α,β-unsaturated N-Acylpyrrole peptidyl derivatives: New proteasome inhibitors

Article abstract of DOI:10.1021/jm100122e

Because of the encouraging results obtained using vinyl ester derivatives, we synthesized and tested a novel series of peptide-based proteasome inhibitors bearing a new pharmacophore unit at the C-terminal. N-Acylpyrrole moiety is a potential substrate for Michael addition by catalytic threonine. Several analogues have demonstrated a selective inhibition of the multicatalytic complex β1 subunits, the capacity to permeate cellular membrane, and good pharmacokinetics properties.

Full text of DOI:10.1021/jm100122e

J. Med. Chem. 2010, 53, 6511–6515 6511
DOI: 10.1021/jm100122e
r,β-Unsaturated N-Acylpyrrole Peptidyl Derivatives: New Proteasome Inhibitors
Anna Baldisserotto,† Valeria Ferretti,‡ Federica Destro,§ Christian Franceschini,† Mauro Marastoni,*,†
Riccardo Gavioli,§ and Roberto Tomatis†
†Department of Pharmaceutical Sciences and Biotechnology Center, ‡Department of Chemistry and Center for Structural Diffractometry, and
§Department of Biochemistry and Molecular Biology, University of Ferrara, I-44100 Ferrara, Italy
Received January 29, 2010
Because of the encouraging results obtained using vinyl ester derivatives, we synthesized and tested a
novel series of peptide-based proteasome inhibitors bearing a new pharmacophore unit at the C-terminal.
N-Acylpyrrole moiety is a potential substrate for Michael addition by catalytic threonine. Several
analogues have demonstrated a selective inhibition of the multicatalytic complex β1 subunits, the
capacity to permeate cellular membrane, and good pharmacokinetics properties.
Introduction
synthetic inhibitors possess a homogeneous structural profile;
they are generally peptide-based compounds with a C-terminal
pharmacophore function required for primary interaction with
catalytic threonine on the enzyme. The peptide component
seems to be important for determining specificity for the three
catalytic sites by secondary interaction with the enzymatic
pockets. Essentially, most of these inhibitors act on the chymo-
trypsin-like activity of the proteasome, although they do under-
go partial inhibition. Recently, research has begun to address
the development of molecules with specific activity for the
proteasome subunits, and already considerable progress has
been made, particularly by Crews et al. who, by varying the
sequence at P2-P4 in epoxomicin, were able to generate
specific inhibitors of the chymotrypsin-like and postacidic acti-
vities, although the physiological consequences of β1-subunit
inhibition still remain to be clarified.14,15 Likewise, new alde-
hyde compounds have shown an interesting selectivity for the
trypsin-like activity, as have vinyl sulfone analogues, although
the foremost proteasome inhibitor to date (bortezomib, Vel-
cade), approved by the U.S. Food and Drug Administration as
a prescription drug for the treatment of multiple myeloma, is
selective for the chymotrypsin-like activity with a partial action
on the β1 site.16,17
We recently reported the design and synthesis of small pep-
tide molecules with selective inhibitory activity toward the three
catalytic sites and good pharmacokinetic properties. In our
early studies, we have identified new oligopeptide inhibitors
with different C-terminal pharmacophore units: amide analo-
gues, compounds with arecholine derivatives, and in particular,
a class of compounds bearing a C-terminal vinyl ester selec-
tive and specific for the β2 activitity of the proteasome. The
pharmacophoric vinyl ester group is able to interact with the
catalytic Thr in the same way as the well-known vinyl sulfone
analogues.18 The best of these derivatives inhibit the β2 subunit
in the nanomolar range, are nontoxic, do not affect cell prolife-
ration, and are capable of modulating the generation of anti-
genic peptides linked by MHC class I molecules. N-Terminal
elongation has also been performed on vinyl ester prototypes,
and in another series we have cyclized the linear peptide por-
tion: both the N-terminal elongated peptides and the cyclic
analogues have been shown to selectively inhibit the β1 subunit.
