Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics

Article abstract of DOI:10.1021/jm9012455

Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal physiological and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer, and metastasis. Furin and related furin-like PCs cleave their substrates at characteristic multibasic consensus sequences, preferentially after an arginine residue. By incorporating decarboxylated arginine mimetics in the P1 position of substrate analogue peptidic inhibitors, we could identify highly potent furin inhibitors. The most potent compound, phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15), inhibits furin with a K_i value of 0.81 nM and has also comparable affinity to other PCs like PC1/3, PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl plague virus (influenza A, H7N1)-infected MDCK cells, inhibitor 15 inhibited proteolytic hemagglutinin cleavage and was able to reduce virus propagation in a long-term infection test. Molecular modeling revealed several key interactions of the 4-amidinobenzylamide residue in the S1 pocket of furin contributing to the excellent affinity of these inhibitors.

Full text of DOI:10.1021/jm9012455

J. Med. Chem. 2010, 53, 1067–1075 1067
DOI: 10.1021/jm9012455
Potent Inhibitors of Furin and Furin-like Proprotein Convertases Containing Decarboxylated P1
Arginine Mimetics
Gero L. Becker,† Frank Sielaff,† Manuel E. Than,‡ Iris Lindberg,§ Sophie Routhier, Robert Day, Yinghui Lu,^
Wolfgang Garten,^ and Torsten Steinmetzer*,†
†Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, D-35032 Marburg, Germany, ‡Leibniz Institute for Age
Research-Fritz Lipmann Institute (FLI), Protein Crystallography Group, Beutenbergstrasse 11, 07745 Jena, Germany, §Department of Anatomy and
Neurobiology, University of Maryland, Baltimore, Maryland 21201, Institut de Pharmacologie de Sherbrooke, Universiteꢀde Sherbrooke, Sherbrooke,
Queꢀbec, Canada, J1H 5N4, and ^Institute of Virology, Philipps University Marburg, Hans-Meerwein-Strasse 2, 35043 Marburg, Germany
Received August 19, 2009
Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal
physiological and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer,
and metastasis. Furin and related furin-like PCs cleave their substrates at characteristic multibasic
consensus sequences, preferentially after an arginine residue. By incorporating decarboxylated arginine
mimetics in the P1 position of substrate analogue peptidic inhibitors, we could identify highly potent
furin inhibitors. The most potent compound, phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15),
inhibits furin with a Ki value of 0.81 nM and has also comparable affinity to other PCs like PC1/3,
PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl
plague virus (influenza A, H7N1)-infected MDCK cells, inhibitor 15 inhibited proteolytic hemagglu-
tinin cleavage and was able to reduce virus propagation in a long-term infection test. Molecular
modeling revealed several key interactions of the 4-amidinobenzylamide residue in the S1 pocket of furin
contributing to the excellent affinity of these inhibitors.
Introduction
anthrax toxin.12 Early endosomal furin also activates several
other bacterial toxins, such as Pseudomonas exotoxin, Shiga-
like toxin-1, and diphtheria toxins.4 Upregulation of PCs was
observed in many tumors, and in some cases, elevated levels of
PC expression could be correlated with enhanced malignancy
and invasiveness, probably via activation of metalloproteases,
angiogenic factors, growth factors, and their receptors.13-16
However, the function of PCs in the regulation of tumor
growth and progression seems to be more complex, because
other reports describe that PCs are also involved in the
activation of proteins with tumor suppressor functions, such
as cadherins.17 PCs are involved in neurodegenerative disor-
ders such as Alzheimer’s disease by activation of R-, β-, and γ-
secretases or via the release of amyloidogenic peptides.18 The
intracellular endoproteolytic PC-catalyzed activation of
membrane-bound MT1-MMP in macrophages is important
for plaque stability in atherosclerosis.19 The cleavage efficacy
of the PCs toward a large number of potential substrates,
some of which are likely to be involved in additional diseases,
has been recently investigated in detail.5 Therefore, PC in-
hibitors might represent potential drugs for the treatment of
these diseases.
Furin is a member of the group of proprotein convertases
(PCs), a family of Ca2þ-dependent multidomain mammalian
endoproteases that contain a catalytic serine protease domain
of the subtilisin type.1 Together with six other members of this
family, PC2, PC1/3, PACE4, PC4, PC5/6, and PC7, furin
possesses a strong preference for substrates containing the
multibasic cleavage motif Arg-X-Arg/Lys-ArgV-X.2-4
Furin and its analogues are responsible for the maturation
of a huge number of inactive protein precursors5,6 and are
therefore involved in many normal physiological processes.
However, several studieshave alsorevealed a function of these
proteases in numerous diseases, such as viral and bacterial
infections, tumorigenesis, neurodegenerative disorders, dia-
betes, and atherosclerosis.3,4 For instance, furin-like PCs can
process the HIV-1 surface protein gp160 into gp120 and gp41,
whichform anenvelope complex necessaryfor the virulence of
HIV-1.7 Additional potential substrates are surface proteins
of highly pathogenic avian influenza viruses of the H5 and H7
subtypes, from the hemorrhagic Ebola and Marburg viruses
or from the measles virus that all must be cleaved atmultibasic
consensus sites for formation of their mature and fusogenic
envelope glycoproteins.8-11 Furin is also involved in the
pathogenicity of Bacillus anthracis because of its ability to
activate the protective antigen precursor, one component of
Compared to other arginine-specific proteases, such as the
trypsin-like serine proteases thrombin and factor Xa, only
moderate progress has been achieved in the field of PC
inhibitors. PCs are inhibited by various naturally occurring
macromolecular protein-based inhibitors; additional bio
engineered inhibitors have been designed by incorporation
of the PC’s consensus sequence into variants of the serpin
R1-antitrypsin, the leech-derived eglin C, and the third domain
*To whom correspondence should be addressed: Institute of Pharma-
ceutical Chemistry, Philipps University Marburg, Marbacher Weg 6,
D-35032 Marburg, Germany. Telephone: þ49-6421-2825900. Fax:
þ49-6421-2825901. E-mail: steinmetzer@staff.uni-marburg.de.
r
2009 American Chemical Society
Published on Web 12/28/2009
pubs.acs.org/jmc

1068 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Becker et al.
of turkey ovomucoid.20,21 Most of the small molecule PC
inhibitors belong to three groups, pure peptides, peptide mime-
tics, or nonpeptidic compounds. Peptides derived from the PC
prodomains22 or identified from a combinatorial library inhibit
furin and some related PCs in the micromolar range.23 Im-
proved activity was obtained with polyarginine24 or poly-
D-arginine-derived analogues; the most potent compound,
nona-D-arginine, inhibits furin with a Ki value of 1.3 nM.25
The first potent peptidomimetic furin inhibitors were developed
by coupling of appropriate multibasic substrate sequences to a
P1 arginyl chloromethyl ketone group. The irreversible inhibi-
tor decanoyl-Arg-Val-Lys-Arg-CMK has now been used by
many groups as a reference to study the effects of furin and
related PCs.9 Other groups developed ketone-based transition
state analogues, which most likely inhibit furin via formation of
a reversible hemiketal.26 Although these ketone-derived inhibi-
tors are valuable biochemical tools, especially for X-ray analy-
sis27 and for preliminary in vivo studies (for example, with fowl
plaque virus8), they are less suited for drug design. Ketones are
often prone to racemization at the P1 CR and can be attacked
by numerous nucleophiles, which limits their stability in vivo.28
A boroarginine-derived transition state inhibitor was used for
the determination of the crystal structure of Kex2, a furin
analogue protease from yeast.29
Scheme 1. Synthesis of Inhibitors 3-8a
a Reagents and conditions: (a) trityl chloride resin, 2 equiv of diamine,
dry THF, 2 h; (b) Fmoc SPPS, double couplings with 4 equiv of amino
acid (or phenylacetic acid), HOBt and HBTU, respectively, and 8 equiv
of DIPEA; (c) TFA/TIS/H2O (95/2.5/2.5, v/v/v), 2 h; (d) 3 equiv of
1H-pyrazole-1-carboxamidine HCl, 4 equiv of DIPEA in a DMF/1 M
3
Na2CO3 solution (1/1, v/v), 16 h.
