Peptidyl vinyl ester derivatives: New class of selective inhibitors of proteasome trypsin-like activity

Article abstract of DOI:10.1021/jm040905d

The proteasome is a multicatalytic proteinase complex which plays a central role in intracellular protein degradation. We report here the synthesis and biological activities of a new class of specific proteasome inhibitors selective for trypsin-like activ

Full text of DOI:10.1021/jm040905d

5038
J. Med. Chem. 2005, 48, 5038-5042
Brief Articles
Peptidyl Vinyl Ester Derivatives: New Class of Selective Inhibitors of
Proteasome Trypsin-Like Activity
Mauro Marastoni,‡,* Anna Baldisserotto,‡ Silvia Cellini,§ Riccardo Gavioli,§ and Roberto Tomatis‡
Department of Pharmaceutical Sciences and Biotechnology Centre, and Department of Biochemistry and Molecular Biology,
University of Ferrara, I-44100 Ferrara, Italy
Received December 14, 2004
The proteasome is a multicatalytic proteinase complex which plays a central role in intracellular
protein degradation. We report here the synthesis and biological activities of a new class of
specific proteasome inhibitors selective for trypsin-like activity. These tripeptide-based
compounds bearing a C-terminal vinyl ester are nontoxic, and do not affect cell proliferation,
but are able to modulate the generation and presentation of immunogenic peptides presented
by MHC class I molecules.
Introduction
26S proteasome represents the multicatalytic pro-
teinase of the ubiquitin/adenosine triphosphate-depend-
ent proteolytic pathaway.1 This large enzymatic complex
is found in the cytosol and nucleus of eukaryotic cells
and plays a central role in the selective degradation of
intracellular proteins.2-4 In a modular structure of the
enzyme, the 20S proteasome is the proteolytic chamber
formed by four stacked rings, where each of the two
inner rings is composed of seven different â subunits.
The â1 subunit contains a post-acidic-like (PGPH) active
site, â2 has trypsin-like (T-L) activity, and â5 is associ-
ated with a chymotrypsin-like (ChT-L) function. All the
proteolytic sites utilize the γ-hydroxyl group as nucleo-
phile and the R-amine as proton donor/acceptor of the
N-terminal threonine residue in the catalytic cycle.
Proteasomes removed abnormal proteins and play a
role in cell-cycle progression and apoptosis, both in
transcription factor activation and in the generation of
peptides presented by the major histocompatibility
complex I (MHC-I). Thus, the proteasome represents a
potential target for the development of therapeutic
agents for the treatment of pathologies such as cancer,
inflammation, immune diseases, and others.5-7 Several
classes of inhibitors have been identified, and these
compounds represent a valuable tool for the identifica-
tion of the role of the ubiquitin-proteasome pathway
in cellular processes.8 In addition, proteasome inhibitors
could be used as therapeutic agents to arrest tumor cell
proliferation and as modulators of antigen presentation.
The most common inhibitors are peptide-based com-
pounds with a C-terminal function, able to interact with
enzymatic catalytic threonine.9-18
Figure 1. Structures of the vinyl ester pseudotripeptide
inhibitors.
tives.19,20 The more effective of these molecules, which
carry an N-terminal 1,2,5,6-tetrahydropyridine-3-car-
bonyl (Guv, guvacine) group, have shown an interesting
inhibition of the tryptic- and chymotryptic-like activities
of the proteasome and favorable pharmacokinetic prop-
erties. In this report, we describe the synthesis and
biological characterization of a new series of peptide-
based inhibitors bearing a C-terminal leucine vinyl ester
(Leu-VE) as pharmacophore, potentially able to interact
with catalytic threonine (Figure 1). The ethyl acrylate
group can function as a substrate of the γ-hydroxy
threonine side chain in Michael addition in the same
way as has been suggested for the well-known peptide
vinyl sulfone inhibitors.21 The dipeptidic structural core,
as reported in our previous work,19 presents two leucine
residues in derivatives 1-4, while the more hydrophilic
sequence Val-Ser is found in compounds 5-8 at posi-
tions P3 and P2, respectively. The N-terminal position
is functionalized with the following six-membered
We recently reported the identification of some trip-
eptidic sequences functionalized with arecoline deriva-
* To whom correspondence should be addressed, Prof. Mauro
Marastoni, Dipartimento di Scienze Farmaceutiche, Via Fossato di
Mortara 17-19, Universita` di Ferrara, I-44100 Ferrara, Italy. Tel. +39-
532-291281. Fax +39-532-291296. E-mail: mru@unife.it.
