꢀ8650, Japan
Corresponding Author: Satoshi Shuto
Phone & Fax: +81ꢀ11ꢀ706ꢀ3769. Eꢀmail: shu@pharm.hokudai.ac.jp
#These authors contributed equally.
Abstract
The natural product belactosin A (1) with a transꢀcyclopropane structure is a useful prototype compound for developing
potent proteasome (core particle, CP) inhibitors. To date, 1 and its analogs are the only CP ligands bind to both the
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nonꢀprimed S1 pocket as well as the primed substrate binding channel, however, these molecules harbor a high IC50
value of more than 1 ꢁM. We have performed structure activity relationship studies, hereby elucidating unnatural
cisꢀcyclopropane derivatives of 1 that exhibit high potency to primarily block the chymotrypsinꢀlike active site of the
human constitutive (cCP) and immunoproteasome (iCP). The most active compound 3e reversibly inhibits cCP and iCP
similarly with an IC50 of 5.7 nM. Xꢀray crystallographic analysis of the yeast proteasome in complex with 3e revealed
that the ligand accommodates predominantly to the primed substrate binding channel and covalently binds to the active
site threonine residue via its βꢀlactone ringꢀopening.
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Introduction
The ubiquitinꢀproteasome system is the major pathway for systematic degradation of intracellular proteins1 and
involved in many physiologically important cellular processes such as signal transduction2, immune reponse3 or cell
cycle progression.4 Since proteasome inhibition causes cell cycle arrest and induces apoptosis, the 20S proteasome core
particle (CP) is an attractive target molecule of anticancer drugs and autoimmune disorders.5 In fact, the dipeptide
boronic acid bortezomib (Figure 1) is in our days a prescriptive drug against various cancer types and currently
represents the best known blockbuster with annual sales of more than $ 1 Mrd.6
Eukaryotic CPs contain three active β subunits (β1, β2, and β5, which are responsible for the caspaseꢀlike (CꢀL),
trypsinꢀlike (TꢀL) and chymotrypsinꢀlike (ChTꢀL) activity, respectively),7 whereas vertebrates present an even more
elaborate version of proteasomes,8 harboring three different types of the CP, the constitutive (cCP), immuno (iCP) and
thymoproteasome (tCP), which all of them have their own set of active sites9: the cCP incorporates subunits β1c, β2c,
and β5c, the iCP β1i, β2i, and β5i, and the tCPs β1i, β2i, and β5t. The mechanism of peptide bond cleavage follows a
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universal principle among all active sites, however, it is the singularity of each substrate binding channel which
determines the chemical nature of the specificity (S) pockets and accommodates the ligand’s side chains (P sites) in
respect to their amino acid progression. Interestingly, the iCP is induced by interferonꢀγ and primarily expressed in
hematopoietic cells10 while the cCP is expressed in all kind of cells. Thus, it is a wellꢀknown fact that blocking either
the cCP or iCP has a major impact on specific biological potencies.3, 5a
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H
H
OH
O
O
O
H
OH
O
H
H
O
H
H
OH
O
N
N
O
N
N
H
N
N
N
H
N
H
S
O
O
O
O
O
O
O
O
OH
OH
O
HN
O
Cl
lactacystin
omuralide
(clasto-lactacystin)
salinosporamide A
(marizomib)
carfilzomib
O
H
N
H2N
N
H
N
O
H
O
O
CO2H
O
O
O
O
O
O
O
N
N
OH
H
O
1
O
N
H
B
belactosin A ( )
N
N
N
H
N
O
OH
H
H
N
O
O
R1
N
N
H
H
n
CO2R2
OMe
PR-957
bortezomib
belactosin C (n = 1, R1 = R2 = H)
bis-benzyl-protected homobelactosin C
(hBelC, n = 2, R1 = Cbz, R2 = Bn)
Figure 1. Known proteasome inhibitors.
Belactosin A (1), which is a naturally occurring Streptomyces sp. tripeptide metabolite and consists of Lꢀalanine,
3ꢀ(transꢀ2ꢀaminocyclopropyl)ꢀLꢀalanine (transꢀ3,4ꢀmethanoꢀLꢀornithine) as well as a chiral carboxyꢀβꢀlactone moiety,
was initially identified as a CP inhibitor by Asai and coꢀworkers.11 It prevents cell cycle division in tumor cells at the
G2/M stage due to its proteasomal inhibition and is therefore a novel lead for developing potent anticancer agents.11b
Inactivation of the CP by belactosin A takes place by acylation of the active site threonine 1 (Thr1) residue via
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ringꢀopening of its strained βꢀlactone, as confirmed by Xꢀray crystallographic analysis of bisꢀbenzylꢀprotected
homobelactosin C (hBel) bound to the yeast 20S proteasome (yCP).12 Furthermore, the structural investigations of the
yCP:hBel complex revealed that the inhibitor occupies the S1 specificity pocket with its (S)ꢀsecꢀbutyl side chain
(pseudoꢀisoleucine) whereas the remaining part of the ligand points towards the primed substrate binding site,12
contrary to all currently identified CP inhibitors.13 Although many structureꢀactivity relationship (SAR) studies of
proteasome ligand complexes have been reported, there exists only limited knowledge of inhibitors binding to the
primed substrate binding channel.12b Thus, detailed analysis using belactosin analogs as a prototype are important to
exploit both, the primed and nonꢀprimed specificity channels as presented in this work.
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The class of belactosins all harbors a cyclopropan, a chemical moiety that is extensively used in medicinal chemistry
due to their unique steric and electronic properties and their ability to function as alkene bioisosteres without the
associated metabolic liability.14 Since the impact of the cyclopropane in these natural products has so far not been
addressed, we aimed to characterize the restriction of the orientation of the ꢀAla and the key βꢀlactone moieties by
L
including this ringꢀmotif and to address its binding profile and mode of action by elucidating the threeꢀdimensional
structure of the ligand in complex with the yCP. We thus designed and synthesized a series of belactosin A derivatives
with stereochemical diversity in which the central aminocyclopropylꢀLꢀalanine (methanoꢀLꢀornithine) part was
substituted with a corresponding stereoꢀ or regioisomeric unnatural cyclopropane amino acid. Principal focus was to
clarify that the cis/Lꢀantiꢀisomer 2 (Figure 2) exhibiting the cyclopropane structure in cisꢀconfiguration is a more potent
proteasome inhibitor than the natural belactosin A having the cyclopropane structure in trans.15
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Figure 2. Proteasome inhibitors developed by us using belactosin A as a prototype.
