Identification and Optimization of Novel Cathepsin C Inhibitors Derived from EGFR Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b00631

In the course of developing the biochemistry to chemistry activity-based protein profiling (BTC-ABPP) method, we herein unexpectedly discovered that the epidermal growth factor receptor irreversible inhibitor WZ4002 also functioned as a low micromolar inhibitor of cathepsin C (CatC), a promising target for the treatment of numerous inflammatory and autoimmune diseases. Building on from this discovery, and following structure-activity relationship investigations guided by computational modeling, a novel series of pyridine scaffold compounds were developed as irreversible CatC inhibitors, further culminated in identifying a highly potent and selective inhibitor 22, which displays good metabolic stability and oral bioavailability. In vivo studies revealed that compound 22 clearly displays the ability to inhibit CatC, consequently leading to efficient inhibition of downstream neutrophil serine proteases in both bone marrow and blood. The overall excellent profile of compound 22 made it an interesting candidate for further preclinical investigation.

Full text of DOI:10.1021/acs.jmedchem.9b00631

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Article
Identification and Optimization of Novel Cathepsin
C Inhibitors Derived from EGFR Inhibitors
Weijie Hou, Huan Sun, Yongfen Ma, Chunyan Liu, and Zhiyuan Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00631 • Publication Date (Web): 30 May 2019
Downloaded from http://pubs.acs.org on May 30, 2019
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Identification and Optimization of Novel Cathepsin
C Inhibitors Derived from EGFR Inhibitors
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‡,†
‡,†
†
†
,†
Weijie Hou, Huan Sun, Yongfen Ma, Chunyan Liu, and Zhiyuan Zhang*
†National Institute of Biological Sciences (NIBS), Beijing 102206, China
ABSTRACT: In the course of developing the biochemistry to chemistry activity-based
protein profiling (BTC-ABPP) method, we herein unexpectedly discovered that the EGFR
irreversible inhibitor WZ4002 also functioned as a low micromolar inhibitor of cathepsin C
(CatC), a promising target for the treatment of numerous inflammatory and autoimmune
diseases. Building on from this discovery, and following structure–activity relationship (SAR)
investigations guided by computational modeling, a novel series of pyridine scaffold compounds
were developed as irreversible CatC inhibitors, further culminated in identifying a highly potent
and selective inhibitor 22, which displays good metabolic stability and orally bioavailability. In
vivo studies revealed that compound 22 clearly displays the ability to inhibit CatC, consequently
leads to efficient inhibition of downstream neutrophil serine proteases in both bone marrow and
blood. The overall excellent profile of compound 22 made it an interesting candidate for further
pre-clinical investigation.
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INTRODUCTION
Cathepsin C (CatC), also known as dipeptidyl peptidase 1 (DPP1), is an important
1
lysosomal cysteine protease that can activate granule associated proinflammatory serine
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proteases including neutrophil elastase (NE), cathepsin G (CatG), and proteinase 3 (PR3). Upon
such activation, these neutrophil serine proteases (NSPs) are secreted into the extracellular space,
where their hydrolysis activity protects host cells from invading pathogens.2, 3 However, in
certain cases, excessive recruitment of neutrophils can increase the secretion of NSPs to such an
extent that they become destructive even after the clearance of pathogens have been completed,
thereby driving the pathogenesis of several disorders. Specifically, NSPs participate in the
progression of a variety of chronic inflammatory diseases such as rheumatoid arthritis,4, 5
sepsis,6 and anti-neutrophil cytoplasmic antibody (ANCA)-associated necrotizing crescentic
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glomerulonephritis (NCGN). Further, chronic respiratory disorders including COPD (chronic
obstructive pulmonary disease; the fourth leading cause of death globally in 2010 and rose to the
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third position in 2016 ), bronchiectasis, and cystic fibrosis are also now understood to be
NSPs–related. Owing to its function in activating NSPs, CatC is now viewed as a promising
therapeutic target for a variety of NSPs-related diseases.11
Biological studies have shown that CatC knock-out (KO) mice, and CatC inhibitor treated
wild-type (WT) mice, demonstrate protection from smoke-induced pulmonary inflammation and
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associated alveolar destruction. These observations support the hypothesis that CatC is viewed
as a therapeutic target against COPD. Human CatC is known to occur as a homo-tetrameric
complex comprising four identical subunits, each of which contains a light chain, a heavy chain,
1
and an exclusion domain (unique to CatC amongst the cathepsin family proteases). As a
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peptidase, CatC removes dipeptide moieties from the N-terminal of the substrates. Studies have
shown that the aspartic acid at position 1 of the N-terminal exclusion domain keeps the
exopeptidase activity of CatC via its interaction with α-amino groups of peptide substrates;
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moreover, Cys234 has been demonstrated as a key catalytic residue in the active site.14
A number of CatC inhibitors have been developed in recent decades; most of these are
peptide mimics derived from known preferred dipeptide substrates of CatC, including
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16, 17
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irreversible inhibitors such as diazoketones, vinyl sulfone,
O-acyl-hydroxamates, as well
as reversible covalent inhibitors with nitrile groups18-21 as warheads. In the development of CatC
inhibitors, metabolic stability given by their peptidic and electrophilic nature may be a serious
issue.14, 22 To date, only two drug candidates entered clinical trials: GSK-279366023-25
(
terminated in phase I) and INS1007/AZD798626, 27 (currently in phase II) (Figure 1), and their
chemotypes are dipeptide derived.
O
N
O
H
O
N
H
N
N
HN
H2N
N
O
O
O
O
GSK-2793660
INS1007/AZD7986
Figure 1. Chemical structures of GSK-2793660 and INS1007/AZD7986.
In the course of a BTC-ABPP study using an activity-based probe derived from an
irreversible EGFR inhibitor (Canertinib) to study off-target effects, we identified a probe-
modified peptide from CatC, thus indicating binding to this protease potentially via to the
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catalytic cysteine. Subsequently, results from both enzymatic assay and cell assays confirmed
that Canertinib was indeed a CatC inhibitor, and a small screen of other irreversible EGFR
inhibitors identified WZ4002 as an additional CatC inhibitor. Docking studies informed our
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proposal of a plausible binding mode for WZ4002 within the CatC active site, and subsequent
rational design efforts based on this mode, alongside SAR studies, ultimately enabled the
identification of a low nanomolar inhibitor. Extensive in vitro/in vivo assays demonstrate that the
candidate compound 22 from our study is highly metabolically stable, exhibits potent CatC
inhibitory effects, and results in the anticipated inhibition on the activation of downstream NSPs
in mice.