The 26S proteasome, a multicatalytic and multisubunit
threonine protease complex (2.4 MDa), is implicated in many
biological processes, including the generation of antigenic
peptides presented by major histocompatibility complex class
I (MHC I) molecules and degradation of most cytosolic
proteins in mammalian cells.1-6 The 26S proteasome consists
of a central 20S core containing three different active sites (β1,
β2, and β5) and two 19S regulatory complexes. On the basis of
the preferential cleavage of short fluorogenic peptidic sub-
strates, the various activities of the proteasome have been
assigned to individual subunits and classified as “chymotryp-
tic-like” (β5), “tryptic-like’ (β2)”, and “peptidyl-glutamyl pep-
tide hydrolyzing”, PGPHa (β1).7,8
As previously mentioned, proteasome plays an essential role
in protein turnover in living cells, and deregulation of the
ubiquitin-proteasome pathway in humans causes several dis-
eases, such as cancer and neurodegenerative, autoimmune, and
metabolic disorders. Furthermore, proteasome inhibition has a
negative influence on the stability of many proteins, especially
those involved in cell cycle regulation. Indeed, most cells become
sensitive to apoptosis after treatment with proteasome inhibi-
tors.9,10 Interestingly, tumor cells are usually more sensitive to
proteasome inhibition than their normal counterparts. Indeed,
healthy cells display cell-cycle arrest when treated with protea-
some inhibitors but, unlike tumor cells, are not as susceptible to
apoptosis.11,12 Thus, selective inhibitors of catalytic proteasome
subunits are appealing targets for drug development.13
Many inhibitors of the ubiquitin-proteasome pathway that
are able to interact with the 20S catalytic core of the multi-
catalytic complex and that can readily enter cells and selectively
inhibit this degradation pathway are currently available. These
*To whom correspondence should be addressed. Phone: þ39-532-
291281. Fax: þ39-532-291296. E-mail: mru@unife.it.
a Abbreviations: Boc, tert-butoxycarbonyl; ChT-L, chymotrypsin-like;
Fmoc, fluorenylmethyloxycarbonyl; HATU, O-(7-azabenzotriazolyl)tetra-
methyluronium hexafluorophosphate; HOBt, N-hydroxybenzotriazole;
NMM, N-methylmorpholine; PGPH, peptidylglutamyl peptide hydrolyz-
ing; TFA, trifluoroacetic acid; T-L, trypsin-like; WSC, water-soluble car-
bodiimide (1-ethyl-3-(30-dimethylaminopropyl)carbodiimide); Z, benzyl-
oxycarbonyl-N-hydroxysuccinimide; VAP, vinylacylpyrrole.
r
2010 American Chemical Society
Published on Web 08/05/2010
pubs.acs.org/jmc

6512 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Baldisserotto et al.
Figure 1. Structures of the novel pseudotripeptide inhibitors.
All of the vinyl ester derivatives have also demonstrated good
resistance to proteolysis in plasma and an ability to permeate
cell membranes.19-25
Fmoc solid phase strategy, and the final reaction with the
C-terminal pharmacophore yielded the desired 1-8. All pro-
ducts were purified by preparative RP-HPLC, and structural
verification was achieved by mass spectrometry and NMR
spectroscopy (Table 1S in Supporting Information).
Herein we report the synthesis and biological activity of a
novel class of peptide-based derivatives containing central
sequences derived from those yielding the best results in prior
experiments (Leu-Leu-Leu and Val-Ser-Leu) and a new phar-
macophore unit formed from an R,β-unsaturated N-acylpyr-
role group on the terminal leucine. The new pharmacophore
can function as a substrate of the γ-hydroxythreonine side
chain in Michael addition (Figure 1) in a similar way to the
vinyl ester function but more effectively. On the basis of pre-
viously data, a benzyloxycarbonyl group (Z) (1,5), a 3-hydroxy-
2-methylbenzoic group (2,6), or a Z N-protected 8-amino-
octanoic acid (4,8) was inserted at the N-terminal.
