Identification of new peptide amides as selective cathepsin L inhibitors: The first step towards selective irreversible inhibitors?

Article abstract of DOI:10.1016/j.bmcl.2013.03.041

A small library of peptide amides was designed to profile the cathepsin L active site. Within the cathepsin family of cysteine proteases, the first round of selection was on cathepsin L and cathepsin B, and then selected hits were further evaluated for binding to cathepsin K and cathepsin S. Five highly selective sequences with submicromolar affinities towards cathepsin L were identified. An acyloxymethyl ketone warhead was then attached to these sequences. Although these original irreversible inhibitors inactivate cathepsin L, it appears that the nature of the warhead drastically impact the selectivity profile of the resulting covalent inhibitors.

Full text of DOI:10.1016/j.bmcl.2013.03.041

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2968–2973
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of new peptide amides as selective cathepsin L
inhibitors: The first step towards selective irreversible inhibitors?
a
ˇ ˇ b
Ana Torkar , Brigita Lenarcic , Tamara Lah a,b, Vincent Dive c, Laurent Devel c,
⇑
a
ˇ
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia
b Faculty of Chemistry and Chemical Technology, University of Ljubljana, Cesta v Mestni log 88a, 1000 Ljubljana, Slovenia
c Service d’Ingénierie Moléculaire des Protéines (SIMOPRO), Institut de Biologie et de Technologies de Saclay (iBiTec-S), Commissariat à l’Énergie Atomique (CEA), CE-Saclay, Bât. 152,
F-91191 Gif-sur-Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A small library of peptide amides was designed to profile the cathepsin L active site. Within the cathepsin
family of cysteine proteases, the first round of selection was on cathepsin L and cathepsin B, and then
selected hits were further evaluated for binding to cathepsin K and cathepsin S. Five highly selective
sequences with submicromolar affinities towards cathepsin L were identified. An acyloxymethyl ketone
warhead was then attached to these sequences. Although these original irreversible inhibitors inactivate
cathepsin L, it appears that the nature of the warhead drastically impact the selectivity profile of the
resulting covalent inhibitors.
Received 17 January 2013
Revised 8 March 2013
Accepted 11 March 2013
Available online 20 March 2013
Keywords:
Cysteine protease
Cathepsin L
Ó 2013 Elsevier Ltd. All rights reserved.
Cathepsin B
Reversible inhibitors
Acyloxyethyl ketone inhibitors
The endopeptidase cathepsin L (CatL) belongs to the papain-like
cysteine proteases, which comprise 11 human cysteine cathepsins:
cathepsins B, C, H, F, K, L, O, S, V, W and X/Z. In spite of the great
similarities in the primary and tertiary structures among the
cathepsins, these proteases have distinct enzymatic properties, as
reflected in their different substrate specificities. Consequently
the cathepsins have different roles in normal and pathological
states,1,2 including cancers.3–5 Human6 and animal7 studies have
clearly shown over-expression of CatL in various pathological pro-
cesses, along with some of these other proteases.8–10 CatL and CatB
have been implicated in several steps of tumorigenesis and cancer
progression.5,11,12 Simultaneously increased protein levels of CatL
and CatB have been reported in breast, gastrointestinal, head and
neck, and lung cancers, and in glioblastoma and other cancers.6
In glioblastoma multiformae, which represent the most invasive
stage of glial tumour astrocytomas, CatB has been shown to be rel-
evant for tumour-cell invasion,13,14 apoptosis15 and angiogenesis.
Multivariate analyses have shown that CatB also has a strong im-
pact on prognosis.16 In contrast, the role of elevated CatL17,18 is less
clear, although it appears to be related to tumour-cell invasion, and
more likely to tumour-cell apoptosis9,19 and resistance to ther-
apy.20 A tool for the detection and selective inhibition of the active
forms of CatL would help to define its role in cancer progression, as
well as in other pathologies.
The papain-like cysteine proteases share highly conserved ac-
tive sites, with a catalytic dyad that is made up of a key nucleophile
(Cys-25) and a general acid (His-159). The superposition of various
X-ray structures of these cysteine cathepsins has provided support
0
for only three well-defined substrate-binding sites: S2, S1 and S1 .
