Article-2019-Recent progress on inhibitors of the type II transmembrane serine proteases, hepsin, matriptase and matriptase-2

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Recent progress on inhibitors of the type II
transmembrane serine proteases, hepsin,
matriptase and matriptase-2
Vishnu C. Damalanka*,1 & James W. Janetka**,1
1
Department of Biochemistry & Molecular Biophysics, 660 South Euclid Avenue, Campus Box 8231, Washington University School
of Medicine, St. Louis, MO 63110, USA
*Authors for correspondence: Tel.: +1 314 362 0509; [email protected]
**Authors for correspondence: [email protected]
Members of the type II transmembrane serine proteases (TTSP) family play a vital role in cell growth and
development but many are also implicated in disease. Two of the well-studied TTSPs, matriptase and hepsin proteolytically process multiple protein substrates such as the inactive single-chain zymogens pro-HGF
and pro-macrophage stimulating protein into the active heterodimeric forms, HGF and macrophage stimulating protein. These two proteases also have many other substrates which are associated with cancer
and tumor progression. Another related TTSP, matriptase-2 is expressed in the liver and functions by regulating iron homoeostasis through the cleavage of hemojuvelin and thus is implicated in iron overload
diseases. In the present review, we will discuss inhibitor design strategy and Structure activity relationships
of TTSP inhibitors, which have been reported in the literature.
First draft submitted: 4 September 2018; Accepted for publication: 11 January 2019; Published online:
4 April 2019
Keywords: benzamidine • hepsin • HGF • HGFA • inhibitor • ketobenzothiazole • ketothiazole • matriptase •
matriptase-2 • MET • MSP • peptide • RON • structure–activity relationship • TTSP • Type II transmembrane serine
protease
Type II transmembrane family of S1 trypsin-like serine proteases
Type II transmembrane serine proteases (TTSPs) were first discovered at the turn of the millennium and now they
have grown to include several related proteases defining a still emerging class of cell surface proteolytic enzymes [1].
TTSPs are a small subfamily of the PA clan or superfamily of serine proteases (peptidases or proteinases), which are
localized into cellular membranes with the active serine protease domain positioned on the extracellular surface.
These proteases play a critical role in the regulation of a wide range of pericellular biological processes and
cell surface activities. These enzymes function by the post-translational proteolytic processing of inactive protein
substrates (zymogens) outside the cell via amide bond hydrolytic cleavage. The resultant N-terminal and C-terminal
peptide fragments in some cases are then biologically functional alone or re-associate into covalent heterodimeric
structures, but in both scenarios result in zymogen activation [1,2]. The human TTSP family contains seventeen
individual members having several common structural features including a small N-terminal cytoplasmic domain,
contains the conserved catalytic active-site triad His57, Asp102 and Ser195 (chymotrypsin numbering) residues at
C-terminal extracellular S1 trypsin-like serine protease domain of the chymotrypsin fold, a transmembrane domain
and a ‘stem region’ that encompasses between 1 and 11 highly conserved structural domains. These structural
domains are derived from six different types; sea urchin sperm protein, enterokinase, agrin (SEA) domain, lowdensity lipoprotein receptor class A domain, group A scavenger receptor (SA) domain, merpin/A5 antigen/receptor
protein phosphotage mu (MAM) domain, Cis/Clr, urchin embryonic growth factor, bone morphogenetic protein-1
(CUB) domain and frizzled domain (for corin family). These structural domains appear to contribute to both the
auto activation of the TTSP and for making key binding interactions with substrates away from the active site [3–7].
Based on the overall full-length structures relating mainly to the makeup of the stem region domains, the TTSP
family is divided into four subfamilies in vertebrates: HAT (human airway trypsin-like protease)/DESC (differ-
C 2019 Newlands Press
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ISSN 1756-8919
Review
Damalanka & Janetka
entially expressed in squamous cell carcinoma) subfamily that contains HAT, HAT like 2, 3, 4, 5, DESC1 and
TMPRSS11A, hepsin/TMPRSS (trans membrane protease serine) subfamily comprising hepsin, enteropeptidase;
MSPL (mosaic serine protease large-form), TMPRSS2, TMPRSS3, TMPRSS4 and TMPRSS5/spinesin; matriptase subfamily encompassed of four members, which are matriptase, matriptase-2, matriptase-3 and the unique
polyserase-1; and corin family [1–3,5].
