Development of substrate analogue inhibitors for the human airway trypsin-like protease HAT

Article abstract of DOI:10.1016/j.bmcl.2011.06.033

A series of substrate analogue inhibitors of the serine protease HAT, containing a 4-amidinobenzylamide moiety as the P1 residue, was prepared. The most potent compounds possess a basic amino acid in the d-configuration as P3 residue. Whereas inhibitor 4 (K_i 13 nM) containing proline as the P2 residue completely lacks selectivity, incorporation of norvaline leads to a potent inhibitor (15, K_i 15 nM) with improved selectivity for HAT in comparison to the coagulation proteases thrombin and factor Xa or the fibrinolytic plasmin. Selected inhibitors were able to suppress influenza virus replication in a HAT-expressing MDCK cell model.

Full text of DOI:10.1016/j.bmcl.2011.06.033

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4860–4864
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of substrate analogue inhibitors for the human airway
trypsin-like protease HAT
Frank Sielaff a, Eva Böttcher-Friebertshäuser b, Daniela Meyer a, Sebastian M. Saupe a, Ines M. Volk a,
Wolfgang Garten b, Torsten Steinmetzer a,
⇑
a Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
b Institute of Virology, Philipps University Marburg, Hans-Meerwein-Str. 2, D-35043 Marburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substrate analogue inhibitors of the serine protease HAT, containing a 4-amidinobenzylamide
Received 19 May 2011
Revised 8 June 2011
Accepted 9 June 2011
Available online 21 June 2011
moiety as the P1 residue, was prepared. The most potent compounds possess a basic amino acid in the D-
configuration as P3 residue. Whereas inhibitor 4 (Ki 13 nM) containing proline as the P2 residue com-
pletely lacks selectivity, incorporation of norvaline leads to a potent inhibitor (15, Ki 15 nM) with
improved selectivity for HAT in comparison to the coagulation proteases thrombin and factor Xa or
the fibrinolytic plasmin. Selected inhibitors were able to suppress influenza virus replication in a HAT-
expressing MDCK cell model.
Keywords:
HAT
Protease inhibitor
Serine protease
Influenza
Ó 2011 Elsevier Ltd. All rights reserved.
Hemagglutinin cleavage
Human influenza viruses cause acute infection of the respira-
tory tract that affects millions of people during seasonal outbreaks
and occasional pandemics worldwide.1 Currently, two drugs tar-
geting the viral neuraminidase (NA) and the M2 channel blockers
amantadine and rimantadine are approved for the treatment of
influenza. Resistance to these drugs have been observed and exac-
erbate the situation.2,3
In addition, HAT and TMPRSS2 also support the multicycle influ-
enza virus replication in HAT- or TMPRSS2-expressing MDCK
cells.6 Additionally, we could demonstrate that HAT is proteolyti-
cally active on the cell surface, whereas it seems that TMPRSS2
cleaves HA0 in the secretory pathway within the cell.8 Due to its
location on the cell surface and its better accessibility HAT might
be a potential target for the treatment of influenza infections. Re-
cently, a first peptidic arginal-derived HAT inhibitor with a Ki value
of 54 nM has been developed.9
The replication cycle of influenza viruses is initiated by its sur-
face glycoprotein hemagglutinin (HA). HA mediates the binding of
the virus to sialic acid containing receptors of the host cells and,
after endocytosis, the fusion of the viral envelope with the endo-
some membrane. This process is termed uncoating and enables
the release of the viral genomic RNA into the cytosol of the host
cell.4 HA is synthesized as HA0 precursor and has to be cleaved
by host endoproteases into disulfide-linked HA1 and HA2 subunits
to become fusogenic and is thus a crucial step for infectivity and
spread of influenza viruses. The HAs of most influenza strains,
including the H1, H2 and H3 subtypes, which typically infects hu-
mans, contain a single arginine as the P1 residue at its cleavage
site.5 Recently it was shown that the trypsin-like serine proteases
HAT (human airway trypsin-like protease or TMPRSS11D),
TMPRSS2 (epitheliasin or transmembrane protease serine 2) and
TMPRSS4 (CAP 2 or transmembrane protease serine 4), which are
expressed in the human respiratory tract, efficiently cleave the
HA0 of various influenza strains with a monobasic cleavage site.6,7
HAT belongs to the type II transmembrane serine proteases and
has a mosaic-like structure with its protease domain in the C-ter-
minal part.10 So far no X-ray structure of HAT is available. How-
ever, the sequence of its protease domain has significant
similarity to other trypsin-like serine proteases, such as the clot-
ting proteases thrombin11 and factor Xa12 or the fibrinolytic uroki-
nase (uPA).13 These proteases are all inhibited by substrate
analogue structures containing a 4-amidinobenzylamide as a
decarboxylated P1 arginine mimetic in combination with a P3 ami-
no acid in the D
-configuration.14,15 Starting from a homology mod-
el16–18 of HAT based on the X-ray structure of DESC119 (2oq5.pdb)
we assumed that such 4-amidinobenzylamide derivatives should
be suitable HAT inhibitors.
A first screen with available compounds, like the previously de-
scribed factor Xa inhibitor 112 and the uPA inhibitors 2 and 313 re-
vealed also some inhibition of HAT with Ki-values >50 nM. These
analogues have been previously used for preliminary experiments
to demonstrate the inhibition of proteolytic activation and
propagation of influenza viruses in HAT-expressing MDCK cells.20
⇑
Corresponding author. Tel.: +49 6421 2825900; fax: +49 6421 2825901.
E-mail address: steinmetzer@staff.uni-marburg.de (T. Steinmetzer).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.033