Applying systematic SAR studies on the primed substrate binding site by using the originally identified
cisꢀcyclopropane analog of belactosin A as the lead compound, we looked for highly potent compounds, whose
proteasome inhibitory activities are comparable to those of bortezomib16 and carfilzomib.17 Furthermore, we
investigated various biological properties of the developed synthetic molecules, such as selectivity for constitutive CP
subunits, inhibitory effects on the subunit β5i, cellꢀgrowth inhibition and stability. Furthermore, the covalent binding
mode of the designed inhibitors was clarified by applying Xꢀray crystallographic analysis using the yCP.
Results and Discussion
Design of Compounds. A comparison of the structure of 3a18 with that of 2 reveals that the carboxy group is replaced
with a vinyl group and a Cbz group is introduced into the terminal amino group of the Ala moiety (Figure 2). This
structural change was thought to make 3a more potent than 2, and we therefore aimed to modify these two moieties
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further to investigate the SAR in detail. Thus, we synthesized new compounds 4aꢀ17a (Figure 3ꢀa), in which the
terminal Ala moiety of 3a was replaced with a variety of acyl and amino acyl groups as well as other new compounds
3bꢀj (Figure 3ꢀb), in which the vinyl group of 3a was substituted by hydrophobic groups such as ethyl, phenyl or
alkylꢀtethered aromatic rings. Furthermore, we created compounds 7e and 16e (Figure 3ꢀc) having a phenethyl group at
the vinyl moiety of 3a significantly alters the CP binding profile as described in detail below.
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O
a)
N
H
H
N
O
X
O
4a: isopropanoyl
5a: Piv
8a:
9a:
Ala
Cbz-D-Ala
X =
6a: Bz
7a: 2-naphthoyl
10a:
11a:
Cbz-Gly
Cbz-Leu
12a: Cbz-Phe
13a: Cbz-Trp
14a: Ac-Ala
15a:
16a:
17a:
Bz-Ala
2-naphthoyl-Ala
Boc-Ala
O
b)
O
N
H
H
N
O
Ph
O
N
H
O
O
Y
3g:
CH2(1-Nph)
3b: Et
Y =
3h: CH2(2-Nph)
3c:
Ph
3i: CH2CH2(1-Nph)
3j: CH2CH2(2-Nph)
3d: Bn
3e: CH2CH2Ph
3f: CH2CH2CH2Ph
(Nph = naphthyl)
c)
O
N
H
H
O
N
O
X
7e: 2-naphtoyl
16e: 2-naphthoyl-Ala
X =
Figure 3. Newly designed compounds as proteasome inhibitors.
Synthesis. We previously developed the chiral cyclopropane units consisting of a cisꢀunit 18 (Scheme 1), its
transꢀisomer, and their two enantiomers, which are highly effective for a series of stereochemically diverse
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cyclopropane compounds.14bꢀd, 19 In fact, these units allowed for the productive synthesis of a series of stereoꢀ and
regioisomers of belactosin A15 and identified the lead compound 3e18 with significant proteasome inhibitory potency
(see below).
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In this study, all of the target compounds (Figure 3) were prepared using the cisꢀunit 18. As summarized in Scheme 1,
after conversion of the unit 18 into the (S)ꢀtꢀbutanesulfinyl imine 19,15 its reaction with various Grignard reagents
afforded the corresponding adduct I diastereoselectively, which could be further converted into II via Curtius
rearrangement. The target compounds III were obtained from II by condensation with the known chiral
carboxyꢀβꢀlactone20 and acylation of the terminal amino group.
Scheme 1. Synthetic plan of target compounds
OTBDPS
OTBDPS
CHO
O
N
S
18
19
t-Bu
diastereoselective
Grignard reaction
OTBDPS
Pg'
H
N
H
N
O
S
N
H
Pg
O
R2
I
II
t-Bu
R2
O
N
H
N
H
O
R1
R2
III
A large amount of the cisꢀunit 18 in high optical purity was required and we focused on the method reported by
Charette:21 the chiral cisꢀalcohol 21 was enantioselectively prepared applying an asymmetric SimmonsꢀSmith reaction
of a 2ꢀbutenediol derivative 20 using a chiral ligand. This reaction was suitable for largeꢀscale synthesis, and we
successfully prepared about 30 g of 21 (quantitative yield) in 92% ee. Furthermore, we found that, after Swern
oxidation of 21, the desired unit 18 was obtained in 98% ee by recrystallization from hexane (Scheme 2).
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Scheme 2. Synthesis of chiral cyclopropane unit 18 with high optical purity
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Having enough compound 18 available, we next synthesized the Nꢀsubstituted target compounds 4aꢀ17a
(NꢀBocꢀprotected compound 26a was the common intermediate as shown in Scheme 3). Compound 22a, prepared
according to our previous protocol,15 was converted into the aldehyde 23a. After Pinnick oxidation of 23a, the resulting
carboxylic acid was treated with diphenyl phosphoryl azide (DPPA)/Et3N in CH2Cl2 to form the corresponding acyl
azide. A Curtius rearrangement yielded the cyclopropylamine derivative 24a. Condensation of 24a and the known
βꢀlactone unit 2520 afforded 26a. After removal of the Boc group of 26a, Nꢀacylation with acid chlorides gave the target
compounds 4aꢀ7a. The Nꢀaminoacylꢀtype target compounds 9aꢀ13a and 17a were obtained by applying the mixed
anhydride method. Compounds 8a and 14aꢀ16a were prepared from 17a.