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RESULTS AND DISCUSSION
Chemistry. The preparation of this series compounds was achieved using the synthetic routes
illustrated in Scheme 1–6. The synthetic routes of compounds 1–8 started from 3-nitrophenol. 3-
Nitrophenol was regioselectively introduced to the C-2 position of 2,3,6-trichloropyridine to
afford the intermediate 36. Amination of C-6 with different substituted anilines was
accomplished through palladium-catalyzed Buchwald-Hartwig coupling. The nitro group in 38a–
f was reduced by iron power/ammonium chloride. Acylation of 39a–f by acryloyl chloride gave
rise to compounds 1, 2, 4, and 40d–f. The subsequent step of BOC de-protection afforded
compounds 3–6. The analogs of 10–11 were obtained in a similar manner, using an appropriate
substituted pyridine in step a of Scheme 2.
The synthetic routes of compounds 14, 17–20 and 21–26 are illustrated in Scheme 3 and 4, and
started from 3-bromo-2,6-dichloropyridine. For preparation of compounds 14, and 17-20,
appropriate phenols were regioselectively introduced to the C-2 position of 3-bromo-2,6-
dichloropyridine to afford the intermediate 47a–e. Amination of C-3 with pyrrolidine, and
subsequent amination of C-6 with 2-methoxy-4-(4-methylpiperazin-1-yl) aniline, were
accomplished through palladium-catalyzed Buchwald-Hartwig coupling. The nitro group in 49a–
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e was reduced by PtO /H . Acylation of 50a–e by acryloyl chloride gave rise to compounds 14,
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2
and 17–20. The analogs of 21–26 and 27–31 were obtained in a similar manner, using the
appropriate amines in step b of Scheme 4 and appropriate substituted anilines in step a of scheme
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1
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, respectively. Compounds 32 and 33 were synthesized as shown in scheme 6.
Scheme 1. Synthesis of compounds 1–8a
NO2
NO2
NH2
37a-39a R6 2-methoxy-4-(4-methylpiperazin-1-yl)
NH2
37a-f
NO2
Cl
37b-39b R6 4-(4-methylpiperazin-1-yl)
R6
Cl
O
O
O
N
37c-39c R6 4-morpholine
37d-39d R6 4-(4-(tert-butoxycarbonyl)piperazin-1-yl)
Cl
Cl
Cl
a
N
b
N
N
OH Cl
R6
c
R6
7e-39e R6 4-(2-tert-butoxycarbonyl)aminoethyl
3
Cl
N
H
N
H
37f-39f R6 2-(2-tert-butoxycarbonyl)aminomethyl
34
35
36
38 a-f
39 a-f
O
O
O
NH
NH
NH
O
O
O
Cl
Cl
N
R6
N
Cl
R6
N
R6
e
f
d
N
N
H
H
N
H
R6 2-methoxy-4-(4-methylpiperazin-1-yl)
R6 4-(4-methylpiperazin-1-yl)
6
1
2
4
4
4
4
3 R 4-piperazine
7 R6 4-(4-cyclopropylmethyl)piperazin-1-yl)
8 R6 4-(4-cyclopentylmethyl)piperazin-1-yl)
5 R6 4-(2-aminoehtyl)
R6 4-morpholine
6 R6 3-aminomethyl
0d R6 4-(4-(tert-butoxycarbonyl)piperazin-1-yl)
0e R6 4-(2-tert-butoxycarbonyl)aminoethyl
0f R6 2-(2-tert-butoxycarbonyl)aminomethyl
a
Reagents and conditions: (a) Cs CO , DMF, 110℃; (b) Cs CO , BINAP, Pd(OAc) , dioxane, microwave,
2
3
2
3
2
1
30℃, 1h; (c) Fe, NH Cl, EtOH/THF/H O, 80℃; (d) Acryloyl chloride, TEA, DCM, 0℃, 1h; (e) TFA,
4 2
DCM, RT, 2h; (f) (bromomethyl)cyclopropane or (bromomethyl)cyclopentane, K CO , 70℃, 4h.
2
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Scheme 2. Synthesis of compounds 10 and 11a
O
NH2
NO2
NO2
NH
NO2
Cl
N
O
R7
O
N
O
N
O
N
R7
R7
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N
R7
R7
N
N
N
N
N
N
a
b
c
d
OH Cl
N
H
Cl
N
H
N
H
41a R7 = CF3
1b R7 = CN
OMe
OMe
OMe
34
42a R7
= CF3
43a R7 = CF3
3b R7 = CN
44a R7 = CF3
44b R7 = CN
4
2b R7 = CN
10
7
4
R
= CF3
4
11 R7 = CN
a
Reagents and conditions: (a) Cs CO , DMF, 110℃; (b) 2-methoxy-4-(4-methylpiperazin-1-yl)aniline,
2
3
Cs CO , BINAP, Pd(OAc) , dioxane, microwave, 130℃, 1h; (c) Fe, NH Cl, EtOH/THF/H O, 80℃; (d)
2
3
2
4
2
Acryloyl chloride, TEA, DCM, 0℃, 1h.
Scheme 3. Synthesis of compounds 14 and 17-20a
NO2
NO2
NO2
3
NH2
3
3
3
4
2
4
2
4
2
R8
5
4
2
R8
R8
5
Cl
NO2
3
R8
5
N
1
O
6
Br
4
2
1
6
O
1
6
O
5
1
O
6
N
N
N
N
R8
Br
N
N
N
N
N
N
d
5
a
b
c
N
Cl
1
OH
6
N
Cl
Cl
H
N
OMe
45
46a-e
47a-e
48a-e
H
4
9a-e
OMe
50a-e
4
6a-50a R8
H
O
46b-50b R8 2-Me
6c-50c R8 6-Me
46d-50d R8 4-Me
46e-50e R8 4-OMe
4
NH
3
4
2
R8
8
14 R
H
5
8
N
1
O
17 R 2-Me
18 R8 6-Me
e
6
N
N
N
9 R8 4-Me
20 R8 4-OMe
1
N
H
OMe
a
Reagents and conditions: (a) Cs CO , DMF, 110℃; (b) pyrrolidine, Cs CO , BINAP, Pd (dba) , toluene,
2
3
2
3
2
3
1
10 ℃ ; (c) 2-methoxy-4-(4-methylpiperazin-1-yl)aniline, Cs CO , BINAP, Pd(OAc) , dioxane,
2 3 2
microwave, 130℃, 1h; (d) PtO , H , MeOH/THF, RT, overnight; (e) Acryloyl chloride, TEA, DCM, 0℃,
2
2
1
h.