Outside of these regions, further sub-sites are not considered to
be actual binding sites, but are more appropriately considered to
be areas in which the substrate residues find their most favourable
binding positions.1
Most of the synthetic cysteine protease inhibitors reported to
date are covalent inhibitors that exploit this extremely well-de-
fined structural context.21 Indeed, these inhibitors incorporate a
peptide or pseudo-peptide segment in their structure that corre-
sponds to the sequences of good substrates, along with an addi-
tional reactive electrophilic moiety, or ‘warhead’, that can
covalently modify the catalytic cysteine21,22 (e.g., Fig. 1, compound
A). Irreversible23–26 and reversible27–31 inhibitors are obtained
according to the chemical nature of this electrophile. Although a
number of potent cysteine protease inhibitors have been described
in the literature, achieving selectivity towards only one member of
the cathepsin family remains a challenge. In any series of covalent
inhibitors, addressing the selectivity for a given target requires the
optimisation of both the initial affinity binding step and the subse-
quent inactivation step. A direct evaluation of libraries of covalent
Abbreviations: AOMK, acyloxymethyl ketone; Cat, cathepsin.
⇑
Corresponding author. Tel.: +33 01 69089565; fax: +33 01 69089071.
E-mail address: laurent.devel@cea.fr (L. Devel).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.041

A. Torkar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2968–2973
2969
the identification of a relatively selective irreversible CatK inhibi-
tor.33 In this context, we have here evaluated the feasibility of such
a strategy to access selective covalent inhibitors of CatL.
Compared to the approach described by Choe et al.33 that relied
on the screening of fluorogenic substrates, we evaluated a library
of simple di-peptide and tri-peptide amides. This approach is sup-
ported by the work of Brinker et al.34 that led to the identification
of a peptide amide as a potent CatL inhibitor (Fig. 1, compound B;
Ki 130 nM) that can interact within the non-primed region of the
active site without being cleaved at its C-terminal position. As a
starting point, we re-explored and optimised the affinity and selec-
tivity profile of the CbzPheLys sequence, a motif that is classically
encountered in irreversible inhibitors and series of activity-based
probes.21,24 Thus, from the pseudo-peptide amide 1 (Fig. 1), chem-
ical diversity was introduced alternatively at each position (P1, P2
and P3). Several substrates and inhibitors of cysteine cathepsins
have a basic residue in the P1 position.24,33,35,36 To evaluate the pre-
cise impact of a P1 lysine on the affinity and selectivity profile to-
wards CatL, basic residues that varied in length and/or flexibility,
and also uncharged residues, were tested in the P1 position (Table 1,
compounds 2–12). The substrate specificity profiles for CatL show
a preference for bulky hydrophobic and aromatic residues in the P2
position.24,33–35,37 On the assumption that a substituted phenyl
moiety would be better tolerated within the S2 sub-site of CatL
compared to other cysteine proteases, several aromatic side chains
that varied in length and substitution were introduced in the P2 po-
sition (Table 1, compounds 13–23). Finally, as already mentioned,
the S3 and S4 substrate-binding sites of CatL are not considered
to be actual binding sites, which suggests that they are susceptible
to accommodate a large panel of various residues that might be
either hydrophobic or basic.35,38 The carboxybenzoyl moiety was
therefore replaced by various acyl moieties, and by randomly se-
lected amino acids (Table 2, compounds 24–45). This small library
Figure 1. Structures of the covalent inhibitors of the cysteine proteases (A), the
peptide amide as a potent CatL inhibitor (B), and the starting structure of the
reversible inhibitor, compound 1.
inhibitors for these targeted enzymes can be envisaged, but access
to a large set of compounds often remains difficult to implement,
mainly due to the sophisticated chemistry that is used to incorpo-
rate the reactive moieties.32 In contrast, an indirect approach
would imply initial profiling of the targeted enzyme active site
with peptide or pseudo-peptide sequences, and then providing
the selected segments with the necessary warhead. Such an ap-
proach represents a major advantage for easy and rapid screening
of a large set of compounds with high chemical diversity using
standard peptide chemistry. However, this strategy does not allow
the simultaneous profiling of primed and non-primed regions.