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Scheme 3. Synthesis of target compounds 4aꢀ17a and 3ba
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aReagents and conditions: (a) HCl, THF/H2O, 0 °C; (b) Boc2O, Et3N, THF; (c) TBAF, THF; (d) Swern ox., 66% (4
steps); (e) Pinnick ox.; (f) DPPA, Et3N, CH2Cl2, 0 °C to rt; (g) toluene, reflux; (h) KOH, THF/H2O; (i) 25, PivCl, Et3N,
CH2Cl2, 0 °C to rt, 48% (5 steps); (j) TFA, CH2Cl2, 0 °C to rt; (k) RCl, Et3N, CH2Cl2, 0 °C, yields (2 steps) were as
follows respectively, 4a (R = isopropanoyl) quant., 5a (R = Piv) 81%, 6a (R = Bz) 78%, 7a (R = 2ꢀnaphthoyl) 92%; (l)
RꢀOH, PivCl, Et3N, CH2Cl2, 0 °C, yields (2 steps) were as follows respectively, 9a (R = CbzꢀDꢀAla) quant., 10a (R =
CbzꢀGly) 96%, 11a (R = CbzꢀLeu) 64%, 12a (R = CbzꢀPhe) 66%, 13a (R = CbzꢀTrp) 65%, 17a (R = BocꢀAla) quant.;
(m) TFA, CH2Cl2, 0 °C, quant. (8a); (n) Pd/C, H2, THF; (o) TFA, CH2Cl2, 0 °C; (p) CbzCl, Et3N, CH2Cl2, 0 °C, 66% (3
steps, 3b); (q) RCl, Et3N, CH2Cl2, 0 °C, yields were as follows respectively, 14a (R = Ac) 59%, 15a (R = Bz) quant.,
16a (R = 2ꢀnaphthoyl) 95%.
Compounds 3cꢀj (Figure 3ꢀb), which have various hydrophobic groups instead of the vinyl moiety were synthesized
successively, following the protocol for 3a as shown in Scheme 415, 18; the ethyl derivative 3b was obtained via
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hydrogenation of 17a (Scheme 3). The synthesis of 7e and 16e is shown in Scheme 5 and Scheme 6, respectively.
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Scheme 4. Synthesis of target compounds 3cꢀja
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aReagents and conditions: (a) RMgX, toluene, reflux; (b) RMgX, CH2Cl2; (c) HCl, THF/H2O, 0 °C; (d) FmocOSu,
Na2CO3, THF/H2O; (e) HCl, AcOEt/MeOH, yields (4 steps) were as follows, respectively, 28c (R = Ph) 60%, 28d (R =
Bn) 56%, 28e (R = phenethyl) 35%, 28f (R = phenylpropyl) 26%; (f) HCl, AcOEt/MeOH; (g) FmocOSu, Na2CO3,
THF/H2O, yields (3 steps) were as follows respectively, 28i (R = 1ꢀnaphthylethyl) 36%, 28j (R = 2ꢀnaphthylethyl) 48%;
(h) DMP, CH2Cl2, yields (4 steps) were as follows respectively, 29g (R = 1ꢀnaphthylmethyl) 20%, 29h (R =
2ꢀnaphthylmethyl) 20%; (i) Pinnick ox.; (j) DPPA, Et3N, CH2Cl2, 0 °C to rt; (k) tꢀBuOH, reflux, yields (4 steps for
30cꢀf,i,j and 3 steps for 30g,h) were as follows respectively, 30c (R = Ph) 70%, 30d (R = Bn) 74%, 30e (R = phenethyl)
70%, 30f (R = phenylpropyl) 62%, 30g (R = 1ꢀnaphthylmethyl) 64%, 30h (R = 2ꢀnaphthylmethyl) 64%, 30i (R =
1ꢀnaphthylethyl) 59%, 30j (R = 2ꢀnaphthylethyl) 52%; (l) K2CO3, MeOH; (m) CbzꢀAlaꢀOH, PivCl, Et3N, CH2Cl2, 0 °C
to rt, yields (2 steps) were as follows respectively, 31c (R = Ph) 74%, 31d (R = Bn) 70%, 31e (R = phenethyl) 84%, 31f
(R = phenylpropyl) 88%, 31g (R = 1ꢀnaphthylmethyl) 97%, 31h (R = 2ꢀnaphthylmethyl) 89%, 31i (R =
1ꢀnaphthylethyl) 94%, 31j (R = 2ꢀnaphthylethyl) 98%; (n) TFA, CH2Cl2; (o) 25, PivCl, Et3N, CH2Cl2, 0 °C to rt, yields
(2 steps) were as follows respectively, 3c (R = Ph) 100%, 3d (R = Bn) 100%, 3e (R = phenethyl) 86%, 3f (R =
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phenylpropyl) 87%, 3g (R = 1ꢀnaphthylmethyl) 74%, 3h (R = 2ꢀnaphthylmethyl) 82%, 3i (R = 1ꢀnaphthylethyl) 76%,
3j (R = 2ꢀnaphthylethyl) 83%.
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Scheme 5. Synthesis of target compound 7ea
aReagents and conditions: (a) K2CO3, MeOH; (b) 2ꢀnaphthoyl chloride, Et3N, CH2Cl2, 0 °C; (c) TFA, CH2Cl2; (d) 25,
PivCl, Et3N, CH2Cl2, 0 °C to rt, 48% (4 steps).
Scheme 6. Synthesis of target compound 16ea
aReagents and conditions: (a) Pd/C, H2, TFA/THF, 0 °C; (b) 2ꢀnaphthoyl chloride, Et3N, CH2Cl2 0 °C, 76% (2 steps).
Inhibitory effect on the ChTꢀL activity of human cCP. The inhibitory effect of compounds on the ChTꢀL activity of
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human 20S proteasomes was measured using the chromophoric substrate SucꢀLLVYꢀAMC. The results for the
compounds 4aꢀ17a modified at the Ala moiety of the lead compound 3a are summarized in Table 1.22 Interestingly,
modifications of the Ala moiety in the ligands exhibit a strong effect on the proteasome inhibition profiles with IC50
values ranging between 25 ꢀ 300 nM. The inhibitory effects of the Nꢀacylated compounds 4a, 5a, and 6a were weak
(IC50 = 130ꢀ230 nM) due to the lack of the alanineꢀmoiety, while the Nꢀ(2ꢀnaphthoyl) compound 7a could compensate
this absence and recapture its potent inhibitory effect (IC50 = 60 nM). Removal of the hydrophobic Cbz group in 3a, i.e.
the Nꢀalanyl derivative 8a, significantly decreased the activity due to lack of the hydrophobic interaction (IC50 = 300
nM).