containing a symmetric P1 diamine moiety as well as their
guanylated analogues (3-12) were prepared on trityl chlor-
ide resin by standard Fmoc SPPS. Initially, the resin was
loaded with the diamine, followed by coupling of the P2-P5
residues. The peptides were cleaved from resin to yield the
unprotected amines. These were finally modified by treat-
ment with 1H-pyrazole-1-carboxamidine35 in a mixture of a
Na2CO3 solution with DMF (Scheme 1).
Excellent potency was described for a series of nonpeptidic
multibasic 2,5-dideoxystreptamine derivatives, which inhibit
furin with Ki values of <10 nM and have negligible affinity to
trypsin-like serine proteases.30 Very recently, several nonpep-
tidic inhibitors with micromolar affinities were identified by
high-throughput screening.31
In the past several years, numerous groups have developed
substrate analogue inhibitors for trypsin-like serine proteases
containing decarboxylated P1 arginine mimetics, e.g., for the
clotting proteases thrombin32,33 and factor Xa.34 Such ana-
logues are not susceptible to degradation by carboxypeptidases
and do not contain a negatively charged P1 carboxylate group,
which should repel the inhibitor from the serine nucleophile in
the enzyme’s active site. Therefore, we incorporated some of
these P1 residues in tetrapeptide derivatives derived from
furin’s consensus sequence and could identify the 4-amidino-
benzylamide group as an excellent arginine replacement.
Herein, we report the activity of these compounds on furin
inhibition; for selected compounds, we also describe their
specificity toward the PC family and certain trypsin-like serine
proteases. A model in complex with furin reveals several
interactions of the P1 group that might contribute to the
excellent potency of these compounds. One inhibitor was used
to inhibit the replication of a highly pathogenic avian influenza
virus (HPAIV)a of subtype H7 in a cellular assay.
The P5-P2 segments of inhibitors 13-18 were prepared
by standard Fmoc SPPS on 2-chlorotrityl chloride resin
(Scheme 2). After liberation of the side chain-protected
intermediate 2 from resin, 4-amidinobenzylamine 2HCl (1)
3
was coupled in solution using a PyBOP/DIPEA mixture in
the presence of excess Cl-HOBt. Final side chain deprotec-
tion provided inhibitor 15. In analogy, 1-Boc-4-(amino-
methyl)piperidine was coupled to fragment 2, followed by
cleavage of protecting groups to yield 13, which was con-
verted by treatment with 1H-pyrazole-1-carboxamidine and
DIPEA to inhibitor 14. The synthesis of compounds 16-18
was performed in analogy to that of inhibitor 15, using
decanoic or acetic acid in the final coupling step.
Determination of Inhibition Constants. Our initial work was
focused on the identification of a more druglike P1 residue to
replace the arginine ketone moieties used in previously de-
scribed furin inhibitors. The peptide sequence of the inhibitors
was derived from the chloromethyl ketone decanoyl-Arg-Val-
Lys-Arg-CMK,7 whereas the P2 residue was changed to argi-
nine to prevent a second guanylation of an unprotected Lys side
chain during reactions with 1H-pyrazole-1-carboxamidine,
which would lead to homoarginine derivatives. In addition,
the N-terminal decanoyl group was substituted with a hydro-
phobic phenylacetyl group to facilitate UV detection, especially
when intermediates without aromatic systems were to be
analyzed on HPLC, e.g., analogues with aliphatic diamines in
P1 position. The structures of the inhibitors synthesized are
summarized in Table 1.
Results
Chemistry. A combined solution- and solid-phase ap-
proach was used for inhibitor synthesis. All compounds
a Abbreviations: Ac, acetyl; Amba, 4-amidinobenzylamide; Cbz,
carboxybenzyl; DCM, dichloromethane; Dec, decanoyl; DIPEA, diiso-
propylethylamine; DMF, dimethylformamide; ER, endoplasmic reticu-
lum; FCS, fetal calf serum; FPV, fowl plaque virus; HA, all forms of the
hemagglutinin; HBTU, O-benzotriazolyl-N,N,N0,N0-tetramethyluro-
nium hexafluorophosphate; HOBt, hydroxybenzotriazole; HPAIV,
highly pathogenic avian influenza virus; LPAI, low-pathogenic or
apathogenic influenza virus strains; MDCK, Madin-Darby canine
kidney; PDB, Protein Data Bank; PFU, plaque-forming units; Phac,
phenylacetyl; PyBOP, (benzotriazolyl)-N-oxy-pyrrolidinium phospho-
nium hexafluorophosphate; SPPS, solid-phase peptide synthesis; TFA,
trifluoroacetic acid; TIS, triisopropylsilane; THF, tetrahydrofuran.
Initially, we incorporated an agmatine moiety in the P1
position, which directly corresponds to a decarboxylated
arginine. As described for substrate analogue thrombin
inhibitors, we also prepared the shorter and longer ana-
logues36 and purified their corresponding amines (e.g., 5),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1069
Scheme 2. Synthesis of Inhibitors 13-15a
a Reagents and conditions: (a) loading of 2-chlorotrityl chloride resin, Fmoc-Arg(Pbf)-OH, 4 equiv of DIPEA, dry DCM, 2 h; (b) Fmoc SPPS (for
conditions, see Scheme 1); (c) 1% TFA in DCM, 2 ꢀ 30 min; (d) 1, 1.1 equiv of PyBOP, 3 equiv of 6-Cl-HOBt, 3 equiv of DIPEA in DMF, 2 h; (e) TFA/
TIS/H2O (95/2.5/2.5, v/v/v), 2 h; (f) 1-Boc-4-(aminomethyl)piperidine, 1.1 equiv of PyBOP, 3 equiv of 6-Cl-HOBt, 3 equiv of DIPEA in DMF, 2 h;
(g) 3 equiv of 1H-pyrazole-1-carboxamidine HCl, 4 equiv of DIPEA in DMF, 16 h.