Such an approach was recently reported for CatK, which led to
Table 1
Effects of the P1 and P2 substituent on affinity towards CatL and CatB
b
P1
P2
Kia
(lM) CatL
Kia
(
lM) CatB
FB
1
2
3
4
5
6
7
8
–(CH2)4–NH2
–(C6H5)
–(C6H5)
–(C6H5)
–(C6H5)
–(C6H5)
–(C6H5)
–(C6H5)
–(C6H5)
–(C6H5)
–(C6H5)
–(C6H5)
–(C6H5)
4.2
2.6
27
3.5
28
26
28
14
64
14
17
16
3406
1547
1734
2460
ND
2787
349
ND
810
595
64
703
NDc
107
12
–(CH2)4–NH–CH3
–(CH2)3–CH(OH)–NH2
–(CH2)3–NH2
–NH2
–(CH2)–(4-NH2–C6H4)
–(CH2)3–NH–(C@NH)–NH2
–(CH2)-1H-Imidazol
–(CH2)2–S–CH3
–(CH2)–(CH)–(CH3)2
–(CH)–(CH3)2
NDc
111
310
NDc
NDc
9
7096
4341
ND
10
11
12
–CH3
ND
13
14
15
16
17
18
19
20
21
22
23
–(CH2)4–NH2
–(CH2)4–NH2
–(CH2)4–NH2
–(CH2)4–NH2
–(CH2)4–NH2
–(CH2)4–NH2
–(CH2)4–NH2
–(CH2)4–NH2
–(CH2)4–NH2
–(CH2)4–NH2
–(CH2)4–NH2
–(C6H4)–p-(C6H5)
2.4
77
38
58
389
245
113
74
39
56
32
21
34
156
11
66
28
325
18
24
96
–(C6H4)–p-(3-NH2–C6H4)
–(C6H4)–p-(3-Cl–C6H4)
–(C6H4)–p-(3-CH3–C6H4)
–(C6H4)–p-(2-CH3–C6H4)
–(C6H4)–p-(4-CH3–C6H4)
–(C6H4)–p-(C6H4)–p-(C6H5)
–(C6H4)–p-(2-Thiophen)
1H-Imidazol
1.8
1.7
2.5
23
1.7
2.6
0.12
3.1
4.2
1.4
1H-Indol
–(4-OH–C6H4)
100
135
Bold values refer to selectivity factors of 3 orders of magnitude by comparison with those of only 2 orders or less.
a
Ki values were determined at a minimum of three concentrations of inhibitors (measured in duplicates).
FB = Ki(CatB)/Ki(CatL).
ND, not determined.
b
c

2970
A. Torkar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2968–2973
Table 2
Table 2 (continued)
Effects of the P3 substituent on the affinity towards CatL and CatB
b
P3
Kia
(lM) CatL
Kia
(lM) CatB
FB
41
42
0.39
0.45
835
2141
2633
1185
b
P3
Kia
(l
M) CatL
Kia
(l
M) CatB
FB
43
0.74
1303
1761
1
4.2
1
3406
312
811
312
24
25
20
3387
169
44
45
0.89
1494
3545
1679
506
26
27
9
1
84
9
301
301
7
28
29
2.9
2.3
26
9
Bold values refer to selectivity factors of 3 orders of magnitude by comparison with
those of only 2 orders or less.
