Design and synthesis of potent, selective inhibitors of matriptase

Article abstract of DOI:10.1021/ml3000534

Matriptase is a member of the type II transmembrane serine protease family. Several studies have reported deregulated matriptase expression in several types of epithelial cancers, suggesting that matriptase constitutes a potential target for cancer therapy. We report herein a new series of slow, tight-binding inhibitors of matriptase, which mimic the P1-P4 substrate recognition sequence of the enzyme. Preliminary structure-activity relationships indicate that this benzothiazole-containing RQAR-peptidomimetic is a very potent inhibitor and possesses a good selectivity for matriptase versus other serine proteases. A molecular model was generated to elucidate the key contacts between inhibitor 1 and matriptase.

Full text of DOI:10.1021/ml3000534

Letter
pubs.acs.org/acsmedchemlett
Design and Synthesis of Potent, Selective Inhibitors of Matriptase
†
†
‡
†
‡
́
Eloïc Colombo, Antoine Desilets, Dominic Duchene, Felix Chagnon, Rafael Najmanovich,
́
̂
́
Richard Leduc,† and Eric Marsault*,†
‡
†Department of Pharmacology and Department of Biochemistry, Faculty of Medicine and Health Sciences, Universite
́
de
Sherbrooke, 3001 12e Avenue Nord, Sherbrooke PQ, J1H5N4, Canada
S
* Supporting Information
ABSTRACT: Matriptase is a member of the type II transmembrane serine
protease family. Several studies have reported deregulated matriptase expression
in several types of epithelial cancers, suggesting that matriptase constitutes a
potential target for cancer therapy. We report herein a new series of slow, tight-
binding inhibitors of matriptase, which mimic the P1−P4 substrate recognition
sequence of the enzyme. Preliminary structure−activity relationships indicate
that this benzothiazole-containing RQAR-peptidomimetic is a very potent
inhibitor and possesses a good selectivity for matriptase versus other serine
proteases. A molecular model was generated to elucidate the key contacts
between inhibitor 1 and matriptase.
KEYWORDS: matriptase, type II transmembrane serine protease, slow tight-binding inhibitor
ell surface proteolysis is an important mechanism for the
degradation or generation of biological effectors that
studies have shown that the proteolytic activity of matriptase
C
must be tightly regulated during development, and deregulated
matriptase activity has been linked to various pathologies. For
example, a rare genetic skin disorder, autosomal recessive
ichtyosis with hypotrichosis (ARIH), was found to be caused by
mutations in the matriptase coding region, leading to the
production of an inactive protease.9,10 Elevated levels of
matriptase in osteoarthritis are thought to facilitate the
induction of cartilage destruction,11 while lowered levels have
been detected in colonic epithelia of inflammatory bowel
disease patients.12 Matriptase is overexpressed in a variety of
epithelial cancers13 and causes malignant transformations when
orthotopically overexpressed in the skin of mice, suggesting a
causal role in human carcinogenesis.14 The latter findings
suggest that deregulation of matriptase expression or activity is
involved in the initiation and/or progression of cancer.
Combined with its localization at the cellular surface of
epithelial cells, matriptase appears as an attractive therapeutic
target for the design and optimization of selective inhibitors to
better understand its role in pathologies such as cancer. Several
groups have been interested in the development of such
inhibitors via different strategies.15−24
engage various signaling pathways. Recently, a novel family of
proteolytic enzymes called type II transmembrane serine
proteases (TTSPs) has been associated with crucial roles in
numerous physiological processes.1 In humans, the 17 members
of this family are divided into four subfamilies: HAT/DESC,
Hepsin/TMPRSS, Corin, and matriptase.1 These transmem-
brane proteases are structurally defined by a cytoplasmic
amino-terminal region, a transmembrane domain, a stem region
that contains various functional domains, and a carboxy-