Biological evaluation of the new pseudotripeptides was
carried out to assess their inhibition of the trypsin-like, chy-
motrypsin-like, and postacidic activities of the proteasome.
Their inhibitory capacity was assayed on proteasomes purified
from lymphoblastoid cell lines (LCL), using fluorogenic sub-
strates specific for the three main proteolytic activities of the
enzyme complex.28,29 All compounds were assayed at different
concentrations (from 0.001 to 10 μM), and the IC50 values were
obtained after 30 min of incubation (Table 1) and subsequently
compared with the well-known proteasome inhibitors epoxo-
micin and MG132 (the values are reported as the average of
three independent determinations). The results obtained show
that these new N-acylpyrrole R,β-unsaturated peptide based
derivatives possess a unique biological profile. In general, none
of the compounds inhibited the chymotrypsin-like or the tryp-
sin-like activities of the proteasomes isolated from LCL. Like-
wise, inhibition of the β2 subunit was relatively unpronounced,
with IC50 of about 10 μM. Only 1, 2, and 8 showed a mild inhi-
bitory capacity of the chymotryptic activity with IC50 slightly
below 10 μM. The different biological results of the new
Results and Discussion
R,β-Unsaturated N-acylpyrrole peptidyl derivatives were
synthesized following the strategy reported in Scheme 1 by a
mixed solution-solid phase synthesis approach. NR-Pro-
tected leucine-vinyl-acylpyrrole (Boc-Leu-VAP) was pre-
pared from the corresponding aldehyde26 by reaction with
pyrrolylmethylenetriphenylphosphorane without racemiza-
tion. The ylide was synthesized as described in the literature.27
The N-terminal functionalized dipeptides were assembled by

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6513
compounds regarding ChT-L activity seem to be independent of
the N-terminal substituent, although more hydrophobic se-
quences appear to be preferred. Interestingly, the introduction
of the new C-terminal pharmacophore unit produced an appre-
ciable decrease in β2 and β5 activity toward the amino acid
sequences that showed a favorable profile for the same catalytic
tasks in previous series. However, selective inhibition of the
postacidic activity of proteasomes was generally found to be
higher than that reported for the well-known proteasome
inhibitors epoxomicin and MG132. In particular, derivatives
2, 4, 7, and 8 displayed postacidic inhibition IC50 in the frac-
tional micromolar range. Compound 8 represents the most
potent inhibitor of the series. A structure-activity analysis
emphasized that the N-acylpyrrole group is a favorable sub-
strate of the γ-hydroxythreonine side chain in the β1 subunit.
The biological response to the presence at the N-terminal of the
benzyloxycarbonyl group (Z) in 1 and 5 was found to be the
lowest in terms of potency. Hence, a good balance between the
hydrophilic/lypophilic characteristics of the amino acid chain
and the N-terminal substituent seems to promote the interaction
with the β1 subunit, thereby confirming that a bulky substituent
at the N-terminal favors β1-specific inhibition. After 1 and 2 h of
incubation, the inhibitory profile of the new compounds was
preserved, although accompanied by progressive reduction of
the IC50, a profile that seems to indicate a reversible inhibition.
Selected inhibitors 2, 4, 7, and 8 were assayed to evaluate
their ability to permeate the cell membrane of LCLs. After cell
treatment, proteasomes were purified and assayed for their
proteolytic activity using specific substrate for T-L, ChT-L,
and postacidic (PGPH) activities. The results obtained were
comparable to those observed in the in vitro assay (Table 1).
The susceptibility of these selected vinylacylpyrrole deriva-
tives to enzymatic hydrolysis was determined by incubation at
37 °C in culture medium (RPMI) in the presence of 10% fetal
calf serum and in human plasma.30 The pseudopeptides show
great stability in the cell culture medium and strong enzymatic
resistance to human plasma proteases.
