Structure-based design of β1i or β5i specific inhibitors of human immunoproteasomes

Article abstract of DOI:10.1021/jm500716s

Mammalian genomes encode seven catalytic proteasome subunits, namely, β1c, β2c, β5c (assembled into constitutive 20S proteasome core particles), β1i, β2i, β5i (incorporated into immunoproteasomes), and the thymoproteasome-specific subunit β5t. Extensive r

Full text of DOI:10.1021/jm500716s

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Article
Structure-based design of either #1i or #5i
specific inhibitors of human immunoproteasomes
Gerjan de Bruin, Eva M. Huber, Bo-Tao Xin, Eva J. van Rooden, Karol Al-
Ayed, Kyung Bo Kim, Alexei F. Kisselev, Christoph Driessen, Mario van der
Stelt, Gijsbert A. van der Marel, Michael Groll, and Herman S. Overkleeft
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500716s • Publication Date (Web): 09 Jul 2014
Downloaded from http://pubs.acs.org on July 11, 2014
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Structureꢀbased design of either β1i or β5i specific
inhibitors of human immunoproteasomes
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Gerjan de Bruin†&, Eva M. Huber§&, BoꢀTao Xin†, Eva J. van Rooden†, Karol AlꢀAyed†,
KyungꢀBo Kim≠, Alexei F. Kisselev#, Christoph Driessen‡, Mario van der Stelt†, Gijsbert A.
van der Marel†, Michael Groll§, Herman S. Overkleeft†*
†
From Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Proteomics
§
Centre, Einsteinweg 55, 2333 CC Leiden, The Netherlands, Center for Integrated Protein
Science at the Department Chemie, Lehrstuhl für Biochemie, Technische Unversität
München, 85748 Garching, Germany, ‡Department of Hematology and Oncology,
≠
Kantonsspital St. Gallen, 9007 St. Gallen, Switzerland, Department of Pharmaceutical
Science, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA
#Department of Pharmacology and Toxicology and Norris Cotton Cancer Center, Geisel
School of Medicine at Dartmouth, 1 Medical Centre Drive HB7936, Lebanon, New
Hampshire 03756, United States of America
* To whom correspondence should be addressed
&Gerjan de Bruin and Eva M. Huber contributed equally to this work
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KEYWORDS Immunoproteasome, Proteasome inhibitors, Epoxyketone, Vinyl Sulfone,
Multiple Myeloma
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ABSTRACT Mammalian genomes encode seven catalytic proteasome subunits, namely β1c,
β2c, β5c (assembled into constitutive 20S proteasome core particles), β1i, β2i, β5i
(incorporated into immunoproteasomes) and the thymoproteasomeꢀspecific subunit, β5t.
Extensive research in the past decades has yielded numerous potent proteasome inhibitors
including compounds currently used in the clinic to treat multiple myeloma and mantle cell
lymphoma. Proteasome inhibitors that selectively target combinations of β1c/β1i, β2c/β2i or
β5c/β5i are available, yet ligands truly selective for a single proteasome activity are scarce. In
this work we report the development of cellꢀpermeable β1i as well as β5i selective inhibitors,
which outperform existing leads in terms of selectivity and/or potency. These compounds are
the result of a rational design strategy using known inhibitors as starting points and
introducing structural features according to the Xꢀray structures of the murine constitutive and
immunoproteasome 20S core particles.
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Introduction
Proteasomes are large, multiꢀcatalytic complexes that partake in the degradation of
cytosolic, nuclear and misfolded ER proteins in most kingdoms of life.1 The 20S core
particles (CP) in which proteolysis takes place are C2ꢀsymmetrical barrelꢀshaped multiꢀ
protein complexes composed of four stacked rings of seven subunits each: the two inner rings
consisting of seven homologous yet distinct βꢀsubunits (β1ꢀβ7) are flanked by two outer rings
composed of the αꢀsubunits 1ꢀ7.2 The proteolytic activity resides within three catalytic
subunits (one copy in each βꢀring), namely β1c (also referred to as caspaseꢀlike, cleaving
preferentially after acidic residues), β2c (trypsinꢀlike, with a preference for basic residues)
and β5c (chymotrypsinꢀlike, preferring neutral, hydrophobic residues at the cleavage site2ꢀ3).
Immuneꢀcompetent cells express next to the constitutive proteasome (cCP) the interferonꢀγꢀ
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inducible subunits β1i (LMP2), β2i (MECLꢀ1) and β5i (LMP7).4 Upon cytokine stimulation
these subunits replace their constitutive proteasome counterparts (β1c, β2c and β5c,
respectively) to form immunoproteasomes (iCPs).
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The substrate preference of cCP catalytic subunits considerably overlaps with their iCP
analogues, though subtle differences have been observed.5 In particular, whereas β1c prefers
acidic residues at P1 (Asp or Glu; the amino acid residue residing at the Cꢀterminus of the
scissile peptide bond), β1i is biased to hydrophobic side chains at this position.6 This
divergence of substrate preference has been connected to processing and presentation of
antigenic peptides from cytosolic/nuclear sources.
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Proteasome inhibitors take up a prominent position both in fundamental and applied
biomedical research. Based on the structural diversity of natural CP inhibitors, numerous
synthetic compounds have been developed. Most of them are peptideꢀbased and feature an
electrophile that reacts with the Nꢀterminal nucleophilic threonine hydroxyl group of catalytic
proteasome subunits to form a covalent linkage. Depending on the nature of the electrophilic
trap the covalent bond is transient (aldehydes, boronic acids), semiꢀpermanent (betaꢀlactones)
or permanent (vinyl sulfones, epoxyketones).7 Subunit specificity is largely governed by the
nature of the peptide fragment (amino acid sequence) of a given inhibitor, though examples
exist in which substitution of the electrophilic head group on an otherwise unaltered
compound also impacts the proteasome inhibition profile.8 CP inhibitors are broadly used to
study the proteolytic system in more detail and to modulate physiological processes, in
particular cell proliferation. The finding that proteasomes are valid therapeutic targets in
various cancers led to the development of the peptide boronic acid bortezomib (Velcade)9 as a
drug for the treatment of multiple myeloma (MM) and mantle cell lymphoma. This
breakthrough was recently followed by the FDA approval of the peptide epoxyketone
carfilzomib (Kyprolis)10 as a secondꢀgeneration CP inhibitor for the treatment of MM. Thus,
both reversible and irreversible proteasome inhibitors are relevant candidates for drug
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development strategies. Currently, several proteasome inhibitors are in clinical trials,
including marizomib (salinosporamide A, NPIꢀ0052), delanzomib (CEPꢀ18770)11, ixazomib
(MLNꢀ9708)12, and oprozomib (ONXꢀ0912, PRꢀ047)13.