TTSPs plays an important roles in a wide range of biological processes including epithelial differentiation,
homeostasis, iron metabolism, hearing and blood pressure regulation [1–3,8–11]. These proteases are differentially
expressed and, in some cases, overexpressed in several forms of cancer. Furthermore, early studies on this class
of proteins revealed that the dysregulated expression and increased activity of TTSPs was found in multiple
tumor types. In recent studies from genetic engineering and those investigating the functional roles of TTSPs in
cancer development and progression, several individual members have emerged as new and exciting therapeutic
targets [5,9,12,13]. Similar to their substrates and other serine proteases, all TTSPs are synthesized as inactive zymogens
that require activation by proteolysis either through auto activation or by another extracellular protease [2].
HGF plays a vital role in oncogenesis as well as cancer disease progression and metastasis. The in active form
of HGF is secreted by tumor associated fibroblasts as a single-chain form, pro-HGF [14]. Active two-chain HGF is
produced in a two-step process: first from the proteolytic cleavage of pro-HGF at Arg494-Val495 by the TTSPs
matriptase and hepsin, or the plasma serine protease HGF activator (HGFA) [15–20], and second by disulfide
bond formation between the N-terminal and C-terminal peptide products. HGF is the only known ligand for the
oncogenic receptor tyrosine kinase (RTK) MET. Binding of activated HGF to the extracellular surface of MET leads
to receptor dimerization, the transphosphorylation of the intracellular kinase domain and subsequent activation
of downstream Ser/Thr kinase signaling pathways ultimately leading to cell survival, proliferation, epithelial to
mesenchymal transformation, angiogenesis and invasion of malignant cells [21], the hallmarks of metastatic cancer.
Interestingly pro-HGF can also bind MET but is unable to activate it thus acting as a natural antagonist for the
receptor. Thus, the processing of Pro-HGF by the TTSPs can occur through soluble pro-HGF or membrane bound
pro-HGF. Inhibition of HGF activation has been well studied as a potential therapeutic intervention in cancer [22–
25]. Inhibitors of the main, most efficient and physiologically relevant HGF activation proteases; matriptase, hepsin
and HGFA block the processing of pro-HGF and thus inactivate the HGF/MET signaling pathway. As mentioned
above, pro-HGF is capable of binding the MET receptor but not activating it, so pro-HGF acts as a MET antagonist
and therefore inhibiting the formation of active HGF results in the dual inhibition of MET signaling [26].
Remarkably, the same three proteases that activate HGF; matriptase, hepsin and HGFA all share another
common growth factor substrate called macrophage stimulating protein (MSP), which is the activating ligand for
the oncogenic RON kinase, a closely related RTK to MET. Like HGF, MSP is secreted as a single-chain inactive
form pro-MSP which is proteolytically processed at Arg483-Val484 to yield the active two-chain form of MSP.
While structurally similar to pro-HGF, in contrast pro-MSP is not capable of binding RON until activated by
one are more of matriptase, hepsin or HGFA. Therefore, the processing requires binding of the TTSP or HGFA
with the soluble form of MSP. Similar to HGF/MET, the MSP/RON signaling pathway has also been implicated
in the development and tumor progression of various solid tumors but most well-studied in breast cancer [27,28].
The expression and activity levels of HGFA, matriptase and hepsin are elevated or upregulated in patients who are
deemed at high risk for progression [29]. Additionally, co-overexpression of both MET and RON has been seen
in cancer patient samples from multiple tumor types [30,31], illuminating the relevance and importance of these
growth factor activating TTSP family of proteases in a variety of cancers.