Scheme 1. Synthesis of 1-8a
The degradation half-lives of the N-terminal-extended
analogues shown in Table 1 were determined as described in
the experimental protocols.
Since proteasomes play a key role in cell viability and
proliferation, we evaluated the proapoptotic and antiproli-
ferative activities of two selected compounds on a lympho-
blastoid cell line and on Burkitt’s lymphoma cells. The two
cell lines were treated with different concentrations (from 0.1
to 10 μM) of 2 and 8 for 3 days, and the cellular effects of the
compounds were evaluated by Trypan blue exclusion and cell
counts. Both pseudotripeptides failed to induce cell death and
to inhibit cell proliferation (data not shown).
The eight molecules are superimposed (Figure 1S in Support-
ing Information), using the “flexible alignment” procedure
implemented in the MOE system of programs [MOE 2000.10
version, Chemical Computing Group Inc., 2008.2009.10]. The
van der Waals surface of the least (6) and the most active (8)
molecule is represented by pink and green dots, respectively. One
of the critical points determining the ligand-proteasome inter-
action appears to be the molecular size. Actually, the residues
delimiting the β1-binding pocket delineate a quite elongated
˚
active site, spanning a distance of about 18 A, where the Thr1
residue is located in the middle.31 To obtain a deeper insight into
the structural aspects of the inhibitor-enzyme interaction
mode, 8 was chosen to perform a docking simulation using a
proteasome X-ray structure retrieved from the literature.32 In
Figure 2, a schematic diagram of the inhibitor-protein interac-
tions is shown; because of its great conformational freedom, the
molecule can easily accommodate itself in the binding site
a Xaa = Leu, Ser. Xbb = Leu, Val. Y = Z, HMB, Z-NH-(CH2)5-
CO-, Z-NH-(CH2)7-CO.
Table 1. Inhibition of Proteasome Subunits and Enzymatic Stability of R,β-Unsatured N-Acylpyrrole Derivatives
isolated enzyme LCL, IC50 (μM)a in vivo inhibition LCL, IC50 (μM)a
half-life (min)
plasma
compd
epoxomycin
MG132
T-L
ChT-L
PGHP
T-L
ChT-L
PGHP
0.284
1.077
>10
>10
>10
>10
>10
>10
>10
>10
0.005
0.002
7.890
3.630
>10
>10
>10
>10
>10
5.080
4.560
>10
1
2
3
4
5
6
7
8
Z-Leu-Leu-Leu-VAP
0.091
0.065
0.155
0.062
0.122
0.178
0.097
0.036
HMB-Leu-Leu-Leu-VAP
Z-NH-(CH2)5-Leu-Leu-Leu-VAP
Z-NH-(CH2)7-Leu-Leu-Leu-VAP
Z-Val-Ser-Leu-VAP
>10
>10
5.600
>10
0.135
0.116
>360
>360
HMB-Val-Ser-Leu-VAP
Z-NH-(CH2)5-Val-Ser-Leu-VAP
Z-NH-(CH2)7-Val-Ser-Leu-VAP
>10
>10
>10
>10
0.147
0.058
>360
>360
a The values reported are the average of three independent determinations.

6514 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Baldisserotto et al.
Figure 2. Schematic view of the ligand 8-proteasome interactions. Hydrogen bonds are drawn as dotted lines. Polar and apolar residues of
the binding site are represented as pink and green circles, respectively.
matching also the pocket dimensions. In contrast, 6, whoseequi-
librium geometry is shown in Figure 2S in Supporting Informa-
tion, is quite rigid, suggesting that the alchilic chain, present in
the most active molecules, can play an important role in enabling
the inhibitors to adapt to the binding site. A second docking
simulation has been performed in order to better understand the
selectivity of molecule 8 toward the β1 catalytic site. The struc-
ture used is the 2-spiro-lactacystin/proteasome complex,33 in
view of the fact that the inhibition properties of the spiro-lactone
and of molecule 8 toward the β5 subunit are strictly comparable.