‡ Department of Pharmaceutical Sciences and Biotechnology Centre.
§ Department of Biochemistry and Molecular Biology.
10.1021/jm040905d CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/01/2005

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 5039
Scheme 1
Table 1. Subsites of Proteasome Inhibition and Metabolic
rings: benzyloxycarbonyl (Z) in 1 and 5, N-(tert-butoxy-
carbonyl)guvacine (Boc-Guv) in 2 and 6, guvacine in 3
and 7, and 3-hydroxy-2-methylbenzoyl (HMB) in 4 and
8 derivatives. These cyclic substituents were chosen for
their different physicochemical characteristics, based on
our previous biological evidence19,20 and due to the fact
that they are easily inserted into a peptide sequence.
Stability of Vinyl Ester Tripeptides
IC50 (µM)a
half-life (min)
compound
T-L ChT-L PGPH medium plasma
1 Z-Leu-Leu-Leu-VE
0.28
2.45 >10
4.36 >10
0.86 >10
4.21 >10
>10
>10
6.54 >10
>10
>360
>360
>360
>360
>360
>360
>360
>360
292
198
2 Boc-Guv-Leu-Leu-Leu-VE 0.19
3 Guv-Leu-Leu-Leu-VE
4 HMB-Leu-Leu-Leu-VE
5 Z-Val-Ser-Leu-VE
6 Boc-Guv-Val-Ser-Leu-VE 0.11 >10
7 Guv-Val-Ser-Leu-VE
8 HMB-Val-Ser-Leu-VE
0.21
0.041
0.071 >10
>360
>360
238
Results and Discussion
154
0.38
0.033 >10
>360
>360
Synthesis. Vinyl ester tripeptides were synthesized
following the strategy reported in Scheme 1 by the
classical solution method using C-terminal stepwise
elongation. NR-Boc-protected leucine vinyl ester was
prepared from the corresponding aldheyde22 by reaction
with [(ethoxycarbonyl)methylidene]triphenylphosphorane
without racemization.23 WSC/HOBt or HATU were
employed for the coupling steps, benzyloxycarbonyl was
introduced as the succinimidyl ester (Z-OSu), and Boc
was removed by TFA. All products were purified by
preparative RP-HPLC, and structural verification was
achieved by mass spectrometry and NMR spectroscopy.
Biological Results. We tested the inhibitory capac-
ity of compounds 1-8 on purified 20S proteasome using
fluorogenic substrates specific for the three major pro-
teolytic activities of the proteasome.24 All vinyl ester
derivatives were assayed at different concentrations
(from 0.001 to 10 µM) for their capacity to inhibit the
tryptic-like (T-L), chymotryptic-like (ChT-L), and post-
acidic (PGPH) activities in vitro of the proteasomes
purified from lymphoblastoid cell lines. All compounds
inhibited the tryptic activity of the proteasome (Table
1), suggesting that the pharmacophore leucine ethyl
acrylate (Leu-VE) at the C-terminus may represent a
functional group which could be used for the design of
a The values reported are the average of two independent
determinations.
a new class of proteasome inhibitors. The central
dipeptidic sequence seems to play a role in determining
the specificity of this class of compounds. In particular,
compounds 1-4, containing the hydrophobic Leu-Leu,
show the capacity to inhibit both tryptic- and chymo-
tryptic-like activities, while compounds 5-8, containing
Val-Ser, show the capacity to inhibit only the tryptic-
like activity, thereby suggesting that they preferentially
bind to the â2 subunit responsible for such activity.