261
113
a
Ki values were determined at a minimum of three concentration of inhibitors
(measured in duplicates).
b
30
31
2.4
2.6
1020
706
425
272
FB = Ki(CatB)/Ki(CatL).
of pseudo di-peptides and tri-peptides was synthesised on rink
amide lanterns (MimotopesÒ) using standard Fmoc-based solid-
phase peptide synthesis. To access differently substituted biphe-
nyl-like side chains in the P2 position, post-modification was car-
ried out on the 4-iodo-phenyl side-chain on a solid support,
using Suzuki coupling conditions and under microwave irradia-
tion37 (see Supplementary data for details). The peptides were then
individually cleaved from the lanterns using trifluoroacetic acid/
triisopropylsilane/water (95/2.5/2.5), and purified by reverse-
phase HPLC. All of the peptides were dissolved in dimethylsulph-
oxide as trifluoroacetic acid salts and characterised by ion-spray
mass spectrometry, with their purities assessed by analytical HPLC
(see Supplementary data, Table S1). The concentration of each of
the stock solutions was determined by amino-acid measurement.
In the primary screening, these peptide amides were evaluated in
kinetic studies, using recombinant CatL and CatB (see Supplemen-
tary data, experimental procedures). For these compounds, a selec-
tivity factor FB corresponding to Ki(CatB)/Ki(CatL) was defined
(Tables 1 and 2). The selected inhibitors that showed significant
selectivity (FB >1000) and affinity profiles towards CatL were fur-
ther tested on two other cysteine cathepsins, CatK and CatS. This
led the determination of factors FS and FK corresponding to Ki(-
CatS)/Ki(CatL) and Ki(CatK)/Ki(CatL), respectively (Table 3).
32
33
2.7
6.8
1757
134
651
20
34
35
5.5
5
80
15
30
148
36
4.6
860
187
37
38
6.1
7.8
51
8.4
623
80
Among all of the compounds with a variable P1 position (Table 1,
compounds 2–12), only compounds 2 and 4 showed potency and
selectivity comparable to peptide 1 towards CatL. These two com-
pounds have an N-methylated lysine side-chain and a shorter side-
chain in the P1 position, respectively. A long and flexible basic side
chain in the P1 position was required to maintain substantial affin-
39
40
3.2
2.5
2035
135
636
54

A. Torkar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2968–2973
2971
Table 3
Selectivity and affinity profiles for compounds 1, 20, 41–44 towards CatL, CatB, CatS and CatK
b
c
d
Kia
(
lM) CatL
Kia
(
lM) CatB
Kia
(
lM) CatS
Kia
(lM) CatK
FB
FS
FK
1
4.2
3406
39
835
1185
1303
1494
56
28
20
12
8.5
41.1
47
811
325
2141
2633
1761
1679
13
233
51
27
11
11
20
41
42
43
44
0.12
0.39
0.45
0.74
0.89
24
639
213
147
284
200
1638
473
198
319
46
Bold values refer to selectivity factors of 3 orders of magnitude by comparison with those of only 2 orders or less.
a
Ki values were determined from a minimum of four concentration of inhibitors (measured in duplicates).
FB = Ki(CatB)/Ki(CatL).
FS = Ki(CatS)/Ki(CatL).
FK = Ki(CatK)/Ki(CatL).
b
c
d
Table 4
Association constants (kass) of irreversible inhibitors A-AOMK, 46–49 for CatL and CatB
kass (sꢀ1 Mꢀ1) CatL
kass (sꢀ1 Mꢀ1) CatB
kass CatL/kass CatB
a
a
A-AOMK
2.96 ꢁ 105
3.92 ꢁ 105
2.69 ꢁ 105
0.38 ꢁ 105
7.8
46
0.57 ꢁ 105
6.9
47
1.36 ꢁ 105
2.0
48
8.75 ꢁ 105
0.76 ꢁ 105
11.5
49
2.52 ꢁ 105
8.22 ꢁ 105
0.3
a
kass were determined by the progression of the curve analysis under the pseudo-first-order conditions, with at least 10-fold molar excess of the inhibitor over the enzyme.