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Although bortezomib and carfilzomib have been developed to primarily target the
chymotrypsinꢀlike active sites β5c and β5i, which are the most important ones for the antiꢀ
tumor effects, they also coꢀinhibit the subunits β1c/β1i as well as β2c/β2i at higher
concentrations.10a, 14 We have contributed to this research field by the development of potent,
cell permeable ligands specific for one catalytic subunit (β13a, β23b or β58a, 15) of the cCP
together with its iCP counterpart. These compounds demonstrated that cytotoxicity in MM
cell lines cannot be reached by exclusive blockage of β5c/β5i, but requires coꢀinhibition of
either β1c/β1i or β2c/β2i.3a, b In the present study we aimed to distinguish between cCP and
iCP catalytic subunits, especially given that in various lymphomas iCPs – absent in most
tissues – are the predominant proteasome species. Thus, compounds specific for one out of
the six catalytic subunits from which cCPs and iCPs are assembled, are highly desirable
commodities for research and clinical applications nowadays. This certainly holds true for
oncological research, where identification of the ideal target/dosage combination – which
catalytic active site should be inhibited, and to what extent – needs to be established.
Furthermore, this also applies to the immunological question of which proteolytic center is
involved in the generation of a given antigenic peptide.
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For many years Xꢀray diffraction data from yeast proteasomes (yCP) were used to clarify
the mode of action and binding specificity of CP inhibitors, including approved drugs and
drug candidates.16 Recently, structure elucidation of the murine cCP and iCP complemented
the yeast structures and highlighted the differences between the cCP and iCP.17 Perusal of the
differences between the two structures and their inhibitorꢀbound counterparts provide insight
to structural features by which β1i and β5i may be targeted independently from β1c and β5c,
respectively. Specifically, β5i appears to be able to accommodate a larger amino acid side
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chain at position P1 than β5c, while the opposite holds true for the P3 residue. On the other
hand, the S1 pocket of β1i is more hydrophobic when compared to β1c, while the S3 pocket
of β1i is more restricted in size and more polar compared to its counterpart β1c.
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Here we report on the discovery of inhibitors highly specific for either β5i or β1i in the
presence of all constitutive proteasome and immunoproteasome active sites. The design of our
β5i specific inhibitors is based on two compounds previously developed by ONYX
Pharmaceuticals, namely ONXꢀ091418 (PRꢀ957, 1) and PRꢀ92418a, 19 (2) (Figure 1A). Using a
structureꢀbased design approach we obtained molecules with considerably improved
selectivity for β5i over β5c. Xꢀray crystallographic analysis of the inhibitors in complex with
the yCP provided important insights into the molecular basis of β5i selectivity. For the
development of β1i selective inhibitors our previously reported β1c/β1i specific inhibitor NCꢀ
0013a (3) (Figure 1B) was used as a starting point. In a first optimization round its key
structural element, a proline residue at P3,20 was modified and in a second step structural
features at P1 were varied.
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Altogether, this strategy allowed us to identify either β5i or β1i selective compounds with
high subunit selectivity and potency. All synthesized inhibitors were proven to be cellꢀ
permeable and active in mammalian cancer cell lines and hence, provide an excellent tool kit
for the systematic evaluation of the distinct proteasomal active sites.
Results
Design, synthesis and evaluation of highly specific β5i inhibitors
The Xꢀray structure of 1 covalently bound to the murine cCP and iCP indicates that the
phenyl moiety at position P1 induces a major conformational change in the S1 pocket of β5c,
but only minor alterations in β5i.17a This difference in binding likely confers β5i selectivity to
inhibitor 1 and we reasoned that substitution at P1 of phenylalanine epoxyketone for larger
hydrophobic P1 residues should lead to compounds with enhanced β5i selectivity (Figure
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1A). To determine the optimal P1 residue for this purpose, we synthesized epoxyketone
analogues of 1 featuring an adamantylꢀ (4), biphenylꢀ (5), 2ꢀnaphthylꢀ (6), 1ꢀnaphthylꢀ (7) or
cyclohexylꢀalanine (8) in the P1 site (Table 1). The compounds were evaluated for their
potencies against β5c and β5i in Rajiꢀcell lysates (Bꢀcell lymphoma cell line) by competitive
activity based protein profiling (ABPP) of the β5ꢀsubunits using our β5c/β5i specific probe
BODIPYꢀNC005 (Table 1 and S1, Figures S1 and S2).21 In contrast to Muchamuel et al. who
determined a 30ꢀfold selectivity for β5i over β5c for 118b using an ELISAꢀbased assay in
MOLTꢀ4 cells, we observed a nineꢀfold selectivity with our ABPP technique. The ABPP
method was used for all the herein described compounds and allowed to unambiguously
compare their activity and selectivity. Incorporation of adamantylalanine epoxyketone at P1
of the lead structure (4) resulted in complete loss of affinity. Interestingly, the bulky biphenyl
side chain in 5 did not show increased selectivity but a twoꢀtoꢀthreeꢀfold enhanced potency
for β5c and β5i. Furthermore, while the 2ꢀnaphthylꢀside chain in 6 has both high affinity and
increased selectivity for β5i, the 1ꢀnaphthyl derivative 7 is significantly less potent than
compound 1. Different orientations of the 1ꢀnaphthyl and 2ꢀnaphthyl substituents in the
β5c/β5i active sites might explain this observation. The most prominent improvement in
selectivity was noticed for 8, featuring a cyclohexylalanine residue at P1. While the binding
strength towards β5i remained similar, the affinity for β5c was significantly impaired
compared to the lead structure ONXꢀ0914 (1), hereby enhancing β5i selectivity almost five
times.
