Optimization of a series of dipeptides with a P3 threonine residue as non-covalent inhibitors of the chymotrypsin-like activity of the human 20S proteasome

Article abstract of DOI:10.1016/j.bmcl.2010.09.032

Starting from a tripeptide screening hit, a series of dipeptide inhibitors of the proteasome with Thr as the P3 residue has been optimized with the aid of crystal structures in complex with the β-5/6 active site of y20S. Derivative 25, (β5 IC₅₀

Full text of DOI:10.1016/j.bmcl.2010.09.032

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6581–6586
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Bioorganic & Medicinal Chemistry Letters
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Optimization of a series of dipeptides with a P3 threonine residue as
non-covalent inhibitors of the chymotrypsin-like activity of the human 20S
proteasome
⇑
Christopher Blackburn , Cynthia Barrett, Jonathan L. Blank, Frank J. Bruzzese, Nancy Bump,
Lawrence R. Dick, Paul Fleming, Khristofer Garcia, Paul Hales, Zhigen Hu, Matthew Jones, Jane X. Liu,
Darshan S. Sappal, Michael D. Sintchak, Christopher Tsu, Kenneth M. Gigstad
Millennium Pharmaceuticals Inc., 40 Landsdowne St., Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from a tripeptide screening hit, a series of dipeptide inhibitors of the proteasome with Thr as the
P3 residue has been optimized with the aid of crystal structures in complex with the b-5/6 active site of
y20S. Derivative 25, (b5 IC50 = 7.4 nM) inhibits only the chymotryptic activity of the proteasome, shows
cellular activity against targets in the UPS, and inhibits proliferation.
Received 13 August 2010
Accepted 7 September 2010
Available online 15 September 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Proteasome
Non-covalent inhibitor
Inhibition of the 20S core particle of the proteasome, which
leads to the accumulation of substrate proteins involved in gene
expression, signal transduction, cell cycle control and apoptosis,
is preferentially cytotoxic to cancer cells.1–3 Indeed, inhibition of
the proteasome is a clinically effective anti-cancer therapy, primar-
ily for hematological malignancies.4,5 The active sites of the 20S
core particle located on the b1, b2, and b5 sub-units are classified
as caspase-like, trypsin-like, and chymotrypsin-like, respec-
tively1–3 and induce proteolysis of peptide substrates via the hy-
droxyl nucleophile of the N-terminal threonine residues
Non-covalent inhibitors of the catalytic b sub-units of the 20S
core particle may offer therapeutic advantages such as more wide-
spread tissue distribution due to their more rapid dissociation
kinetics. The few non-covalent inhibitors reported to date include
TMC-95A (Fig. 2), a cyclic peptide natural product that competi-
tively inhibits the chymotrypsin-like activity of the proteasome
with nanomolar potency21 as well as synthetic analogs of moderate
potency22 and linear analogs such as 2.23 A related trimethoxy-
L-
phenylalanine-containing tripeptide, 3, optimized24,25 from a sta-
tine-like HTS hit,26 has been shown to inhibit the chymotrypsin-
like activity reversibly with an IC50 of 15 nM and a binding mode
proposed.27 In addition, a series of 5-methoxy-1-indanone capped
dipeptides (e.g., CVT-659)28 has been reported; although these
compounds were designed to be covalent, our recent X-ray data
with an analogous compound29,30 suggest that N-terminal inda-
nones do not form covalent adducts with the 20S proteasome. Re-
cently, we described in detail the biological properties of a
tripeptide screening hit 4;30 this compound shows comparable po-
tency to bortezomib but is a rapid-equilibrium inhibitor of the b5
(chymotrypsin-like) activity of the constitutive proteasome and
does not inhibit the activity of either the b1 or b2 sites. Compound
c
(Thr1O ).6,7 Various natural and synthetic inhibitors have been de-
scribed8–10 that form covalent adducts with the active site threo-
nine hydroxyls. These include peptide aldehydes and vinyl
sulfones,2 a0b0-epoxyketones (such as Carfilzomib, Fig. 1),11
2-keto-1,3,4-oxadiazoles,12 b-lactones13 (Salinosporamide A, and
Omuralide, for example), b-lactams14,15 and boronic acids.16 The
dipeptide boronic acid bortezomib (1) (PS-341 or VELCADEÒ) is
used clinically to treat multiple myeloma and refractory mantle
cell lymphoma, and is being evaluated for the treatment of other
malignancies.17 Bortezomib is a high affinity slowly reversible
inhibitor (Ki = 0.55 nM; dissociation t1/2 ca. 110 min)10 of the b5
site of the proteasome7 (and to a lesser extent the b1 and b2 sites)
the adduct of which with the N-terminal threonine of 20S has been
characterized by X-ray crystallography.18 Two additional boronic
acids MLN970810,19 and CEP-1887020 are undergoing clinical trials.
4 also inhibits TNFa-induced activation of NFjB-Luc in HEK293
cells consistent with potent proteasome inhibition and is cytotoxic
toward human cancer cell lines. We further showed that although
dipeptide analog 5 has considerably lower enzymatic and cellular
activities, improvements to these non-covalent inhibitors can be
effected by variation of the P1 through P4 residues.29,30 In this Let-
ter we discuss the SAR of a sub-series of dipeptide derivatives
⇑
Corresponding author. Tel.: +1 617 761 6811.
E-mail addresses: blackburn@mpi.com, chris.blackburn@mpi.com (C. Blackburn).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.032

