Synthesis and Evaluation of Macrocyclic Peptide Aldehydes as Potent and Selective Inhibitors of the 20S Proteasome

Article abstract of DOI:10.1021/acsmedchemlett.5b00401

This research explores the first design and synthesis of macrocyclic peptide aldehydes as potent inhibitors of the 20S proteasome. Two novel macrocyclic peptide aldehydes based on the ring-size of the macrocyclic natural product TMC-95 were prepared and e

Full text of DOI:10.1021/acsmedchemlett.5b00401

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Evaluation of Macrocyclic Peptide Aldehydes as
Potent and Selective Inhibitors of the 20S Proteasome
David L. Wilson,† Isabel Meininger,‡,§ Zack Strater,†,§ Stephanie Steiner,† Frederick Tomlin,† Julia Wu,†
Haya Jamali,† Daniel Krappmann,‡ and Marion G. Gotz*,†
̈
†Department of Chemistry, Whitman College, Walla Walla, Washington 99362, United States
‡Research Unit Cellular Signal Integration, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum
Munchen−German Research Center for Environmental Health, Neuherberg 85764, Germany
̈
S
* Supporting Information
ABSTRACT: This research explores the first design and synthesis of macrocyclic
peptide aldehydes as potent inhibitors of the 20S proteasome. Two novel
macrocyclic peptide aldehydes based on the ring-size of the macrocyclic natural
product TMC-95 were prepared and evaluated as inhibitors of the 20S proteasome.
Both compounds inhibited in the low nanomolar range and proved to be selective
for the proteasome over other serine and cysteine proteases, particularly when
compared to linear analogues with similar amino acid sequences. In HeLa cells, both
macrocycles efficiently inhibited activation of nuclear factor-κB (NF-κB) tran-
scription factor by blocking proteasomal degradation of the inhibitor protein IκBα
after cytokine stimulation. Due to their covalent mechanism of binding these
compounds represent a 1000-fold increase in inhibitory potency over previously
reported noncovalently binding TMC-95 analogues. Molecular modeling of the macrocyclic peptides confirms the preference of
the large S3 pocket for large, hydrophobic residues and the ability to exploit this to improve selectivity of proteasome inhibitors.
KEYWORDS: Proteasome, inhibitor, macrocycle, aldehyde, multiple myeloma
proteasome has been of great interest as a therapeutic target for
cancer treatment.4,5
he 26S proteasome is a barrel-shaped, multicatalytic
Tprotease that uses an N-terminal threonine residue to
hydrolyze polyubiquitinated substrates in the ubiquitin−
proteasome pathway (UPP). It consists of a 20S core particle
(CP) and two 19S regulatory caps. The 20S CP of the
proteasome comprises four circular heptamers consisting of α
and β subunits stacked in an α1−7β1−7β1−7α1−7 fashion. The
outer α subunits serve a regulatory function, while the two
inner rings each possess three catalytic activities. These
activities vary in their substrate specificity; in the human
constitutive proteasome the β1 subunit has peptidyl-glutamyl
hydrolyzing activity (PGPH) activity, the β2 subunit has
trypsin-like (TL) activity, and the β5 subunit has chymotrypsin-
like (CL) activity.1
The UPP is responsible for the targeted destruction of many
substrates including regulatory proteins such as the cyclin-
dependent kinase inhibitor p27kip1 and the p53 tumor
suppressor protein. Further, activation of transcription factor
NF-κB is a prototype example for the participation of the UPP
in signal transduction. Upon cell stimulation with cytokines,
and many other agents, cytosolic NF-κB inhibitor proteins such
as IκBα are phosphorylated by the IκB kinase (IKK) complex,
and phosphorylation marks the IκBs for ubiquitin-dependent
proteasomal degradation.2 NF-κB induces expression of many
genes responsible for cell proliferation and resistance to
apoptosis.3 Thus, due to the involvement of many UPP
substrates in the regulation of apoptosis and the cell cycle, the
In particular, the hematologic malignancy multiple myeloma
(MM) has been shown to be receptive to treatment with
proteasome inhibitors.6,7 Proteasome inhibition in plasma cells
results in an unfolded protein response through ER-stress
activated expression of the proapoptotic signaling protein
NOXA, a BH3-only member of the Bcl-2 family of
proteins.8−10 In 2012, carfilzomib became the second FDA
approved proteasome inhibitor for the treatment of multiple
myeloma.11
To date, most synthetic proteasome inhibitors consist of a
short peptide with an electrophilic trap designed to covalently
bind to the nucleophilic Thr1Oγ of the proteasome’s catalytic β
subunits. Electrophilic traps that have been reported include
aldehydes, boronic acids, epoxyketones, α-ketoaldehydes, β-
lactones, and vinyl sulfones.12 Many proteasome inhibitors,
including the FDA approved peptidyl boronic acid bortezomib,
suffer from adverse off-target effects. In the case of bortezomib,
coinhibition of serine proteases essential for neural health leads
to peripheral neuropathy in patients with high dosing
schedules.13,14 For this reason, continued research toward
Received: October 13, 2015
Accepted: January 13, 2016
© XXXX American Chemical Society
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Figure 1. Inhibitor design based on the TMC-95A macrocycle.