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Scheme 4. Synthesis of compounds 21–26a
O
NO2
NH2
NH
NO2
NO2
N
O
N
O
N
O
O
O
N
R9
N
R9
R9
N
R8
N
N
N
Br
N
N
b
c
d
a
N
H
N
H
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
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0
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0
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9
0
Cl
Cl
OMe
OMe
H
OMe
53a-f
47d
51a-f
52a-f
54a R9
2 R9 piperidin-1-yl
1a-53a R9 3-((tert-butyldimethylsilyl)oxy)pyrrolidin-1-yl
3-((tert-butyldimethylsilyl)oxy)pyrrolidin-1-yl
5
O
51b-53b R9 piperidin-1-yl
2
9
51c-53c R9 3-((tert-butyldimethylsilyl)oxy)piperidin-1-yl
54c R
3-((tert-butyldimethylsilyl)oxy)piperidin-1-yl
NH
9
1d-53d R9 morpholino-1-yl
24 R morpholino-1-yl
5
25 R9
26 R9
1e-53e R9 4-methylpiperazin-1-yl
4-methylpiperazin-1-yl
phenyl
5
51f-53f R9 phenyl
N
O
R9
N
N
e
N
H
OMe
2
1 R9 3-hydroxypiperidin-1-yl
23 R9 3-hydroxypyrrolidin-1-yl
a
Reagents and conditions: (a) Cs CO , BINAP, Pd (dba) , toluene, 110℃ or Pd(PPh ) , Na CO (2M),
2
3
2
3
3 4
2
3
1
,2-dimethoxyethane, 80 ℃ ; (b) 2-methoxy-4-(4-methylpiperazin-1-yl)aniline, Cs CO , BINAP,
2
3
Pd(OAc) , dioxane, microwave, 130 ℃ , 1h; (c) PtO , H , MeOH/THF, RT, overnight; (d) Acryloyl
2
2
2
chloride, TEA, DCM, 0℃, 1h; (e) TBAF, THF, 0℃-RT, 2h.
Scheme 5. Synthesis of compounds 27–31a
R10
:
N
N
N
N
O
N
NO2
NH2
55b-57b, 28
O
NO2
NH
55a-57a, 27
O
O
O
N
N
N
N
O
O
R10 NH2
N
N
N
N
N
O
N
O
N
a
N
b
c
N
55c-57c, 29
55d-57d, 30
R10
R10
R10
N
N
Cl
N
H
H
H
55a-e
56a-e
57a-e
N
N
5
1b
27-31
5
5e-57e, 31
a
Reagents and conditions: (a) Cs CO , BINAP, Pd(OAc) , dioxane, microwave, 130℃, 1h; (b) PtO , H ,
2
3
2
2
2
MeOH/THF, RT, overnight; (c) Acryloyl chloride, TEA, DCM, 0℃, 1h.
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Scheme 6. Synthesis of compounds 32–33a
NHBoc
NHBoc
NO2
NH2
NHBoc
F3C
F3C
F3C
a
F3C
b
F3C
c
Cl
NO2
O
N
O
N
N
O
N
O
O
R10
N
R10
Br
F3C
Br
Br
Br
N
N
N
d
e
Cl
OH
Cl
Cl
62a R10 pyridin-1-yl
2b R10 piperidin-1-yl
N
H
Cl
Cl
58
O
6
45
59
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
60
61
3a R10 pyridin-1-yl
3b R10 piperidin-1-yl
6
6
O
NH2
NH
F3C
F3C
N
O
N
O
N
R10
R10
N
N
N
g
f
N
H
N
O
H
O
32 R10 pyridin-1-yl
33 R10 piperidin-1-yl
64a R10 pyridin-1-yl
4b R10 piperidin-1-yl
6
a
Reagents and conditions: (a) Cs CO , DMF, 110 ℃ ; (b) Fe, NH Cl, EtOH/H O, 80 ℃ ; (c) (Boc) O,
2
3
4
2
2
DMAP, TEA, THF, RT, overnight; (d) pyrrolidine or piperidine, Cs CO , BINAP, Pd (dba) , toluene, 110
2
3
2
3
℃
; (e) 2-methoxy-4-(4-methylpiperazin-1-yl)aniline, Cs CO , BINAP, Pd(OAc) , dioxane, microwave,
2 3 2
1
30℃, 1h; (f) TFA, DCM, RT; (g) Acryloyl chloride, TEA, DCM, 0℃, 1h.
Hit Compound Development. In our previous work, CatC was identified as one of the off-
targets of Canertinib; the CatC active site residue Cys234—known to be essential for its
peptidase—was modified by our probe.28 Most irreversible CatC inhibitors reported to date
covalently bind to Cys234 via their electrophilic warheads.14, 22 We therefore asked whether
other known irreversible inhibitors of EGFR could likewise inhibit CatC activity. As shown in
Figure 2, Canertinib and WZ4002 displayed better inhibition of CatC (IC = 2.7 μM and 2.1
5
0
μM, respectively) than the other 3 inhibitors (IC > 10 μM). Both Canertinib and WZ4002 were
5
0
selected as the initial hits for further optimization; and the rest of the paper reports our SAR
development of WZ4002.
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F
O
NH
N
HN
N
N
N
HN
O
N
O
N
O
N
N
N
Cl
N
N
N
NH
N
N
N
H
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
OMe
OMe
OMe
Osimertinib IC50: 32.9 M
WZ4002 IC50: 2.1 M
PF06747775 IC50: >10 M
F
O
F
H
N
HN
N
Cl
O
N
H
HN
Cl
H2N
O
N
N
N
HN
N
O
O
N
O
N
O
O
CN
Canertinib IC50: 2.7 M
Afatinib IC50: >10 M
AZD5248 IC50: 2.9 nM
Figure 2. Chemical structures and CatC enzymatic inhibitory activity of selected irreversible
2
9
inhibitors of EGFR and AZD5248 (a known CatC inhibitor as tool compound).
Understanding the possible binding mode of WZ4002 in the active site of CatC could
provide useful guidance for designing analogs to conduct structure–activity relationship (SAR)
studies. Docking studies with WZ4002 based on the CatC/AZD5248 complex crystal structure
(
PDB ID: 4CDE) identified two inhibitor-acceptor interactions as potentially related to our
observed effects: a potential covalent attachment between the acrylamide fragment of WZ4002
and Cys234 in the CatC active site and a salt-bridge interaction between the piperazine N-4 of
WZ4002 and Asp1 in the exclusion domain (Figure 3). Its predicted binding mode suggested that
improvements in potency could perhaps be achieved via modification of the pyrimidine at C-5
position and/or the phenyl at the C-2 position.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Putative binding mode of WZ4002 (orange) within the active site of CatC (PDB ID:
4
CDE).