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For further optimization we thus chose three side chains: cyclohexyl (highest selectivity),
biphenyl (highest potency) and phenyl (for comparison). We synthesized cyclohexyl (9) and
biphenyl (10) analogues of the phenylꢀbearing compound PRꢀ924 (2) – another β5i selective
inhibitor with 100ꢀfold preference for β5i.19b, 22 9 turned out to be 500ꢀfold selective for β5i
over β5c yet with threefold lower potency compared to 2. Compound 10 appeared less
selective than 2, indicating that a biphenyl substituent at P1 is not suitable for optimal β5i
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selectivity. Next, hybrid compounds featuring elements from both 1 and 2 with various P1
amino acid derivatives were evaluated (11ꢀ22, Tables 1 and S1). From this focused set of
compounds, several β5c/β5i inhibition and selectivity patterns emerge. First, differences in
potency and selectivity between P2 residues such as 4ꢀmethyltyrosine (4MeTyr)ꢀ and
tryptophanꢀderived epoxyketones are marginal. Second, the chirality (D/L) of the alanine in
P3 can have significant influence on β5i selectivity and ꢀpotency. For example, the Nꢀ
acylmorpholine capped compounds 8 and 11 show a similar selectivity profile, however 11
(featuring DꢀAla in P3) is fivefold less active towards β5i and β5c compared to 8 (bearing Lꢀ
Ala in P3). In contrast, the 3ꢀmethylꢀ1Hꢀindene capped inhibitor 12 with LꢀAla in P3 displays
a significant decrease in selectivity but an increase in potency versus its DꢀAla featuring
analogue 13. We conclude that LꢀAla at P3 combines better with an Nꢀacylmorpholine cap,
whereas DꢀAla at P3 requires a 3ꢀmethylꢀ1Hꢀindene Nꢀcap for optimal β5i selectivity.
Compounds 9 and 13 show the highest β5i selectivity, indicating that DꢀAla/3ꢀmethylꢀ1Hꢀ
indene is preferred over LꢀAla/Nꢀacylmorpholine for β5i selectivity.
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As previously reported, subunit selectivity of proteasome inhibitors can be influenced by
the electrophilic trap.8 For example, exchanging the epoxyketone warhead by a vinyl sulfone
in β5ꢀselective inhibitors improves β5ꢀselectivity8a. Based on these results, we decided to
compare the epoxyketones 1, 2, 8 and 9 with their vinyl sulfone counterparts 23, 24, 25 and
26 (Tables 1 and S1). All tested peptide epoxyketones outperform their peptide vinyl sulfone
analogues where it comes to β5i inhibition potency. Of the peptide vinyl sulfone series only
25 is a moderately potent β5i inhibitor, however with four times higher selectivity for β5i
compared to 2.
The six most specific and potent epoxyketone compounds, namely 8, 9, 11, 13, 15 and the
vinyl sulfone 25 were evaluated for selectivity over the other subunits, i.e. β1c/β1i and
β2c/β2i using our ABPP technique in Raji cell lysates with β1c/β1i selective probe BODIPYꢀ
NC001 and panꢀreactive probe BODIPYꢀepoxomicin (Figures 2 and S2, Table S1).21a The
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obtained results disclose that 8 targets β1i and β2i with slightly more potency than 1, while
β1c is completely unaffected. Compound 2 and to a lesser extent 9 also coꢀinhibit β1i in the
low micromolar range, whereas compounds 11, 13, 15 and 25 are highly specific for β5c/β5i
and do not inhibit β1c/β1i and β2c/β2i at concentrations up to 10 µM.
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Next, we tested the cellular potency and selectivity of 8, 9, 11, 13, 15 and 25 in RMPIꢀ8226
(MMꢀcell line) (Figure 3, Table S2). All inhibitors proved to be cellꢀpermeable and selectivity
trends resemble those observed in Raji lysates. Compound 8 exhibits an increased selectivity
for β5i over β5c compared to 1. Notably, it also favors β1i and β2i over β5c, making it a
broadꢀspectrum immunoproteasome inhibitor, when applied at a concentration of 1 µM. To
the best of our knowledge, this is the first proteasome inhibitor able to selectively inhibit all
three iCP subunits over their constitutive counterparts. Although the potency of the Dꢀalanine
containing compounds 11 and 15 towards β5i is decreased by a factor of 3ꢀ5, they are 4 times
more selective for β5i than 1. Moreover, the analogues 9, 13 and 25 are >10 times less
reactive towards β5i compared to 2. However, in case of 13 and 25 all other subunits remain
fully active up to 100 µM (Figure 3, Table S2). In conclusion, compounds 9, 13 and 25 are the
most selective β5i inhibitors known to date.
Xꢀray structures of β5i specific inhibitors in complex with the yeast CP
The compounds 2, 8, 9, 11 and 17 complexed with yCP were further analyzed by Xꢀray
crystallography. Despite the high concentrations used for crystal soakings the DꢀAlaꢀfeaturing
inhibitors 2, 9, 11 and 17 solely target subunit yβ5. In contrast, 1 (P3ꢀLꢀAla) was shown to
inhibit all three active sites of the yCP17a and its cyclohexyl analogue 8 (LꢀAla) occupies at
least the subunits yβ5 and yβ2 (Figures 4A and S3C, Table S4). The P3ꢀDꢀAla residue hence
confers selectivity for the chymotrypsinꢀlike active sites, which is consistent with the ABPP
results in Raji cell lysate and RPMIꢀ8226 cell assays (Figures 2 and 3). Structural modeling
by superposition with the murine substrate binding channels, which are built up of the two
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neighboring subunits β1/2, β2/3 and β5/β6, respectively, provide explanations for this subunit
preference (Sequence numbering according to Huber et al.17a).
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The compounds 11 and 17 do not properly fit into the β1/β2 substrate binding channels due to
clashes of their Nꢀcaps with Glu112 (β2i; 1.8 Å), His114/Tyr114 (β2i/c; 2.7ꢀ3.1 Å) and
Asp120 (β2c; direct contact). Inhibition of the more polar caspaseꢀlike yβ1/β1c active sites is
further disfavored by the hydrophobicity of the ligand. In addition, modeling 11 into the β2/3
substrate binding channel depicts that the Nꢀacylmorpholine cap clashes with Met119 (β3)
(distance 1.3ꢀ2.3 Å), thereby preventing its binding (Figure S3A).
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Compound 2 and its derivative 9 preferentially bind to the subunits yβ5, β5c/i and β1i, a
fact that is mostly due to the favorable interactions between their hydrophobic P1 sites and the
respective protein S1 pockets. Compared to β5 subunits, the affinity for β1 active sites is
reduced by a clash with His116. Polarity reasons and steric hindrance with Met96 (2.3 Å)
further impair binding to the yβ1/β1c active sites. A clash of the 3ꢀmethylꢀ1Hꢀindene cap with
Leu115 (β3; 2.0ꢀ2.3 Å) as well as Asn22 (β2i; 2.6 Å) leads to reduced affinity also for the
β2/3 substrate binding channel (Figure S3A).