Furthermore, it should be noted that the presence of
3-hydroxy-2-methylbenzoyl (HMB in compounds 4 and
8) at the N-terminus increases the inhibitory potency
of this class of compounds. All members of the series
show negligible inhibition of the PGPH-like activity
attributed to the â1 subunit of the proteasome. Vinyl
ester tripeptide 8 is the most potent (IC50 ) 33 nM) and
selective for trypsin-like activity.
The cell membrane permeation of the most represen-
tative compounds, 4 and 8 were tested in live cells. After
cell treatment, proteasomes were purified and assayed
for proteolytic activity using specific substrate for T-L

5040 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Brief Articles
Figure 2. Panel A: Effect of compounds 4 and 8 on cell proliferation. The colon carcinoma cell line HCT116 was cultured for 3
days in the presence or not (-) of the indicated concentrations of compounds 4 and 8. Cell proliferation was evaluated by [3H]-
thymidine incorporation. One representative experiment out of three performed is shown. Panel B: Effect of compounds 4 and 8
on the presentation of an EBV-derived CTL epitope. HLA-A2-positive RG lymphoblastoid cells were cultured for 18 h in the
presence or not (-) of the indicated concentrations of compounds 4 and 8 and used as target of HLA-A2-restricted CLG-specific
CTL cultures. Results are expressed as % specific lysis. One representative experiment out of three performed is shown.
and ChT-L activities. The results obtained were com-
parable to that observed in the in vitro assay (IC50:
0.062 and 0.050 µM, respectively), thereby demonstrat-
ing that compounds 4 and 8 are cell-permeable and able
to inhibit the proteasome in vivo.
proliferative activities of these compounds on tumor cell
lines.26 To this end, two colon-carcinoma cell lines
(COO115, HCT116) were treated with different concen-
trations (from 0.1 to 10 µM) of prototypes 4 and 8 for 3
days, and the cellular effects of the compounds were
evaluated by trypan blue exclusion and cell proliferation
assays. No toxicity was observed (data not shown) while
inhibition of HCT116 cell proliferation was noticed at
elevated concentrations (10 µM) only for compound 4
(Figure 2, panel A). This suggests that inhibitors of the
tryptic-like activity of proteasomes do not induce cell
death or inhibit cell proliferation. Similar results were
obtained with COO115 cells (data not shown).
The critical involvement of the proteasome in MHC
class I-restricted antigen processing was established
with the help of specific inhibitors. It is now widely
recognized that treatment with membrane-permeable
inhibitors of the proteasome strongly affect the genera-
tion of antigenic peptides.27 However, the presentation
of certain CTL epitopes appears to be insensitive to
proteasome inhibition, and some epitopes are generated
in larger amounts in cells subjected to partial inhibition
of proteasomes.28 We also evaluated the effects of
compounds 4 and 8 on the presentation of an EBV-
derived HLA-A2 presented CTL epitope. To this end,
HLA-A2 positive lymphoblastoid cells were treated for
18 h with compounds 4 and 8 and tested in cytotoxicity
assays using HLA-A2-restricted CTLs specific for the
CLG epitope. CLG-specific CTLs showed low recognition
of target cells (Figure 2, panel B) due to poor expression
of the CLG epitope on the surface of EBV-infected B
cells.29 However, a net dose-dependent increase of CLG-
specific killing was observed in cells treated with
compounds 4 and 8. Treatment of HLA-A2-negative cells
To characterize the proteasomal interaction of vinyl
ester tripeptides, derivatives 4 and 8 were analyzed for
reversibility inhibition. In the first assay, the enzyme-
inhibitor (0.1 µM) mixture was preincubated for 1 h,
increasing concentrations of specific T-L substrate were
added, and the enzyme activity was measured after each
addition. In the second experiment, after enzyme-
inhibitor preincubation, the mixture was diluted for 25
or 50-fold prior to the addition of substrate, and the
activity was monitored. In both experiments, small
shifts in the IC50 values were observed (from 0.030 to
0.057 µM for 4 and from 0.031 to 0.040 µM for compound
8), indicating an irreversible inhibition. This suggests
that the vinyl ester group functions as a substrate of
catalytic threonine and, in particular, that the conjugate
double bond is susceptible to Michael’s addition of the
threonine γ-hydroxyl group. Crystallographic data and
molecular modeling studies, such as docking simulation,
are in progress to define the inhibitor-enzyme interac-
tion subsite pockets.