Using the docking study data as a guide, we aimed to develop a highly potent CatC
inhibitor, whilst depleting the activity against EGFR. We first explored the possibility of
eliminating WZ4002's kinase binding capability by focusing on the known ‘hinge binding’
regions of the inhibitor: a hydrogen bond between pyrimidine N1 and EGFR Met793 facilitates
the crucial hinge binding.30 Notably, according to our proposed binding mode, however,
WZ4002's pyrimidine N1 does not contribute to ligand interactions with CatC, suggesting that
replacing N1 with a carbon may eliminate its EGFR inhibitory effects without affecting its
binding affinity for the CatC active site (Figure 3).
We synthesized compound 1 to test this hypothesis, and observed that the EGFR IC of
5
0
compound 1 was reduced by more than 5000-fold (IC > 10 μM) compared to WZ4002 (IC =
5
0
50
1
.7 nM) (Figure 4) while its inhibitory effect against CatC was slightly increased compared to
WZ4002 (IC of 810 nM vs 2.1 μM in the enzymatic assay, and IC = 538 nM in the U937
5
0
50
cell). We also tested the metabolic stability of compound 1: their CL (μL/min/mg protein) in
int
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liver microsomes (human, rat, and mouse) were 5.4, 39.7, and 26.3, respectively (Supporting
Information, Table S1), which strongly encouraged our additional efforts to further develop of
this novel series of compounds.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
NH
NH
N
O
N
O
4
2
N
3
Cl
N
1
Cl
N
N
5
6
3
4
N
2
N
1
N
6
H
H
5
OMe
OMe
1
WZ4002
Cat C Enz IC50: 2.1 M
EGFR WT IC50: 1.7 nM
Cat C Enz IC50: 810 nM
U937 Cell IC50: 537 nM
EGFR WT IC50: >10 M
Figure 4. Hit validation.
Hit to lead. Firstly, 4-methyl-piperazine compound 2, removal of the methoxy group from the
phenyl ring, showed slightly better potency than 1. In general, a salt-bridge provides strong
interactions between a ligand and its receptor, so we made efforts to identify potentially superior
salt-bridge interaction groups. Several analogs with various aniline substitutions at pyridine C-6
position were prepared to compare with the piperazine analog 2. Piperazine compound 3
maintained its inhibitory activity, while the salt-bridge interaction between piperazine and Asp1
was undisrupted (according to the proposed binding mode); however, morpholine compound 4
showed a significant loss in potency (IC > 10 μM), which could be explained by the absence of
5
0
a salt-bridge interaction. Compound 5 and 6—with flexible amine chains at different positions of
the phenyl ring—were synthesized and tested, and 4-fold enzymatic activity reduction were
observed with both compounds as compared to compound 3. Adding a larger alkyl group on the
N-4 with the hope of improving potency, cyclopropylmethyl compound 7 maintained the
potency, while the cyclopentylmethyl group of compound 8 was apparently too bulky to be
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tolerated by the surrounding environment of CatC, leading to a more than 10-fold loss in
potency. Although compound 2 was slightly more potent than 1, its liver microsomes stability in
mouse and rat was poorer (Supporting Information, Table S1). Thus, we decided to continue to
use the C-6 2-methoxy-4-(4-methylpiperazin-1-yl)-aniline moiety in further SAR investigation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. SAR of substitutions at the pyridine core C-6 position
O
NH
O
2
1
Cl
N
R1
3
4
N
6
5
H
a
a
1
1
CatC Enz (IC : nM)
CatC Enz (IC : nM)
Compd
2
R =
Compd
6
R =
5
0
50
NH2
N
N
483±1
2051±101
783±160
p–
p–
p–
N
N
N
N
NH
3
4
499±1.5
7
8
p–
N
N
O
>10,000
>10,000
p–
p–
NH2
5
2150±247
m–
a
Human recombinant CatC isolated enzyme assay. Mean values from a minimum of two experiments.
Reviewing our proposed docking mode, the unfilled space near the C-3 position at pyridine
of compound 1 appeared attractive as a potential site for generating additional desired binding
interactions. We therefore conducted SAR studies for the modification of the C-3 position: the
results are shown in Table 2. Replacing the -Cl with -H (compound 9) or cyano (compound 11)
at pyridine C-3 position had little impact on IC values. Trifluoromethyl compound 10 and
5
0
dimethylamino compound 13 conferred a slight loss (2-fold) of potency. However, replacement
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with a larger pyrrolidine group as in compound 14 resulted in a 5-fold increase in potency.
Another two cyclic amine compounds were also synthesized: 15 with a piperdinyl moiety and 16
with an azepanyl moiety. These two compounds showed similar activity to compound 14.
Compounds 14-16 strongly suggested that the increased potency likely results from additional
hydrophobic interaction between the cyclic amines and the nearby CatC Trp405 residue.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. SAR of substitutions at the pyridine core C-3 position
O
NH
N
O
2
R2
N
1
N
3
4
N
6
H
5
OMe
a
a
2
2
CatC Enz (IC : nM)
CatC Enz (IC : nM)
Compd
R =
Compd
R =
5
0
50
1
9
Cl
H
810±1.5
785±24
13
14
15
N
1520±278
175±77
N
1
0
CF3
1605±782
N
221±15
11
CN
835±45
16
N
271±2
1
2
391.5±21.5
a
Human recombinant CatC isolated enzyme assay. Mean values from a minimum of two experiments.
These encouraging results further supported our proposed binding mode, and led us to
suspect that fine tuning of the phenyl ring bearing the acrylamide could confer yet additional
improvements in potency. As results shown in Table 3, when a methyl group was introduced to
the C-2 position, compound 17 lost almost all CatC inhibitory activity (IC > 10 μM). However,
5
0
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a 4-fold improvement in potency was observed when a methyl group (19) was incorporated into
C-4 position of 14. These results implied that the orientation of acrylamide relative to Cys234
made a significant influence on potency. In addition, the methyl group of compound 19
established the hydrophobic interaction with nearby residues. Compound 18, bearing a methyl
group at the C-6 position, was 3-fold less potent than 14, probably due to the unfavorable angle
between Phenyl-O-Pyridine.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Phenyl ring substituent modifications
O
NH
3
4
2
3
R
5
N
1 O
6
N
N
N
N
H
OMe
a
b
c
3
CatC Enz (IC : nM)
THP1 Cell (IC : nM)
U937 Cell (IC : nM)
Compd
R =
50
50
50
1
1
1
4
7
8
H
175±77
>10000
NTd
NT
NT
NT
2-Me
6-Me
4-Me
4-OMe
505±35
NT
NT
19
20
44±3.8
12.3±7.3
21.3±5.9
40.8±16.9
70±3.0
139.8±41.6
aHuman recombinant CatC isolated enzyme assay. Mean values from a minimum of two experiments.
b
c
CatC cell assay using THP1 cell line. Mean values from a minimum of two experiments. CatC cell assay
d
using U937 cell line. Mean values from a minimum of two experiments. NT = not test.