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C. Blackburn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6581–6586
Figure 1. Covalent proteasome inhibitors.
Figure 2. Non-covalent proteasome inhibitors.
focusing on analogs with threonine residues at the P3 position and
report the crystal structures of representative examples in complex
with yeast 20S.
proteasome were determined. Proteasomal degradation of the
inhibitory protein I , (following its phosphorylation and ubiqui-
tination) activates the NF B transcription factor in cells in re-
sponse to TNF
stimulation.28,30,32 Thus, monitoring the effect of
proteasome inhibitors on the NF B pathway was used to assess
cellular activity. In addition, the long-term effects of these com-
pounds on cell viability were assessed in the human lung cancer
cell line Calu6 by monitoring cellular ATP levels. Testing over one
hundred analogs of compound 5 from the first library did not lead
to any derivatives where both h-Phe residues had been replaced
that showed comparable activity to 5. However, we did identify
O-methyl serine (6) and threonine (7) derivatives (Fig. 3) that
exhibited twofold improvements in inhibitory activity compared
to 5 (Table 1) and considerably improved solubilities33 albeit with
jBa
j
Results and discussion. We approached the optimization of com-
pound 5 by systematically modifying the C-terminal cap (P1 resi-
due), central amino acids (P2, P3) and N-terminal cap (P4) in a
series of compound libraries.29 In our first series of compounds,
we retained 4-methylbenzylamine and 3-phenoxyphenylacetyl27
at the termini while varying the amino acids in an attempt to find
replacements for the hydrophobic homo-phenylalanine (hPhe) res-
idues. Compounds were prepared using automated liquid-phase
peptide synthesis methods employing Boc-protection of the
a
j
N-
example, 4-methylbenzylamine was coupled to the appropriate
Boc-N- -amino acid in the presence of HBTU. After removal of
the Boc protecting group under acidic conditions, the next pro-
a
-amino groups as described in detail previously.30 Thus, for
a
modest cellular activities (NFjB assay).
In the next series of compound libraries, prepared similarly, we
scanned for alternatives to 3-phenoxyphenylacetyl as the N-termi-
nal cap (P4 residue) and noted that analogs 8 and 9, derived from
an isomeric benzoic acid, showed significant improvements in
tected
a-amino acid was coupled, de-protected and capped at
the N-terminus (Scheme 1).31
The inhibitory activities of these analogs on the rate of hydroly-
sis of the b5 substrate Ac-WLA-AMC by human 20S constitutive
enzymatic activity accompanied by sub-lM potencies in the