terminal extracellular serine protease domain of the chymo-
trypsin (S1) fold, characterized by the canonical histidine,
aspartate, and serine catalytic triad essential for proteolytic
activity. Matriptase, one of the most characterized TTSPs to
date, is expressed in epithelial cells where it carries out essential
functions in development, differentiation, and maintenance of
epithelial barrier homeostasis. Matriptase knockout mice die
shortly after birth due to severe dehydration caused by impaired
epidermal barrier function, indicative of a crucial role in
development.2 Among the most recognized matriptase
substrates are pro-hepatocyte growth factor,3 pro-prostasin,4
protease-activated receptor-2 (PAR-2),5 pro-urokinase plasmi-
nogen activator,5 CUB domain-containing protein-1,6 and
platelet-derived growth factor-D.7
In this study, we report a new class of potent and selective
peptidomimetic inhibitors of matriptase based on the P4−P1
(Arg-Gln-Ala-Arg) portion of the activation peptide of
matriptase, to which was linked a carboxy-terminal serine trap
in the form of a ketobenzothiazole group. The ketobenzothia-
zole serine trap was selected to form a covalent and reversible
bond with the catalytic serine residue of the enzyme as reported
Like many other proteases, the inactive zymogen precursor
of matriptase needs to be converted to its active form. This is
achieved via an initial cleavage occurring at residue Gly149
,
followed by an autoproteolytic cleavage at residue Arg614 within
the RQAR614-VVGG sequence of the activation peptide of
matriptase. Matriptase forms complexes with its cognate Kunitz
type serine protease inhibitor, the hepatocyte growth factor
activator inhibitor-1 (HAI-1), which is involved in activation,
inhibition, expression, and trafficking of the enzyme.8 Several
Received: February 28, 2012
Accepted: April 11, 2012
Published: April 11, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
by Costanzo et al. on thrombin.25 The use of a serine trap is
reminiscent of the recently approved HCV NS3-4A protease
inhibitors Boceprevir and Telaprevir.26,27 Herein, we report the
synthesis, inhibitory activity, preliminary structure−activity
relationships (SARs), and selectivity of this new class of
inhibitors and propose a molecular model of inhibitor 1 docked
into the active site of matriptase.
a
Scheme 2. Assembly of Fragments
The inhibitor sequence is based on the natural autoactivation
peptide sequence of matriptase Arg-Gln-Ala-Arg (RQAR)
explored by our group,28 to which was added a ketobenzothia-
zole serine trap (Figure 1).
a
Reagents and conditions: (i) HATU, DIPEA, DMF, r.t. (j) IBX,
DMSO, r.t. (k) HF, anisole, 5 °C. (l) reverse phase prep HPLC.
Figure 1. Chemical structure of inhibitor 1.
of protective groups with HF. Compounds were generally
obtained as a 8:2 mixtures of epimers,25 which were separated
by reverse-phase preparative high-performance liquid chroma-
tography (HPLC). Structural analogues 2−7 (Table 1) were
obtained according to the same synthetic method.
Synthetically, inhibitor 1 and its analogues were assembled
similarly to the method reported by Costanzo et al.25 by
peptide coupling of warhead-functionalized P1 fragment 8 with
protected P4−P2 fragment 10 (Schemes 1 and 2). The
a
Table 1. Preliminary SARs
a
Scheme 1. Synthesis of Tripeptide 11
inhibitor
P4-P3-P2-P1
R1, R2
Ki (nM)
1
2
3
4
5
6
7
R-Q-A-R
R-Q-A-R
R-Q-A-K
Q-A-R
O
0.011tb 0.0004
6124mm 2702
9.5tb 1.3
0.088tb 0.010
1.4tb 0.3
457mm 132
4.6tb 0.8
b
H, OH
O
O
O
O
O
A-R
R
a
Reagents and conditions: (a) HN(Me)OMe, HATU, DIPEA, THF,
R-Q-A-(D)R
r.t. (b) Benzothiazole, n-Buli, THF, −78 °C. (c) NaBH4 MeOH, −20
°C. (d) TFA/DCM 20:80, r.t. (e) Fmoc-Gln(Trt)-OH, HATU,
DIPEA, THF, r.t. (f) Et2NH/DCM 20:80, r.t. (g) Boc-Arg(Mtr)-OH,
EDC-HOBt, DCM, r.t. (h) H2, Pd/C (10%) EtOH, r.t.
a
Ki values were determined as described in the Materials and Methods
(tb, tight binding; mm, mixed model). Measurements of enzymatic
activity were performed in triplicate and represent the means
standard deviations of at least three independent experiments. A 3:2
b
mixture of diastereomers, absolute stereochemistry undetermined.