3
which were obtained as intermediates directly after cleavage
from resin. A similar inhibitory potency toward furin was
observed for the agmatine (6) and noragmatine (4) deriva-
tives, whereas their analogue amines are significantly less
potent and have Ki values of >1 μM. In contrast, for the
longer diaminopentane inhibitor 7, a slightly stronger pote-
ncy compared to that of its guanylated analogue 8 was
found. A poor potency was observed for both diaminoxylene
inhibitors and their guanylated analogues (9-12), whereas
the N-(amidino)piperidine-derived inhibitor 14 binds to
furin with a Ki value of 53 nM. In preliminary investigations,
we could find only a poor inhibition of furin with simple
benzamidine and 4-aminomethylbenzamidine (1) with inhi-
bition constants of 0.75 and 1.2 mM, respectively. There-
fore, we were delighted to observe excellent potency with
4-amidinobenzylamide compound 15. Enzyme kinetic stu-
dies revealed that all compounds summarized in Table 1 act
as reversible competitive inhibitors; as an example, the
Dixon plot for inhibitor 15 is given in Figure 1.
For comparison to the reference inhibitor Dec-Arg-Val-
Lys-Arg-CMK, we also prepared analogues with N-terminal
decanoyl and acetyl groups and a P2 Lys derivative. Whereas
slightly reduced potency was observed for decanoyl com-
pound 16, an excellent Ki value was found for acetylated
inhibitor 17. P2 Lys inhibitor 18 has activity comparable to
that of its Arg analogue 16, which indicates that both
residues are well accepted by furin’s S2 pocket.
Although some differences in substrate specificity exist
between furin-like PCs,5 they all share a strong preference for
similar multibasic cleavage sequences. Therefore, we as-
sumed that these substrate analogue inhibitors also have
the potential to inhibit other PCs. To prove this hypothesis,
we selected five compounds and measured their selectivity
toward related PCs (Table 2).
As expected, other PCs, such as hPACE4, hPC5/6, and
hPC2, were also inhibited in a similar range by the selected
inhibitors, whereas hPC7 and hPC2 were less affected.
Surprisingly, the highest potency for hPC2 was observed
with decanoyl analogue 16. To further analyze selectivity,
three inhibitors with different P1 residues were also tested
toward the trypsin-like serine proteases thrombin, factor Xa,
and plasmin; however, only a marginal inhibition of these
proteases was found.
Inhibition of Proteolytic Hemagglutinin (HA) Cleavage of
Fowl Plague Virus (FPV). The virus surface HA-spike pro-
tein of FPV, which belongs to the highly pathogenic avian
influenza (HPAI) viruses of subtypes H5 and H7, is synthe-
sized as precursor molecule HA0. HA0 is cleaved by furin or
by a furin-like protease, present in all cells, at a multibasic
cleavage site into distal subunit HA1 and membrane-an-
chored subunit HA2. The cleavage occurs during the trans-
port of HA from the ER to the plasma membrane, where
virus assembly and budding take place.37,38 At the beginning
of infection, after receptor binding and endocytosis, the viral
envelope merges with the endosomal membrane. The viral
genome and accessory proteins are delivered into the cell
nucleus, where multiplication of influenza virus progeny
proceeds. The fusion step is essential for each host cell
infection cycle and is absolutely dependent on correctly
cleaved HA. However, it should be mentioned that cleavage
is not required for hemagglutination or virus assembly. We
selected inhibitor 15 to study its effects on the proteolytic
processing of HA0 in FPV-infected cells (Figure 2). FPV-
infected cell cultures were exposed to different concentra-
tions of inhibitor 15 and Dec-Arg-Val-Lys-Arg-CMK,
which was used as a control. When viruses in the cell super-
natant reached a hemagglutination titer of 27 HAU, cell
lysates were prepared and viral proteins analyzed. The level
of cleavage of HA is most reduced with both inhibitors at a
concentration of 50 μM (Figure 2A). Stepwise 2-fold dilu-
tion of the inhibitors is associated with a gradual loss of
noncleaved HA0, which was quantified by using a near-IR
dye-labeled antibody. The IC50 value of compound 15 is
approximately 10 μM and is in a range similar to that found
for the control inhibitor (Figure 2B). Very similar results in
this cellular assay were also obtained with decanoyl inhibitor
16, whereas acetyl analogue 17 had reduced activity (data not
shown). At 50 μM concentrations of inhibitor 15, the virus
particles obtained from inhibitor-treated cells contain con-
siderable (>80%) noncleaved HA (data not shown).

1070 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Becker et al.
Table 1. Inhibition of Furin by Inhibitors of the General Formula
R-Arg-Val-P2-P1
Figure 1. Dixon plot of inhibitor 15. Kinetic measurements were
performed with four different substrate (pyroGlu-Arg-Thr-
Lys-Arg-AMC) concentrations [(b) 5, (O) 12.5, (2) 20, and (4)
50 μM] in the presence of 0.95 nM furin using various inhibitor
concentrations of g10 nM. The dashed line represents 1/Vmax
,
which was obtained from a Michaelis-Menten curve measured at
the same time in parallel on the same 96-well plate (Km = 4.9 μM;
V
max = 28.4 RFU/s).
infection were examined by multiple-cycle replication of
FPV after an inoculation of the cell cultures with a low
multiplicity of infection (MOI). This type of experiment
results in a retardation and reduced virus replication in the
presence of inhibitors.39 MDCK cells were infected by FPV
at a low MOI of 10-5 and incubated in the presence of 25 μM
compound 15 and in the absence of any inhibitor for up to
72 h. FPV propagation in the presence of compound 15 at
25 μM was monitored by virus titration of released virus into
the cell culture supernatants using the standard hema-
gglutination test39 (Figure 3). Virus release appears delayed
by ∼24 h in comparison to that in FPV-infected cell cultures
which were not treated with inhibitor and in which FPV has
more rapidly proliferated. The fact that FPV is still produced
after a period of delay up to nearly the same level as obtained
without inhibitor demonstrates that the viability of the cells
is not noticeably influenced in the presence of inhibitor.
Discussion
A search for suitable replacements of the P1 arginylketones
in a series of peptidic furin inhibitors revealed various alter-
natives among decarboxylated arginine mimetics, known from
previous developments of trypsin-like serine protease inhibi-
tors. The incorporation of 4-amidinobenzylamide, originally
used for the development of the thrombin inhibitor melagatran
and of its hydroxyamidino prodrug ximelagatran,32 noragma-
tine,40 agmatine,36 and 4-(amidomethyl)-N-(amidino)piperi-
dine,41 provided potent inhibitors of furin and related PCs,
such as hPACE4, hPC5/6, and hPC2. The limited selectivity
toward the PC family might be a drawback of these inhibitors
under some circumstances but otherwise could provide some
advantage for special applications, i.e., if PCs are coexpressed
in cells and the inhibition of only one PC may not be sufficient.
The low affinity of the selected inhibitors toward thrombin,
factor Xa, and plasmin, which is in the range of simple
benzamidines, should be advantageous for the maintenance
of the homeostasis of blood without affecting the well-balanced
clotting and fibrinolysis systems. One of the reasons for the
a This compound was synthesized according to Scheme 2 using Fmoc-
Lys(Cbz)-OH for coupling of the P2 amino acid. The Cbz group was
removed in the final step by hydrogenation in 90% acetic acid at room
temperature overnight using Pd/C as a catalyst.