In agreement with the β5ꢀselectivity of the investigated compounds, no clashes with
residues from the β5/6 substrate binding channels in cCP or iCP were identified. Inspection of
the 2FOꢀFC electron density maps, however, revealed pronounced differences in the binding
mode of 1 and 8 compared to 11 and 17 as well as 2 and 9. Compound 8 adopts a
conformation, which is identical to 117a, whereas this does not apply to 11 and 17, the P3ꢀDꢀ
Ala analogues of 1 and 8 (Figures 4 and S3). The inverted chirality induces a sharp (almost
90°) turn in the peptide backbone of 11 and 17 and forces their P3 site as well as their Nꢀ
acylmorpholine cap to undergo unique interactions. By contrast, binding of 2 and its analogue
9, both featuring a DꢀAla at P3 in combination with a 3ꢀmethylꢀ1Hꢀindene cap is comparable
to that of 1 and 8 (Figure 4). Our structureꢀaffinity relationship studies also indicate that a
cyclohexyl side chain in P1 disfavors binding to β5c and thereby enhances selectivity for β5i
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compared to a phenyl group. Notably, structural superpositions of the yCPꢀbound compounds
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1, 2, 8, 9 as well as 17 reveal similar conformations of their P1 site (Figure S3B).
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Development of β1i specific inhibitors
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The crystal structures of the murine cCP and iCP depict differences between the substrate
binding channels of β1c and β1i to exist predominantly in the S1 and S3 pockets.17a As a
result of the substitutions T20V, T31F, R45L and T52A in β1i (relative to β1c), the cleavage
preference for acidic residues at P1 is largely abolished, while processing after hydrophobic
amino acids is enhanced.3c Furthermore, the S1 and S3 pockets appear diminished in size
when compared to β1c and the S3 pocket is more polar than in β1c.
For the development of β1i specific inhibitors, we chose azꢀNC0019c (3), a potent β1c/β1i ꢀ
specific ligand as starting point (Figure 1B). Since the β1c/β1i selectivity of 3 appears to be
governed by the turnꢀinducing P3ꢀproline residue, we evaluated seven analogues of 3 (27ꢀ33)
in which Pro was substituted for proline isosters (Figure 5A). With the aim to increase the
polarity of the P3 residue we selected hydroxyproline (Hyp) (27) and thioproline (Thz) (28).
The optimal ring size was determined by comparing Lꢀazetidineꢀ2ꢀcarboxylic acid (Aze, 4ꢀ
membered ring) (29) and Lꢀpipecolic acid (Pip, 6ꢀmembered ring) (30). Fluorinated proline
analogues 31, 32 and 33 were included to probe the effect of altering cis/trans ratios around
the peptide bonds.23
The analogues 27ꢀ33 were evaluated by ABPP for blockage of β1c and β1i in Raji lysates
(Figure 5B, Table S3). As anticipated, 32 (P3 = Rꢀ4FPro) was found to be about tenfold more
potent than 31 (P3 = Sꢀ4FPro), however, the affinity was not increased compared to 3.
Interestingly, 33 (P3 = 4,4ꢀF2Pro) showed a clear preference for β1i versus β1c, without loss
of potency.
To further enhance β1i selectivity the P1 side chain was modified. During the evaluation of
our β5i specific inhibitors, we observed that compounds bearing a phenyl or cyclohexyl
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residue at P1 inhibit β1i much stronger than β1c (Figure 2B), indicating that the S1 pocket of
β1i is more apolar than that of β1c.17a In addition, neither β1c nor β1i is inhibited by
compounds featuring a biphenyl substituent at P1 (data not shown), a finding that supports
structural data showing that β1c/i have a smaller S1 pocket than β5c/i.17a Hence, we
synthesized a series of analogues of 3 with phenyl or cyclohexylalanine in P1 and Pro or 4,4ꢀ
F2Pro in P3 (Figure 6A). Compound 34 (P1 = Phe) binds to β1i without loss of potency
compared to 3, indicating that bulky residues in P1 are tolerated by β1i (Figure 6C, Table S3).
Substitution of the phenylalanine moiety for cyclohexylalanine (as in 35) further increases the
selectivity by a factor of four. Combining bulky residues in P1 with 4,4ꢀF2Pro in P3 resulted
in the compounds 36 and 37, of which the latter showed 250ꢀfold selectivity for β1i over β1c.
In RPMIꢀ8226 cells as well as Raji lysate, 37 is selective for β1i and as potent as 3 (Figure
6D, Table S2). Finally, we compared 37 with the epoxyketone UKꢀ10124 (38), the so far only
β1i specific inhibitor (MLꢀ60444025 is the only other β1i inhibitor reported, which is equipped
with the reversible boronic acid warhead) (Figure 6B, Table S2). In Raji lysate, the potency of
38 for β1i is comparable to 37, however, it is about twoꢀfold less specific for β1i and also
targets β5c/β5i before β1c (Figure 6C). In conclusion, 37 is the most specific and cellꢀ
permeable nonꢀreversible β1i inhibitor known to date.
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Discussion
The main challenge in proteasomal drug development is the design of subunitꢀspecific
inhibitors. Although compounds for β1c/β1i, β2c/β2i and β5c/β5i, respectively, are
available3a, b, 8a, 26, compounds that target only one of the catalytic active sites are still rare and
often exhibit a rather narrow window of selectivity. Here we describe the development of
improved β5i inhibitors as well as a new class of β1i inhibitors, using design parameters
derived from previously determined crystal structures.2ꢀ3, 27 The coꢀcrystallization of the
β5iinhibitor ONXꢀ0914 (1) with the murine cCP and iCP showed that the subunitꢀselectivity
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of 1 is mainly caused by the P1 phenyl ring.17a This feature causes major structural
rearrangements in the backbone and the S1 pocket of the β5c subunit, whereas β5i is able to
accommodate the aromatic ring without pronounced conformational changes. Hence, we
assumed that larger P1 residues would further disfavor binding to β5c and enhance potency as
well as selectivity for β5i. Indeed, large residues such as biphenyl (5) and 2ꢀnaphtyl (6) in P1
yielded more potent β5i inhibitors. Yet, the 1ꢀnaphthyl derivative (7) resulted in a 40ꢀfold
drop in activity and the adamantyl analogue (4) was not active at all. Interestingly, β5i
selectivity is enhanced by the 2ꢀnaphthyl (6; threeꢀfold) and cyclohexyl (8; fourꢀfold) P1 side
chains. Notably, this is due to the reduced affinity for subunit β5c. These observations
indicate that only certain bulky P1 residues improve β5i selectivity and that the difference in
the S1 pockets between β5c and β5i are not in their linear depth but in their bottom. This is in
agreement with the Xꢀray structures of the murine cCP in complex with ONXꢀ0914 (1), which
showed that Met45 forming the bottom of the S1 pocket is displaced upon ligand docking.