There are two endogenous polypeptide serine protease inhibitors of HGF (and MSP) activation, HAI-1 and
HAI-2. Upregulation of the HGF activating proteases coupled to significantly reduced levels of HAIs in tumors
has been reported to be closely associated with poor survival of patients [32,33]. HAI-1 and HAI-2 potently inhibit
matriptase, hepsin and HGFA at low nanomolar concentrations. In this review, we focus on the recent development
of small molecule and peptide-based synthetic inhibitors of the TTSPs matriptase, matriptase-2 and hepsin, in
addition to the plasma serine protease HGFA, which shares the common substrates of HGF and MSP with hepsin
and matriptase. Like the TTSPs, HAI-1 and HAI-2 are also expressed on the cell surface where they can bind
and inhibit the cell surface proteases effectively and locally. Moreover, HAI-1 and HAI-2 play other important
roles in the regulation of the TTSPs. For example, and ironically, they have been shown to be necessary for the
proper activation of matriptase and hepsin. They are also capable of shedding from the cell surface and have been
reported to promote the shedding of matriptase and hepsin in certain circumstances, which has clear physiological
consequences.
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Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
Review
Matriptase, the most studied members of the TTSP family, is overexpressed in broad range of epithelial tumors
including breast, prostate, colon, uterus, skin and cervix [2,5,13,34,35]. However, some studies have described downregulation of matriptase mRNA in gastric and colorectal carcinomas [36,37]. It was also found that downregulation
of the matriptase inhibitor HAI-1 mRNA in gastric and colorectal carcinomas. Further studies indicated that the
ratio of matriptase to HAI-1 was not expressively different in colorectal carcinomas [36]. Matriptase was initially
identified in breast cancer cell lines and was discovered to be highly expressed in human breast carcinoma cells.
Recent studies have confirmed that matriptase is involved in breast cancer progression through activation of the
HGF/MET and in some cases the MSP/RON signaling pathway, therefore matriptase has been identified as both
a promising therapeutic target and a potential biomarker in this disease [38–42].
Hepsin was initially discovered as a proteolytic enzyme in the liver and is primarily expressed in both normal
human liver and kidney cells. Hepsin is also highly expressed in prostate cancer and renal cell carcinomas [43–46].
Furthermore, expression of hepsin has been reported in several other types of epithelial cancers, including ovarian
and breast [3,47–50]. Hepsin was found to be overexpressed in more than 40% of breast cancers in tumor microarrays,
notably being highly expressed in 60% of triple-negative breast cancers and 40–50% of luminal A, B and HER2+
subtypes [47]. Elevated mRNA levels of hepsin are seen prominently in about 90% of prostate cancers, with tenfold
higher levels in metastatic prostate tumors versus early-stage disease [24,51,52]. These studies have demonstrated that
hepsin is closely associated with prostate cancer progression and metastasis in vivo [53]. Hepsin plays an important
role in stimulating the metastatic spread of primary nonmetastasizing prostate cancers to the bone, liver and lung
as shown in in vivo animal studies. In addition, hepsin activity is also increased in other cancers including ovarian,
and breast cancers [52]. Moreover, hepsin is involved in the disruption of various cell membrane components such
as desmoglein and promoting cell motility [45,51–55,122]. Therefore, hepsin is considered as a potential therapeutic
target for the treatment and prevention of metastatic cancer [41,46,50,56–58].
HGFA is not a TTSP but is located in the plasma but like matriptase and hepsin, belongs to the family of
S1 trypsin-like serine proteases [59] and efficiently process pro-HGF and pro-MSP. HGFA has been extensively
investigated in breast cancer, where its activity is highly upregulated in the tumor microenvironment. This protease
has also been found to be activated in a number of other tumor types including the hematological cancers acute
myeloid leukemia (AML) and multiple myeloma both in cancer cells and patient samples [60]. HGFA is produced
as an inactive zymogen, pro-HGFA by the liver or cancer cells and is converted to its active form by other serine
proteases including thrombin and the kallikrein related (KLK) peptidases [58,61–63]. Unlike matriptase and hepsin
which are mainly localized on epithelial cells, HGFA is present at high concentrations in the systemic circulation
as inactive pro-HGFA and is likely under stricter regulation for both its activation and inhibition by HAI-1 and
HAI-2.