Susceptibility to enzymatic hydrolysis of compounds
1-8 was determined by incubation at 37°C in culture
medium (RPMI) in the presence of 10% foetal calf serum
or in human plasma.25 The half-lives of the derivatives
reported in Table 1 show great stability in cell culture
medium and good enzymatic resistance to human
plasma proteases.
Since proteasomes play a key role in cell viability and
proliferation, we evaluated the pro-apoptotic and anti-

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 5041
Purification of Proteasomes. Partially purified protea-
somes were obtained from lymphoblastoid cell lines, untreated
or treated for 12 h at 37 °C with the inhibitors, as previously
described.30 A subsequent purification was carried out by
affinity chromatography (mAb R-subunit, Affinity). Fractions
containing proteasomes were combined and dialyzed against
25 mM Tris-HCl pH 7.5. Protein concentration was determined
using BCA protocol (Pierce, Rockford, IL).
with compounds 4 and 8 did not affect killing by CLG-
specific CTLs (data not shown). These results suggest
that inhibition of the tryptic activity of the proteasome
favors the generation and presentation of the subdomi-
nant CLG epitope.
Conclusion
Proteasome Subunit Inhibition Assays. Suc-LLVY-
AMC, Boc-LRR-AMC, and Ac-YVAD-AMC (Sigma) were used
to measure chymotrypsin-like, trypsin-like, and postacidic
proteasome activities, respectively. Substrates were incubated
at 37 °C for 30 min with proteasomes, untreated or pretreated
with 0.01-20 mM of test compounds, in activity buffer.
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 and absence of
inhibitor) vs inhibitor concentration, and fitted with the
equation Y ) 100/1 + (X/IC 50)A, where IC50 is the inhibitor
concentration at 50% inhibition, and A is the slope of the
inhibition curve.
Cytotoxicity Assay. HLA-A2-restricted EBV-specific CTL
cultures reacting against the LMP2-derived CLGGLLTMV
(CLG, aa 426-434) epitope were obtained by stimulation of
lymphocytes from the HLA-A2-positive EBV-seropositive donor
RG with CLG-pulsed T2 cells as previously described.29 CTL
cultures were maintained in medium supplemented with 10
U/mL rIL-2 (Chiron, Milan, Italy).
Target cells were treated or not for 18 h with different
concentrations of compounds 4 and 8 and labeled with
Na251CrO4 for 90 min at 37 °C. Cytotoxicity tests were
routinely run at different effector:target ratios in triplicate.
Percentage specific lysis was calculated as 100 × (cpm sample
- cpm medium)/(cpm Triton X-100 - cpm medium). Spontane-
ous release was always less than 20%.
Cell Viability and Proliferation Assay. The colon car-
cinoma cell lines COO115 and HCT116 were cultured in the
presence of compounds 4 and 8 at different concentrations
(from 0.1 to 10 µM), and cell viability was evaluated by trypan
blue exclusion. In the cell proliferation assays, 5 × 104 cells
were seeded in 96-well microtiter plates in the presence or
absence of different concentrations of compounds 4 and 8,
cultured for 48 h, and pulsed with [3H]-thymidine (1 µCi/well)
for the last 16 h. Cells were collected on glass microfiber filters
using a cell harvester, and radioactivity was assessed by a
â-counter.