From a structural point of view binding of both cyclohexyl and phenyl moieties to the S1
pocket of the yβ5 subunit is identical, yet, further analysis essentially requires crystal
structures of the human cCP and iCP (in complex with the investigated compounds).
Removing the aromatic system tends to increase the IC50 values in both the β5c and β5i active
sites, indicating that the πꢀsystem might interact with the sulfur of Met45 and that the strength
of this contact depends on its orientation. Inverting the chirality of the P3 residue can also
significantly impact β5ꢀaffinity and selectivity. Furthermore, the yCP complex structures
provide detailed explanation for the β5ꢀselectivity of the DꢀAla/3ꢀmethylꢀ1Hꢀindene
compounds.
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Based on the observation that our β5i inhibitors preferentially target β1i over β1c at higher
concentrations, we substituted the P1 leucine epoxyketone of the β1c/β1i specific inhibitor
NCꢀ001 for either phenylalanine or cyclohexylalanine epoxyketone. The resulting compounds
were indeed more specific for subunit β1i. Since the mouse cCP and iCP crystal structures
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show subtle differences between the S3 pockets of β1c and β1i, a P3 Pro analogue scan using
3 as a template was performed. This analysis revealed that the 4,4F2ꢀPro (33) induces β1i
selectivity without loss of activity. Combination of the cyclohexylalanine and the 4,4F2ꢀPro
ultimately led to compound 37, which has a >300 fold binding preference for β1i over all
other subunits in RPMIꢀ8226 cells.
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In keeping with the tradition of naming our compounds, we termed the six improved β5i
specific inhibitors LUꢀ005i (8), LUꢀ015i (9), LUꢀ025i (11), LUꢀ035i (13), LUꢀ045i (15), LUꢀ
055i (25), and the β1i specific inhibitor LUꢀ001i (37), in which LU stands for Leiden
University and ‘i’ for immunoproteasome.
The set of improved subunitꢀspecific and cellꢀpermeable β5i and β1i inhibitors represent a
useful tool to investigate the cytotoxic effect of subunitꢀspecific proteasome inhibition in MM
cells and disease models, for which controversial data exist.19b, 22 Future drug design strategies
might concentrate on the development of inhibitors selective for the cCP subunits β1c and
β5c. Two β5c selective compounds have been reported, but their selectivities are modest
(about 10ꢀ20 fold compared to β5i).13, 19b So far, also no compounds that discriminate between
the subunits β2c and β2i are available. Our compounds 1 and 8 inhibit β2i slightly more
potent than β2c and might provide a basis to develop also ligands for one of the two β2ꢀ
subunits, which certainly will help to understand their biological and functional difference, a
question that so far could not be addressed.
Experimental procedures
General procedures
1
All tested compounds are >95% pure on the basis of LCꢀMS and NMR. H and 13C NMR
spectra were recorded on a Bruker AVꢀ400 (400 MHz) or AVꢀ600 (600 MHz) spectrometer.
Chemical shifts are given in ppm (δ) relative to CD3OD or CDCl3 as internal standard. LCꢀ
MS analysis was performed on a Finnigan Surveyor HPLC system with a Gemini C18 50 ×
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4.60 mm column (detection at 200−600 nm) coupled to a Finnigan LCQ Advantage Max
mass spectrometer with ESI. The applied buffers were H2O, MeCN and 1.0% TFA in H2O
(0.1% TFA end concentration). Methods used are: 15 min (0→0.5 min: 10% MeCN;
0.5→10.5 min: 10% → 90% MeCN; 10.5→12.5 min: 90% MeCN; 12.5→15 min: 90% →
10% MeCN) or 12.5 min (0→0.5 min: 10% MeCN; 0.5→8.5 min: 10% → 90% MeCN;
8.5→10.5 min: 90% MeCN; 10.5→12.5 min: 90% → 10% MeCN). HPLC purification was
performed on a Gilson HPLC system coupled to a Phenomenex Gemini C18 5ꢁm 250×10 mm
column and a GX281 fraction collector.
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General procedure for azide couplings
Compounds 4ꢀ37 were prepared via azide coupling of properly protected tripeptide hydrazide
and properly deprotected vinyl sulfone amines and epoxyketone amines. The appropriate
hydrazide was dissolved in 1:1 DMF:DCM (v/v) and cooled to ꢀ30 °C. tBuONO (1.1 equiv)
and HCl (4M solution in 1,4ꢀdioxane, 2.8 equiv) were added and the mixture was stirred for
3h at ꢀ30 °C after which TLC analysis (10% MeOH/DCM, v/v) showed complete
consumption of the starting material. The epoxyketone or vinyl sulfone was added as a free
amine to the reaction mixture as a solution in DMF. DiPEA (5 equiv) was added to the
reaction mixture, and this mixture was allowed to warm to RT slowly overnight. The mixture
was diluted with EtOAc and extracted with H2O (3×). The organic layer was dried over
MgSO4 and purified by flash column chromatography (1ꢀ5% MeOH in DCM) and HPLCꢀ
purification (if necessary). Peptide hydrazides were prepared by hydrazinolysis of peptide
methyl esters synthesized by standard procedures of solution peptide chemistry as described
in Supporting Information.
MorphAcꢀAlaꢀTyr(OMe)ꢀChaꢀEK (8). Compound 8 was obtained by the general protocol
for azide coupling on a 50 µmol scale. Purification by column chromatography (2→4%
MeOH in DCM) provided the title compound (21.88 mg, 74.5%) as a white powder after
lyophilization. 1H NMR (400 MHz, Chloroformꢀd) δ 7.45 (d, J = 7.5 Hz, 1H), 7.10 (d, J = 8.6
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Hz, 2H), 6.85 – 6.69 (m, 3H), 6.27 (d, J = 7.7 Hz, 1H), 4.62 – 4.49 (m, 2H), 4.49 – 4.34 (m,
1H), 3.76 (s, 3H), 3.69 (t, J = 4.5 Hz, 4H), 3.25 (d, J = 5.0 Hz, 1H), 3.03 – 2.91 (m, 3H), 2.90
– 2.81 (m, 2H), 2.49 – 2.38 (m, 4H), 1.85 – 1.51 (m, 6H), 1.49 (s, 3H), 1.35 (d, J = 7.0 Hz,
3H), 1.25 – 1.06 (m, 5H), 0.88 (dd, J = 15.7, 7.9 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ
208.24, 171.99, 170.71, 170.30, 158.66, 130.50, 128.35, 114.07, 67.03, 61.72, 59.15, 55.31,
54.39, 53.86, 52.51, 49.74, 48.43, 38.63, 36.81, 34.42, 34.02, 31.98, 26.42, 26.33, 26.05,
17.84, 16.83. LCꢀMS (linear gradient 10 → 90% MeCN, 0.1% TFA, 15 min): Rt (min): 6.62
(ESIꢀMS (m/z): 587.13 (M+H+)). HRMS: calcd. for C31H46N4O7 587.34393 [M+H]+; found
587.34393.