C. Blackburn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6581–6586
6583
Scheme 1. Reagents and conditions: (i) ArCH2NH2, HBTU, NMM, DMF, 25 °C, 24 h; (ii) 4 N HCl, dioxane, 25 °C, 8 h; (iii) BocNHCH(R2)COOH, HBTU, NMM, DMF, 25 °C, 24 h; (iv)
R3COOH, HBTU, NMM, DMF, 25 °C, 24 h; (v) p-BnOC6H4CHO, NaBH3CN, MeOH, 25 °C, 24 h.
Figure 3. Structures of human 20S inhibitors 6–15.
cellular assays. Threonine derivatives were generally superior to
their O-methylserine counterparts so these derivatives were se-
lected for further optimization at P4. A diverse selection of hydro-
phobic acyl residues was tolerated at this position as illustrated by
to the P1 residue such as methylation at N-1 (compound 16) or the
benzylic position (compound 17) abolished activity (Fig. 4). Like-
wise, extending the benzyl P1 residue by an additional methylene
group was also detrimental; compound 18, for example, has little
activity. Several smaller residues were incorporated at the P1 site
such as ethyl and allyl (analogs 19 and 20, respectively), leading
to >10-fold reductions in potency. Removal of the 40-methyl group
from the P1 benzyl residue also led to a slight reduction in potency
(compare 21 with 9). While introduction of a chlorine atom at the
the b5 inhibitory activities, cellular activities (NFjB assay) and
anti-proliferative effects of analogs 10–14 (Table 1). b-Ketoamide
14 is particularly noteworthy with a comparable profile to 4. Com-
pound 15, however, with a methylene replacement for the P4 car-
bonyl of compound 9 was over 100-fold less potent. Modifications

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C. Blackburn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6581–6586
Table 1
Enzymatic, cellular and anti-proliferative effects of a series of capped dipeptides compared to bortezomib
Compound
Humana 20S-PA28 IC50 (nM) b5c
HEK293 NF
j
B-Luc IC50 (nM) (% max inh)b
Cell viabilityc Calu6 LC50 (nM)
1
4
8.2
16
10 (100)
47 (79)
6.1
140
5
6
7
8
1100
550
450
74
20
75
120
25
20
>50,000 (28)
6100 (40)
24,000 (30)
340 (78)
120 (81)
690 (97)
690 (80)
320 (89)
95 (89)
90 (100)
>50,000 (37)
4400 (86)
>50,000
>25,000
>25,000
15,000
220
9
380
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1500
2300
1200
2300
136
14,000
6600
9000
>25,000
5100
2800
1400
>25,000
290
7.3
3100
9100
>100,000
3200
440
100
68
210
13
31
>50,000
2600 (98)
880 (99)
710 (68)
15,000 (44)
29 (87)
330 (71)
28 (81)
2700
110
7.4
a
Purified human erythrocyte 20S (0.25 nM) was used as a source of constitutive (c) proteasome to measure the peptidase activity of the b5 (chymotrypsin-like) sub-site.
The assay was performed in the presence of 15 M of the fluorogenic substrate Ac-WLA-AMC, 12 nM PA28 , and a titration of each inhibitor.
B-luciferase activity in HEK293 cells was determined by pre-incubation of the cells (10,000 per well) with compound for 1 h followed by stimulation
in the continued presence of compound for 3 h. The IC50 and percentage inhibition of NF B-luciferase activity are given.
l
a
b
Inhibition of NF
j
with 10 ng/ml TNF-
a
j
c
Effects of compounds on the viability of Calu6 cells (2000 per well) were assessed following incubation with compound for 72 h. Viability was assessed by monitoring
cellular ATP levels using the luminescence-based ATPlite assay as described previously.30 In each case, results are mean values of at least three independent experiments.
Figure 4. Structures of human 20S inhibitors 16–25.