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Compounds 9aꢀ13a, in which the CbzꢀLꢀAla of 3a was replaced with other Cbzꢀamino acids, exhibited inhibitory
effects (IC50 = 28ꢀ57 nM) comparable to 3a (IC50 = 49 nM). The Nꢀsubstituted alanyl compounds, NꢀBzꢀAla (15a),
Nꢀ(2ꢀnaphthoyl)ꢀAla (16a), and NꢀBocꢀAla (17a) derivatives, were shown to be potent inhibitors (IC50 = 25ꢀ79 nM),
while the NꢀAcꢀAla derivative 14a had a decreased binding profile (IC50 = 230 nM).
These systematic SAR studies focused on the Ala moiety suggested that Ala structure is not essential for the high
potency (3a versus 7a, 9aꢀ13a) but it seems to function as a linker to place a hydrophobic group attached at the terminal
amino group into the proteasome hydrophobic binding pocket (5a versus 17a). Compound 7a does not include Ala
structure but its large Nꢀ(2ꢀnaphthoyl) group might enable itself to reach the hydrophobic binding pocket. Also as the
hydrophobic moiety, not only an aromatic group but also an aliphatic hydrophobic group, as exemplified by 17a, can be
accommodated in the pocket. Thus, potency of the proteasome inhibition by the compounds summarized in Table 1
would reflect the efficacy of the hydrophobic interactions with the pocket.
Among them, the Nꢀ(2ꢀnaphthoyl)ꢀAla derivative 16a (IC50 = 25 nM) was the most potent compound identified.
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Table 1. Proteasome inhibitory effects of compounds 3aꢀ17a modified at the NꢀCbzꢀalanyl moiety in 3a.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
compound number
R1
ChTꢀL activity, IC50 [nM]a
49 ± 9.5
O
O
N
3a
4a
5a
6a
7a
8a
9a
10a
H
O
230 ± 40
210 ± 25
130 ± 13
60 ± 12
300 ± 70
57 ± 12
O
41 ± 11
O
N
H
O
47 ± 9.2
30 ± 3.1
28 ± 2.6
11a
12a
13a
230 ± 35
79 ± 13
25 ± 1.7
77 ± 12
14a
15a
16a
17a
O
N
H
O
O
O
N
H
O
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belactosin A
bortezomib
1440
4.5
6
7
8
9
aBased on three experiments.
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Due to the lack of structural insights of substrate binding into the primed sites of the CP we performed an analytical
random screen at the vinyl moiety of 3a, which is summarized in Table 2. Notably, this site turned out to have a
significant effect on the IC50 of each of the synthesized compound varying between 5.7 and 200 nM. The order of the
inhibitory potency of these compounds is according to: 3e > 3d > 3j > 3f > 3b > 3a > 3i > 3c > 3g > 3h. Introduction of
a phenyl ring clearly improved the inhibitory activity, in which the best side chain length was identified to be two
carbons (n = 2), i.e. the phenethyl derivative 3e. The IC50 value of 3e was as low as 5.7 nM and comparable to that of
the clinical drug bortezomib (IC50 = 4.5 nM).
Table 2. Proteasome inhibitory effects of compounds 3aꢀj modified at the vinyl moiety in 3a.
compound number
R2
ChTꢀL activity, IC50 [nM]a
49 ± 9.5
3a
3b
3c
3d
38 ± 11
90 ± 20
14 ± 1.2
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24
25
5.7 ± 1.2
35 ± 9.1
130 ± 17
200 ± 44
63 ± 10
29 ± 7.8
1440
3e
3f
3g
3h
3i
3j
belactosin A
bortezomib
4.5
aBased on three experiments.
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Finally, we designed phenethylꢀtype compounds 7e and 16e, where 2ꢀnaphthoyl and 2ꢀnaphthoylꢀAla groups were
selected as potentially effective Nꢀsubstituents based on the results summarized in Table 1, respectively (Table 3). Both
of these compounds revealed superb IC50 values of 33 nM (7e) and 10 nM (15e), respectively, as explained by the
structural results (see below).
Table 3. Proteasome inhibitory effects of compounds 7e and 16e modified at the NꢀCbzꢀalanyl moiety in 3e.
O
N
H
H
N
O
R1
O
R2
compound number
R1
R2
ChTꢀL activity, IC50 [nM]a
49 ± 9.5
O
3a
O
N
H
O
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33 ± 8.4
10 ± 1.0
7e
16e
6
7
8
9
belactosin A
1440
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11
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13
14
15
16
17
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60
Bortezomib
4.5
aBased on three experiments.
Inhibitory effects on the CꢀL and TꢀL activities on human cCP and other proteases. Next, we evaluated the
inhibitory activity of the selected compounds (3a, 7a, 12a, 16a, 3e and 16e) against the CꢀL (βꢀ1) and TꢀL (βꢀ2)
activities of the human cCP using AcꢀnLPnLDꢀAMC and AcꢀRLRꢀAMC as chromophoric substrates, respectively. As
summarized in Table 4, all these compounds inhibit the CꢀL activity > 600 nM (Table 4) in agreement to belactosin
analogs,11b, 12 suggesting that CꢀL inhibition at high concentrations is a common property of this class of proteasome
ligands whereas the TꢀL active sites is not altered >1300 nM (see structural results). It has been reported that
bortezomib inhibits beside the proteasome as a major target also a multitude of serine proteases, thus causing severe
side effects upon application.23 Contrary, evaluation of the inhibitory activity of 3e against proteases such as caspase 3,
cathepsin B and trypsin revealed strong selectivity for the CP and therefore supports further optimization of this class of
compounds for future drug development (Figure 4).
Table 4. Inhibitory activity of selected compounds against ChTꢀL, CꢀL and TꢀL activity of proteasomes and cell growth.