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MorphAcꢀDꢀAlaꢀTyr(OMe)ꢀChaꢀEK (11) Compound 11 was obtained by the general
protocol for azide coupling on a 50 µmol scale. Purification by column chromatography
(1→3% MeOH in DCM) provided the title compound (23.19 mg, 79.0%) as a white powder
after lyophilisation.1H NMR (400 MHz, Chloroformꢀd) δ 7.56 (d, J = 6.9 Hz, 1H), 7.11 (d, J
= 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H), 6.58 (d, J = 8.1 Hz, 1H), 6.36 (d, J = 7.8 Hz, 1H),
4.65 – 4.48 (m, 2H), 4.34 (t, J = 7.1 Hz, 1H), 3.77 (s, 3H), 3.72 (t, J = 4.5 Hz, 4H), 3.27 (d, J
= 5.0 Hz, 1H), 3.10 – 2.91 (m, 4H), 2.86 (d, J = 5.0 Hz, 1H), 2.60 – 2.46 (m, 4H), 1.65 (td, J =
38.0, 37.2, 13.3 Hz, 6H), 1.48 (s, 3H), 1.31 (d, J = 7.0 Hz, 3H), 1.27 – 1.10 (m, 5H), 0.93 –
0.83 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 208.29, 171.77, 170.65, 158.79, 130.54, 128.18,
114.17, 67.06, 61.76, 59.15, 55.34, 54.18, 53.89, 52.46, 49.63, 48.86, 38.48, 37.10, 34.31,
34.02, 32.00, 26.44, 26.28, 26.01, 17.87, 16.81. LCꢀMS (linear gradient 10 → 90% MeCN,
0.1% TFA, 15 min): Rt (min): 6.80 (ESIꢀMS (m/z): 587.27 (M+H+)) HRMS: calcd. for
C31H46N4O7 587.34393 [M+H]+; found 587.34393.
3MeIndAcꢀDꢀAlaꢀTyr(OMe)ꢀChaꢀEK (13). Compound 13 was obtained by the general
protocol for azide coupling on a 50 µmol scale. Purification by column chromatography
(1→2% MeOH in DCM) provided the title compound (23.50 mg, 76.3%) as a white powder
1
after lyophilization. H NMR (400 MHz, Chloroformꢀd) δ 7.45 (t, J = 5.7 Hz, 2H), 7.38 –
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7.29 (m, 2H), 7.12 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 8.5 Hz, 2H), 6.49
(dd, J = 7.4, 2.7 Hz, 2H), 4.70 – 4.47 (m, 3H), 3.70 (s, 3H), 3.58 (s, 2H), 3.24 (d, J = 5.0 Hz,
1H), 3.01 (ddt, J = 22.1, 14.4, 7.6 Hz, 2H), 2.83 (d, J = 5.0 Hz, 1H), 2.51 (s, 3H), 1.78 – 1.45
(m, 5H), 1.43 (s, 3H), 1.38 (d, J = 7.0 Hz, 3H), 1.31 – 0.94 (m, 6H), 0.91 – 0.77 (m, 2H). 13C
NMR (101 MHz, CDCl3) δ 208.31, 172.53, 170.87, 166.10, 158.74, 148.39, 145.57, 142.22,
131.44, 130.54, 128.30, 127.48, 126.89, 123.91, 120.94, 114.09, 59.14, 55.25, 54.38, 52.47,
49.74, 49.10, 38.55, 38.32, 37.00, 34.34, 33.97, 31.99, 26.39, 26.24, 25.98, 18.49, 16.79,
12.50. LCꢀMS (linear gradient 10 → 90% MeCN, 0.1% TFA, 15 min): Rt (min): 10.44 (ESIꢀ
MS (m/z): 616.13 (M+H+)) HRMS: calcd. for C36H45N3O6 616.33811 [M+H]+; found
616.33813.
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MorphAcꢀDꢀAlaꢀTrpꢀChaꢀEK (15). Compound 15 was obtained by the general protocol
for azide coupling on a 50 µmol scale. Purification by column chromatography (1→3%
MeOH in DCM) provided the title compound (23.27 mg, 78.1%) as a white powder after
lyophilisation.1H NMR (400 MHz, Chloroformꢀd) δ 8.35 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H),
7.56 (d, J = 6.9 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.23 – 7.07 (m, 3H), 6.77 (d, J = 7.7 Hz,
1H), 6.28 (d, J = 7.7 Hz, 1H), 4.69 (q, J = 7.3 Hz, 1H), 4.53 – 4.43 (m, 1H), 4.36 (p, J = 7.1
Hz, 1H), 3.81 – 3.62 (m, 4H), 3.32 (dd, J = 14.6, 5.7 Hz, 1H), 3.23 – 3.07 (m, 2H), 2.95 (s,
2H), 2.82 (d, J = 5.0 Hz, 1H), 2.50 (tt, J = 11.7, 7.2 Hz, 4H), 1.73 – 1.57 (m, 4H), 1.45 (s,
5H), 1.31 (d, J = 7.0 Hz, 3H), 1.21 – 1.00 (m, 5H), 0.90 – 0.79 (m, 2H). 13C NMR (101 MHz,
CDCl3) δ 208.33, 171.87, 171.00, 136.31, 127.49, 123.61, 122.40, 119.91, 118.87, 111.42,
110.35, 67.02, 61.74, 59.09, 53.85, 53.80, 52.52, 49.74, 48.85, 38.51, 34.23, 33.94, 31.99,
27.96, 26.41, 26.23, 26.01, 18.02, 16.81. LCꢀMS (linear gradient 10 → 90% MeCN, 0.1%
TFA, 15 min): Rt (min): 6.85 (ESIꢀMS (m/z): 596.20 (M+H+)) HRMS: calcd. for C32H45N5O6
596.34426 [M+H]+; found 596.34418.
3MeIndAcꢀDꢀAlaꢀTrpꢀChaꢀEK (9). Compound 9 was obtained by the general protocol for
azide coupling on a 50 µmol scale. Purification by column chromatography (1→2% MeOH in
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DCM) provided the title compound (23.45 mg, 75%) as a white powder after lyophilization.