Matriptase-2, encoded by the gene TMPRSS6, is structurally most similar to matriptase and is located on the
cell surface of hepatocytes with its substrate hemojuvelin and which is mainly restricted to liver [63–65]. Matriptase-2
plays a pivotal role in the regulation of iron homeostasis in mice and humans. Matriptase-2 cleaves hemojuvelin at
the plasma membrane of hepatocytes, acting de facto as a negative regulator of hepcidin expression [65–68]. Hepcidin,
the key regulator of iron levels in circulation [66,69], binds to the iron transporter ferroportin, causes its degradation
and leads to the reduction of iron transport into the blood stream [70]. Hemochromatosis and β-thalassemia are two
major inherited blood disorders which are also referred to as iron overload diseases. Only limited and ineffective
treatments are available for these ailments which are also invasive and associated with significant side effects [71,72].
The regulation of the hepcidin activating pathway comprises a potential approach to treat both diseases. Therefore,
matriptase-2 is a promising new therapeutic target for both of these iron overload diseases of high unmet medical
need [73,74].
There have been number of recent reports available underlying the role of HGFA, matriptase and hepsin in
cancer progression and metastasis. Antibody-based inhibitors of hepsin [75–78], matriptase [79–81] and HGFA [82,83]
have been disclosed recently. In this present review, we will focus on recent progress in the design and development
of small molecule and peptide inhibitors of the TTSPs matriptase, hepsin and matriptase-2, while also including
the soluble plasma protease HGFA during the past 10 years.
Synthetic inhibitors of hepsin, HGFA, matriptase & matriptase-2
The first small molecule inhibitors of matriptase were reported back in 2001 by Wang et al. [84]. Since then
several x-ray crystal structures of matriptase including those cocrystalized with inhibitors have been solved [85–
88] and several groups of investigators from both industry and academia have discovered several chemical series
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Damalanka & Janetka
H
N
O
O
NH2
NH
N
HN
O
N
N
NH2
NH
O S
O
H2N
NH
O
H2N
Figure 1.
N
N
O
HN
NH2
NH
Nafamostat
HGFA Ki 25 nm
Matriptase Ki 0.02 nm
Hepsin Ki 0.53 nm
O
O
S
HN
O
CJ-672
HGFA Ki 17,800 nm
Matriptase Ki 4.3 nm
Hepsin Ki 1580 nm
NH
Hgfac-221
HGFA Ki 590 nm
Matriptase Ki 7180 nm
Hepsin Ki 553 nm
Benzguanidine and benzamidine containing inhibitors of HGFA, matriptase and hepsin.
of mainly peptidomimetic inhibitors. These include small molecule benzamidine [32,84,87–91], substrate-based
peptidomimetics [32,41,92–95] and cyclic peptides based on the natural product sunflower trypsin inhibitor (SFTI1) [96–98] through rational drug design. Herein, we will strictly focus only on the small molecule and substrate-based
inhibitors.
For clarification, we will be using widely-accepted terminology specifically developed for protease substrates and
their inhibitors [99]. As such, when discussing substrates and inhibitors, amino acid residues in the substrate or
Peptide (P) which are N-terminal to the scissile amide bond (P1-P1 ) are designated P1, P2, P3, etc. while residues
C-terminal to the cleavage site are designated P1 , P2 , P3 – among others. The protease subsites (S) accommodating
the corresponding amino acid residue side chains are designated S1, S2, S3 – among others and S1 , S2 , S3 –
among others, respectively [100]. The S1 trypsin-like proteases, such as HGFA, matriptase, matriptase-2 and hepsin,
specifically recognize substrates that contain basic amino acid side chains in P1 either an Arg or Lys which bind
in the S1 pocket of the active site where the hydrolysis occurs between P1(N-terminal) and P1 (C-terminal) by
catalytic triad (Ser95, His57 and Asp102) [16,101]. The canonical Asp189 in the S1 pocket of all trypsin-like serine
proteases confers the prevalent substrate specificity for Arg or Lys [101]. This attribute is also evident in the design
and optimization of inhibitors for this class of proteases, often requiring a high pKa group and in the case of the
substrate-based inhibitors a strong preference for Arg, to occupy this pocket for potent inhibition.