ity for CatL, as illustrated by the loss of potency for compounds 5
and 6, which have a simple amino function and an aniline-like
side-chain in the P1 position, respectively. Compound 7 has a P1
arginine, and it did not show any selectivity for the CatL active site
over that of CatB. These data are in agreement with the literature,
where it has been reported that a P1 arginine residue promotes the
activity of all cysteine proteases.33 Although uncharged residues
can interact with the CatL S1 sub-site, the corresponding peptides
(compounds 9–12) were less potent towards CatL. Overall, these
data confirm those from the literature: a free lysine group at the

2972
A. Torkar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2968–2973
P1 position is necessary to maintain a favourable affinity and selec-
tivity profile towards CatL.24
orders of magnitude (41–44) were selected for further modifica-
tion. It was hypothesised that only sequences with a marked gain
of selectivity compared to the sequence of reference 1 would sig-
nificantly impact the selectivity profile of the resulting irrevers-
ible inhibitors. The acyloxymethyl ketone (AOMK) warhead was
chosen as a quiescent reactive moiety. Compounds containing
AOMK were first reported by Krantz et al.,39 who claimed that
they are clinically useful inhibitors of cysteine cathepsins as they
are stable in human serum, non-toxic for cells, and soluble and
cell permeant.21,24 Four covalent irreversible inhibitors, AOMK
derivatives of compounds 41–44, compounds 46–49 were thus
synthesised as previously described.40 It appeared that, whatever
was the peptide sequence, the resulting AOMK derivatives inacti-
vate both CatL and CatB in a one-step process. In such situation,
affinity binding constants (Ki) and inactivation rate (kinact) were
not accessible as the adsorptive EI complex is kinetically irrele-
vant. Thus only association constants (kass) towards CatL and CatB
can be determined (Table 4 and see Supplementary data, Fig. S2
for details). Hence no correlation between the reversible and irre-
versible inhibitors can be established in regards to their selectiv-
ity profiles towards CatB and CatL. Our data clearly highlight the
major influence of the warhead on the selectivity profile. Beyond
the steric hindrance of the AOMK moiety, which will impact nec-
essarily on the inhibitor binding, a warhead with a relatively high
intrinsic reactivity will tend to react equally with the conserved
nucleophile that adopts a similar orientation within all the cys-
teine protease active sites. Consequently, this might have unfa-
vourable effects on the selectivity profiles towards these
enzymes. Significantly and despite similar factor of selectivity FB
for compounds 43 and 44 (Table 3), the resulting AOMK inhibi-
tors 48 and 49, respectively, displayed very different profiles of
selectivity (Table 4). Our data clearly suggest that the selected
warhead was not appropriate to draw a general conclusion on
the selectivity changed between the peptides and their AOMK
analogues.
With the exception of compound 17, all of the peptides with a
P2 biphenyl-like side chain (Table 1, compounds 13–18) were as
potent as compound 1 towards CatL, and were significantly more
potent inhibitors of CatB, thus reducing their selectivity factors
with regard to CatL. The same occurred for the compounds with
a P2 triphenyl side chain (compound 19) and with other aromatic
moieties (compounds 21–23). Interestingly, compound 20, which
has a P2 phenyl thiophen side-chain, showed a significant, 35-fold
increase in affinity for CatL compared to reference peptide 1, with a
Ki of 120 nM. Although compound 20 also showed a 87-fold gain in
the potency for CatB, the selectivity factor remained largely in fa-
vour of CatL (FB = 325). Overall, compared to peptide 1 with a P2
phenyl moiety, none of the modifications introduced at the P2 po-
sition resulted in peptides with any significantly increased selec-
tivity for CatL.
In contrast, the P3 and P4 positions appeared to be critical for
the modulation of both the inhibitor affinity and selectivity to-
wards CatL and CatB (Table 2, compounds 24–45). The introduc-
tion of various acyl moieties in the P3 position (compounds 24–
40) only had a moderate impact on the inhibitor potency for CatL.
In contrast, the affinity of these peptides towards CatB ranged
from millimolar concentrations to 26 lM for the most potent, as
compound 28. On the other hand, the inclusion of N-carbo-
xybenzoyl protected amino acids in the P3 position, such as tryp-
tophane in compound 41, leucine in compound 42, arginine in
compound 43 and lysine in compound 44, led to potent inhibitors
with sub-micromolar affinities for CatL. Surprisingly, these modi-
fications had almost no impact on their affinities for CatB. This
led to the identification of four compounds (41–44) with selectiv-
ity factors FB that showed three orders of magnitude in their
selectivity for CatL (Table 2).