F. Sielaff et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4860–4864
4861
of
D-glutamic acid (18). However, all other analogues had reduced
activity; so we decided to keep
study the influence of further P2 substitutions.
D
-arginine in the P3 position to
P3-P2
D-Arg-Gly
D-Ser-Ser
Ki (nM)21
NH
NH2
1
2
3
430
78
Results from Table 3 indicate that valine, isoleucine and alanine
H
N
as well as its close analogues
a-aminobutyric acid and norvaline
P3
S
P2
are suitable P2 residues and provide inhibitors with Ki-val-
ues 6 30 nM. We assumed that some of these analogues could
have an improved selectivity compared to the unspecific proline
derivative 5. Therefore, we selected some inhibitors and deter-
mined their Ki-values against the coagulation proteases thrombin
and factor Xa and the fibrinolysis enzyme plasmin (Table 4). Inhib-
itor 31 is the only compound that has stronger potency against
HAT compared to the other proteases. Especially compounds 4
and 5 have a significantly higher affinity to thrombin and factor
Xa. The inhibitory potency against thrombin could be strongly re-
duced by incorporation of a P2 serine residue (36). A similar effect
was previously observed in a series of analogous urokinase inhib-
itors13; however, inhibitor 36 is still a relatively potent factor Xa
inhibitor. In contrast, all selected compounds from the piperazine
series (18, 19, 21) showed a reduced affinity for factor Xa but were
still relatively potent thrombin inhibitors (Ki <15 nM). It should be
noted that various compounds also inhibit the protease domain of
TMPRSS2 with Ki-values <100 nM (e.g., 20, 53 and 68 nM for deriv-
atives 5, 30 and 31, respectively). Detailed results regarding the
O
O
D-Ser-Lys(Cbz)
51
Therefore, we screened22 and synthesized additional analogues
of this inhibitor type. Based on previous experience with related
thrombin,23 factor Xa12 and urokinase13 inhibitors it was known
that proline is a suitable P2 residue in substrate analogue inhibi-
tors of various trypsin-like serine proteases. Consequently, we pre-
pared a series of P2 proline analogues with several P3 amino acids
in the D-configuration and maintained the important P4 benzylsul-
fonyl15 and the P1 4-amidinobenzylamide24 (Table 1).
Only the replacement of glycine in inhibitor 1 by proline re-
sulted in an approximately 20-fold improved inhibition constant
of compound 5, which could marginally be enhanced by incorpora-
tion of a P3 homo-D-arginine (4, Ki 13 nM). Obviously, the conform-
ationally constrained proline is well accepted by the S2 site of HAT.
However, proline is also a suitable P2 residue for many other
trypsin-like serine proteases and its incorporation often leads to
lack of specificity. Indeed, compound 5 is also a potent inhibitor
of thrombin (3.5 nM), factor Xa (2.4 nM), plasma kallikrein
(8.3 nM), matriptase (55 nM) and matriptase 2 (170 nM), thus
completely lacking any selectivity.25,26 The other P2 proline ana-
logues have reduced potency, however, some of them still inhibit
HAT with Ki-values <100 nM, including several inhibitors with
hydrophobic P3 residues. Based on the Ki-values it seems that
HAT has some preference for positively charged P3 residues,
whereas the acidic
Interestingly, the tert-butyl protected
acid inhibitors 6 and 7 show relatively high potency with Ki-values
<50 nM. Therefore, we used analogues with a free -aspartic- and
-glutamic acid side chain in the P3 position as suitable starting
Table 2
Inhibition of HAT by inhibitors of the general structure
NH
H
N
O
NH2
O
H
N
D
-aspartyl residue (17) is poorly accepted.
S
N
D
-aspartic- or -glutamic
D
O
O
n
O
D
D
Compound
n
P3
Ki (nM)21
point for further modifications and incorporated various pipera-
zine derivatives and other cyclic amines in an additional series
(Table 2).
The strongest inhibitory potency within this series was ob-
tained with a 1-(2-pyrimidyl)-piperazine coupled to the side chain
N
N
O
N
N
N
18
2
2
17
19
38
N
O
N
N
N
N
20
21
1
2
43
68
Table 1
N
Inhibition of HAT by P2 proline inhibitors of the general formula
NH
NH
H
N
O
NH2
P3
22
1
N
73
N
S
O
N
O
O
O
Compound
P3
Ki (nM)21
23
24
1
1
135
149
N
N
N
N
4
5
13
19
D
D
D
D
D
D
D
D
D
D
D
D
D
D
-homo-Arg
-Arg
N
6
36
-Glu(OtBu)
-Asp(OtBu)
-Lys
7
38
8
40
25
1
158
9
53
-Lys(Cbz)
-homo-Phe
-Val
N
N
N
N
N
N
10
11
12
13
14
15
16
17
63
76
26
27
28
29
1
1
1
1
183
202
210
221
O
98
-Phe
108
120
168
311
1425
-Cha
NH
N
-Leu
OH
-Phe(4-Amidino)
-Phe(4-CN)
-Asp
N