Multiple-Cycle Replications of FPV in the Presence and
Absence of Inhibitors. Inhibitory effects on long-term virus

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1071
Table 2. Specificity of Selected Inhibitors for Furin-like PCs and Trypsin-like Serine Proteases
Ki (nM)
inhibitor
furin
hPACE4
hPC5/6
hPC7
hPC1/3
hPC2
thrombin
fXa
plasmin
6
78
53
42
67
85
173
1.6
6.3
3.6
>10000
>10000
6154
53
70
>10000
>10000
312
102000
50000
23000
nda
83000
123000
40000
nda
97000
>1000000
6000
14
15
16
17
0.81
1.6
1.0
0.6
3.0
2.4
0.75
3.65
1.7
968
5131
55
1388
nda
nda
nda
nda
a Not determined.
Figure 3. Inhibition of multiple-cycle replication of FPV in cell
culture in the absence (black bars) and in the presence of inhibitor 15
(white bars). Cultures of MDCK cells were inoculated with the
Rostock strain of FPV at an MOI of 10-5 PFU per cell and
incubated in DMEM without FCS at 37 ꢀC. The inhibitor was
added to the medium, reaching a final concentration of 25 μM. At
different times (10, 18, 32, 48, 60, and 72 h postinfection), the
amount of virus released into the medium was measured by hema-
gglutination titration (HAU), and at 10 h, no released virus was
detected in either group. Mean values of four independent experi-
ments are shown.
Figure 2. Inhibition of fowl plague virus hemagglutinin cleavage by
compound 15 and Dec-Arg-Val-Lys-Arg-CMK as a control. (A)
Confluent MDCK cell cultures were infected with egg-grown
influenza virus A/FPV/Rostock/34 (H7N1) at a multiplicity of
infection of 10 per cell. Inhibition of HA cleavage (PSKKRK-
KRVGLFG) at different inhibitor concentrations was analyzed after
cell lysis 16 h postinfection. Proteins from virus-infected cells were
subjected to SDS-PAGE and Western blotting. Viral proteins were
immunochemically detected by using rabbit anti-FPV serum and
the ECL reaction kit (Pierce): HA0 (82 kDa), nucleoprotein NP
(56 kDa), and HA1 (∼50 kDa). HA2 (32 kDa) was not detectable by
the antiserum used. (B) Quantification of HA cleavage inhibition.
Three independent experiments for both inhibitors were performed
using a modified Western blot analysis technique allowing quanti-
fication by a near-IR dye-labeled second monoclonal antibody
applicable for the LI-COR Odyssey Image System. The maximum
amount of HA0 obtained by inhibition with 50 μM Dec-Arg-Val-
Lys-Arg-CMK was normalized to 100% cleavage inhibition. Other
HA0 band intensities at different concentrations were measured and
equalized by standardization of each HA0 value correlating with the
corresponding nucleoprotein NP band (56 kDa).
X-ray structureof mouse furinincomplex withthe irreversible
inhibitor Dec-Arg-Val-Lys-Arg-CMK.27 Therefore, we re-
placed the arginyl-chloromethyl ketone group in PDB entry
1p8j with 4-amidinobenzylamide followed by energy minimi-
zation. As only three amino acid residues vary between the
catalytic domains of the mouse and human enzymes, none
being in the proximity of the catalytic cleft, the modeled
complex between inhibitor 18 and the mouse enzyme should
also be representative of the human enzyme (Figure 4).
On the basis of the modeled structure, we could identify
several interactions between the P1 residue and furin which
may explain the high potency of the 4-amidinobenyzlamide
inhibitors. The amidino group bindstothe carbonyl oxygen of
Ala292 and makes salt bridges to the carboxyl side chains of
Asp306 and Asp258. In addition, a weak hydrogen bond is
formed between the carbonyl oxygen of Pro256 and the
surrounding water molecules, connecting the amidinobenzyl
moiety via the Ca2þ ion to the carboxyl side chains of Glu331
and Asp301. The benzyl ring is sandwiched between the main
chains of Trp254 and Gly255 and the main chains of Gly294
and Asn295, similar to the guanidino group of the P1 Arg seen
in the experimental structure. Several other hydrogen bonds
are formed between the enzyme and the inhibitor, which are
identical to the X-ray structure. The P2 Lys amino group
binds to the carboxyl residue of Asp154, to the side chain
amide of Asn192, and weakly to the carbonyl of Asp191. In
addition, hydrogen bonds are formed to a cluster of water
different affinities toward PCs and trypsin-like proteases is
most likely the different requirements for the configuration of
the P3 residue. Most of the potent noncovalent substrate
analogue trypsin-like serine protease inhibitors contain a P3
residue in the D-configuration, whereas the PC inhibitors
described here possess an L-amino acid at this position.
Very often, peptidic structures suffer from poor stability
due to rapid proteolysis. However, because of the missing
C-terminal carboxyl group, we assume that the inhibitors
described in Table 1 are resistant to cleavage by carboxy-
peptidases, whereas the N-terminal acetylation should stabi-
lize them against aminopeptidases. However, at present, we
have no information regarding the stability of these com-
pounds toward endoproteases.
To gain a first idea regarding the binding mode of the
4-amidinobenzylamide in the S1 pocket, we modeled the
complex of inhibitor 18 bound to furin, based on the known

1072 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Becker et al.
Figure 4. Stereoview of the modeled complex between inhibitor 18 (Dec-Arg-Val-Lys-Amba) and mouse furin. (A) The inhibitor (ball-and-
stick model) is shown in front of the solid surface of the catalytic domain, colored according to its calculated negative (-15 e/kT, red) and
positive (15 e/kT, blue) electrostatic potential. Most of the amino acids responsible for the strong negative surface potential in the vicinity of the
active site are labeled. Asp306 (denoted with an asterisk) is actually located below the enzyme surface at the bottom of the S1 pocket, making a
direct contact with the amidino moiety of the inhibitor (see also panel B). (B) Stick model of the surrounding residues together with the inhibitor
(ball-and-stick model, carbon colored light green). Furin carbons are colored gray, oxygens red, and nitrogens blue. The catalytic residues are
shown as thicker dark gray sticks, and the calcium 2 ion at the bottom of the S1 pocket is shown as a purple sphere. For the sake of clarity, water
molecules were omitted and only important enzyme residues involved in interactions with the inhibitor and/or being responsible for the strong
negative surface potential are labeled. Strong H-bond and salt bridge contacts discussed in the text are indicated by dotted green lines. Panel B
was slightly rotated with respect to panel A to improve the visibility. A PDB file of the modeled complex is available as Supporting Information.
This figure was prepared using Grasp,50 MolScript,51 and Raster3D.52
molecules. A P2 Arg side chain should easily be accommodated
by a slight rearrangement of those water molecules and, again
after a slight structural rearrangement, potentially by addi-
tional interaction(s) with the carboxyl side chains of Asp228
and Asp191. The backbone of the P3 Val forms a short
antiparallel β-sheet to Gly255, reminiscent of similar inter-
actions between the D-P3 residue in trypsin-like serine protease
inhibitors and the protease residue Gly216. However, in the
case of substrate analogue furin inhibitors, an L-configuration
of the P3 residue is required, because the space above the indole
ring of Trp254 is filled by the side chain of Leu227 and is
therefore not accessible for the side chain of a P3 amino acid in
the D-configuration. The β-sheet mentioned above is completed
by a hydrogen bond between the P1 amide NH group and the
carbonyl group of Ser253. The NH group and carbonyl oxygen
of the P4 Arg and the decanoyl carbonyl group are involved in
hydrogen bonds with surrounding water molecules, bridging
interaction with the amide NH group of Glu257. The P4
guanidine group makes salt bridges to Asp264 and Glu236. It
forms additional tight hydrogen bonds with the carbonyl
oxygen of its own P5 residue and the OH group of Tyr308,
connecting it again with the carboxyl side chain of Glu236.