In spite of the topologically comparable non-primed regions be-
tween the CatL and CatB active sites, we successfully identified five
original short peptides that can interact potently with CatL while
maintaining an excellent selectivity profile towards CatL (com-
pounds 20, 41–44). The affinities of these compounds towards
two other members of the papain-family proteases, CatS and CatK,
were also determined, as shown in Table 3. Interestingly, despite
the canonical cathepsin cysteine specificity for aromatic residues
in the P2 position, it appeared that elongating the P2 phenyl moiety
with a thiophen heterocycle did not have the same effects on CatL
and CatB. There was a marked gain of affinity for CatL compared to
CatK and CatS, where there were almost no effects on affinity (Ta-
ble 3, compound 20). This pseudo-peptide thus shows favourable
affinity and selectivity for CatL with selectivity factors of two or-
ders of magnitude over CatB, CatS and CatK. Comparisons of their
S2 regions did not highlight any marked topological differences
that might explain these data (Supplementary data, Fig. S1). Com-
pared to peptide 1, an additional N-carboxybenzoyl-protected ami-
no acid in the P3 position led to compounds with slightly improved
potency toward CatS, but which showed lower affinity for CatK
(Table 3, compounds 41–44). When analysing the topological dif-
ferences between the S3 regions of CatL, CatB, CatS and CatK, it ap-
peared that CatK, and to a lesser extent CatB, has a narrower S3
sub-site compared to CatL and CatS (Supplementary data,
Fig. S1). These observations suggested that a bulky P3 substituent
would not correctly enter the S3 sub-sites in CatK and CatB, which
might explain the poor affinity of compounds 41–44 towards CatK
and CatB. However, although they show poor selectivity factors
with regard to CatS, compounds 41–44 retained their selectivity
for CatL versus CatK and CatB.
In the present study, we have identified original di-peptide and
tri-peptide motifs as potent and selective reversible CatL inhibi-
tors. These peptide sequences might be useful for the future devel-
opment of covalent inhibitors directed towards CatL. However,
further covalent reversible and irreversible warheads have to be
screened to evaluate the precise impact of such peptide sequence
optimisation. Finally, since most of the cathepsins are intracellular
proteases, the real selectivity profile of inhibitors has to be as-
sessed in cellular assays. Selective inhibitors against purified
cathepsins might display different and in some cases diminished
selectivity in cells due to lysosomotropism, which is especially true
for peptide sequence containing basic residue such as compounds
43 and 44.41
Acknowledgments
This study was supported by Grants of Bilateral Collaboration
between the CEA and the Research Agency of Slovenia (ARRS): BI
FR/CEA10-12-002 to T.L. and V.D., and by ARRS Project J1-0295
to T.L. The CEA supported Ana Torkar with a scholarship during
her stay in France.
Supplementary data
Supplementary data (the synthesis of the di-peptides and tri-
peptides and the acyloxymethyl ketones, the in vitro enzyme as-
says, the analytical characterisation of all of the synthesised mole-
cules, and the superimposition of the cathepsin active sites)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2013.03.041.
To follow the idea of progressing from reversible peptides to
covalent inhibitors with optimised affinity and selectivity toward
CatL, only the compounds displaying selectivity factors FB of three

A. Torkar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2968–2973
2973
24. Blum, G.; Mullins, S. R.; Keren, K.; Fonovic, M.; Jedeszko, C.; Rice, M. J.; Sloane,
B. F.; Bogyo, M. Nat. Chem. Biol. 2005, 1, 203.
References and notes
25. Mendieta, L.; Picó, A.; Tarragó, T.; Teixidó, M.; Castillo, M.; Rafecas, L.; Moyano,
A.; Giralt, E. Chem. Med. Chem. 2010, 5, 1556.
26. Verhelst, S. H.; Witte, M. D.; Arastu-Kapur, S.; Fonovic, M.; Bogyo, M.
ChemBioChem 2006, 7, 943.