The results are graphically displayed in Figure 3S in Supporting
Information where the docked molecule is shown superimposed
to the crystallographic ligand and surrounded by the interacting
residues. It is interesting to note that the middle part of molecule
8 can mimic the disposition of N/O atoms of the smaller
lactacystin molecule, a fact that could reasonably explain the
similar biological behavior. In contrast, the terminal N-acylpyr-
role moiety, which in the previous simulation appears to be
involved in hydrophobic contacts with the surrounding residues,
here is no longer implied in any interaction, actually pointing
toward the inner part of the proteasome channel. These findings
appear to support the idea that the C-teminal fragment plays an
important role in the definition of the β1-β5 selectivity.
Fluka (Switzerland). Crude pseudopeptides were purified by pre-
parative reversed-phase HPLC using a Water Delta Prep 4000
system with a Waters PrepLC 40 mm assembly column C18 (30 cm
ꢀ 4 cm, 300 A, 15 μm spherical particle size column). The column
˚
was perfused at a flow rate of 50 mL/min with a mobile phase
containing solvent A (10% v/v, acetonitrile in 0.1% TFA), and a
linear gradient from 0 to 50% of solvent B (60% v/v, acetonitrile in
0.1% TFA) in 25 min was adopted for the elution of compounds.
HPLC analysis was performed by a Beckman System Gold with a
Hypersil BDS C18 column (5 μm; 4.6 mm ꢀ 250 mm). Analytical
determination and capacity factor (K0) of the peptides were deter-
mined using HPLC conditions in the above solvent system (solvents
A and B) programmed at flow rates of 1 mL/min using the following
linear gradients: (a) from 0% to 100% B in 25 min and (b) from
10% to 70% B in 25 min. All pseudopeptides showed less than 1%
impurities when monitored at 220 and 254 nm. Molecular weights
of compounds were determined by a ESI Micromass, ZMD2000
mass spectrometer. 1H NMR spectroscopy was obtained on a
Bruker AC 200 spectrometer. The purity of tested compounds
was determined by combustion elemental analyses conducted by
the Microanalytical Laboratory of the Chemistry Department,
University of Ferrara, with a Yanagimoto MT-5 CHN recorder
elemental analyzer. All tested compounds possess a purity of at least
95% of the theoretical values.
Proteasome Subunit Inhibition Assays. Partially purified pro-
teasomes were obtained from lymphoblastoid cell lines, un-
treated or treated for 12 h at 37 °C with the inhibitors, as
previously described.34
Conclusions
Suc-LLVY-AMC, Boc-LRR-AMC, and Z-LLE-AMC (Sigma)
were used to measure chymotrypsin-like, trypsin-like, and postaci-
dic proteasome activities, respectively. Substrates were incubated at
37 °C with proteasomes, untreated or pretreated with 0.001-10 μM
test compounds, in activity buffer for 30 min. Fluorescence was
determined by a fluorimeter (Spectrafluor Plus, Tecan, Salzburg,
Austria) using an excitation of 360 nm and emission of 465 nm.
Activity was evaluated in fluorescence units, and the inhibitory
activity of the compounds is expressed as IC50. The data were
plotted as percentage control (the ratio of percentage conversion in
the presence or absence of inhibitor) vs inhibitor concentration and
fitted with the equation Y = 100/[1 þ (X/IC 50)A], where IC50 is the
inhibitor concentration at 50% inhibition and A is the slope of the
inhibition curve.
In this work we have designed, synthesized, and defined the
biological profile of a new series of R,β-unsaturated N-acylpyr-
rolic, peptide-based proteasome inhibitors. The best derivatives
of this series interact selectively for the β1 catalytic site of the
proteasome and display good pharmacokinetic properties. Thus,
this new class of inhibitors may represent a useful tool for eluci-
dating the mechanisms underlying antigen production by the
ubiquitin-proteasome system and may have future applications
in new therapeutic protocols aimed at increasing the generation
and presentation of poorly expressed CTL epitopes.