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Having successfully improved the potency from a low micromolar initial hit WZ4002 to
low nanomolar compounds in enzymatic and cellular assays, we next turned our attention to
metabolic stability issue. As a starting point, compound 19 suffered from poor stability in liver
microsomes (Table 4a) and in plasma (data not shown). The liver microsomes stability data
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Supporting Information, Table S1) suggested that pyridine C-3 substituents apparently strongly
influenced metabolic stability. To address this issue, we conducted optimization of substitutions
at pyridine C-3 position.
First, we synthesized compound 21, in which an additional hydroxyl was introduced to the
meta-position of compound 19. Compound 21 was rapidly metabolized in all 3-tested liver
microsomes systems; noting that it had slightly increased potency over compound 19. We then
replaced the pyrrolidine with a piperidine to obtain compound 22: excitingly, compound 22
exhibited an improvement in liver microsomes stability (human/rat/mouse liver microsomes
CL = 1.8, 6.3, and 10.8 μL/min/mg, Table 4a) and was more potent than compound 19 in both
int
enzymatic and cellular assays. Next, adding a hydroxyl group to the meta-position of the
piperidine as in compound 23 led to a slight improvement in metabolic stability in mouse liver
microsomes but reduced stability in both human and rat liver microsomes. When the piperidine
was replaced by morpholine as in compound 24 or 4-methylpiperazine as in compound 25, the
enzymatic activity decreased about 3-fold compared to compound 22; further, while the stability
of compound 25 was maintained, compound 24 showed was less stable in rat and mouse liver
microsomes. A planar structure phenyl group was also introduced to the pyridine C-3 position
(compound 26), however this led to a 9-fold loss in inhibitory activity.
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. Further optimization at the pyridine C-3 position (a) and C-6 position (b)
O
(a)
NH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
2
R4
N
1
N
3
4
N
6
5
H
OMe
CatC Enz
THP1 cell
U937 cell
HLM
CLint
RLM MLM
Compd
R4=
d
d
d
a
b
c
CLint
23.1
ND
CLint
26.7
ND
(IC : nM)
(IC : nM)
(IC : nM)
5
0
50
50
1
9
N
44±3.8
32.3±13.9
17.3±0.9
12.3±7.3
28.4±19.3
1.9±0.9
40.8±16.9
49.4±10.0
3.4±1.4
4.3
NDf
1.8
OH
2
1e
N
2
2
N
6.3
10.8
OH
2
3e
34.1±8.4
4.9±2.5
7.4±1.7
4.9
9
6
N
N
O
2
4
5
6
59.3±9.8
46.5±0.3
3.9±1.2
2.8±1.2
NTg
5.9±1.7
4.7±0.7
NT
1.9
3.7
NT
10
2
4
3
N
N
2
2
154.0±16.2
NT
NT
aHuman recombinant CatC isolated enzyme assay. Mean values from a minimum of two experiments.
b
c
CatC cell assay using THP1 cell line. Mean values from a minimum of two experiments. CatC cell assay
d
using U937 cell line. Mean values from a minimum of two experiments. CL : μL/min/mg protein,
int
e
f
g
determined in human/rat/mouse microsomes. it was racemic. ND = not detected at 0 min. NT = not test.
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O
O
O
(
b)
NH
NH
NH
F3C
F3C
N
O
N
O
O
2
N
N
N
N
1
N
N
N
N
3
4
R5
N
6
N
N
5
H
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
OMe
OMe
32
33
CatC Enz
THP1 cell U937 cell
HLM RLM MLM
CLint
5
Compd
R =
d
d
d
a
b
c
CLint
CLint
(
IC : nM)
(IC : nM) (IC : nM)
50
50
50
NDe
2
2
7
8
N
N
17.6±2.9
28.6±3.6
2.7±1.7
5.8±1.1
2.9
15
N
N
N
6.5±1.5
NTf
10.0±3.4
NT
6.0
NT
13
22
OMe
O
N
N
2
9
135.4±10.6
NT
NT
OMe
OMe
N
N
N
30
15.6±2.8
1.0±0.4
NT
2.2±0.6
NT
2.5
NT
4.1
NT
4
N
3
1
307.1±12.1
NT
N
32
—
—
4±0.4
6.7±1.1
1.8±0.2
11.5±2.0
2.4±0.5
3.9
5.2
11
866
3
3
2.3±1.1
15.1
15.4
aHuman recombinant CatC isolated enzyme assay. Mean values from a minimum of two experiments.
b
c
CatC cell assay using THP1 cell line. Mean values from a minimum of two experiments. CatC cell assay
d
using U937 cell line. Mean values from a minimum of two experiments. CL : μL/min/mg protein,
int
e
f
determined in human/rat/mouse microsomes. ND = not detected at 0 min. NT = not test.
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The effect of pyridine C-6 substituents on the metabolic stability was also evaluated in all
three liver microsomes systems (Table 4b). Removing the methoxy group from the phenyl
(compound 27) or replacing the methoxyphenyl with a methoxypyridine (compound 28) caused
poorer stability than 22 in liver microsomes of all three species. Compound 29, with an acetyl
group at its N4-position that disrupts the salt-bridge interaction with CatC, only exhibited a
moderate decrease in potency (about 8-fold less potent than 22). The change in potency might be
explained by a likely hydrogen bond occurring between the carbonyl of 29 and CatC Asp1 that
was predicted in docking simulations. Replacement of piperazine (compound 22) with N,N-
dimethylpiperidin (compound 30) had little effect on potency and liver microsomes metabolic
stability. Compound 27 implied that the modification of ortho-position at phenyl probably
improved the metabolic stability, so an N-atom substitution analog was prepared (compound 31),
for which a dramatic loss (about 20-fold) in potency was observed. Although introduction of
trifluoromethyl to the 4-position of phenyl at pyridine C-2 increased the potencies (compound 32
and 33), these modifications made the compounds metabolically unstable in liver microsomes.