synthesis of inhibitor 1 is shown as an example. First, fragment
8 carrying the serine trap was prepared from the corresponding
Weinreb amide 7, by addition of in situ generated 2-lithio-
benzothiazole.25 The resulting ketobenzothiazole was reduced
in the same operation with NaBH4 as a means of protecting the
electrophilic keto group, and then, the Boc group was
deprotected by acidolysis. On the other hand, L-Ala benzyl
ester tosylate was coupled with Fmoc-Gln-OH using HATU to
afford dipeptide 9. After Fmoc removal, the crude dipeptide
was coupled with Boc-Arg(Mtr)-OH in the presence of 1-ethyl-
3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC) and N-hydroxybenzotriazole (HOBt) to give the
corresponding fully protected tripeptide. Benzyl ester hydro-
genolysis then generated the desired fragment 10. Subsequent
coupling of tripeptide 10 with warhead 8 provided intermediate
11 (Scheme 2). The tetrapeptide scaffold 11 was then oxidized
with 2-iodoxybenzoic acid (IBX),29 followed by final acidolysis
First, the inhibitory activity of tetrapeptide 1 toward
matriptase was characterized. The progress curve for hydrolysis
of a fluorogenic substrate by matriptase (1 nM) in the presence
of compound 1 (2.5 nM) displays a biphasic curve with a rapid
initial phase and a slower, steady-state phase (data not shown),
suggesting reversible slow, tight-binding, or irreversible
inhibition.
To further evaluate the inhibitor profile, the dissociation of
the enzyme:inhibitor complex (EI) was investigated using
dilution experiments.30 Figure 2 reports the comparison of the
dissociation curves for compound 1 (Figure 2A) and
irreversible inhibitor Glu-Gly-Arg chloromethyl ketone (EGR-
CMK) (Figure 2B). The dissociation curve of EGR-CMK
displays a linear product versus time relationship, indicative of
irreversible inhibition. Conversely, the dissociation curve of
531
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ACS Medicinal Chemistry Letters
Letter
simultaneously deleted, remains a tight-binding inhibitor with
127-fold reduced potency as compared to 1 (Ki = 1.4 vs 0.011
nM). Finally, inhibitor 6, in which the P4−P2 tripeptide
portion is removed, is dramatically less potent, with a Ki of 457
nM, over 30000-fold less potent than inhibitor 1. Additionally,
6 no longer behaves as a tight-binding inhibitor but as an
inhibitor possessing a mixed mode of inhibition as determined
by global fitting analysis for different modes of inhibition (see
the Supporting Information). Altogether, these results confirm
the importance of residues at the P4−P2 positions for potent
inhibition of matriptase.
The selectivity profile of inhibitor 1 for matriptase versus
other serine proteases, including TTSPs, was subsequently
determined (Table 2). Indeed, the selectivity of most published
a
Table 2. Selectivity Profile
proteases
Ki (nM)
selectivity (Ki other/Ki matriptase)
matriptase
matriptase-2
hepsin
0.011tb 0.0004
3.3tb 1.0
Figure 2. Dissociation of EI complex. Matriptase and increasing
concentrations of (A) RQAR-Benzo or (B) EGR-CMK were
preincubated for 20 min at room temperature and diluted 2000
times in reaction buffer containing 400 μM Boc-QAR-AMC. The final
concentration of matriptase is 0.25 nM and is varied as indicated for
inhibitors. The proteolytic activity in the reaction buffer was measured
as described in the Materials and Methods (see the Supporting
Information).
300
1.1tb 0.3
100
TMPRSS11D
trypsin
8.4tb 2.6
764
0.97tb 0.17
637mm 131
NI (10 μM)
88
thrombin
furin
>30,000
a
Ki values were determined as described in the Materials and Methods
(tb, tight binding; mm, mixed model; and NI, no inhibition).
Enzymatic measurements were performed in triplicate and represent
inhibitor 1 shows an exponential shape, suggesting dissociation
of the EI complex. Together, these data confirm the formation
of a slow, tight-binding, and reversible complex between
inhibitor and enzyme, as initially designed.
the mean
standard deviation of at least three independent
experiments.
To further characterize matriptase inhibition by compound 1,
the inhibition constant (Ki) was determined using the Morrison
equation for reversible tight-binding inhibition.31 In these
conditions, the RQAR-ketobenzothiazole inhibitor 1 showed
high potency for matriptase, with a Ki of 0.011 nM (Table 1). A
preliminary analysis of SARs was subsequently performed by
exploring the critical P1 position. To confirm the importance of
the keto group for matriptase inhibition, we measured the
inhibitory activity of a reduced form of the RQAR-
ketobenzothiazole toward matriptase (compound 2, Table 1).
Reduced analogue 2 (3:2 mixture of diastereomers at the
alcohol position, undetermined absolute stereochemistry)
displayed very weak inhibition, as expected. Indeed, a
stoichiometric ratio of I/E > 1000 was required to observe
substantial inhibition, as testified by a Ki of 6.1 μM, which
contrasts with the much more potent oxidized form 1 and is
consistent with a functional serine trap mechanism.