These newly described inhibitors should be useful tools for
further studies of the physiological role of furin-like PCs. In a
first application, compound 15 inhibited the multiple-cycle
replication of FPV in cell culture and suppressed the activation
of HA precursor HA0 in FPV-infected cells with a potency
similar to that observed for the chloromethyl ketone-based
control inhibitor. However, in contrast to the nanomolar Ki
values, a significantly higher inhibitor concentration of ap-
proximately 10 μM was required in the cellular assay to
produce a 50% inhibition of HA0 cleavage. The reduced
potency could be related to the predominant intracellular
localization of furin within the trans-Golgi network,42 which
makes the protease poorly accessible to such multibasic and
polar inhibitors. A strong discrepancy between the potent in
vitro activity and significantly reduced efficacy in cellular assays
was also found for many other furin inhibitors.25,30,43-45 In
contrast, relatively small differences were determined for a
recently discovered series of more hydrophobic dicoumarols;
the obtained IC50 values from cellular assays were only slightly
increased compared to their Ki values, which were in the range
between 1 and 20 μM.31
Despite equipotent activity between inhibitor 15 and the
chloromethyl ketone inhibitor, we believe that the 4-amidino-
benzylamide derivatives have a significant advantage because
of their improved stability. We could not detect any change in
the elution profile of inhibitor 15 by HPLC, when a stock

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1073
solution of the inhibitor was stored in water at room tem-
perature over a period of 2 weeks. The synthesis and incor-
poration of the chemically stable 4-amidinobenzylamide
group into peptidic inhibitors are less laborious than the
preparation of arginyl ketone derivatives. Therefore, a more
simple optimization of these PC inhibitors will be possible in
the future. Our work is presently focused on the design of
inhibitors with different selectivity profiles and improved
physicochemical properties, including the use of well-known
benzamidine prodrug strategies, to enhance cell perme-
ability and bioavailability. We assume that these are impor-
tant prerequisites for further enhancing the activity of the
4-amidinobenzylamide-derived furin inhibitors in cellular
assays and identification of suitable candidates for evaluation
in animal models.
tert-BuOH in water: white solid (yield, 1.452 g, 1.38 mmol); purity
>97% based on HPLC at 220 nm; retention time, 64.6 min; MS
calcd m/z 1051.49, m/z found 1052.5, (M þ H)þ.
Phac-Arg-Val-Arg-3-(amido)propylamine 3TFA (3). 1,3-Dia-
3
minopropane (50.03 μL, 0.6 mmol, 4 equiv) was dissolved in 1.2
mL of dry THF and poured immediately onto trityl chloride
resin (0.1 g, resin loading of 1.5 mmol/g). The mixture was
shaken for 2 h at RT, followed by treatment with a DCM/
MeOH/DIPEA mixture (17/2/1, v/v/v, 3 ꢀ 1 min), and was
washed several times with DCM, DMF, and DCM. The synth-
esis proceeded by automated Fmoc SPPS as described for the
manual synthesis of compound 2. The product was cleaved by
shaking the resin in a TFA/TIS/H2O mixture (95/2.5/2.5, v/v/v)
for 2 h at RT, followed by filtration and precipitation with cold
ether. The precipitate was washed twice with ether and dried.
The product was purified by preparative HPLC and lyophilized
from 80% tert-BuOH in water: white solid (yield, 39 mg, 0.041
mmol); retention time, 22.2 min; MS calcd m/z 603.4, found m/z
604.6, (M þ H)þ.
Experimental Section
Analytical HPLC experiments were performed on a Shimadzu
˚
The synthesis of inhibitors 5, 7, 9, and 11 was performed in an
identical manner; for analytical data, see the Supporting Infor-
mation.
LC-10A system [column, Nucleodur C18, 5 μm, 100 A, 4.6 mm ꢀ
€
250 mm (Machery-Nagel, Duren, Germany)] with a linear gra-
dient of acetonitrile (from 1 to 70% over 69 min, detection at
220 nm) containing 0.1% TFA at a flow rate of 1 mL/min. The
final inhibitors were purified to >95% purity (detection at
220 nm) by preparative HPLC (pumps, Varian PrepStar model
218 gradient system; detector, ProStar model 320; fraction col-
lector, Varian model 701) using a C8 column [Nucleodur, 5 μm,
Phac-Arg-Val-Arg-3-(amido)propylguanidine 3TFA (4). To
3
obtain compound 4, the corresponding amine was prepared
via the manner described for compound 3. The precipitated
amine was dissolved in a mixture of 1 mL of a 1 M Na2CO3
solution/DMF mixture (1/1, v/v) followed by addition of
1H-pyrazole-1-carboxamidine HCl (68.1 mg, 0.45 mmol) and
3
˚
100 A, 32 mm ꢀ 250 mm (Macherey-Nagel)] and a linear gradient
DIPEA (106 μL, 0.6 mmol). The mixture was stirred for 16 h at
RT, and the solvent was removed in vacuo. The residue was
purified by preparative HPLC and lyophilized from 80% tert-
BuOH in water: white solid (yield, 40 mg, 0.041 mmol); retention
time, 23.4 min; MS calcd m/z 645.4, found m/z 646.5, (M þ H)þ.
The synthesis of inhibitors 6, 8, 10, and 12 was performed by
an identical procedure; for analytical data, see the Supporting
Information.
of acetonitrile containing 0.1% TFA at a flow rate of 20 mL/min.
All inhibitors were finally obtained as TFA salts after lyophiliza-
tion. The molecular mass of the synthesized compounds was
determined using a QTrap 2000 ESI spectrometer (Applied
1
Biosystems). The H and 13C NMR spectra were recorded on
an ECX-400 instrument (Jeol Inc.) at 400 and 100 MHz, respec-
tively, and are referenced to internal solvent signals.
Reagents for synthesis (Fmoc amino acids, coupling reagents,
and reagents for synthesis) were obtained from Orpegen,
Novabiochem, Iris, Fluka, Aldrich, or Acros. The automated
solid-phase peptide synthesis was performed in a Syro 2000
instrument (MultiSynTech GmbH, Witten, Germany).
Phac-Arg-Val-Arg-4-(amidomethyl)piperidine 3TFA (13).
3
Compound 2 (105.2 mg, 0.1 mmol, 1 equiv), 1-Boc-4-(amino-
methyl)piperidine (Fluka, 21.43 mg, 0.1 mmol, 1 equiv), PyBOP
(57.3 mg, 0.11 mmol, 1.1 equiv), 6-Cl-HOBt (20.9 mg, 0.3 mmol,
3 equiv), and DIPEA (51.36 μL, 0.3 mmol, 3 equiv) were
dissolved in 0.5 mL of DMF, and the mixture was stirred for
2 h at RT. The solvent was removed in vacuo to yield a brownish
oil. This was dissolved in a TFA/TIS/H2O mixture (95/2.5/2.5,
v/v/v), stirred for 3 h at RT, precipitated with cold ether, washed
twice with ether, and dried. The precipitate was purified by
preparative HPLC and lyophilized from 80% tert-BuOH in
water: white solid (yield, 55 mg, 0.056 mmol); retention time,
23.1 min; MS calcd m/z 643.43, found m/z 644.5, (M þ H)þ.