C. Blackburn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6581–6586
6585
meta-position of the benzylic P1 residue, as in compound 22, was
unfavorable, ortho-chlorobenzyl derivatives 23 and 24 and ortho-
methylthiophene derivative 25 showed high inhibitory activities
in enzymatic and cellular assays.
Many of the SAR trends noted above can be explained from
X-ray structures of the inhibitors in complex with the b5 site of
yeast open gate 20S.34 To date, we have solved 30 such structures29
and reported four of these in the open literature.30 The co-crystals
of compounds 24 and 25, obtained at 2.6 and 2.85 Å resolution,
respectively35 have not been reported previously and their impor-
tant features are illustrated in Figures 5–7. The same general
binding mode that we reported earlier30 involving b-sheet-type
H-bonding interactions was observed (Fig. 5). These backbone
interactions include H-bonding between the amide flanked by
the P1 and P2 residues and Thr21 and Gly47 of the b5 site account-
ing for the loss of activity of N-methylated derivative 16. As shown
in Figure 6, a benzylic residue can fit tightly into the S1 binding
pocket and diminished hydrophobic interactions accounts for the
lower potency of the allyl and ethyl derivatives 19 and 20. Addi-
tional hydrophobic interactions with sub-pockets within S1 are ob-
served for the 40-methyl compounds;30 for example, compound 9 is
threefold more potent than unsubstituted analog 21. Similarly,
ortho-substituents on a P1 benzyl residue can access an additional
sub-pocket contributing to the high potencies of chloro-com-
pounds 23 and 24 and ortho-methyl thiophene 25 (Fig. 6). The
p-system of the hPhe residue of these compounds forms a ledge
interaction with Gly47-Gly48 within the shallow S2 binding pocket
that we have discussed previously,30 while the threonine residue
partially occupies the well defined S3 specificity pocket. In con-
trast, the S4 pocket is very large and ill-defined accounting for
the wide range of hydrophobic substituents tolerated at this
position.
The reduced activity of the secondary amine capped analog 15
can be attributed to a weaker hydrogen bonding interaction be-
tween the nitrogen and Asp-114 compared to its analog 9 or a less
optimal projection of the benzyl group toward the P4 pocket. The
value of the 4-benzyloxybenzoyl P4 residue appears to be associ-
ated with an induced fit effect (Fig. 7). Thus, in comparison to com-
pound 24, where the P4 residue partially occupies the S4 binding
pocket, the benzyloxybenzoyl group of 25 projects farther into
the binding pocket accessing additional interactions with Pro94
and Tyr96 in the adjacent b-6 site. By forcing apart Tyr5 and
Figure 6. Molecular surface of the 20S b5 site showing the S1 sub-pocket being
filled by the ortho-chloro substituent in P1 for compound 24 (left panel), compared
to the ortho-methyl substituent for compound 25 (right panel). Atoms are shown in
ball-and-stick representation with carbon in yellow, nitrogen in blue, oxygen in red,
chlorine in green, and sulfur in orange. Figure made using PyMOL Molecular
Graphics System (DeLano Scientific, Palo Alto, CA).
Figure 7. Superposition of the structures for compounds 24 and 25 bound to the
b5/b6 active site of 20S highlighting the S4 pocket and changes in the position of
Tyr96. All residues labeled are from the b6 sub-unit. Coloring scheme is as
described above, with carbon atoms for the compound 24 structure shown in two
shades of yellow and for the compound 25 structure in two shades of green.
Figure 5. Schematic representation of the b5/b6 active site of 20S with compound
24 bound. Key hydrogen bonds between the inhibitor and the protein are shown as
dashed lines colored magenta, with distances indicated in Angstroms.
Tyr96 the latter can form a
group of compound 25.
r–p edge interaction with the P4 aryl