Overall, compound 22 exhibited the best combination of strong potency and liver microsomes
stability, so it was selected as a potential candidate for further in vitro evaluation.
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Molecular docking. To better understand the SAR results observed for our compounds, we
performed molecular docking of compound 22 within the active site of CatC based on the
CatC/AZD5248 complex crystal structure (PDB ID: 4CDE). The acrylamide formed two
hydrogen bond donor-acceptor motifs respectively with Asn380 and Gln228, and these two
hydrogen bonds enabled the β-carbon atoms of acrylamide to readily reach the sulfhydryl group
of Cys234 (within 4.0 Å), thereby facilitating a Michael addition to form the covalent bond
(Figure 5 and Supporting Information Table S2). A salt-bridge interaction was also observed
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between the piperazine N-4 of compound 22 and the Asp1 residue of the CatC exclusion domain,
and a hydrophobic interaction was predicted between the piperidine and Trp405 (with a 4–5 Å
distance between the two rings). The methyl group of the phenyl ring generated a slight
hydrophobic interaction with nearby residues, and also forced the acrylamide to retain its proper
orientation relative to Cys234 for Michael addition to form the covalent bond.
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0
Figure 5. Putative binding mode of compound 22 (orange) within the active site of CatC (PDB
ID: 4CDE).
In vitro/in vivo profiling. As our compounds are derived from an EGFR inhibitor, several
compounds with favorable CatC activity were selected for evaluation of their EGFR inhibitory
activity (Supporting Information, Figure S1). For all tested compounds, no inhibition of EGFR
WT kinase was observed (when tested up to 10 μM). To evaluate the cathepsin selectivity for our
compounds, assays with compounds 19, 22, and 24 were conducted against human recombinant
enzymes Cat B, Cat L, and Cat S. These three compounds exhibited generally high cathepsin
selectivity (Table 5).
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Table 5. Cathepsin selectivity of compound 19, 22 and 24
Compd
19
CatC Enz IC50a
44 nM
Cat B Enz IC50a
3～10 μM
Cat L Enz IC50a
3～10 μM
Cat S Enz IC50a
> 10 μM
0
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17 nM
3～10 μM
3～10 μM
> 10 μM
59 nM
3～10 μM
> 10 μM
> 10 μM
a
Human recombinant CatC/CatB/CatL/CatL isolated enzyme assay.
We also tested the inhibitory activity against cytochrome P450s using five recombinant
human cytochrome P450 isoforms (1A2, 2D6, 3A4, 2C9, and 2C19) (Supporting Information,
Table S3). For compound 22 and 24, the IC values were higher than 10 μM, suggesting a low
50
potential to be involved in drug-drug interactions. In contrast, compound 19 showed weak
inhibition against 1A2 and 2C19, with IC values of 8.75 and 9.91 μM, respectively; the other 3
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isoforms had IC values over 10 μM. Compound 22 was then processed into mouse in vivo PK
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studies, which demonstrated desirable results upon oral delivery, with a half-life of 3.06 h and
bioavailability of 22.3% (Table 6). Tissue distribution studies showed that lung had the highest
compound concentration and kidney had the second highest compound concentration
(Supporting Information, Figure S2). On the basis of its overall favorable profiles, compound 22
was thusly selected as a potential drug candidate for further in vivo profiling.
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Table 6. Pharmacokinetic parameters for intravenous (IV), intraperitoneal (IP) and oral (PO)
administration of 22 in C57BL/6 mice
Route
IVa
AUC (hr*ng/mL)
t (hr)
tmax (hr)
Cmax (ng/mL)
F%
—
inf
1/2
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1
1
1
1
1
1
1
1
1
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2
2
2
2
2
2
2
2
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3
3
3
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3
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4
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4
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4
4
4
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5
5
5
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0
1
2
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0
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2
3
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8
9
0
1225
1243
1.62
1.19
3.06
—
0
—
IPb
766
184
20.3
22.3
POc
1364
0.5
a
b
c
IV dose: 2 mg/kg. IP dose: 10 mg/kg. PO dose: 10 mg/kg. All administrated with 0.5% Methocel™,
and 0.1%Tween 80 in citrate buffer, pH 3. Values were the mean values of male for mouse (n = 3).
In vivo efficacy of compound 22. In chronic inflammatory diseases, CatC has been linked with
11
activation of neutrophil elastase (NE), cathepsin G (CatG), and proteinase 3 (PR3). These three
serine proteases are major components of neutrophil granules, which are secreted and which
cause both inflammation and destruction of extra-cellular matrices. Specifically, it has been
found that CatC functions to peptidolytically remove N-terminal dipeptides from pro-NSPs
during the promyelocyte stage of neutrophil maturation in bone marrow.31, 32 Then, the activated
NSPs (packaged in granules) in mature neutrophils are eventually released from bone marrow
into the circulating blood. It has been reported that a continuous very high fractional inhibition of
the CatC activity is required to effectively prevent the activation of NSPs in vivo.33, 34
In vitro, 22 showed concentration dependent inhibition of CatC in both THP1 and U937
cell-based assays, with IC values of 2 and 3 nM, respectively. Subsequently, we tested whether
5
0
the treatment of 22 could inhibit the activity of CatC and NSPs in vivo. First, the effect of oral
administration of 22 on CatC inhibition was examined in mice. Within 15 min of oral
administration of 22 at a dose (20 mg/kg), the blood concentration of 22 was 200 ng/ml, and
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CatC activity in blood was inhibited by 75%. And time-course activity studies showed
statistically significant inhibition (80%) of CatC by 22 in blood lasting for 8 hours post
administration (Supporting Information, Figure S3). Next, the effect of compound 22 on the
activation of NE, PR3, and CatG were assessed in vivo by oral administration of 22 twice daily
to mice (6.7, 20, and 60 mg/kg) over a 6-day time course. In Figure 6, dose-dependent decreases
of CatC activity were observed in both bone marrow and blood.
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Bone marrow
200
50
100
Blood
1
5
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0
0
6.7 20 60
0 6.7 20 60
0 6.7 20 60
0 6.7 20 60 (mg/kg)
0 6.7 20 60
0 6.7 20 60
0 6.7 20 60
0 6.7 20 60 (mg/kg)
Cat C
NE
Pr 3
Cat G
Cat C
NE
Pr 3
Cat G
Figure 6. In vivo profiling data (mice) of compound 22. Compound 22 inhibited CatC and the
activation of NE, PR3, and CatG in bone marrow and blood, and did so in a dose-dependent
manner. Compound 22 was administered orally twice daily to naive mice (6.7, 20, and 60 mg/kg)
for 6 days. The activity inhibition data of compound treating groups were showed as percentage
activity of vehicle mean (set to 100% activity).