O
N
H
H
N
O
R1
O
R2
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cell growth
IC50 [ꢀM]
HCT116
0.91
IC50 [nM]a
compound
number
R1
R2
ChTꢀL activity
49 ± 9.5
CꢀL activity
1600
TꢀL activity
> 3000
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31
O
O
N
H
3a
7a
O
60 ± 1.2
670
1900
11.2
30 ± 3.1
1780
2190
4.16
12a
25 ± 1.7
5.7 ± 1.2
10 ± 1.0
4.5
690
2420
1320
1920
2.61
1.82
0.79
0.01
16a
3e
O
O
N
H
O
> 3000
130
> 3000
1880
16e
bortezomib
aBased on three experiments.
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Figure 4. Effect of 3e on various proteases compared to the CP.
Inhibitory effects on human β5i of the iCP. New studies on the selective inhibition of the iCP demonstrated a
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therapeutic benefit, particularly in autoimmune diseases and put the iCP in the limelight as a novel drug target.5a, 9a, 24
Yet, there have been no reports on the binding properties of the class of belactosins and its derivatives to the
immunoproteasome. We thus evaluated the inhibitory effects of belactosin A and 3e on human β5i via an activeꢀsite
ELISA method according to Kuhn et al.17b Bortezomib, lactacystin and PRꢀ957 were used as controls beside analysis of
belactosin A and 3e. Figure 5 displays the binding profile for each of the compounds for the human cCP and iCP.
Remarkably, lactacystin and PRꢀ957 have a strong tendency to either inhibit β5c or β5i, respectively,5a whereas
belactosin A and its derivative 3e block the ChTꢀL sites of each CPꢀtype equally, similar as bortezomib. These data
represent for the first time a comparison on the binding probabilities of ligands which are selective for both, the
nonꢀprimed and primed sites of the proteasome. Interestingly, the results demonstrate that at least 3e do not show
apparent alterations in their affinity to a specific type of the CP (see structural results).
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Figure 5. Activeꢀsite ELISA analysis of 20S proteasomes or 20S immunoproteasomes incubated with various
inhibitors; concentration of compounds was 0.3 ꢀM, except for lactacystin (2.5 ꢀM).
Inhibitory Effect on Cell Growth. Next, we investigated the cell growth inhibitory effects of selected compounds (3a,
7a, 12a, 16a, 3e, 16e) on HCT116. As summarized in Table 4, all of these compounds showed cell growth inhibitory
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effects with IC50 values of 0.79 ꢀ 11.2 ꢀM as compared to bortezomib (IC50 = 0.01ꢁM). Furthermore, we examined the
cell cycle distribution of HCT116 cells. As shown in Figure 6, 3e inhibited cell cycle progression at the G2/M stage like
bortezomib which is also revealed for most of the compounds (see supporting information).
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Figure 6. Cell cycle distribution analysis of HCT116 cells treated with (a) DMSO, (b) 3e or (c) bortezomib.
In parallel, we investigated the proteasome inhibitory effect of these inhibitors in a cellular system by Western blot
analysis. The tumor suppressor gene product p53 is a wellꢀknown proteasome target.25 26 As shown in Figure 7, p53
was clearly accumulated in HCT116 cells treated with belactosin derivatives. Furthermore, cleavage of
poly(ADPꢀribose) polymerase (PARP), a prominent marker of apoptosis,27 was observed in the treated cells, indicating
that the apoptotic pathway is activated in the cells. These results suggest that CP blockage by our designed balactosin
derivatives induces G2/M cell cycle arrest and causes an accumulation of p53, which result in HCT116 cell apoptosis,
thus explaining the observed cell growth inhibitory effects.
Figure 7. Immunoblot analysis of p53 and cPARP in HCT116 cells treated with bortezomib (B), belactosin derivatives
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(3a, 7a, 12a, 16a, 3e, 16e), or DMSO (control, D).
4
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7
8
9
Stability in Aqueous Medium. So far, all potent inhibitors identified in this study were only poorly soluble in aqueous
medium, thus we employed 33e (Table 5) by removal of the Nꢀterminal Cbz group in 3e. In contrast to previous
belactosin analogues, 33e could be incubated in 0.1 M triethylammonium acetate (TEAA) buffer (pH 7.4), allowing to
record a timeꢀcourse by HPLC, hereby determining its halfꢀlife (t1/2). As summarized in Table 5, the t1/2 of 33e in pH 7.4
buffer was approximately 10 h, indicating that it is chemically much more stable than other βꢀlactone type proteasome
inhibitors (omuralide, 13 min; salinosporamide A, 56 min).28
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Table 5. The chemical stability of 33e, omuralide and salinosporamide A.
omuralide
13 mina
salinosporamide A
56 mina
33e
10 h
t1/2 in pH 7.4 buffer (37 °C)
aFrom ref. 28
Mode of Proteasome Inhibition. An interesting approach for the conversion of a reversible into an irreversible β–
lactone inhibitor is exemplified by omuralide7 versus salinosporamide A (Sal A).29 Sal A exhibits essentially the same
scaffold as omuralide, though it displays a chloroethyl group in the P2 site. This latter moiety is responsible for the
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irreversible mode of action, since after ester bond formation with Thr1Oγ a second reaction occurs resulting in a
tetrahydrofuran ring. Belactosins invert the classic concept of previously described β–lactone compounds as identified
in the yCP crystal structure in complex with hBel.12a It was shown that this class of belactosins, which lack the
γꢀlactamꢀring, bind to the CP in a reversed fashion, targeting predominantly the primed site of the substrate binding
channel. To date, its mode of action to either bind reversible or irreversible has not been addressed. We therefore
preincubated the CP with 3e and measured the CP activity before and after dialysis in comparison to bortezomib and
carfilzomib. As shown in Figure 8 and in agreement with previous reports17a, 28, 30 bortezomib and carfilzomib exhibit
different mode of actions to block the proteasome activity either reversible or irreversible. The inhibitory effect of 3e
was attenuated after dialysis and therefore classifies belactosines as slowly reversible CP inhibitors, however with
dissociation rates even less than bortezomib.