27. Lynas, J. F.; Hawthorne, S. J.; Walker, B. Bioorg. Med. Chem. Lett. 2000, 10, 1771.
28. Myers, M. C.; Shah, P. P.; Beavers, M. P.; Napper, A. D.; Diamond, S. L.; Smith, A.
B., III; Huryn, D. M. Bioorg. Med. Chem. Lett. 2008, 18, 3646.
29. Shah, P. P.; Myers, M. C.; Beavers, M. P.; Purvis, J. E.; Jing, H.; Grieser, H. J.;
Sharlow, E. R.; Napper, A. D.; Huryn, D. M.; Cooperman, B. S.; Smith, A. B. I.;
Diamond, S. L. Mol. Pharm. 2008, 74, 34.
30. Asaad, N.; Bethel, P. A.; Coulson, M. D.; Dawson, J. E.; Ford, S. J.; Gerhardt, S.;
Grist, M.; Hamlin, G. A.; James, M. J.; Jones, E. V.; Karoutchi, G. I.; Kenny, P. W.;
Morley, A. D.; Oldham, K.; Rankine, N.; Ryan, D.; Wells, S. L.; Wood, L.;
Augustin, M.; Krapp, S.; Simader, H.; Steinbacher, S. Bioorg. Med. Chem. Lett.
2009, 19, 4280.
31. Kishore kumar, G. D.; Chavarria, G. E.; Charlton-Sevick, A. K.; Arispe, W. M.;
Macdonough, M. T.; Strecker, T. E.; Chen, S. E.; Siim, B. G.; Chaplin, D. J.;
Trawick, M. L.; Pinney, K. G. Bioorg. Med. Chem. Lett. 2010, 20, 1415.
32. Arastu-Kapur, S.; Ponder, E. L.; Fonovic, U. P.; Yeoh, S.; Yuan, F.; Fonovic, M.;
Grainger, M.; Phillips, C. I.; Powers, J. C.; Bogyo, M. Nat. Chem. Biol. 2008, 4, 203.
33. Choe, Y.; Leonetti, F.; Greenbaum, D. C.; Lecaille, F.; Bogyo, M.; Bromme, D.;
Ellman, J. A.; Craik, C. S. J. Biol. Chem. 2006, 281, 12824.
34. Brinker, A.; Weber, E.; Stoll, D.; Voigt, J.; Muller, A.; Sewald, N.; Jung, G.;
Wiesmuller, K. H.; Bohley, P. Eur. J. Biochem. 2000, 267, 5085.
35. Biniossek, M. L.; Nagler, D. K.; Becker-Pauly, C.; Schilling, O. J. Proteome Res.
2011, 10, 5363.
36. Puzer, L.; Cotrin, S. S.; Alves, M. F.; Egborge, T.; Araujo, M. S.; Juliano, M. A.;
Juliano, L.; Bromme, D.; Carmona, A. K. Arch. Biochem. Biophys. 2004, 430, 274.
37. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
1. Turk, D.; Guncar, G. Acta Crystallogr., Sect. D 2003, 59, 203.
2. Turk, B.; Turk du, S. A.; Turk, V. EMBO J. 2012, 31, 1630.
3. Gocheva, V.; Zeng, W.; Ke, D.; Klimstra, D.; Reinheckel, T.; Peters, C.; Hanahan,
D.; Joyce, J. A. Genes Dev. 2006, 20, 543.
4. Lah, T. T.; Duran Alonso, M. B.; Van Noorden, C. J. Expert Opin. Biol. Ther. 2006, 6,
257.
5. Mohamed, M. M.; Sloane, B. F. Nat. Rev. Cancer 2006, 6, 764.
6. Lah, T.; Obermajer, N.; Duran Alonso, M. B.; Kos, J. In The Cancer Degradome:
Proteases and Cancer Biology; Edwards, D., Hoyer-Hansen, G., Blasi, F., Sloane, B.
F., Eds.; Springer: New York, 2008; p 585.