Figure 2. Minimum energy conformation of 7 (a) and 8 (b) bound to the CL active site. The molecular surface map of the binding pocket (green =
nonpolar, purple = polar) shows the P3 residue more adequately filling the S3 pocket. Furthermore, the biaryl ether macrocycle is well accommodated
by the shallow S2 and S4 pockets.
potent and selective inhibitors of the 20S proteasome is of great
interest.
and S4 pockets suggest that a macrocyclic peptide aldehyde
with a suitable peptide sequence will yield a potent and
selective inhibitor of the 20S proteasome. Other desirable
pharmacological properties introduced by the macrocycle
include increased metabolic stability, cellular uptake, and tissue
distribution.20,21
The size and position of the macrocycle was based on the
natural product TMC-95. The inhibitors 7 and 8 incorporate a
para−meta biphenyl ether linkage that results in a 17-
membered macrocycle, mimicking the ring size and structure
of TMC-95A while avoiding the synthetic challenges associated
with the oxidized tryptophan and biaryl linkage. Previous
studies by Kaiser et al. have shown that the stereospecific
hydroxyl groups of TMC-95A are not essential for proteasome
binding.22
In accordance with previously synthesized biaryl ether
analogues of TMC-95A, a modified Phe at P4 and Tyr at P2
were used to generate the ether macrocycle.23 Leu was selected
for the P1 position based on the peptide sequence of
carfilzomib and MG-132 and is thought to form favorable
interactions with Met45 in the S1 pocket of the β5 subunit.24
The P3 position was varied between two hydrophobic residues,
Leu and Phe(OMe). The P3 Leu residue of 7 mimics the
sequence of MG-132 and carfilzomib, while the Phe(OMe)
residue of 8 was chosen for its increased hydrophobic bulk. A
SAR study by Adams et al. determined that bulky, hydrophobic
residues in the P3 position can dramatically increase inhibitor
potency of peptide aldehydes as much as 1000-fold when
compared to inhibitors with smaller, hydrophobic residues in
the P3 position.15 A subsequent SAR study by Momose et al.
Compounds 7 and 8 (Figure 1) reported in this study
represent the first macrocyclic peptide aldehydes with
selectivity for the 20S proteasome. The compounds were
designed to achieve the potency and reversible binding
mechanism of peptide aldehydes such as MG-132 (Cbz-Leu-
Leu-Leu-H, Ki = 4 nM15) as well as incorporate a macrocycle to
increase their selectivity. The macrocyclic natural product
TMC-95A (Figure 1), a noncovalent reversible inhibitor of the
proteasome, is known for its potency and high selectivity. In
crystallographic studies, Groll et al. found that the large,
aromatic macrocycle of TMC-95A fills the S2 and S4 pockets of
the proteasome.16 This confers high specificity because the
large, nonspecific S2 pocket and shallow, hydrophobic S4 pocket
of the proteasome can accommodate the sterically demanding
macrocycle, which most other proteases cannot.12,13 Addition-
ally, the macrocyclic clamp forces the inhibitor backbone into
an extended β-sheet like conformation, which avoids loss of
entropy upon binding.
A series of biaryl ether (BIA) analogues of TMC-95A were
previously reported by Groll et al. (Figure 1). However, they
show severely reduced potency, which is most likely due to
their noncovalent mechanism of inhibition.17 Li et al. have
demonstrated successful inhibition of the Plasmodium falcipa-
rum proteasome using macrocyclic biaryl ethers.18 A SAR by
Abell et al. on macrocyclic peptide aldehyde inhibitors of ovine
calpain found that the addition of an aldehyde to a peptide
macrocycle can increase potency as much as ∼1000-fold.19 The
results of Abell et al. combined with the proteasome’s flexible S2
B
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Letter
a
showed Phe(OMe) to be potent in the P3 position for peptide
aldehyde inhibitors of the proteasome.25 Since the N-terminus
of the inhibitor has negligible interactions with the
proteasome,16 we decided to incorporate a Cbz-group for its
synthetic stability.