Benzguanidine analogs
Nafamostat as an irreversible and promiscuous benzguanidine serine protease inhibitor marketed as an anticoagulant drug. Investigators have utilized Nafamostat and the benzamidine sulfonamide series of protease inhibitors, first discovered as thrombin inhibitors [102–105], in the design of potent and selective inhibitors for
matriptase such as the benzamidine sulfonamide compound CJ-672 [106]. For example, Janetka and coworkers
developed new benzguanidine inhibitors (Nafamostat analogs) of matriptase, hepsin and HGFA [32]. Nafamostat
displays potent activity against all three proteases HGFA, matriptase and hepsin (Figure 1) [107]. It was found that
several analogs with para or meta substitutions on the aromatic rings that are summarized in Table 1, failed to
improve potency over Nafamostat. Di-substituted analog 9 (HGFA IC50 0.3 μM) had similar potency relative to
Nafamostat [32]. It appears that the level of activity for these analogs is directly related to the reactivity of the ester
and not to increased binding to HGFA.
Benzamidine derivatives
In the same paper as the Nafamostat analogs discussed above, Franco et al. reported small molecule benzamidine
sulfonamide analogs of HGFA, matriptase and hepsin. Genentech developed Hgfac-221 (Figure 1) as an inhibitors
of Factor VIIa, but which also inhibited HGFA with a Ki of 0.59 μM. It was found that this compound also
inhibits matriptase and hepsin with Ki values of 7.2 and 0.55 μM, respectively [32]. CJ-672 is a potent and
selective matriptase inhibitor (Ki = 4.3 nM). It was identified based on the thrombin inhibitors NAPAP and
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Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
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Table 1. Structure and biological activity of benzguanidine analogs of nafamostat against HGFA.
H
N
R
O
NH2
NH
O
1, 4-10
Compound
R
1 (Nafamostat)
HGFA IC50 (μ M)
Compound
0.15
7
R
HGFA IC50 (μ M)
Ref.
6.8
[32]
3.0
[32]
0.3
[32]
0.60
[32]
O
N
O
O
H2N
NH
4 (camostat)
32
8
O
O2N
O
5
1.1
9
H2N
O
NO2
6
0.82
10
O
O2N
Cl
O
TAPAP [106,108,109]. A cocrystal structure of CJ-672 bound to matriptase (Protein Data Bank [PDB] ID: 2GV7)
was solved with resolution of 2.2 Å [1]. This structure was employed in the rational design of novel benzamidine
sulfonamide HGFA, matriptase and hepsin inhibitors. In general, it was reported that analogs of this benzamidine
derived series potently inhibit matriptase and hepsin while weakly inhibiting HGFA.
Amidino phenylalanine benzamidine sulfonamide analogs 11-23 shown in Table 2, showed weak inhibition
against HGFA relative to matriptase and hepsin. Compound 13, 3-S-aminopiperidine showed no activity against
HGFA up to 50 μM concentration nor did the diastereomeric 3-R-aminopiperidine derivative 19. The S isomer
13 is more selective for matriptase (Ki 1.2 μM) than the R isomer 19 (Ki 6.7 μM), indicating a stereochemical
preference in the S3 binding pocket of matriptase. The R isomer is a slightly better inhibitor of hepsin with a Ki
of 2.6 μM. Piperazinyl benzamidine analogs 12, 14, 16 and 17 showed potent and selective binding affinity to
hepsin with Ki values 1.0, 0.048, 0.67 and 1.2 μM, respectively. Compound 14 containing the bromobenzyl urea
of the piperazine displayed 100-fold selectivity relative to matriptase (Ki 4.6 μM) and >300-fold compared with
HGFA (Ki 16.2 μM). The authors rationalized that the increase in activity toward hepsin is a result of the Pro60
in the pocket which allows more space than matriptase to bind the large bromobenzyl group.
Compounds 11, 13,15,18, 20, 21, 22 and 23 (piperidinyl and piperazinyl) showed good potency toward
matriptase with Ki values 0.3, 1.2, 0.48, 1.5, 1.8, 0.62, 0.12 and 0.76 μM, respectively. Compound 22, containing
the tri-isopropyl phenyl sulfonamide derived from CJ-672 but having a truncated piperidine showed a 23-fold
decrease in matriptase activity (Ki 0.12 μM) and 11-fold decrease in hepsin potency (Ki 8.1 μM) compared with CJ672. The decrease in activity of matriptase for 22 relatives to CJ-672 is likely due to increased distance between
S3 Asp side-chain and the free amine of piperidine, which limits the electrostatic charge–charge interaction.