Table 1 reports the influence of structural variations of
inhibitor 1 on matriptase inhibition. To ascertain the
importance of stereochemistry at the P1 position, analogue
R-Q-A-(D)R 7 was tested and displayed a 400-fold lower
inhibition. Next, it is known that the TTSPs have a preference
for basic residues (Lys or Arg) in position P1.28 Accordingly,
matriptase displays a preference for an Arg residue in position
P1 by almost 3 orders of magnitude over Lys (3 vs 1, Ki 0.011
vs 9.5 nM). Furthermore, to better ascertain the respective
contribution of the P4, P3, and P2 residues on the inhibitory
profile, the peptidic portion was truncated by one, two, and
three residues starting from the N-terminal extremity
(analogues 4−6). Deletion of the P4 residue gave a compound
that conserves the profile of a tight-binding inhibitor, yet with
an 8-fold decreased potency as compared to 1 (4, Ki = 0.088 vs
0.011 nM). Inhibitor 5, in which the P4 and P3 moieties were
matriptase inhibitors has not been reported versus other
TTSPs. Experimental Ki values were determined as described in
the Materials and Methods (see the Supporting Information)
for inhibitor 1 against other TTSPs (hepsin, matriptase-2, and
TMPRSS11D) and serine proteases (trypsin and thrombin).
Selectivity was expressed as the ratio of Ki values. Compound 1
was found to be highly selective for matriptase versus other
enzymes: trypsin (88-fold), hepsin (100-fold), matriptase-2
(300-fold), TMPRSS11D (764-fold), thrombin (>30000-fold),
and furin (no inhibition). This high level of selectivity of the
RQAR-benzothiazole sequence for matriptase relative to other
trypsin-like proteases is remarkable. Although at this stage the
structural reasons for such selectivity are not elucidated,
additional studies, including molecular modeling and crystallog-
raphy, are underway to provide an explanation for this level of
selectivity.
To understand the preferred mode of docking of inhibitor 1
in the active site of matriptase and rationalize SARs, a molecular
model of inhibitor 1 docked in the published X-ray structure of
matriptase was built (Figure 3). According to this docking
model, the side chain of residue Arg in P1 is highly stabilized in
a network of hydrogen bonds, which includes a salt bridge with
matriptase residue Asp799, a hydrogen bond with Ser800, and
hydrogen bonds with the backbone amide of Gly827 and Gly829
.
This may account for the preference of Arg over Lys in P1,
particularly since the S1 pocket seems to be best suited to
accommodate the side chain of Arg instead of Lys, which is
longer and possesses reduced hydrogen bond capability as
compared to Arg. Residue Ala in P2 of inhibitor 1 lays over
Phe708, which separates the S2 and the S4 pockets. This pocket
is quite nonpolar due to the presence of the Phe708 residue. It
can also accommodate larger residues, in agreement with our
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dx.doi.org/10.1021/ml3000534 | ACS Med. Chem. Lett. 2012, 3, 530−534

ACS Medicinal Chemistry Letters
Letter
Funding
We thank Universite
Institutes of Health Research for financial support (CIHR,
́
de Sherbrooke and the Canadian
MOP-97964). CREPSUS (Centre de Recherche en Pharma-
cologie Structurale de l'Universite de Sherbrooke) is gratefully
́
acknowledged for a scholarship to D.D.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Professors B. Guerin and Y. Dory are acknowledged for sharing
purification equipment.
■
ABBREVIATIONS
■
DIPEA, N,N-diisopropylethylamine; EDC, 1-ethyl-3-(3-dime-
thylaminopropyl) carbodiimide hydrochloride; HOBt, N-
hydroxybenzotriazole; IBX, 2-Iodoxybenzoic acid; THF,
tetrahydrofuran; DCM, dichloromethane; EtOH, ethanol;
TFA, trifluoroacetic acid; MeOH, methanol; DMF, dimethyl-
formamide; DMSO, dimethylsulfoxide; n-Buli, n-butyllithium;
HPLC, high-performance liquid chromatography
Figure 3. Docking of inhibitor 1 in the active site of matriptase.
Matriptase32 is shown in gray and residues within the active site.
Inhibitor 1 is shown in orange. Catalytic triad residues are shown in
red. The image was generated using Pymol.33 Numbers refer to
matriptase numbering.
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■
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures, characterization data, biological methods,
and molecular modeling procedures. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Eric.Marsault@Usherbrooke.ca.
■
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