4-Amidinobenzylamine 2HCl (1). Ten grams (32 mmol) of
3
Boc-4-acetylhydroxyamidinobenzylamide34 was dissolved in
500 mL of 90% acetic acid and treated with 650 mg of catalyst
(10% Pd/C) under an atmosphere of hydrogen. The mixture was
hydrogenated over a period of 48 h; the catalyst was removed by
filtration, and the solvent was removed in vacuo. The remaining
Boc-4-amidinobenzylamide acetate was dissolved in 150 mL of
3
water and treated with 40 mL of concentrated HCl. After the
mixture had been stirred for 1.5 h at room temperature (RT), the
solvent was removed in vacuo and the residue was lyophilized
from water: white solid (yield, 6.8 g, 30.6 mmol); MS calcd m/z
149.09, found m/z 150.0, (M þ H)þ; 1H NMR (400 MHz, D2O)
δ 4.46 (s, 2 H), 7.80-8.01 (2d, 4 H, aromatic); 13C NMR (100
MHz, D2O) δ 42.64 (CH2), 128.65, 129.26, 129.62, 138.69
(aromatic), 166.47 (carbon amidino group).
Phac-Arg-Val-Arg-4-(amidomethyl)-N-amidinopiperidine
3
3TFA (14). Compound 13 (25 mg, 0.02 mmol, 1 equiv) and 1H-
pyrazole-1-carboxamidine HCl (9.8 mg, 0.06 mmol, 3 equiv)
3
were dissolved in 0.5 mL of DMF and DIPEA (10.03 μL, 0.08
mmol, 4 equiv). The mixture was stirred for 16 h at RT. The
solvent was removed in vacuo, and the product was purified by
preparative HPLC and lyophilized from 80% tert-BuOH in
water: white solid (yield, 19.5 mg, 0.019 mmol); retention time,
Phac-Arg(Pbf)-Val-Arg(Pbf)-OH (2). Fmoc-Arg(Pbf)-OH
(1.509 g, 2.325 mmol, 1 equiv) was dissolved in 15 mL of dry
DCM and 1.548 mL (9.3 mmol, 4 equiv) of DIPEA, and the
mixture was immediately poured onto 2-chlorotrityl chloride resin
(1.5 g, 1 equiv, resin loading of 1.55 mmol/g). The mixture was
shaken for 2 h at RT followed by treatment with a DCM/MeOH/
DIPEA mixture (17/2/1, v/v/v, 3 ꢀ 1 min), washed several times
with DCM, DMF, and DCM, and dried in vacuo. The synthesis
proceeded by manual Fmoc SPPS using a 4-fold excess of Fmoc-
amino acid, HOBt and HBTU, respectively, and 8 equiv of
DIPEA. The phenylacetic acid was coupled in accordance with
the amino acids. The protected peptide was cleaved from the
resin with 1% TFA in DCM (2 ꢀ 30 min) at RT. The solvent
was removed in vacuo and the peptide lyophilized from 80%
24.4 min; MS calcd m/z 685.45, found m/z 343.9, (M þ 2H)2þ
.
Phac-Arg-Val-Arg-4-(amidomethyl)benzamidine 3TFA (15).
3
Compound 2 (105.2 mg, 0.1 mmol, 1 equiv), compound 1
(22.21 mg, 0.1 mmol, 1 equiv), PyBOP (57.3 mg, 0.11 mmol,
1.1 equiv), 6-Cl-HOBt (20.9 mg, 0.3 mmol, 3 equiv), and DIPEA
(51.36 μL, 0.3 mmol, 3 equiv) were dissolved in 0.5 mL of DMF,
and the mixture was stirred for 2 h at RT. After completion of
the reaction (HPLC control), the solvent was removed in vacuo
to give a brownish oil. The synthesis was continued in a manner
similar to that described for compound 13: white solid (yield,
70 mg, 0.069 mmol); retention time, 24.2 min; MS calcd m/z
678.41, found m/z 679.4, (M þ H)þ.

1074 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Becker et al.
The synthesis of inhibitors 16-18 was performed as described
for 15 using decanoic or acetic acid for coupling of the P5
residue. Inhibitor 18 was prepared by coupling of Fmoc-Lys-
(Cbz)-OH as the P2 residue; the Cbz group was removed in the
final step by hydrogenation in 90% acetic acid at room tem-
perature overnight using Pd/C as a catalyst. For analytical data,
see the Supporting Information.
Enzyme Kinetics with Furin. The inhibition constants of re-
combinant soluble human furin25 were determined at RT accord-
ing to the method of Dixon46 using a Safire 2 fluorescence plate
reader (Tecan) (λex = 380 nm; λem = 460 nm) and pyroGlu-
Arg-Thr-Lys-Arg-AMC as the substrate (Bachem) in 100 mM
HEPES buffer (pH 7.0) containing 0.2% Triton X-100, 2 mM
CaCl2, 0.02% sodium azide, and 1 mg/mL BSA. The enzyme
concentration used in the assay was 0.95 nM, and the substrate
concentrations were normally 5, 20, and 50 μM; for inhibitor 15, a
fourth substrate concentration of 12.5 μMwasused(Figure1). The
lowest inhibitor concentration was at least 10 times higher than the
enzyme concentration used to avoid tight binding conditions.
Information about enzyme kinetic studies with PC2, PC1/3,
PACE4, PC5/6, and PC7 is given in the Supporting Informa-
tion. The Ki values for the trypsin-like serine proteases throm-
bin, factor Xa, and plasmin were determined from Dixon plots
as described previously.47
Modeling. Inhibitor 18 was manually docked into the active site
cleft of furin and energy-minimized using MAIN.48 Basically, the
P1 arginine group of the Dec-Arg-Lys-Arg-CMK-inhibited mouse
furin crystal structure (PDB entry 1p8j)27 was replaced with
4-amidinobenzylamide, and the entire inhibitor was locally en-
ergy-minimized using a force field based on the parameters of Engh
and Huber49 while the amidino moiety was kept coplanar with the
benzyl ring and the coordinates of the enzyme and the Ca2þ ion
were rigidly retained. During minimization, the P4 Arg side chain
was forced to retain its crystallographically clearly defined con-
formation (but apparently somewhat unfavored by the force field),
as previously described.27
(6) Izidoro, M. A.; Gouvea, I. E.; Santos, J. A.; Assis, D. M.; Oliveira,
V.; Judice, W. A.; Juliano, M. A.; Lindberg, I.; Juliano, L. A study
of human furin specificity using synthetic peptides derived from
natural substrates, and effects of potassium ions. Arch. Biochem.
Biophys. 2009, 487, 105–114.
(7) Hallenberger, S.; Bosch, V.; Angliker, H.; Shaw, E.; Klenk, H. D.;
Garten, W. Inhibition of furin-mediated cleavage activation of
HIV-1 glycoprotein gp160. Nature 1992, 360, 358–361.
(8) Stieneke-Grober, A.; Vey, M.; Angliker, H.; Shaw, E.; Thomas,
G.; Roberts, C.; Klenk, H. D.; Garten, W. Influenza virus hema-
gglutinin with multibasic cleavage site is activated by furin, a
subtilisin-like endoprotease. EMBO J. 1992, 11, 2407–2414.