1H NMR (400 MHz, Chloroformꢀd) δ 8.53 – 8.48 (m, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.43 (t, J
= 7.2 Hz, 2H), 7.38 – 7.28 (m, 3H), 7.17 – 7.02 (m, 4H), 6.61 (d, J = 6.9 Hz, 1H), 6.53 (d, J =
7.7 Hz, 1H), 4.77 (q, J = 7.0 Hz, 1H), 4.55 – 4.45 (m, 2H), 3.54 (s, 2H), 3.29 (dd, J = 14.7,
6.5 Hz, 1H), 3.24 – 3.11 (m, 2H), 2.80 (d, J = 5.0 Hz, 1H), 2.48 (s, 3H), 1.72 – 1.38 (m, 9H),
1.33 (d, J = 7.0 Hz, 3H), 1.20 – 0.97 (m, 5H), 0.80 (d, J = 11.4 Hz, 2H). 13C NMR (101 MHz,
CDCl3) δ 208.40, 172.66, 171.31, 166.10, 148.10, 145.58, 142.28, 136.31, 131.61, 127.48,
127.39, 126.84, 123.91, 123.74, 122.22, 120.90, 119.75, 118.79, 111.38, 110.35, 59.15, 53.85,
52.53, 49.92, 49.27, 38.33, 34.31, 33.90, 31.99, 27.80, 26.38, 26.19, 25.98, 18.46, 16.83,
12.48. LCꢀMS (linear gradient 10 → 90% MeCN, 0.1% TFA, 15 min): Rt (min): 10.36 (ESIꢀ
MS (m/z): 625.13 (M+H+)). HRMS: calcd. for C37H44N4O5 625.33845 [M+H]+; found
625.33844.
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3MeIndAcꢀAlaꢀTyr(OMe)ꢀPheꢀVS (25)
Compound 25 was obtained by the general protocol for azide coupling on a 50 µmol scale.
Purification by column chromatography (1→3% MeOH in DCM) provided the title
compound (15.11 mg, 47.3%) as a white powder after lyophilization. Isolated with 16% cis
isomer. Peaks reported correspond to trans isomer. 1H NMR (400 MHz, Chloroformꢀd) δ 8.24
(s, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.45 (dd, J = 6.3, 2.3 Hz, 2H), 7.38 – 7.30 (m, 3H), 7.24 –
7.09 (m, 5H), 7.06 (dd, J = 7.3, 1.9 Hz, 2H), 6.95 (d, J = 8.6 Hz, 1H), 6.76 (d, J = 2.2 Hz,
1H), 6.68 (dd, J = 15.0, 4.4 Hz, 2H), 6.32 (d, J = 5.7 Hz, 1H), 6.15 (dd, J = 15.1, 1.7 Hz, 1H),
4.92 (dt, J = 12.1, 6.0 Hz, 1H), 4.81 – 4.68 (m, 1H), 4.23 (q, J = 6.7 Hz, 1H), 3.63 – 3.43 (m,
2H), 3.35 (dd, J = 14.6, 5.1 Hz, 1H), 3.22 – 3.09 (m, 1H), 2.76 (d, J = 7.6 Hz, 2H), 2.70 (s,
3H), 2.54 – 2.44 (m, 3H), 1.42 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 173.04,
171.06, 166.84, 148.99, 146.31, 145.34, 142.25, 136.68, 136.17, 130.83, 129.98, 129.79,
129.36, 128.75, 128.62, 127.76, 127.54, 126.99, 126.94, 124.03, 123.48, 122.50, 121.09,
119.97, 118.47, 111.70, 109.63, 54.34, 50.90, 50.30, 43.75, 42.65, 39.50, 38.36, 26.96, 17.43,
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12.71. LCꢀMS (linear gradient 10 → 90% MeCN, 0.1% TFA, 12.5 min): Rt (min): 7.78 (ESIꢀ
MS (m/z): 639.13 (M+H+)). HRMS: calcd. for C36H38N4O5S 639.26357 [M+H]+; found
639.26355.
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N3GlyꢀAlaꢀ4,4ꢀF2ProꢀNleꢀChaEK (37). Compound 37 was obtained by the general
protocol for azide coupling on a 50 µmol scale. Purification by column chromatography
(0→2% MeOH in DCM) provided the title compound (14.01 mg, 46%) as a white powder
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after lyophilization. Complex NMR due to a 2.5:1 ratio of rotamers. H NMR (400 MHz,
Chloroformꢀd, peaks of major rotamer) δ 7.08 (d, J = 7.3 Hz, 1H), 6.95 (d, J = 7.7 Hz, 1H),
6.13 (d, J = 7.9 Hz, 1H), 4.78 (dd, J = 9.3, 5.5 Hz, 1H), 4.67 (t, J = 7.1 Hz, 1H), 4.63 – 4.53
(m, 1H), 4.32 (td, J = 7.7, 5.6 Hz, 1H), 4.26 – 4.07 (m, 1H), 4.00 (d, J = 4.2 Hz, 2H), 3.91 –
3.79 (m, 1H), 3.28 (d, J = 5.0 Hz, 1H), 3.04 – 2.91 (m, 1H), 2.89 (d, J = 5.0 Hz, 1H), 2.65 –
2.48 (m, 1H), 1.87 – 1.54 (m, 10H), 1.51 (s, 3H), 1.39 (d, J = 6.9 Hz, 3H), 1.35 – 1.07 (m,
9H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (151 MHz, CDCl3, all peaks reported) δ 209.85,
208.58, 172.22, 171.73, 171.56, 171.28, 168.78, 168.67, 168.58, 166.34, 126.18 (t, J = 241.6
Hz), 59.24, 59.07, 59.03, 58.91, 58.06, 56.08, 53.73, 53.57 (t, J = 33 Hz), 52.59, 52.54,
52.50, 52.17, 49.95, 48.70, 47.79, 46.82, 38.58, 35.62 (t, J = 25.7 Hz), 34.48, 34.23, 34.09,
32.21, 32.02, 31.97, 31.32, 28.05, 27.37, 26.45, 26.42, 26.31, 26.03, 22.47, 22.23, 18.08,
16.89, 16.69, 16.65, 14.03, 13.97. . LCꢀMS (linear gradient 10 → 90% MeCN, 0.1% TFA,
12.5 min): Rt (min): 7.91 (ESIꢀMS (m/z): 612.27 (M+H+)). HRMS: calcd. for C28H43F2N7O6
612.33157 [M+H]+; found 612.33154.