7. Reinheckel, T.; Deussing, J.; Roth, W.; Peters, C. Biol. Chem. 2001, 382, 735.
8. Reiser, J.; Adair, B.; Reinheckel, T. J. Clin. Invest. 2010, 120, 3421.
9. Goulet, B.; Sansregret, L.; Leduy, L.; Bogyo, M.; Weber, E.; Chauhan, S. S.;
Nepveu, A. Mol. Cancer Res. 2007, 5, 899.
10. Lankelma, J. M.; Voorend, D. M.; Barwari, T.; Koetsveld, J.; Van der Spek, A. H.;
De Porto, A. P.; Van Rooijen, G.; Van Noorden, C. J. Life Sci. 2010, 86, 225.
11. Joyce, J. A.; Baruch, A.; Chehade, K.; Meyer-Morse, N.; Giraudo, E.; Tsai, F. Y.;
Greenbaum, D. C.; Hager, J. H.; Bogyo, M.; Hanahan, D. Cancer Cell 2004, 5,
443.
12. Sevenich, L.; Werner, F.; Gajda, M.; Schurigt, U.; Sieber, C.; Muller, S.; Follo, M.;
Peters, C.; Reinheckel, T. Oncogene 2010, 30, 54.
13. Levicar, N.; Nuttall, R. K.; Lah, T. T. Acta Neurochir. (Wien) 2003, 145, 825.
14. Gole, B.; Duranalonso, M. B.; Dolenc, V.; Lah, T. Pathol. Oncol. Res. 2009, 15, 711.
15. Malla, R.; Gopinath, S.; Alapati, K.; Gondi, C. S.; Gujrati, M.; Dinh, D. H.;
Mohanam, S.; Rao, J. S. PLoS ONE 2010, 5, e13731.
16. Colin, C.; Voutsinos-Porche, B.; Nanni, I.; Fina, F.; Metellus, P.; Intagliata, D.;
Baeza, N.; Bouvier, C.; Delfino, C.; Loundou, A.; Chinot, O.; Lah, T.; Kos, J.;
Martin, P. M.; Ouafik, L.; Figarella-Branger, D. Acta Neuropathol. 2009, 118, 745.
17. Strojnik, T.; Kavalar, R.; Trinkaus, M.; Lah, T. T. Cancer Detect. Prev. 2005, 29,
448.
18. Gole, B.; Huszthy, P. C.; Popovic, M.; Jeruc, J.; Ardebili, Y. S.; Bjerkvig, R.; Lah, T.
T. Int. J. Cancer 2012, 131, 1779.
19. Kenig, S.; Frangez, R.; Pucer, A.; Lah, T. Apoptosis 2011, 16, 671.
20. Zheng, X.; Chu, F.; Chou, P. M.; Gallati, C.; Dier, U.; Mirkin, B. L.; Mousa, S. A.;
Rebbaa, A. Am. J. Physiol. Cell Physiol. 2009, 296, C65.
38. Fujishima, A.; Imai, Y.; Nomura, T.; Fujisawa, Y.; Yamamoto, Y.; Sugawara, T.
FEBS Lett. 1997, 407, 47.
39. Krantz, A.; Copp, L. J.; Coles, P. J.; Smith, R. A.; Heard, S. B. Biochemistry 1991, 30,
4678.
40. Kato, D.; Boatright, K. M.; Berger, A. B.; Nazif, T.; Blum, G.; Ryan, C.; Chehade, K.
A.; Salvesen, G. S.; Bogyo, M. Nat. Chem. Biol. 2005, 1, 33.
41. Falgueyret, J. P.; Desmarais, S.; Oballa, R.; Black, W. C.; Cromlish, W.; Khougaz,
K.; Lamontagne, S.; Masse, F.; Riendeau, D.; Toulmond, S.; Percival, M. D. J. Med.
Chem. 2005, 48, 7535.
21. Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Chem. Rev. 2002, 102, 4639.
22. Lecaille, F.; Kaleta, J.; Bromme, D. Chem. Rev. 2002, 102, 4459.
23. Schaschke, N.; Assfalg-Machleidt, I.; Machleidt, W. ChemBioChem 2008, 9, 1721.