We also found that the activation of downstream NE, PR3, and Cat G was also inhibited, in
a dose-dependent manner, both in bone marrow and in blood (Figure 6). Oral administration of
compound 22 (60 mg/kg; twice daily) was tolerated, and we detected no difference in white
blood cell count (Supporting Information, Figure S4) between the vehicle-treated and 60 mg/kg
treatment groups, thus suggesting that compound 22 might not exert direct effects on the
maturation or release of neutrophil into the blood circulation. Collectively, these in vivo studies
robustly confirmed that compound 22 can block CatC enzymatic activity and can, further, inhibit
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the activation of downstream NSPs (both in bone marrow and in blood samples). These results
demonstrated that compound 22 could potentially be a therapeutic compound for the treatment of
neutrophil-associated inflammatory diseases.
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1
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Recently, Aghdassi et.al reported that deficiency of cathepsin C ameliorated severity of
3
5
acute pancreatitis in mice. Next, we examined the potential protective effect of compound 22
on the pancreas using a caerulein-induced acute pancreatitis mouse model. In this model, mice
th
were orally administrated 22 or AZD5248 twice daily at 20 mg/kg for 6 days. On the 6 day,
acute pancreatitis was induced via hourly intraperitoneal injections of caerulein. Compound 22
and AZD5248 both caused significant inhibition of CatC enzymatic activity in the pancreas, and
displayed a considerable pancreas-protecting effect based on assessment of photomicrographs of
H&E stained pancreas sections (Figure 7). On the basis of its attractive performance, both in
vitro and in vivo, we have initiated further druggability evaluations of compound 22 that are
currently underway.
(a)
1
50
00
##
1
**
**
50
0
m
e
a
cl
ehi
22
248
in+
h
5
S
D
e
r
V
Z
+
A
u
l
ae
n
in+
i
e
r
e
r
C
u
l
u
l
ae
C
ae
C
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(
b)
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Figure 7. Compound 22 reduced the severity of acute pancreatitis: (a) CatC activity in
homogenates prepared from mice pancreas samples. Compound 22 and AZD5248 were
administered orally twice daily at 20 mg/kg. The activity inhibition data of the compound-treated
groups are shown as the percentage activity of a sham mean (set to 100% activity). Values are
means ± SEM. Statistical significance was considered: ## p < 0.05 versus Sham; ** p < 0.05
versus caelurein + vehicle group. (b) Pancreas histology representative photomicrographs.
Hematoxylin and eosin staining of formalin-fixed sections of pancreas tissue. Scale bar: 50 μm.
CONCLUSION
Starting from hit compound WZ4002 (a low micromolar CatC inhibitor), structure–activity
relationship (SAR) investigations were performed guiding by a plausible binding mode of
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WZ4002 within the CatC active site. These efforts ultimately led to the discovery of a novel
series of CatC inhibitors with significant improvement in enzymatic/cellular potency,
culminating finally in the identification of compound 22 as a highly potent and selective
irreversible inhibitor of CatC. In vitro and in vivo profiling assays demonstrated that compound
1
1
1
1
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1
1
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1
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0
1
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3
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9
0
1
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0
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0
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2
2 is an extremely promising drug candidate, and further evaluations of compound 22 as a pre-
clinical candidate are currently in progress.
EXPERIMENTAL SECTION
Biology: Recombinant human cathepsin C (1071-CY-010), cathepsin L (952-CY-010),
Mca-RPKPVE-Nval-WRK (Dnp)-NH (ES002) and Z-Leu-Arg-AMC (ES008) were purchased
2
from R&D (Akron, OH, US). Recombinant human cathepsin B (10483-H08H) and cathepsin S
(10487-H08H) were purchased from Sino Biological Inc. (Beijing, CN). Human THP1 cells and
U937 cells were purchased from ATCC (Manassas, VA, USA) and maintained in RPMI 1640
culture medium. The medium was supplemented with 10% fetal bovine serum (12483-020) and
100-unit/mL penicillin-streptomycin (15140-122), which were bought from Life Technology
(
Beijing, CN). Human and mouse liver microsomes were purchased from BD Biosciences
Beijing, CN). Gly-Phe-AFC (SMAFC041) was bought from SM Biochemicals (Anaheim, CA,
(
US). Cisbio HTRF KinEASE-TK kit (62TK0PEC) was purchased from Cisbio (Shanghai, CN).
All the other chemicals were purchased from Sigma-Aldrich (Shanghai, CN). The assay and cell
culture plates were purchased from Corning (Shanghai, CN). C57BL/6 mice matched for gender
and age were purchased from Vitalriver (Beijing, CN). All experiments on mice were conducted
according to institutional and national animal regulations. Animal protocols were approved by
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the ethics committee of national institute of biological sciences. Mice were used at the age of 6–8
weeks.
In vitro CatC enzyme assay. The CatC enzyme assay was performed in black 384-well
plate. Firstly, rhCatC (440 nM) was activated by rhCatL (300 nM) in activation buffer (25 mM
MES, 5 mM DTT, pH 6.0) at RT for 2 h and then activated rhCatC was diluted to 4.4 nM in
assay buffer (25 mM MES, 50 mM NaCl, 5 mM DTT, 0.01% (v/v) Triton X100, pH 6.0).14 μL
activated rhCatC and 1μL compound in 10% DMSO or 10% DMSO were incubated at RT for 3
h, and 5 μL substrate (Gly-Phe-AFC) was added to final concentration of 100 μM. After 60 min
reaction, the product of the reaction was measured in the Enspire plate reader using Ex λ400 nm
and Em λ505 nm for AFC. A standard irreversible CatC inhibitor AZD5248 by AstraZeneca was
used as an inhibitor control in the assay.
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CatC cell assay. The cell assay was carried out in a 96-well clear black plate. 30 μL of 1.0
6
× 10 /mL THP-1 or U937 cells in medium were added to all wells. Then 10 μL of 5% (v/v)
DMSO positive controls or 10 μL of 3-fold serial diluted compound were dispensed into the
appropriate well. The plate was preincubated at 37℃ for 4 h in a cell culture incubator, and then
1
0 μL of H-Gly-Phe-AFC (100 μM) was added to start the assay reaction. The plate was further
incubated for 60 min at 37℃ and measured as above CatC enzyme assay.