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Figure 8. ChTꢀL activity of the 20S proteasome treated with 3e, bortezomib and carfilzomib before and after washꢀout.
Crystal structure analysis of yCP in complex with 3e. In order to gain insights into the mode of action of the newly
identified highly potent cisꢀbelactosin analogue, we coꢀcrystallized 3e with the yCP and determined its structure to 2.8
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Å resolution (Rfree = 22.4%, Table ST1). Using inhibitor concentrations in the mM range for crystal soaking
experiments, the belactosin derivative targeted predominantly the β5 active sites of the yCP, whereas subunits β1 and
6
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9
β2 display the ligand only partially defined in the electron density map with low occupancy / high DebyeꢀWaller factors.
The improper binding profile of 3e to the CꢀL and TꢀL sites is due to the restricted size of their primed specificity
pockets as well as the sterically demanding cyclopropane group and the constrained conformation of the ligand, in
agreement with the kinetic results (Table 4). Contrary, the FoꢀFcꢀdifference electron density map of the ChTꢀL activity
clearly depicts 3e covalently bound by βꢀlactone opening and acylation of the catalytic Nꢀterminal Thr1Oγ as observed
for the βꢀlactones omuralide and hBel.7, 12a Moreover, the (S)ꢀsecꢀbutyl side chain of 3e proved to be the only side chain
facing into the nonꢀprimed site as observed in the yCP:hBel complex,12b thus forming a multitude of VanꢀderꢀWaals
interactions in the S1 specificity pocket, while the remaining part of the ligand is oriented towards the primed substrate
binding channel (Figure 9ꢀa).
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In general, once bound at the active site of the CP, βꢀlactones generate a C4ꢀhydroxyl group which in the case of
omuralide is stabilized through a hydrogen bond with the Thr1 and occupies the position formerly taken by a
nucleophilic water molecule in the unligated enzyme, hereby preventing acylꢀenzyme hydrolysis by disfavoring the
BürgiꢀDunitz trajectory (Figure 9ꢀb). Contrary, 3e performs a major structural rearrangement once bound to the yCP
compared to omuralide: the newly generated hydroxyl group at C4 is Hꢀbonded to Arg19O, hence pointing into the
opposite direction, just as observed in the CP:hBel crystal structure.12 The cyclopropane ring of 3e adopts a position
similar to that of the C4ꢀOH of omuralide, preventing addition of the nucleophilic water molecule and thus explaining
the mode of binding to act as a proteasome inhibitor, which however is different to omuralide. Notably, the binding
position, the stereochemistry of 3e and its cyclopropane ring together force the remainder of this compound into the
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primed site of the CP. In agreement to the random screen for improving the binding preference towards R2 (Table 2),
the phenethyl moiety is well defined in the electron density map and protrudes into a specificity pocket composed of
Ser96, Tyr112, Asp114, Ser115 and Val127 of subunit β5, thus explaining its low IC50 value. Interestingly, most of these
proteasome residues are conserved between the human cCP and iCP and thus do not distinguish between these two
proteasome types in accordance with the ELISA analysis shown in Figure 5. In contrast, the Alaꢀmoiety of 3e is the
only part of the ligand which is flexible in the crystal structure, whereas the NꢀCbz group performs distinct
VanꢀderꢀWaals interactions with Ile24 of β4 as well as Tyr129 and Phe132 of the adjacent β4’ꢀsubunit, respectively
(Figure 9ꢀa). These structural observations again confirm the functional data, showing that Nꢀacyl compounds lacking
the Alaꢀmoiety can only be recaptured in their potency by introducing a spacious Nꢀ(2ꢀnaphtoyl) group (7a versus 4a,
Table 1).
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Next, we performed a structural superposition of yCP bound to 3e and hBel (Figure 9ꢀc). Both ligands adopt a unique
arrangement in the primed substrate binding channel which in some parts significantly differ to each other: though both
ligands exhibit a similar binding mode to the active site Thr1Oγ and that of the P1 residues as well as a comparable
orientation of the phenethyl side chain versus the benzyl ester moiety in hBel, the alanine moiety of the two inhibitors
is oriented almost orthogonal (Figure 9ꢀc). This unique binding of the Nꢀterminal tail of 3e is explained by the bent
conformation caused by the cisꢀconfiguration of the cyclopropane structure, which is absent in hBel. However, the
phenyl ring from the Cbz group of both ligands are directed towards a lipophilic pocket formed by the adjacent subunits
β4 and β4’, hereby getting stabilized by strong hydrophobic interactions. These findings coincide with the functional
data showing that removal of the hydrophobic Cbz group in the belactosin derivatives increased the IC50 value from 49
nM (3a) to 300 nM (8a, Table 1)). Hence, the performed crystallographic studies elucidate important insights into the
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design and flexibility of future proteasome inhibitors exploiting the primed substrate binding channel. Once the Thr1Oγ
ester bond is formed via the strained βꢀlactone opening of the ligand, stringent conformational rearrangements of the
inhibitor are restricted due to the rigidity of the cisꢀcyclopropane ring linker. In addition, the binding affinity of future
belactosin derivatives might be further improved by exploiting the identified hydrophilic pocket stabilizing the
phenethyl side chain as well as the distal hydrophobic pocket interacting with the Cbz group of 3e, which are both
located solely in the primed substrate binding channel.
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Figure 9. 3e and its binding mode to the β5 subunit. (a) Crystal structure of the ChTꢀL active site in complex with 3e
(PDB code 4J70). The backbone of the proteasomal subunit β5 is colored in grey, subunits β4 and β4’ in dark grey, and
shown in coil representation; the ligand is represented in a stick model. The 2FoꢀFc electron density map (colored in
blue), in which 3e and Thr1 has been omitted prior phase calculations, is contoured from 1 σ (Thr1 is colored in black).
The various sets of Hꢀbonds between 3e and β5 main chain atoms Gly47, Thr1N and Arg19O are depicted as black
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dashed lines. (b) The structural superposition of 3e and omuralide7 as well as (c) 3e and hBel12a bound to the ChTꢀL
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active site are represented in yellow and the corresponding CPꢀinhibitor in grey, respectively.