Experimental Section
General. Amino acids, amino acid derivatives, resins, and
chemicals were purchased from Bachem, Novabiochem, or
Growth Inhibition Assays. The cells were seeded in duplicate in
a 24-well plate at a density of 50 ꢀ 103 cells/mL in medium

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6515
containing 10% FCS. A lymphoblastoid cell line (LCL) and
Burkitt’s lymphoma JiJoye cells were cultured in medium alone
or in medium containing 5 or 14 at different concentrations
(from 0.1 to 10 μM), and cell viability and proliferation were
evaluated by Trypan blue exclusion and cell counting for 3 days.
Enzymatic Stability Assays. The stability of selected new vinyl
ester inhibitors under proteases degradation was studied in
human plasma. Test compounds were incubated with plasma
(0.6 mL) in a total volume of 1.5 mL of 10 mM Tris-HCl buffer,
pH 7.5. Incubation was performed at 37 °C for 360 min. The
incubation was terminated by addition of ethanol (0.2 mL), the
mixture was poured at 21 °C, and after centrifugation (5000 rpm
for 10 min) aliquots (20 μL) of the clear supernatant were
injected into an RP-HPLC column. HPLC was performed as
described in analytical determinations. The degradation half-
life (T1/2) was obtained by a least-squares linear regression
analysis of a plot of the logarithmic inhibitor concentration
versus time, using a minimum of five points.
(14) Elofsson, M.; Splittgerber, U.; Myung, J.; Mohan, R.; Crews, C. M.
Towards subunit-specific proteasome inhibitors: synthesis and evalua-
tion of peptide R0,β0-epoxyketones. Chem. Biol. 1999, 6, 811–822.
(15) Myung, J.; Kim, K. B.; Dantuma, N. P.; Crews, C. M. Lack of
proteasome active site allostery as revealed by subunit-specific
inhibitors. Mol. Cell 2001, 7, 411–420.
(16) Cusack, J. C., Jr.; Liu, R.; Houston, M.; Abendroth, K.; Elliott, P. J.;
Adams, J.; Baldwin, A. S., Jr. Enhanced chemosensitivity to CPT-11
with proteasome inhibitor PS-341: implications for systemic nuclear
factor-kappaB inhibition. Cancer Res. 2001, 61, 3535–3540.
(17) Orlowski, R. Z.; Stinchcombe, T. E.; Mitchell, B. S.; Shea, T. C.;
Baldwin, A. S.; Stahl, S.; Adams, J.; Esseltine, D.-L.; Elliott, P. J.;
Pien, C. S.; Guerciolini, R.; Anderson, J. K.; Depcik-Smith, N. D.;
Bhagat, R.; Lehman, M. J.; Novick, S. C.; O’Connor, O. A.;
Soignet, S. L. Phase I trial of the proteasome inhibitor PS-341 in
patients with refractory hematologic malignancies. J. Clin. Oncol.
2002, 20, 4420–4427.
(18) Bogyo, M.; McMaster, J. S.; Gaczynska, M.; Tortorella, D.;
Goldberg, A. L.; Ploegh, H. Covalent modification of the active
site threonine of proteasomal beta subunits and the Escherichia coli
homolog HslV by a new class of inhibitors. Proc. Natl. Acad. Sci. U.
S.A. 1997, 94, 6629–6634.
(19) Marastoni, M.; Baldisserotto, A.; Cellini, S.; Gavioli, R.; Tomatis, R.
Peptidyl vinyl ester derivatives: new class of selective inhibitors of
proteasome trypsin-like activity. J. Med. Chem. 2005, 48, 5038–5042.
(20) Marastoni, M.; Baldisserotto, A.; Trapella, C.; Gavioli, R.; Tomatis,
R. P3 and P4 position analysis of vinyl ester pseudopeptide proteasome
inhibitors. Bioorg. Med. Chem. Lett. 2006, 16, 3125–3130.