Metabolic Stability Assays. The degradation kinetics of
new inhibitors were studied in culture medium (RPMI) and
human plasma. An amounr of 0.1 mL of a solution of each
compound (10 mg/mL in acetonitrile/H2O 1:1) was added to 1
mL of RPMI containing 20% FCS. Alternatively, test com-
pounds 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 different durations: up to 360 min in
the case of human plasma, and up to 2 days in the case of
RPMI containing 20% FCS. The incubation was terminated
by addition of ethanol (0.2 mL), the mixture poured at 21 °C,
and, after centrifugation (5000 rpm for 10 min), aliquots (20
µL) of the clear supernatant were injected into 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.
In this work, we have designed, synthesized, and
defined the biological profile of a new series of peptide-
based proteasome inhibitors. Prototypes of these vinyl
ester tripeptide derivatives are potent and selective for
tripsyn-like activity, are cell permeable, and are stable
in in vitro degradation assays. This new class of inhibi-
tors may represent a useful instrument to understand
the mechanisms underlying antigen production by the
ubiquitin-proteasome system and, in perspective, may
have 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
Fluka (Switzerland). Crude pseudopeptides were purified by
preparative reversed-phase HPLC using a Water Delta Prep
4000 system with a Waters PrepLC 40 mm Assembly column
C18 (30 × 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 × 250 mm). Analytical determination and capacity
factor (K′) of the peptides was determined 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 weight of compounds was determined by a ESI
1
Micromass, ZMD2000 mass spectrometer. H NMR spectros-
copy was obtained on a Bruker AC 200 spectrometer.
Chemistry. General Procedures. Coupling with WSC/
HOBt. The amino component (1 mmol), NMM (1 mmol), HOBt
(1.1 mmol), and WSC (1.1 mmol) in this order were added to
a solution of the carboxyl component (1 mmol) in DMF (10 mL)
at 0 °C. The reaction mixture was stirred for 1 h at 0 °C and
18 h at room temperature; then the solution was diluted with
EtOAc (100 mL) and washed consecutively with 0.1 N HCl,
brine, NaHCO3, and brine. The organic phase was dried
(MgSO4), filtered, and evaporated to dryness. The residue was
treated with Et2O, and the resulting solid was separated by
centrifugation.
Coupling with HATU. The deprotected vinyl ester trip-
eptide (1 mmol), NMM (1 mmol), and HATU (1 mmol) were
added to a solution of the Boc-Guv (1 mmol) in DMF (3 mL)
at 0 °C. The reaction mixture was stirred for 1 h at 0 °C and
24 h at room temperature; then the solution was diluted with
AcOEt (70 mL) and treated as described above.
Introduction of Z. Z-OSu (0.5 mmol) was added to a
solution of the tripeptide vinyl ester (0.5 mmol) in DMF (5 mL)
at 0 °C. The mixture was stirred for 1 h at 0 °C and 15 h at
room temperature and then evaporated. The resulting target
compounds were purified by preparative HPLC.
TFA Deprotection. tert-Butyloxycarbonyl protection was
removed by treating intermediates with aqueous 90% TFA (1:
10, w/v) for 30-40 min. After evaporation, the residue was
treated with Et2O, and resulting solid was separated by
centrifugation.
Acknowledgment. Financial support of this work
was given by the University of Ferrara, the Ministero
dell′Universita` e della Ricerca Scientifica e Tecnologica
(MURST), the Associazione Italiana per la Ricerca sul
Cancro (AIRC), and the Istituto Superiore di Sanita`

5042 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Brief Articles
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for linguistic assistance.
Appendix
Abbreviations used: Boc, tert-butoxycarbonyl; HATU,
O-(7-azabenzotriazolyl)tetramethyl uronium hexafluo-
rophosphate; HOBt, N-hydroxybenzotriazole; NMM,
N-methylmorpholine; TFA, trifluoroacetic acid; WSC
(water soluble carbodiimide), 1-ethyl-3-(3′-dimethylami-
nopropyl)carbodiimide; Z, benzyloxycarbonyl-N-hydroxy-
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Supporting Information Available: Analytical data of
intermediates and products 1-8 are available free of charge
via the Internet at http://pubs.acs.org.
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