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Conclusions
We performed the first systematic SAR study of proteasome inhibitors that bind to the primed substrateꢀbinding site
and the S1 binding site, using the unnatural cisꢀcyclopropane belactosin analog 3a to identify a series of highly potent
proteasome inhibitors. Among them, 3e showed marked proteasome inhibitory activity as potent as bortezomib. The
Xꢀray crystallographic analysis of the yeast proteasome in complex with 3e revealed its covalent binding mode and
allows future optimization of this interesting class of CPꢀinhibitors for further characterization in clinical phase studies.
Supporting Information
Supporting Information Available: Experimental details of synthesis, biological evaluation, Xꢀray crystallographic
analysis and table listing combustion analysis data for target compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
Abbreviations Used
AMC, aminomethylcoumarin; CP, core particle; DMP, DessꢀMartin periodinane; ELISA, enzymeꢀlinked
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immunosorbent assay; Fmoc, 9ꢀfluorenylmethyloxycarbonyl; HPLC, highꢀpressure liquid chromatography; Me,
methyl; Piv, pivaloyl; Ph, phenyl; Suc, succinyl; TEAA, triethylammonium acetate; TFA, trifluoroacetic acid; THF,
tetrahydrofuran
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References
1.
(a) Orlowski, M. The multicatalytic proteinase complex, a major extralysosomal proteolytic system. Biochemistry
1990, 29, 10289ꢀ10297; (b) Ciechanover, A. The ubiquitinꢀproteasome proteolytic pathway. Cell 1994, 79, 13ꢀ21.
2.
(a) Fuchs, S. Y. The role of ubiquitinꢀproteasome pathway in oncogenic signaling. Cancer Biol. Ther. 2002, 1,
337ꢀ341; (b) Chen, J. J.; Lin, F.; Qin, Z. H. The roles of the proteasome pathway in signal transduction and neurodegenerative
diseases. Neurosci. Bull. 2008, 24, 183ꢀ194.
3.
Muchamuel, T.; Basler, M.; Aujay, M. A.; Suzuki, E.; Kalim, K. W.; Lauer, C.; Sylvain, C.; Ring, E. R.; Shields, J.;
Jiang, J.; Shwonek, P.; Parlati, F.; Demo, S. D.; Bennett, M. K.; Kirk, C. J.; Groettrup, M. A selective inhibitor of the
immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nat. Med.
2009, 15, 781ꢀ787.
4.
King, R. W.; Deshaies, R. J.; Peters, J. M.; Kirschner, M. W. How proteolysis drives the cell cycle. Science 1996,
274, 1652ꢀ1659.
5.
(a) Huber, E. M.; Groll, M. Inhibitors for the Immunoꢀ and Constitutive Proteasome: Current and Future Trends in
Drug Development. Angew. Chem. Int. Ed. 2012, 51, 8708ꢀ8720; (b) Adams, J. The proteasome: a suitable antineoplastic
target. Nat. Rev. Cancer 2004, 4, 349ꢀ360.
26
ACS Paragon Plus Environment

Page 27 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
6.
(a) Bross, P. F.; Kane, R.; Farrell, A. T.; Abraham, S.; Benson, K.; Brower, M. E.; Bradley, S.; Gobburu, J. V.;
Goheer, A.; Lee, S. L.; Leighton, J.; Liang, C. Y.; Lostritto, R. T.; McGuinn, W. D.; Morse, D. E.; Rahman, A.; Rosario, L. A.;
Verbois, S. L.; Williams, G.; Wang, Y. C.; Pazdur, R. Approval summary for bortezomib for injection in the treatment of
multiple myeloma. Clin. Cancer Res. 2004, 10, 3954ꢀ3964; (b) Kane, R. C.; Farrell, A. T.; Sridhara, R.; Pazdur, R. United
States Food and Drug Administration approval summary: bortezomib for the treatment of progressive multiple myeloma after
one prior therapy. Clin. Cancer Res. 2006, 12, 2955ꢀ2960; (c) Kane, R. C.; Dagher, R.; Farrell, A.; Ko, C. W.; Sridhara, R.;
Justice, R.; Pazdur, R. Bortezomib for the treatment of mantle cell lymphoma. Clin. Cancer Res. 2007, 13, 5291ꢀ5294.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7.
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, 463ꢀ471.
8.
Groll, M.; Clausen, T. Molecular shredders: how proteasomes fulfill their role. Curr. Opin. Struct. Biol. 2003, 13,
665ꢀ673.
9.
(a) Huber, Eva M.; Basler, M.; Schwab, R.; Heinemeyer, W.; Kirk, Christopher J.; Groettrup, M.; Groll, M.
Immunoꢀ and Constitutive Proteasome Crystal Structures Reveal Differences in Substrate and Inhibitor Specificity. Cell 2012,
148, 727ꢀ738; (b) Murata, S.; Sasaki, K.; Kishimoto, T.; Niwa, S.ꢀi.; Hayashi, H.; Takahama, Y.; Tanaka, K. Regulation of
CD8+ T Cell Development by ThymusꢀSpecific Proteasomes. Science 2007, 316, 1349ꢀ1353.
10.
Parlati, F.; Lee, S. J.; Aujay, M.; Suzuki, E.; Levitsky, K.; Lorens, 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
chymotrypsinꢀlike activity of the proteasome. Blood 2009, 114, 3439ꢀ3447.
11.
(a) Asai, A.; Hasegawa, A.; Ochiai, K.; Yamashita, Y.; Mizukami, T. Belactosin A, a novel antitumor antibiotic
acting on cyclin/CDK mediated cell cycle regulation, produced by Streptomyces sp. J. Antibiot. 2000, 53, 81ꢀ83; (b) Asai, A.;
27
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Page 28 of 33
1
2
3
Tsujita, T.; Sharma, S. V.; Yamashita, Y.; Akinaga, S.; Funakoshi, M.; Kobayashi, H.; Mizukami, T. A new structural class of
4
5
6
proteasome inhibitors identified by microbial screening using yeastꢀbased assay. Biochem. Pharmacol. 2004, 67, 227ꢀ234.