Scheme 2. Solid Phase Synthesis of Peptide Aldehydes
To further explore interactions at the P3 position, QM/MM
simulations were carried out on 7 and 8 to determine their
preferred binding geometries. Using the Amber12:EHT force
field and the crystal structure collected by Harshbarger et al.,26
the minimum energy conformation of the β4β5 dimer bound to
7 and 8 was calculated. Each inhibitor was modeled as a
hemiacetal adduct of the catalytic Thr1. As expected, EM
calculations showed both inhibitors adopting almost identical
geometries with the biaryl ether macrocycle extending into the
shallow S2 and S4 pockets and the P1 and P3 side chains
penetrating deep into the S1 and S3 pockets. The primary
difference between 7 and 8 was the extent to which they filled
the spacious S3 pocket (Figure 2). The smaller Leu side chain
of 7 did not fill the large, hydrophobic S3 pocket as well as the
Phe(OMe) side chain of 8, suggesting that 8 would bind more
tightly and have higher specificity for the proteasome than 7.
Additionally, the P1 Leu of both inhibitors is situated
approximately 3.4 Å from the Met45 residue, which is in
good agreement with previous findings that Met45 is
responsible for controlling the β5 subunit’s specificity for
hydrophobic residues.24 We chose to specifically target the CL
activity of the β5 subunit since it has been found to be most
essential for proteasome function.27 Based on both previous
SAR studies and our QM/MM simulations exploring S3/P3
interactions, we hypothesized that 8 would be a more potent
and specific inhibitor for the 20S proteasome.
a
Reagents and conditions: (a) (i) 20% v/v piperidine, DMF, rt, 30
min; (ii) Fmoc-Leu-OH, HATU, DIPEA, DMF, rt, 6 h; (b) (i) 20% v/
v piperidine, DMF, rt, 30 min; (ii) Fmoc-Tyr-OH, HATU, DIPEA,
DMF, rt, 2 h; (c) (i) 20% v/v piperidine, DMF, rt, 30 min; (ii) Fmoc-
Phe(4-OMe)−OH or Fmoc-Leu-OH, HATU, DIPEA, DMF, rt, 2 h;
(d) (i) 20% v/v piperidine, DMF, rt, 30 min; (ii) 3 (1.5 equiv),
HATU, DIPEA, rt, 2 h. (e) K2CO3, CaCO3, 3 Å molecular sieves,
DMF, 45 °C, 4 days; (f) (i) LiAlH4, THF, 0 °C, 30 min; (ii) KHSO4
(sat.), K, Na tartrate (sat.), THF, rt, 40 min.
amino acid sequence similar to compound 8. The linear
tetrapeptide 9 was synthesized by solid phase approach on a
Weinreb amide with Fmoc-protection chemistry (not shown in
scheme).
The synthesis of 7 and 8 was achieved by a convergent
approach (Schemes 1 and 2) of N-terminal Cbz-protected
Fluorometric kinetic enzyme inhibition assays were per-
formed to measure Ki values for 7 and 8 for the three
proteasomal activities as well as the serine protease
chymotrypsin and cysteine proteases cathepsin B and m-
calpain (Table 1). In addition we tested proteasome inhibitor
standard MG-132 (Cbz-Leu-Leu-Leu-H), the linear analogue 9,
bortezomib and carfilzomib as a reference for 7 and 8 since
reported Ki values vary depending on assay conditions. With
respect to the three proteasome activities, both inhibitors as
well as MG-132 showed high selectivity for the CL active site
stemming from the hydrophobic P3 residues. Comparing
inhibitory potency for the CL activity, 7 (Ki = 241 nM) is
less potent than MG-132 (Ki = 63.5 nM), while 8 (Ki = 54.5
nM) is as potent as MG-132 and 4-fold more potent than 7.
These data confirm our initial hypothesis that the peptide
sequence of 8 could achieve improved binding compared to 7.
Linear analogue 9 showed 2-fold higher potency compared to
macrocycle 8, likely due to the less flexible design of the
macrocycle. No inhibition was detected for either 7 or 8 for the
PGPH activity at concentrations up to 100 μM, due to
unfavorable interactions between the hydrophobic P3 residues
and the charged Lys45 residue in the β1 subunit. The TL
activity was moderately inhibited by 7 but not inhibited by 8.