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C. Blackburn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6581–6586
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j
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rable cellular potencies to those of tripeptide 4. It should be noted
that all the analogs described here are highly selective inhibitors of
the b5 sub-unit with no significant inhibitory activity (IC50
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>20
examples showed only modest inhibitory activity at a concentra-
tion of 100
M for ten unrelated proteases.36 The cytotoxic LC50
values of these compounds in Calu6 cells also correlate with their
20S b5 potencies, indicating that selective inhibition of the chymo-
trypsin-like site of the proteasome is sufficient to inhibit cell pro-
liferation. Although none of the compounds were as potent as
bortezomib, five examples gave cytotoxicity LC50 values below
500 nM and compound 25, with an LC50 of 95 nM, showed superior
lM) of human b1 and b2 sub-units. In addition, representative
l
activity to that of 4 (Table 1) tracking the NFjB-Luc activity in
HEK293 cells. These data further support the notion that selective
non-covalent inhibition of the b5 (chymotrypsin-like) site of the
proteasome is sufficient to inhibit the degradation of TNF
a-depen-
dent NF B activity and the proliferation of cancer cells. Several
j
compounds reported here have comparable or greater potencies
than any previously reported non-covalent inhibitor. In addition,
co-crystals of compounds 24 and 25 bound to the b5 active site
of yeast 20S unequivocally demonstrate the binding mode of this
series of compounds and providing further opportunities for
optimization.
21. (a) Koguchi, Y.; Kohno, J.; Nishio, M.; Takahashi, K.; Okuda, T.; Ohnuki, T.;
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Acknowledgements
24. García-Echeverría, C.; Imbach, P.; France, D.; Furst, P.; Lang, M.; Noorani, M.;
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We are grateful to Drs. Marion Schmidt and Dan Finley for pro-
viding the yeast 20S expression strains and to Dr. Juan Gutierrez
and Zhi Li their assistance with preparation of the yeast 20S en-
zymes. We gratefully acknowledge the analytical chemistry assis-
tance of David Lok and Nina Molchanova.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.032.
29. Blackburn, C.; Achab, A.; Blank, J.; Bump, N.; Bruzzese, F.; Dick, L.; Fleming, P.;
Garcia, K.; Gigstad, K.; Hales, P.; Herman, L.; Jones, M.; Li, Z.; Liu, J.;
Mishechkina, A.; Nagayoshi, M.; Sappal, D.; Sintchak, M.; Tsu, C. Abstracts,
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G.; Podar, K.; Hideshima, T.; Velankar, M.; Mitsiades, C.; Mitsiades, N.; Yasui,
H.; Letai, A.; Ovaa, H.; Berkers, C.; Nicholson, B.; Chao, T.-H.; Neuteboom, S. T.
32. Palombella, V. J.; Rando, O. J.; Goldberg, A. L.; Maniatis, T. Cell 1994, 78, 773.
33. Representative turbidometric solubilities were obtained by sequential
dilutions of 10 mM DMSO stock solutions into 50 mM potassium
phosphate buffer at pH 6.8. Measurements were made on
Nepheloskan Ascent plate reader equipped with quartz halogen lamp
emitting between 580 and 630 nm giving the following solubility results: 4:
M; 5; 3 M; 6: 50 M; 7: 50 M; 8: 50 M; 9: 25 M; 10: 12 M; 11:
M; 12: 100 M.
a Labsystems
a
6
l
l
l
l
l
l
l
50
l
l
34. Given the close correlation in potencies of proteasome inhibitors from various
classes for the human and yeast 20S enzymes,30 we are confident optimizing
compounds for human 20S inhibitory activity based on X-ray structures
obtained with yeast 20S.
35. Co-crystals of the y20S proteasome with inhibitors 24 and 25 were solved with
resolutions of 2.85 Å and 2.5 Å, respectively (see Supplementary data).
36. Protease selectivity was assessed by testing the inhibitory activities of
representative compounds on a panel of purified human enzymes including
cathepsin B and L, coagulation factor b-XIIa, chymotrypsin, elastase, plasmin,
thrombin, tissue plasminogen activator, trypsin and calpain I.