7
8
9
12.
(a) Groll, M.; Larionov, O. V.; Huber, R.; De Meijere, A. Inhibitorꢀbinding mode of homobelactosin C to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
proteasomes: New insights into class I MHC ligand generation. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 4576ꢀ4579; (b)
Korotkov, V. S.; Ludwig, A.; Larionov, O. V.; Lygin, A. V.; Groll, M.; de Meijere, A. Synthesis and biological activity of
optimized belactosin C congeners. Org. Biomol. Chem. 2011, 9, 7791ꢀ7798.
13.
(a) Borissenko, L.; Groll, M. 20S proteasome and its inhibitors: crystallographic knowledge for drug development.
Chem. Rev. 2007, 107, 687ꢀ717; (b) Groll, M.; Kim, K. B.; Kairies, N.; Huber, R.; Crews, C. M. Crystal structure of
epoxomicin : 20S proteasome reveals a molecular basis for selectivity of alpha ',beta 'ꢀepoxyketone proteasome inhibitors. J.
Am. Chem. Soc. 2000, 122, 1237ꢀ1238; (c) Groll, M.; Berkers, C. R.; Ploegh, H. L.; Ovaa, H. Crystal structure of the boronic
acidꢀbased proteasome inhibitor bortezomib in complex with the yeast 20S proteasome. Structure 2006, 14, 451ꢀ456; (d)
Groll, M.; Huber, R.; Potts, B. C. M. Crystal structures of salinosporamide A (NPIꢀ0052) and B (NPIꢀ0047) in complex with
the 20S proteasome reveal important consequences of betaꢀlactone ring opening and a mechanism for irreversible binding. J.
Am. Chem. Soc. 2006, 128, 5136ꢀ5141; (e) Groll, M.; Schellenberg, B.; Bachmann, A. S.; Archer, C. R.; Huber, R.; Powell, T.
K.; Lindow, S.; Kaiser, M.; Dudler, R. A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel
mechanism. Nature 2008, 452, 755ꢀ758; (f) Hines, J.; Groll, M.; Fahnestock, M.; Crews, C. M. Proteasome inhibition by
fellutamide B induces nerve growth factor synthesis. Chem. Biol. 2008, 15, 501ꢀ512; (g) Clerc, J.; Groll, M.; Illich, D. J.;
Bachmann, A. S.; Huber, R.; Schellenberg, B.; Dudler, R.; Kaiser, M. Synthetic and structural studies on syringolin A and B
reveal critical determinants of selectivity and potency of proteasome inhibition. Proc. Natl. Acad. Sci. U. S. A. 2009, 106,
6507; (h) Blackburn, C.; Barrett, C.; Blank, J. L.; Bruzzese, F. J.; Bump, N.; Dick, L. R.; Fleming, P.; Garcia, K.; Hales, P.;
28
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1
2
3
4
5
6
7
8
9
Hu, Z. Optimization of a series of dipeptides with a P3 threonine residue as nonꢀcovalent inhibitors of the chymotrypsinꢀlike
activity of the human 20S proteasome. Bioorg. Med. Chem. Lett. 2010, 20, 6581ꢀ6586; (i) Grawert, M. A.; Gallastegui, N.;
Stein, M.; Schmidt, B.; Kloetzel, P. M.; Huber, R.; Groll, M. Elucidation of the alphaꢀketoꢀaldehyde binding mechanism: a
lead structure motif for proteasome inhibition. Angew. Chem. Int. Ed. Engl. 2011, 50, 542ꢀ544; (j) Gallastegui, N.; Beck, P.;
Arciniega, M.; Huber, R.; Hillebrand, S.; Groll, M. Hydroxyureas as Noncovalent Proteasome Inhibitors. Angew. Chem. Int.
Ed. 2012, 51, 247ꢀ249; (k) Huber, E. M.; Basler, M.; Schwab, R.; Heinemeyer, W.; Kirk, C. J.; Groettrup, M.; Groll, M.
Immunoꢀand Constitutive Proteasome Crystal Structures Reveal Differences in Substrate and Inhibitor Specificity. Cell 2012,
148, 727ꢀ738.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
14.
(a) Armstrong, P. D.; Cannon, J. G.; Long, J. P. Conformationally Rigid Analogues of Acetylcholine. Nature 1968,
220, 65ꢀ66; (b) Kazuta, Y.; Hirano, K.; Natsume, K.; Yamada, S.; Kimura, R.; Matsumoto, S.; Furuichi, K.; Matsuda, A.;
Shuto,
S.
Cyclopropaneꢀbased
conformational
restriction
of
histamine.
(1S,2S)ꢀ2ꢀ(2ꢀaminoethyl)ꢀ1ꢀ(1Hꢀimidazolꢀ4ꢀyl)cyclopropane, a highly selective agonist for the histamine H3 receptor, having
a cisꢀcyclopropane structure. J. Med. Chem. 2003, 46, 1980ꢀ1988; (c) Watanabe, M.; Kazuta, Y.; Hayashi, H.; Yamada, S.;
Matsuda, A.; Shuto, S. Stereochemical diversityꢀoriented conformational restriction strategy. Development of potent
histamine H3 and/or H4 receptor antagonists with an imidazolylcyclopropane structure. J. Med. Chem. 2006, 49, 5587ꢀ5596;
(d) Watanabe, M.; Kobayashi, T.; Hirokawa, T.; Yoshida, A.; Ito, Y.; Yamada, S.; Orimoto, N.; Yamasaki, Y.; Arisawa, M.;
Shuto, S. Cyclopropaneꢀbased stereochemical diversityꢀoriented conformational restriction strategy: histamine H3 and/or H4
receptor ligands with the 2,3ꢀmethanobutane backbone. Org. Biomol. Chem. 2012, 10, 736ꢀ745.
15.
Yoshida, K.; Yamaguchi, K.; Sone, T.; Unno, Y.; Asai, A.; Yokosawa, H.; Matsuda, A.; Arisawa, M.; Shuto, S.
Synthesis of 2,3ꢀ and 3,4ꢀmethanoamino acid equivalents with stereochemical diversity and their conversion into the
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