Neither compound 7 or 8 inhibited the serine protease
chymotrypsin, and both compounds showed similar inhibition
of the clan CA cysteine protease m-calpain to MG-132 (Ki =
653 nM) with Ki = 2028 nM for 7 and Ki = 2983 nM for 8.
Inhibitor 8 is ∼55-fold and analogue 9 is ∼61-fold more
selective for the proteasome over m-calpain, which renders
these more specific than 7 and MG-132 that display 8- and 10-
fold selectivity, respectively. Importantly, both macrocyclic
Scheme 1. Synthesis of Enantiomerically Pure Phenylalanine
Analogue
a
a
Reagents and conditions: (a) NBS, AIBN, CCl4, reflux, 24 h; (b)
diethyl acetamidomalonate, NaH, DMF, rt, 4 h; (c) HCl, reflux, 24 h;
(d) Ac2O, NaHCO3, dioxane, H2O, rt, 12 h; (e) acylase I, pH 7.5, 37
°C, 20 h; (f) Cbz-OSu, NaHCO3, dioxane, H2O, rt, 12 h.
Phe(3-F, 4-NO2) 3 coupled to a resin-bound C-terminal
tripeptide. Compound 3 was first used by Boger et al. and
Janetka et al. to form biaryl ether macrocycles with a Tyr
residue in the i + 2 position by an intramolecular SNAr
mechanism.28,29 In this study, the improved chemoenzymatic
synthesis of 3 described by Vergne et al.30 allowed access to the
enantiomerically pure phenylalanine analogue, which was then
Cbz protected. The C-terminal tripeptides were synthesized by
solid phase peptide synthesis on a Weinreb amide resin using
commercially available, Fmoc-protected amino acids. After
coupling the modified Phe (3), the resin bound tetrapeptides 5
and 6 were cyclized according to the procedure developed by
Boger et al.28 The macrocycle was cleaved from the resin using
lithium aluminum hydride to generate a peptide aldehyde from
a Weinreb amide.31 For proof of concept we also prepared
linear analogue 9 (Cbz-Phe-Phe(4-OMe)-Phe-Leu-H) with an
C
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Table 1. Inhibitory Potency of 7 and 8 in Comparison to 9, MG-132, bortezomib, and carfilzomib
Ki in nM
20S proteasome
a
a
a
a
a
a
inhibitor
CL-activity
TL-activity
8,040
PGPH-activity
chymotrypsin
cathepsin B
3047
m-calpain
b
b
7
8
9
241 62.2
54.5 4.9
28.0 9.9
63.5 21.5
24.0 2.8
25.5 12.0
N.I.
N.I.
1
2028 319
2983 621
1708 99
653 211
b
b
b
N.I.
N.I.
N.I.
30345 900
2285 1,700
230 206
c
c
b
N.T.
N.T.
N.I.
b
b
MG-132
N.I.
3700
N.I.
c
c
c
c
c
bortezomib
carfilzomib
N.T.
N.T.
N.I.
N.I.
N.I.
c
c
c
c
c
N.T.
N.T.
N.I.
N.I.
N.I.
a
b
c
Assay protocols are provided in the Supporting Information. No inhibition at 100 μM inhibitor concentration. Not tested.
Figure 3. Macrocyclic peptides 7 and 8 inhibit proteasomal IκBα degradation and NF-κB activation in HeLa cells. (A,B) IκBα phosphorylation
(S32/36) and degradation as well as NF-κB DNA binding were analyzed by Western blot and EMSA, respectively. β-ACTIN was used as loading
control. (C−E) HeLa cells were incubated and analyzed as in A and B with increasing concentrations (0.1, 0.3, 1, 3, 10, and 20 μM) of compounds 7,
8, or MG-132 before TNFα stimulation.
aldehydes showed better selectivity for the proteasome over
MG-132 as well as linear analogue 9 when compared to the
clan CA protease cathepsin B suggesting that the peptide
macrocycle is responsible for increased specificity of 7 and 8
over the linear peptide aldehydes. In particular, 8 was
determined to be 550-fold more selective for the proteasome
than for cathepsin B compared to MG-132’s 4 and compound
9’s 80-fold selectivity. As the CL active site has a preference for
binding large hydrophobic residues in the peptide substrate,
which correlates to the peptide sequence specificity of cysteine
proteases such as calpains and lysosomal cathepsins, the
increased selectivity for proteasomal inhibition of macrocycles
7 and 8 compared to the linear MG-132 and 9 is particularly
advantageous. Although our assays indicate that bortezomib
and carfilzomib are more potent and selective inhibitors of the
20S proteasome (bortezomib Ki = 24.0 nM, carfilzomib Ki =
25.5 nM), due to their different modes of action boronic acid
and epoxyketone warheads are expected to possess higher
potency and selectivity than aldehydes. Our results suggest that
cyclizing warhead-containing linear analogues could further
enhance specificity.