2-Oxotetrahydropyrimidin-1(2H)-yl benzimidamide analogs
Tetrahydropyrimidin-2(1H)-one cyclic ureas have been used as a unique platform for the development of trypsinlike serine protease inhibitors, which were first employed for Factor Xa [110] and recently by Galemmo and colleagues
in the design of ‘triplex’ cyclic urea benzamidine inhibitors of matriptase, hepsin and HGFA [89]. They rationally
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Damalanka & Janetka
Table 2. Inhibitory activity data of benzamidine sulfonamide analogs of HGFA, matriptase and hepsin.
11-23
X
HGFA Ki (μ M)
Matriptase Ki
(μ M)
Hepsin Ki (μ M)
Ref.
11
C
10.1
0.3
3.3
[32]
12
N
14.0
2.6
1.0
[32]
13
C
⬎50
1.2
2.3
[32]
14
N
16.2
4.6
0.048
[32]
15
C
20
0.48
4.7
[32]
16
C
⬎50
2.5
0.67
[32]
17
N
⬎50
14.9
1.2
[32]
18
N
⬎50
1.5
3.5
[32]
Compound
R1
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R2
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Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
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Table 2. Inhibitory activity data of benzamidine sulfonamide analogs of HGFA, matriptase and hepsin (cont.).
11-23
X
HGFA Ki (μ M)
Matriptase Ki
(μ M)
Hepsin Ki (μ M)
Ref.
19
C
⬎50
6.7
2.6
[32]
20
N
18.2
1.8
7.0
[32]
21
N
⬎50
0.62
2.4
[32]
22
N
5.5
0.12
7.4
[32]
23
N
22.2
0.76
7.4
[32]
Compound
R1
R2
designed tetrahydropyrimidin phenylamidine cyclic urea analogues 24–30 that can specifically access the S3 subsite
of these proteases. These compounds exhibit strong inhibition activities against HGFA, matriptase and hepsin
(Table 3). The design of this series takes advantage of three conserved structural features a) phenylamidine group
has strong ionic interaction with the conserved Asp189 in the S1 pocket, b) the carbonyl group of cyclic urea core
forms an H-bond interaction with Gly216-NH, 3) a lipophilic interaction in the S4 pocket with Trp 215. First,
they examined the substitution on the phenylamidine and benzylamidine with compounds 24–27. Compound
24 is most active against matriptase (Ki 0.83 μM), hepsin (Ki 3.4 μM) and HGFA (Ki 9.8 μM) compared with
others and the amidine group at meta-position showed improved activities than para-position as summarized in
Table 3. Acyclic compound 30 derived from 24 shows decreased activity toward all three proteases; HGFA (6-fold),
matriptase (49-fold) and hepsin (16-fold). Therefore, constraining the urea into a cyclic urea significantly improves
the activity. Compound 28 (SRI 31215) has an insertion of methylene between tetrahydropyrimidin-2(1H)-one
and the N-benzylpiperidine, which leads to additional binding with Trp215 in the S4 pocket. Compound 28
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Table 3. Inhibitory activity data of 2-oxotertahydropyrimidine against HGFA, matriptase and hepsin.
24-30
Compound
Substitutions
HGFA Ki (μ M)
Matriptase Ki (μ M)
Hepsin Ki (μ M)
Ref.
24
meta X = 0, Y = 0
9.8
0.83
3.4
[89]
25
para X = 0, Y = 0
46.5
48.9
61.2
[89]
26
meta X = 1, Y = 0
⬎100
13.5
15.1
[89]
27
para X = 1, Y = 0
39.6
15.4
20.8
[89]
28 (SRI 31215)
meta X = 0, Y = 1
0.48
0.53
0.54
[89]
29
meta X = 0, Y = 2
1.11
0.51
2.12
[89]
54.6
53.1
40.5
[89]
30
Tyr 146
Ser/Ala
190
S4
Hepsin
Matriptase
HGFA
Trp 215
Asp/Thr 217
S3
Asp 189
S1
Figure 2. Comparison of the binding mode of compound 28 from the model in matriptase.