The synthesis of compound 1ꢀ3 has been described elsewhere.3a, 18a Synthetic procedures
and analytical data for remaining compounds are provided in Supporting Information.
Biological analysis. Materials. Activity based probes were synthesized as described
previously.21a, 28
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Competition Assays in Cell Lysate. Lysates of Raji cells were prepared by sonication in 3
volumes of lysis buffer containing 50 mM Tris pH 7.5, 1 mM DTT, 5 mM MgCl2, 250 mM
sucrose, 2 mM ATP, and 0.025% digitonin. Protein concentration was determined by the
Bradford assay. Cell lysates (diluted to 10ꢀ15 ꢁg total protein in buffer containing 50 mM Tris
pH 7.5, 2 mM DTT, 5 mM MgCl2, 10% glycerol, 2 mM ATP) were exposed to the inhibitors
for 1 h at 37 °C prior to incubation with AzidoBODIPYꢀMeTyrꢀPheꢀLeuꢀVS (BODIPYꢀ
NC005; 0.1ꢀ0.5 ꢁM; to probe β5), BODIPYꢀepoxomicin (0.5 ꢁM; to probe all subunits, used
for β2ꢀprofiling) or BODIPYꢀFLꢀAlaꢀProꢀNleꢀLeuꢀEK (BODIPYꢀNC001; 1 ꢁM; to probe β1
activity. This probe does bind slightly to β5i, so lysates were treated for 1 h with NC005VS8a
(5 ꢁM), prior to probe incubation for an additional 1 h at 37 °C, followed by 3 min boiling
with a reducing gelꢀloading buffer and fractionation on 12.5% SDSꢀPAGE. Inꢀgel detection of
residual proteasome activity was performed in the wet gel slabs directly on a ChemiDoc™
MP System using Cy3 settings to detect BODIPYꢀNC005 and BODIPYꢀepoxomicin and Cy2
settings to detect BODIPYꢀNC001. Intensities of bands were measured by fluorescent
densitometry and normalized to the intensity of bands in mockꢀtreated extracts. Average
values of at least two or three independent experiments were plotted against inhibitor
concentrations. IC50 (inhibitor concentrations giving 50% inhibition) values were calculated
using GraphPad Prism software.
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Competition Assays in living RPMIꢀ8226 cells. RPMIꢀ8226 were cultured in RPMIꢀ1640
media supplemented with 10% fetal calfs serum, GlutaMAX™, penicillin, streptomycin in a
5% CO2 humidified incubator. 5ꢀ8 × 105 cells/mL were exposed to inhibitors for 1 h at 37 °C.
Cells were harvested and washed with twice with PBS. Cell pellets were treated with lysis
buffer (50 µL: 50 mM Tris pH 7.5, 2 mM DTT, 5 mM MgCl2, 10% glycerol, 2 mM ATP,
0.05% digitonin) on ice for 1.5 h, followed by centrifugation at 14000 rpm for 15 min.
Proteasome inhibition in the obtained cell lysates was determined using the method described
above. Intensities of bands were measured by fluorescent densitometry and divided by the
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intensity of bands in mockꢀtreated extracts. Gels were stained by Coomassie Brilliant Blue,
which was used to correct for gel loading differences. Average values of two or three
independent experiments were plotted against inhibitor concentrations. IC50 (inhibitor
concentrations giving 50% inhibition) values were calculated using GraphPad Prism software.
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Crystallographic analysis.
yCP crystals were grown by hanging drop vapour diffusion as previously described.29 Crystal
drops were incubated for 12 h with 5 µl of cryo protectant and 0.5 µl of inhibitor
(concentration: 50 mM in DMSO). Diffraction datasets were collected using synchrotron
radiation of λ=1.0 Å at the beamline X06SA, Swiss Light Source (SLS), Villigen,
Switzerland. Structure determination was performed as previously reported.29 For model
building the programs SYBYL, MAIN30 and COOT31 were used. Refinement with
REFMAC532 yielded excellent R factors as well as r.m.s.d. bond and angle values.
Coordinates were confirmed to fulfill the Ramachandran plot (Table S4). Figures were
prepared with PyMOL.
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ASSOCIATED CONTENT
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Assays of compounds 1ꢀ26 in Raji lysates, pIC50 values and standard errors of all compounds
in cell lysates and intact cells, structures of activity based probes, supplementary structural
data, Xꢀray data table, complete synthetic details and characterization of all compounds and
synthetic intermediates, NMR spectra and LCꢀMS traces of compounds 8, 9, 11, 13, 15, 25,
37. This material is available free of charge via the Internet at http://pubs.acs.org.
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ACCESSION CODES
4QLT (yCPꢀ2), 4QLQ (yCPꢀ8), 4QLU (yCPꢀ9), 4QLS (yCPꢀ11), 4QLV (yCPꢀ17)
AUTHOR INFORMATION
Corresponding Author
* Phone: +31ꢀ 71ꢀ5274342; fax: +31ꢀ71ꢀ527ꢀ4307; eꢀmail:
h.s.overkleeft@chem.leidenuniv.nl.
Author Contributions
&Gerjan de Bruin and Eva M. Huber contributed equally to this work
Funding Sources
This work was supported by the Netherlands Genomics Centre (Zenith Grant, to HSO) by the
Deutsche Forschungsgemeinschaft (DFG, grant GR1861/10ꢀ1, to MG).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
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We thank the staff of the beamline X06SA at the Paul Scherrer Institute, Swiss Light Source,
Villigen (Switzerland) for assistance during data collection. We are grateful to R. Feicht for
largeꢀscale purification of yeast 20S proteasomes.
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ABBREVIATIONS
ABPP, activity based protein profiling; biphe, biphenylalanine; BODIPY, boronꢀ
dipyrromethene, (4,4ꢀdifluoroꢀ5,7ꢀdimethylꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene); cCP, constitutive
proteasome, DiPEA, N,Nꢀdiisopropylethylamine; EA, ethyl acetate; ek, epoxyketone; ER,
endoplasmatic reticulum; Cha, cyclohexylalanine; HBTU, 2ꢀ(1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀ
tetramethyluronium hexafluorophosphate; HCTU, 2ꢀ(6ꢀchloroꢀ1Hꢀbenzotriazolꢀ1ꢀyl)ꢀ1,1,3,3ꢀ
tetramethyluronium hexafluorophosphate; iCP, immunoproteasome; MM, multiple myeloma;
vs, methyl vinyl sulfone; pent, pentane; yCP, yeast proteasome.
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