(21) Marastoni, M.; Baldisserotto, A.; Trapella, C.; Gavioli, R.; To-
matis, R. Synthesis and biological evaluation of new vinyl ester
pseudotripeptide proteasome inhibitors. Eur. J. Med. Chem. 2006,
41, 978–984.
(22) Baldisserotto, A.; Marastoni, M.; Trapella, C.; Gavioli, R.; Ferretti,
V.;Pretto,L.;Tomatis, R. Glutaminevinyl ester proteasome inhibitors
selective for trypsin-like (beta2) subunit. Eur. J. Med. Chem. 2007, 42,
586–592.
(23) Baldisserotto, A.; Marastoni, M.; Lazzari, I.; Trapella, C.; Gavioli,
R.; Tomatis, R. C-Terminal constrained phenylalanine as a phar-
macophoric unit in peptide-based proteasome inhibitors. Eur. J.
Med. Chem. 2208, 43, 1403–1411.
(24) Baldisserotto, A.; Marastoni, M.; Fiorini, S.; Pretto, L.; Ferretti,
V.; Gavioli, R.; Tomatis, R. Vinyl ester-based cyclic peptide
proteasome inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 1849–
1854.
Acknowledgment. Financial support of this work was pro-
ꢀ
vided by the University of Ferrara, the Ministero dell’Universita
e della Ricerca Scientifica e Tecnologica (MURST), the Asso-
ciazione Italiana per la Ricerca sul Cancro (AIRC), and the
ꢀ
Istituto Superiore di Sanita (Progetto AIDS). Thanks are also
due to Anna Forster for linguistic assistance.
Supporting Information Available: General synthetic proce-
dures, 1H NMR of inhibitors 2 and 8, Table 1S listing analytical
data of 1-8, experimental procedure for molecular modeling
studies, and Figures 1S, 2S, and 3S from molecular modeling
studies. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) Coux, O.; Tanaka, K.; Goldberg, A. L. Structure and functions of
the 20S and 26S proteasomes. Annu. Rev. Biochem. 1996, 65, 801–
847.
(25) Baldisserotto, A.; Marastoni, M.; Gavioli, R.; Tomatis, R. New
cyclic peptide proteasome inhibitors. Bioorg. Med. Chem. Lett.
2009, 19, 1966–1969.
(26) Fehrentz, J. A.; Pothion, C.; Califano, J. C.; Loffet, A.; Martinez, J.
Synthesis of chiral N-protected R-amino aldehydes by reduction of
N-protected N-carboxyanhydrides (UNCAs). Tetrahedron Lett.
1994, 35, 9031–9034.
€
€
(2) Baumeister, W.; Walz, J.; Zuhl, F.; Seemuller, E. The proteasome
paradigm of a self-compartmentalizing protease. Cell 1998, 92,
367–380.
(3) Voges, D.; Zwickl, P.; Baumeister, W. The 20S proteasome: a
molecular machine designed for controlled proteolysis. Annu. Rev.
Biochem. 1999, 68, 1015–1068.
€
(4) Lowe, J.; Stock, D.; Zwicki, P.; Baumeister, W.; Huber, H. Crystal
(27) Matsunaga, S.; Qin, H.; Sugita, M.; Okada, S.; Kinoshita, T.;
Yamagiwa, N.; Shibasaki, M. Catalytic asymmetric epoxidation of
R,β-unsaturated N-acylpyrroles as monodentate and activate ester
equivalent acceptors. Tetrahedron 2006, 62, 6630–6639.
(28) Hendil, K. B.; Uerkvitz, W. The human multicatalytic proteinase:
affinity purification using a monoclonal antibody. J. Biochem.
Biophys. Methods 1991, 22, 159–165.
structure of the 20S proteasome from the archaeon T. acidophilum
˚
at 3.4 A resolution. Science 1995, 268, 533–539.
(5) Pamer, E.; Cresswell, P. Mechanisms of MHC class I-restricted
antigen processing. Annu. Rev. Immunol. 1998, 16, 323–358.