Data taken from [89].
displays strong potency relative to 24 for HGFA (Ki 0.48 μM), matriptase (Ki 0.53 μM) and hepsin (Ki 0.54 μM).
It was found that further insertion of ethylene group as in compound 29 proved to be less effective. It was shown that
compound 28 blocks fibroblast-induced activation of HGF/MET signaling pathways in colon tumor cells, which
is a direct consequence of its inhibition of the cleavage pro-HGF to HGF [26]. The compound 28 pharmacophore
binding model with matriptase (PDB 2GV6), hepsin 1Z8G) and HGFA (2WUC) was developed (Figure 2). The
benzamidine group was constrained in the S1 pocket and minimized showing strong ionic interaction between the
amidine group and Asp 189 in the S1 pocket; the methyl group of cyclic urea faces toward and favorably interacts
with amino acid Asp 217 (matriptase), Thr 217 (hepsin) and Asp 217 (HGFA) [89].
Compounds 31–42 containing various substituents on the urea ring are highlighted in Table 4. These analogs
directed to the S3 su-pocket have on off-target selectivity. Compared with Compound 28, 31 which has an OH
group at the R position displayed improved potency for matriptase (Ki 0.28 μM) and hepsin (Ki 0.21 μM) but
decreased activity for HGFA (Ki 0.88 μM). Compounds 28, 32, 36, 40, 41 and 42 are triplex inhibitors of HGFA,
matriptase and hepsin, similar to the selectivity profile of endogenous polypeptide inhibitor HAI-1 but weaker
in potency. Compound 34 showed the best potency for HGFA (Ki 0.16 μM) relative to matriptase and hepsin.
Compound 28 was tested for its pharmacokinetic properties where it showed good stability in mouse and human
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Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
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Table 4. Inhibitory activity data of 2-Oxotetrahydropyrimidin-1(2H)-yl benzamides against HGFA, matriptase and hepsin.
31-42
Compd
R
HGFA Ki (μ M)
Matriptase Ki (μ M)
Hepsin Ki (μ M)
Ref.
31
0.88
0.28
0.21
[89]
32
0.23
0.58
0.69
[89]
33
2.49
0.81
0.76
[89]
34
0.16
0.85
0.92
[89]
35
1.92
1.54
0.76
[89]
36
0.43
0.85
0.56
[89]
37
1.1
0.47
1.1
[89]
38
0.38
1.28
1.12
[89]
39
1.08
1.51
0.31
[89]
40
0.35
0.33
0.72
[89]
41
0.52
0.52
0.67
[89]
42
0.44
0.35
0.53
[89]
microsomes 2283 ml/h, <70% of hepatic circulation with an in vivo half-time of 5.8 h in mice when dosed IV but
had no oral bioavailability. Compound 28 has been shown to be an effective inhibitor of fibroblast-induced MET
activation and it overcomes HGF-dependent resistance to EGFR inhibitors in colon and lung [111] tumor cells [26,89].
Compound 28 is a good starting point for further chemistry optimization but is also currently considered adequate
as a drug for using in in vivo models for cancer [89].
Bisbenzamidine derivatives
Gutschow et al. reported the bisbenzamidine derivatives of matriptase and matriptase-2, shown in Table 5 [112].
They designed and synthesized 27 bisbenzamidine analogs and evaluated the activity of matriptase and matriptase2 [112]. They docked oxamide-containing bisbenzamidine 43 into the active site of matriptase (PDB ID: 4JZI) and
observed ionic interactions with carboxylate of Asp189 and hydrogen bonding with His57 and Cys58 in the S2
future science group
10.4155/fmc-2018-0446
Review
Damalanka & Janetka
Table 5. Inhibitory data of bisbenzamidine derivatives of matriptase and matriptase-2.
43-49
Compound
R
Matriptase Ki (nM)
Matriptase-2 Ki (nM)
43
0.492
6.23
[112]
Ref.