4862
F. Sielaff et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4860–4864
Table 3
expression of the proteolytic domain of TMPRSS2 in Escherichia coli
Modifications in the P2 position
and inhibition studies with these substrate analogue inhibitors will
be published in due course.27
NH
NH2
The relatively selective HAT inhibitor 31, its close analogue 30
and the most potent compound from the piperazine series (18)
were selected to inhibit influenza virus propagation in cell culture.
For this assay MDCK cells were used which express HAT under
doxycycline-induced transcriptional activation.8 These MDCK cells
were infected with various human influenza strains and incubated
with the respective inhibitors for 24 h to allow multiple cycles of
viral replication. Subsequently infected cells and comet-like spread
of infection were immunostained using antibodies against the viral
nucleoprotein (NP) as described previously.20 As expected, multi-
cycle replication of viruses is observed in doxycycline-treated cells
in the absence of inhibitors, whereas no virus spread is visible in
cells lacking doxycycline-induced expression of HAT (Fig. 1).
O
H
H
N
N
S
O
P2
O
NH
H2N
NH
Compound
P2
Ki (nM)21
30
31
32
33
34
35
36
37
38
39
40
41
Abua
Nvab
Val
Ile
Ala
Lys(Cbz)
Ser
Leu
Phe
Asp
14
15
22
23
30
34
34
47
57
At a concentration of 1
compound 5, which serves as control,8 show only a negligible ef-
fect against all three tested virus strains, whereas at 10 M all
lM (not shown) selected inhibitors and
l
422
457
512
compounds, especially inhibitors 18 and 30, strongly suppress
multicycle virus replication.
Arg
Asp(OtBu)
The synthesis of the inhibitors was performed according to pre-
viously described methods12,13,26 and is exemplarily described only
for the most potent analogue 4 and its precursors 8 and 9 and for
inhibitor 18 (Schemes 1 and 2). Briefly, benzylsulfonylchloride (42)
a
a
-Aminobutyric acid.
b
Norvaline.
was introduced to H-D-Lys(Cbz)-OH followed by coupling of H-Pro-
4-amidinobenzylamide  2HCl29 (44) to give inhibitor 9. Cleavage
Table 4
Specificity of selected compounds
of the Cbz-group provided inhibitor 8, which was converted into
the
D-homo-Arg analogue 4 by reaction with 1H-pyrazole-1-
Compound
HAT21
Ki (nM)
carboxamidine.30
Thrombin28
FXa28
Plasmin28
Inhibitor 18 was synthesized according to Scheme 2 starting
with inhibitor 6. Cleavage of the tert-butyl group provided com-
pound 45, which was used for PyBOP mediated coupling of 1-(2-
pyrimidyl)-piperazine to give analogue 18.
In summary, the replacement of glycine by proline in the initial
inhibitor 1 improved the Ki-value by a factor of 20, but leads to
poor selectivity. In fact, thrombin and factor Xa are even more
inhibited than HAT by various compounds. Interestingly, the incor-
poration of P2 norvaline provided an inhibitor (31) with similar
potency against HAT and improved selectivity against the other
tested three trypsin-like serine proteases. Furthermore, this
4
5
6
13
19
36
40
17
38
68
14
15
22
30
34
0.39
3.5
6.7
1.0
5.2
9.8
13.5
29
68
66
74
1610
2.7
2.4
31
17.5
39
1520
63
23,130
1160
114
3030
2110
2810
590
8
52
18
19
21
30
31
32
34
36
129
397
565
2.1
37
21
1.4
14.5
161
Figure 1. Inhibition of influenza virus propagation by inhibitor treatment in MDCK–HAT cells. Cells were infected with different influenza A viruses and incubated with or
without doxycycline (Dox) in the presence of 10 M of inhibitors 5, 31, 30 and 18 at 37 °C. After 24 h the cells were immunostained with influenza virus NP-specific
l
antibodies. Infection of MDCK–HAT cells treated or not with doxycycline in the absence of inhibitors are used as control. Immunostained cells were counted using a
microscope, and the percentage of infected cells compared to control (100%) is given below each well.