In order to assess the potency of the new macrocyclic
peptides in cellular inhibition of the 26S proteasome, we
evaluated the effects of 7 and 8 on NF-κB signaling and
compared these to MG-132, which potently inhibits stimulus-
induced NF-κB activation.32 For this, we incubated the human
cervix carcinoma cell line HeLa with compound 7 or 8 or MG-
132 (20 μM each) for 2 h prior to stimulation with the pro-
inflammatory cytokines TNFα or IL-1β (Figure 3A,B). Cellular
effects on NF-κB signaling were monitored by determining
phosphorylation and degradation of the NF-κB inhibitor IκBα
by Western blot as well as NF-κB DNA binding by
electrophoretic mobility shift assays (EMSA). Just like the
well-characterized proteasomal inhibitor MG-132, 7 and 8
severely impaired TNFα and IL-1β induced IκBα degradation
and concomitant NF-κB activation. IKKβ catalyzes phosphor-
ylation of IκBα at serine 32/36, which marks the inhibitor for
UPP. All three proteasome inhibitors promote strong
accumulation of phosphorylated IκBα, providing evidence
that the macrocyclic peptides 7 and 8 are acting at the level
of the proteasome in the inhibition of NF-κB signaling.
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To compare the potency of 7 and 8 and MG-132 in cellular
proteasome inhibition, we monitored dose-dependent effects
on TNFα induced NF-κB signaling (Figure 3C−E). As evident
from the inhibition of IκBα degradation and NF-κB DNA
binding as well as accumulation of phosphorylated IκBα, all
three proteasome inhibitors impaired NF-κB signaling at 300
nM with maximal inhibition observed at approximately 10 μM.
A long exposure of IκBα in Western blot revealed an
accumulation of high molecular weight IκBα adducts in 7, 8,
and MG-132 cells. Previous results have shown that these
adducts represent polyubiquitinated IκBα species,33 and their
accumulation confirms that inhibition of IκBα degradation is
directly caused by inhibition of the proteasome. Thus, our
results demonstrate that the macrocyclic peptides 7 and 8 are
effectively inhibiting the proteasome and thus canonical NF-κB
activation inside the cells.
ACKNOWLEDGMENTS
■
The authors would like to thank the Arkansas Statewide Mass
Spectrometer facility for providing high resolution analysis.
ABBREVIATIONS
■
Ac2O, acetic anhydride; AIBN, azobis(isobutyronitrile); Cbz,
carboxybenzyl; Cbz-OSu, N-(carboxybenzyloxy) succin-imide;
CCl4, carbon tetrachloride; DIPEA, N,N-diisopropylethyl-
amine; DMF, dimethylformamide; ER, endoplasmic reticulum;
Fmoc, fluorenylmethyloxycarbonyl; HATU, 1-[bis-
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxid hexafluorophosphate; MOE, molecular
operating envrionment; NBS, N-bromosuccinimide; O-Me,
O-methyl; SAR, structure−activity relationship; THF, tetrahy-
drofuran
In summary, the high potency of 7 and 8 combined with
their selectivity, cellular stability, and efficacy suggest that
further research into macrocyclic peptidyl inhibitors may yield
synthetically facile cancer therapeutics with reduced side effects.
Compared to the previously reported noncovalent macrocyclic
peptide BIA-1a (Figure 1), the covalently binding macrocyclic
aldehydes reported here are 1000-fold more potent.17,23 In
addition to being the first macrocyclic peptide aldehydes with
specificity for the proteasome, we found that introduction of a
macrocycle significantly increases selectivity for the proteasome
over intracellular cysteine proteases, such as calpains and
cathepsins.
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* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00401.
General methods; synthesis of 7, 8, and 9; NMR spectra
and HRMS of final compounds; in vitro kinetic assays;
cell culture; Western blot and electrophoretic mobility
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: gotzmg@whitman.edu. Phone: (509) 527-5957.
Author Contributions
§These authors contributed equally to this work. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Funding
Research reported in this publication was supported by the
National Institute of General Medical Sciences of the National
Institutes of Health under Award Number R15GM113198. The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes
of Health. This research was also supported by a Cottrell
College Science Award from Research Corporation and a Louis
B. Perry Award from Whitman College.
Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acsmedchemlett.5b00401
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