(9) Garten, W.; Hallenberger, S.; Ortmann, D.; Schafer, W.; Vey, M.;
Angliker, H.; Shaw, E.; Klenk, H. D. Processing of viral glycopro-
teins by the subtilisin-like endoprotease furin and its inhibition by
specific peptidylchloroalkylketones. Biochimie 1994, 76, 217–225.
(10) Volchkov, V. E.; Feldmann, H.; Volchkova, V. A.; Klenk, H. D.
Processing of the Ebola virus glycoprotein by the proprotein
convertase furin. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5762–5767.
(11) Volchkov, V. E.; Volchkova, V. A.; Stroher, U.; Becker, S.;
Dolnik, O.; Cieplik, M.; Garten, W.; Klenk, H. D.; Feldmann,
H. Proteolytic processing of Marburg virus glycoprotein. Virology
2000, 268, 1–6.
(12) Gordon, V. M.; Klimpel, K. R.; Arora, N.; Henderson, M. A.;
Leppla, S. H. Proteolytic activation of bacterial toxins by
eukaryotic cells is performed by furin and by additional cellular
proteases. Infect. Immun. 1995, 63, 82–87.
(13) Bassi, D. E.; Lopez De Cicco, R.; Mahloogi, H.; Zucker, S.;
Thomas, G.; Klein-Szanto, A. J. Furin inhibition results in absent
or decreased invasiveness and tumorigenicity of human cancer
cells. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10326–10331.
(14) Scamuffa, N.; Siegfried, G.; Bontemps, Y.; Ma, L.; Basak, A.;
Cherel, G.; Calvo, F.; Seidah, N. G.; Khatib, A. M. Selective
inhibition of proprotein convertases represses the metastatic
potential of human colorectal tumor cells. J. Clin. Invest. 2008,
118, 352–363.
(15) Khatib, A. M.; Bassi, D.; Siegfried, G.; Klein-Szanto, A. J.; Ouafik,
L. Endo/exo-proteolysis in neoplastic progression and metastasis.
J. Mol. Med. 2005, 83, 856–864.
(16) Bassi, D. E.; Fu, J.; Lopez de Cicco, R.; Klein-Szanto, A. J.
Proprotein convertases: “Master switches” in the regulation of
tumor growth and progression. Mol. Carcinog. 2005, 44, 151–161.
€
(17) Muller, E. J.; Caldelari, R.; Posthaus, H. Role of subtilisin-like
convertases in cadherin processing or the conundrum to stall
cadherin function by convertase inhibitors in cancer therapy.
J. Mol. Histol. 2004, 35, 263–275.
Acknowledgment. This work was supported by the
Deutsche Forschungsgemeinschaft (SFB 593 TPB2 and TH
862/1-4), by NIH (DA05084 to I. Lindberg) and by grants
from the Canadian Institutes of Health Research (CIHR) and
(18) Bennett, B. D.; Denis, P.; Haniu, M.; Teplow, D. B.; Kahn, S.;
Louis, J. C.; Citron, M.; Vassar, R. A furin-like convertase
mediates propeptide cleavage of BACE, the Alzheimer’s β-secre-
tase. J. Biol. Chem. 2000, 275, 37712–37717.
(19) Stawowy, P.; Meyborg, H.; Stibenz, D.; Borges Pereira Stawowy,
N.; Roser, M.; Thanabalasingam, U.; Veinot, J. P.; Chretien, M.;
Seidah, N. G.; Fleck, E.; Graf, K. Furin-like proprotein conver-
tases are central regulators of the membrane type matrix metallo-
proteinase-pro-matrix metalloproteinase-2 proteolytic cascade in
atherosclerosis. Circulation 2005, 111, 2820–2827.
(20) Bontemps, Y.; Scamuffa, N.; Calvo, F.; Khatib, A. M. Potential
opportunity in the development of new therapeutic agents based on
endogenous and exogenous inhibitors of the proprotein conver-
tases. Med. Res. Rev. 2007, 27, 631–648.
(21) Basak, A. Inhibitors of proprotein convertases. J. Mol. Med. 2005,
83, 844–855.
ꢁ
ꢀ
ꢀ
the Ministere du Developpement economique, de l’Innova-
tion et de l’Exportation (MDEIE) to RD. We thank Petra
€
Neubauer-Radel for excellent technical assistance and Sarah
Fehling for data analysis of the cell assays.
Supporting Information Available: A table containing MS and
HPLC data of all final inhibitors, conditions of enzyme kinetic
studies with PC2, PC1/3, PACE4, PC5/6, and PC7, conditions
of cellular assays for detecting virus inhibition, and a PDB file of
inhibitor 18 modeled in complex with mouse furin. This material
is available free of charge via the Internet at http://pubs.acs.org.
(22) Basak, A.; Lazure, C. Synthetic peptides derived from the pro-
segments of proprotein convertase 1/3 and furin are potent inhibi-
tors of both enzymes. Biochem. J. 2003, 373, 231–239.
References
(23) Apletalina, E.; Appel, J.; Lamango, N. S.; Houghten, R. A.;
Lindberg, I. Identification of inhibitors of prohormone convertases
1 and 2 using a peptide combinatorial library. J. Biol. Chem. 1998,
273, 26589–26595.
(24) Cameron, A.; Appel, J.; Houghten, R. A.; Lindberg, I. Polyarginines
are potent furin inhibitors. J. Biol. Chem. 2000, 275, 36741–36749.
(25) Kacprzak, M. M.; Peinado, J. R.; Than, M. E.; Appel, J.; Henrich,
S.; Lipkind, G.; Houghten, R. A.; Bode, W.; Lindberg, I. Inhibition
of furin by polyarginine-containing peptides: Nanomolar inhibi-
tion by nona-D-arginine. J. Biol. Chem. 2004, 279, 36788–36794.
(26) Angliker, H. Synthesis of tight binding inhibitors and their action
on the proprotein-processing enzyme furin. J. Med. Chem. 1995,
38, 4014–4018.
(1) van de Ven, W. J.; Voorberg, J.; Fontijn, R.; Pannekoek, H.; van
den Ouweland, A. M.; van Duijnhoven, H. L.; Roebroek, A. J.;
Siezen, R. J. Furin is a subtilisin-like proprotein processing enzyme
in higher eukaryotes. Mol. Biol. Rep. 1990, 14, 265–275.
(2) Rockwell, N. C.; Krysan, D. J.; Komiyama, T.; Fuller, R. S.
Precursor processing by kex2/furin proteases. Chem. Rev. 2002,
102, 4525–4548.
(3) Fugere, M.; Day, R. Cutting back on pro-protein convertases: The
latest approaches to pharmacological inhibition. Trends Pharma-
col. Sci. 2005, 26, 294–301.
(4) Thomas, G. Furin at the cutting edge: From protein traffic to
embryogenesis and disease. Nat. Rev. Mol. Cell Biol. 2002, 3, 753–766.
(5) Remacle, A. G.; Shiryaev, S. A.; Oh, E. S.; Cieplak, P.; Srinivasan, A.;
Wei, G.; Liddington, R. C.; Ratnikov, B. I.; Parent, A.; Desjardins,
R.; Day, R.; Smith, J. W.; Lebl, M.; Strongin, A. Y. Substrate
cleavage analysis of furin and related proprotein convertases. A
comparative study. J. Biol. Chem. 2008, 283, 20897–20906.