EGFR kinase assay. The EGFR kinase assays were performed in black 384-well plate
using Cisbio HTRF KinEASE-TK kit. The assay buffer contained 50 mM HEPES (pH7.0), 0.02%
NaN , 0.01% BSA, 0.1 mM Orthovanadate, 10 mM MgCl , 2.5 mM DTT and 6.25 nM SEB.
3
2
EGFR kinases was first incubated with compounds for 2h respectively, then ATP/peptide
substrate mixture was added to initiate the reaction. The final concentration EGFR WT was 0.1
nM. The final concentration of biotin-labeled peptide substrate was 1.4 μM, and ATP was 20 μM
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for EGFR WT. After 30 min reaction at RT, the detection reagents were added. The TR-FRET
signal was measured on PerkinElmer Envision using Ex λ 320 nm and Em λ 615 nm/665 nm.
Data analysis. Data were analyzed using GraphPad Prism (GraphPad Software, Inc., San
Diego, CA, USA). The curves were fitted using a non-linear regression model with a sigmoidal
dose response.
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Selectivity profile of Cathepsin B/L/S. Selectivity assay was conducted in black 384-well
plate. Briefly, compounds and recombinant human Cathepsin enzymes were incubated at RT for
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0 min followed by reaction with substrates for further 30 min respectively. Z-Leu-Arg-AMC (as
substrate of cathepsin B and L) was measured by PerkinElmer Envision using Ex λ 380 nm and
Em λ 460 nm. MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH (as substrate of
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cathepsin S) was measured at Ex λ 320 nm and Em λ 405 nm using Enspire plate reader. E-64, a
natural inhibitor of cysteine proteases, was used as an inhibitor control in three assays.
Compounds stability test in liver microsomes assays. Compounds were incubated with
human or mouse liver microsomes and NADPH in 0.05 M Phosphate buffer (pH=7.4) at 37℃
for 0-60 min. The reaction was quenched and the amount of the remaining compound was
analyzed using LC-MS/MS.
Pharmacokinetics study in mice. Following intravenous (IV), intraperitoneal (IP), or oral
administration (PO) of 22 to C57BL/6 mice (n=3), blood was sampled through eye puncture at
various time points. Compound concentrations in the blood samples were analyzed by LC-
MS/MS. Pharmacokinetic parameters were determined from individual animal data using non-
compartmental analysis in phoenix 64 (winNonlin 6.3).
Mice in vivo Study. C57BL/6 mice (20–25g) were randomly divided into 4 groups (n=10)
and kept at 5 animals per cage. Compound 22 was administered orally twice daily at 6.7, 20, 60
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mg/kg for 6 days. The matched vehicle controls (0.5% Methocel™, 0.1% Tween 80 in 100 mM
citrate buffer, pH 3) were also administered twice daily for 6 days. Mice were weighed and the
dose adjusted accordingly on each day. Blood was sampled through eye puncture at 14 h
intervals after the end of the administration. Then mice were terminated and bone marrow cells
were immediately extracted from femurs and tibias using ice cold RPMI 1640 medium. Blood
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cells and bone marrow cells were lysed according to the protocols previously described with
some modifications. Briefly, cell lysis buffer contained 50 mM Tris (pH 7.4), 750 mM NaCl, 1%
(v/v) Triton X-100 and cell lysates were mildly treated by ultrasonic in ice. Following cell lysis,
the supernatants were kept at -80℃ until NSPs activity analyses.
Analysis of downstream NSPs activities. The analysis was performed as previously
described. Briefly, cell lysates were diluted into according protein concentration using different
assay buffer.
The assay buffer used were 25 mM MES, 50 mM NaCl, 5 mM DTT, pH 6.0 for CatC, 50
mM Tris, 750 mM NaCl, pH 7.4 for NE and PR3, and 50 mM Tris, 750 mM NaCl, 5 mM
EDTA, pH 7.4 for CatG. Blood lysates were diluted into 5 fold higher than bone marrow lysates.
Lysates were added to 384-well plates together with DMSO control or protease inhibitors
(inhibitor AZD5248 by AstraZeneca for CatC, avelestat for NE, sivelestat for PR3, Cathepsin G
inhibitor 1 for CatG). Synthetic peptide substrates (Gly-Phe-AFC (SMBiochemicals) for CatC,
methoxysuccinyl-Ala-Ala-Pro-Val-AMC (Sigma) forNE, aminobenzoyl-Val-Ala-Asp-Cys-Ala-
Asp-Gln-ethylenediamine 2,4-dinitrophenyl (Peptide Synthetics) for PR3, and N-succinyl-Ala-
Ala-Pro-Phe-pNA (Sigma) for CatG) were added before plates were read at fluorescence or
absorbance. Assay kinetics was monitored for up to 90 min. Data were analyzed using GraphPad
Prism (GraphPad Software, Inc., San Diego, CA, USA).
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Induction of Pancreatitis. The acute pancreatitis was induced by hourly intraperitoneal
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injections of caerulein (50 μg/kg/body weight) for up to 8 h. C57BL/6 mice (20–25 g) were
randomly divided into 4 groups (n=5). Compound 22 and AZD5248 were administered orally
twice daily at 20 mg/kg for 6 days. The matched vehicle controls (0.5% Methocel™, 0.1%
Tween 80 in 100 mM citrate buffer, pH 3) were also administered twice daily for 6 days. On the
6th day, mice were induced pancreatitis after compound administration and after the last
caerulein injection, mice were administered compound. The sham-treated group was performed
with the same as vehicle controls except that saline was administered instead of caerulein. Mice
were killed at 1h after the last caerulein injection. Tissue was harvested immediately after
sacrificing the animals. A portion of pancreas samples were frozen in liquid nitrogen for
preparation of homogenate. For histology, pancreas was fixed in 4.5% formalin for paraffin
embedding and stained with hematoxylin and eosin. Serum samples were prepared and stored at
-20℃.
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Molecule docking. The molecular docking was performed using MOE 2015.10 software.
The co-crystal structure of CatC in complex with AZD5248 (PDB ID: 4CDE) was used for
docking studies. The optimized structure of 22 was generated by MOE 2015.10. Before docking
simulation, ligands and protein were prepared with the standard protocol using MOE 2015.10. 22
was inserted into the substrate-binding pocket of CatC to replace AZD5248. The complex of 22-
CatC was used for geometrical optimization. All docking calculations were performed using
default settings.
Chemistry. The reagents and solvents for reactions were reagent-grade and used without
further purification. Glass apparatus for reaction dewater and deoxygenize under nitrogen
atmospheric and high temperature. Reactions sensitive to water and air rigorously followed
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