(6) Rock, K. L.; Goldberg, A. L. Degradation of cell proteins and the
generation of MHC class-I presented peptides. Annu. Rev. Immu-
nol. 1999, 17, 739–779.
(29) Gavioli, R.; Gallerani, E.; Fortini, C.; Fabris, M.; Bottoni, A.;
Canella, A.; Bonaccorsi, A.; Marastoni, M.; Micheletti, F.; Cafaro,
A.; Rimessi, P.; Caputo, A.; Ensoli, B. HIV-1 tat protein modulates
the generation of cytotoxic T cell epitopes by modifying protea-
some composition and enzymatic activity. J. Immunol. 2004, 6,
3838–3843.
(7) Walz, J.; Erdmann, A.; Kania, M.; Typke, D.; Koster, A. J.;
Baumeister, J. 26S proteasome structure revealed by three-dimen-
sional electron microscopy. J. Struct. Biol. 1998, 121, 19–29.
€
(8) Groll, M.; Ditzel, L.; Lowe, J.; Stock, D.; Bochtler, M.; Bartunik,
˚
H. D.; Huber, R. Structure of 20S proteasome from yeast at 2.4 A
resolution. Nature 1997, 386, 463–471.
(30) Manfredini, S.; Marastoni, M.; Tomatis, R.; Durini, E.; Spisani, S.;
Pani, A.; Marceddu, T.; Musiu, C.; Marongiu, M. E.; La Colla, P.
Peptide T-araC conjugates: solid-phase synthesis and biological activ-
ity of N4-(acylpeptidyl)-ara C. Bioorg. Med. Chem. 2000, 8, 539–547.
(31) Borissenko, L.; Groll, M. 20S proteasome and its inhibitors:
crystallographic knowledge for drug development. Chem. Rev.
2007, 107, 687–717.
(32) Groll, M.; Kim, K. B.; Kairies, N.; Crews, C. Crystal structure of
epoxomicin:20S proteasome reveals a molecular basis for selec-
tivity of R,β-epoxyketone proteasome inhibitors. J. Am. Chem.
Soc. 2000, 122, 1237–1238.
(33) Groll, M.; Balskus, E. P.; Jacobsen, E. N. Structural analysis of
spiro β-lactone proteasome inhibitors. J. Am. Chem. Soc. 2008,
130, 4981–4983.
(34) Gavioli, R.; Vertuani, S.; Masucci, M. G. Proteasome inhibitors
reconstitute the presentation of cytotoxic T-cell epitopes in
Epstein-Barr virus-associated tumors. Int. J. Cancer 2002, 101,
532–538.
(9) Golab, J.; Bauer Thomas, M.; Daniel, V.; Naujokat, C. Role of the
ubiquitin-proteasome pathway in the diagnosis of human dis-
eases. Clin. Chim. Acta 2004, 340, 27–40.
(10) An, B.; Goldfarb, R. H.; Simon, R.; Dou, Q. P. Novel dipeptidyl
proteasome inhibitors overcome Bcl-2 protective function and
selectively accumulate the cyclin-dependent kinase inhibitor p27
and induce apoptosis in transformed, but not normal, human
fibroblasts. Cell Death Differ. 1998, 5, 1062–1075.
(11) Orlowski, R. Z.; Eswara, J. R.; Lafond-Walker, A.; Grever, M. R.;
Orlowski, M.; Dang, C. V. Tumor growth inhibition induced in a
murine model of human Burkitt’s lymphoma by a proteasome
inhibitor. Cancer Res. 1998, 58, 4342–4348.
(12) Kisselev, A. F.; Golberg, A. L. Proteasome inhibitors: from
research tools to drugs candidates. Chem. Biol. 2001, 8, 739–758.
(13) Orlowski, R. Z.; Small, G. W.; Shi, Y. Y. Evidence that inhibition
of p44/42 mitogen-activated protein kinase signaling is a factor in
proteasome inhibitor-mediated apoptosis. J. Biol. Chem. 2002, 277,
27864–27871.