(27) Henrich, S.; Cameron, A.; Bourenkov, G. P.; Kiefersauer, R.;
Huber, R.; Lindberg, I.; Bode, W.; Than, M. E. The crystal
structure of the proprotein processing proteinase furin explains
its stringent specificity. Nat. Struct. Biol. 2003, 10, 520–526.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1075
(28) Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Irreversible
inhibitors of serine, cysteine, and threonine proteases. Chem. Rev.
2002, 102, 4639–4750.
(40) Gustafsson, D.; Elg, M.; Lenfors, S.; Borjesson, I.; Teger-Nilsson,
A. C. Effects of inogatran, a new low-molecular-weight thrombin
inhibitor, in rat models of venous and arterial thrombosis, throm-
bolysis and bleeding time. Blood Coagulation Fibrinolysis 1996, 7,
69–79.
(29) Holyoak, T.; Wilson, M. A.; Fenn, T. D.; Kettner, C. A.; Petsko,
˚
G. A.; Fuller, R. S.; Ringe, D. 2.4 A resolution crystal structure of
€
(41) Steinmetzer, T.; Batdorsdhjin, M.; Kleinwachter, P.; Seyfarth, L.;
the prototypical hormone-processing protease Kex2 in complex
with an Ala-Lys-Arg boronic acid inhibitor. Biochemistry 2003, 42,
6709–6718.
€
Greiner, G.; Reissmann, S.; Sturzebecher, J. New thrombin in-
hibitors based on D-cha-Pro-derivatives. J. Enzyme Inhib. 1999, 14,
203–216.
(30) Jiao, G. S.; Cregar, L.; Wang, J.; Millis, S. Z.; Tang, C.; O’Malley,
S.; Johnson, A. T.; Sareth, S.; Larson, J.; Thomas, G. Synthetic
small molecule furin inhibitors derived from 2,5-dideoxystrept-
amine. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19707–19712.
(31) Komiyama, T.; Coppola, J. M.; Larsen, M. J.; van Dort, M. E.;
Ross, B. D.; Day, R.; Rehemtulla, A.; Fuller, R. S. Inhibition of
furin/proprotein convertase-catalyzed surface and intracellular
processing by small molecules. J. Biol. Chem. 2009, 284, 15729–
15738.
(32) Gustafsson, D.; Bylund, R.; Antonsson, T.; Nilsson, I.; Nystrom,
J. E.; Eriksson, U.; Bredberg, U.; Teger-Nilsson, A. C. A new oral
anticoagulant: The 50-year challenge. Nat. Rev. Drug Discovery
2004, 3, 649–659.
€
(42) Schafer, W.; Stroh, A.; Berghofer, S.; Seiler, J.; Vey, M.; Kruse, M.
L.; Kern, H. F.; Klenk, H. D.; Garten, W. Two independent
targeting signals in the cytoplasmic domain determine trans-Golgi
network localization and endosomal trafficking of the proprotein
convertase furin. EMBO J. 1995, 14, 2424–2435.
(43) Jean, F.; Thomas, L.; Molloy, S. S.; Liu, G.; Jarvis, M. A.; Nelson,
J. A.; Thomas, G. A protein-based therapeutic for human cyto-
megalovirus infection. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
2864–2869.
(44) Komiyama, T.; VanderLugt, B.; Fugere, M.; Day, R.; Kaufman,
R. J.; Fuller, R. S. Optimization of protease-inhibitor interactions
by randomizing adventitious contacts. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 8205–8210.
€
(33) Steinmetzer, T.; Sturzebecher, J. Progress in the development of
synthetic thrombin inhibitors as new orally active anticoagulants.
Curr. Med. Chem. 2004, 11, 2297–2321.
(45) Jean, F.; Stella, K.; Thomas, L.; Liu, G.; Xiang, Y.; Reason, A. J.;
Thomas, G. R1-Antitrypsin Portland, a bioengineered serpin
highly selective for furin: Application as an antipathogenic agent.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7293–7298.
€
(34) Schweinitz, A.; Sturzebecher, A.; Sturzebecher, U.; Schuster, O.;
Sturzebecher, J.; Steinmetzer, T. New substrateanalogue inhibitors
€
€
of factor Xa containing 4-amidinobenzylamide as P1 residue: Part
(46) Dixon, M. The determination of enzyme inhibitor constants.
1. Med. Chem. 2006, 2, 349–361.
Biochem. J. 1953, 55, 170–171.
€
€
(35) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. 1H-Pyrazole-
1-carboxamidine hydrochloride an attractive reagent for guanyla-
tion of amines and its application to peptide synthesis. J. Org.
Chem. 1992, 57, 2497–2502.
(47) Sturzebecher, J.; Prasa, D.; Hauptmann, J.; Vieweg, H.; Wikstrom,
P. Synthesis and structure-activity relationships of potent throm-
bin inhibitors: Piperazides of 3-amidinophenylalanine. J. Med.
Chem. 1997, 40, 3091–3099.
€
€
(36) Wiley, M. R.; Chirgadze, N. Y.; Clawson, D. K.; Craft, T. J.;
Giffordmoore, D. S.; Jones, N. D.; Olkowski, J. L.; Schacht, A. L.;
Weir, L. C.; Smith, G. F. Serine-protease selectivity of the throm-
bin inhibitor D-Phe-Pro-agmatine and its homologs. Bioorg. Med.
Chem. Lett. 1995, 5, 2835–2840.
(37) Garten, W.; Klenk, H. D. Cleavage activation of the influenza virus
hemagglutinin and its role in pathogenesis. In Avian Influenza;
Klenk, H. D., Matrosovich, M., Stech, J., Eds.; Karger: Basel,
Switzerland, 2008; Vol. 27, pp 156-167.
(38) Klenk, H. D.; Garten, W. Host cell proteases controlling virus
pathogenicity. Trends Microbiol. 1994, 2, 39–43.
(48) Turk, D. Weiterentwicklung eines Programmes fur Molekulgra-
phik und Elektronendichte-Manipulation und seine Anwendung
€
auf verschiedene Protein-Strukturaufklarungen. Ph.D. Thesis,
€
€
Technische Universitat Munchen, Munich, Germany, 1992.
(49) Engh, R. A.; Huber, R. Accurate bond and angle parameters for
X-ray protein-structure refeinement. Acta Crystallogr. 1991, A47,
329–400.
(50) Nicholls, A.; Sharp, K. A.; Honig, B. Protein folding and associa-
tion: Insights from the interfacial and thermodynamic properties of
hydrocarbons. Proteins 1991, 11, 281–296.
(51) Kraulis, P. J. MOLSCRIPT: A program to produce both detailed
and schematic plots of protein structures. J. Appl. Crystallogr.
1991, 24, 946–950.
(39) Garten, W.; Stieneke, A.; Shaw, E.; Wikstrom, P.; Klenk, H. D.
Inhibition of proteolytic activation of influenza virus hemaggluti-
nin by specific peptidyl chloroalkyl ketones. Virology 1989, 172,
25–31.
(52) Merritt, E. A.; Bacon, D. J. Raster3D: Photorealistic molecular
graphics. Methods Enzymol. 1997, 277, 505–524.