F. Sielaff et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4860–4864
4863
NH2
NH2
NH
O
O
NH
H
N
O
H2N
O
O
NH
OH
S
OH
O
NH
O
H
N
O
Cl
O
S
HN
S
N
O
O
42
44
HN
O
HN
O
a
b
O
HN
O
O
O
9
43
NH2
NH
NH2
NH
O
O
NH
NH
O
O
H
N
H
O
O
N
S
N
S
N
O
O
c
d
NH2
HN
NH
NH2
8
4
Scheme 1. Synthesis of inhibitors 4, 8 and 9. Reagents and conditions: (a) 2.2 equiv trimethylsilylchloride, 2.2 equiv diisopropylethylamine reflux in dry dichloromethane for
1 h followed by addition of 1.1 equiv 42 and 1.1 equiv diisopropylethylamine at 0 °C for 15 min, then room temperature for 3 h, (b) 1 equiv 44, 1.1 equiv HBTU, 1.1 equiv
HOBt, 3 equiv diisopropyethylamine in DMF at room temperature, 12 h, (c) 10% Pd/C, H2 atmosphere, in 90% acetic acid, room temperature for 24 h, (d) 3 equiv 1H-pyrazole-
1-carboxamidine hydrochloride, 4.5 equiv diisopropylethylamine in DMF, 16 h. All final compounds were purified by reversed phase HPLC to a purity of >95% according to
HPLC analysis and UV detection at 220 nm and obtained as lyophilized powders.
NH2
NH
NH2
NH
NH2
NH
O
O
NH
O
H
N
O
NH
O
O
H
N
O
NH
O
S
N
N
O
H
N
S
O
N
O
O
a
b
S
O
N
O
O
N
O
N
HO
N
6
45
18
Scheme 2. Reagents and conditions: (a) trifluoroacetic acid for 1 h, (b) 1.1 equiv 1-(2-pyrimidyl)-piperazine, 1 equiv PyBOP, 2 equiv diisopropylethylamine, room
temperature, 6 h.
3. Gong, J.; Fang, H.; Li, M.; Liu, Y.; Yang, K.; Liu, Y.; Xu, W. Curr. Med. Chem. 2009,
16, 3716.
the piperazine series were effective in suppressing replication of H1
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and H3 influenza viruses in a HAT-expressing MDCK cell model.
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6. Böttcher, E.; Matrosovich, T.; Beyerle, M.; Klenk, H. D.; Garten, W.; Matrosovich,
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Acknowledgment
The authors would like to thank Robert Etges for correcting the
English version of the manuscript.
9. Wysocka, M.; Spichalska, B.; Lesner, A.; Jaros, M.; Brzozowski, K.; Legowska, A.;
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.033.
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experiments.
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