44
2.28
5.2
[112]
45
0.816
5.7
[112]
46
0.719
1.16
[112]
47
0.397
4.39
[112]
48
0.558
3.68
[112]
49
0.526
1.64
[112]
pocket. These inhibitors showed more potency toward matriptase relative to matriptase-2. Introduction of sterically
hindered diazepane compound 47, slightly improved the potency for the both the proteases over 43. Compounds
48 and 49, which are the more flexible inhibitors, didn’t show much effect in changing matriptase activity (Ki
0.558 and 0.526 μM over 43, 0.492 μM) but did for matriptase with a twofold to fourfold increase in activity.
Most of these low molecular weight inhibitors exhibited strong inhibition for matriptase with good selectivity over
matriptase-2. To improve the selectivity of matriptase-2 one might need to design the molecules to target residues
Asp97 and His99 in the upper part of S3/S4 pocket of matriptase-2 [112].
Protease substrate-based peptide inhibitors of matriptase & matriptase-2
A common strategy in the design of protease inhibitors is to duplicate the first four or so residues to the P1 or
N-terminal portion of the protein substrate cleavage site and then attach an electrophilic group or ‘warhead’ at the
C-terminal portion of the molecule intended to react with the catalytic Ser102 [113,114]. These small peptides are
called substrate-based or mechanism-based inhibitors. Marsault and coworkers followed this strategy for designing
the first mechanism/substrate-based inhibitors of matriptase using the electrophilic ketone ketobenzothiazole (kbt)
10.4155/fmc-2018-0446
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future science group
Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
Review
Table 6. Activity data of peptidomimetic inhibitors of matriptase.
50-56
Compound
P4 -P3 -P2 -P1
R1
50
R-Q-A-R
51
R-Q-A-R
52
R-Q-A-K
53
Q-A-R
0.088
[115]
54
A-R
1.4
[115]
55
R
457
[115]
56
R-Q-A-(D)R
4.6
[115]
H
R2
OH
Matriptase Ki (nM)
Ref.
0.011
[115]
6124
[115]
9.5
[115]
warhead group at the carboxy-terminus to form a covalent and reversible bond with catalytic serine residue [94,115].
The structural variations of P1-P4 which were utilized are shown in Table 6. Compound 50, RQAR-kbt showed
high potency for matriptase, with a Ki value of 0.011 nM. The authors confirmed the importance of the keto
group at the P1-P1 scissile bond by measuring the activity of a reduced form compound 51 which displayed very
weak inhibition. Replacing the P1 Arg with Lys decreases the activity 900-fold and was also demonstrated that
the stereochemistry also plays key role in the P1 pocket; compound 56 RQA-(D)R-kbt displayed a 400-fold lower
inhibition that the L-isomer. The tetrapeptide P4-P1 RQAR-kbt (Ki 0.011 nM) was shortened to P3-P1 and P2-P1
and just the P1 Arg to ascertain the structure activity relationships, where it was observed an 8-fold decrease in
activity when the P4 Arg is eliminated then 16-fold in the dipeptide AR-kbt, then >320-fold drop in potency for
R-kbt with a Ki of only 457 nM.
Najmanovich and coworkers reported a library of tetrapeptide inhibitors for matriptase and matriptase-2 with
the combinations of 20 natural amino acids at positions P4, P3 and P2 [116]. They have made the modifications
related to RQAR in P2 (RQPR, RQYR and RQFR), P3 (RYAR and RRAR) and P4 (LQAR and SQAR), shown
in Table 7. In all cases, these compounds have higher potency for matriptase over matriptase-2. Compounds 50,
57 and 63 showed strong potency for matriptase with Ki values 0.011, 0.061 and 0.092 nM but these inhibitors
are less potent for matriptase-2. Interestingly, compounds 67, 68 and 69 showed eightfold, 12-fold and twofold
more potency for matriptase-2 over matriptase. These SAR data reveals matriptase prefers small amino acids, such
as Ala and Pro in P2 position and matriptase-2 prefers aromatic residues such as Phe, Tyr and Trp.
This same research group also recently published on tetrapeptide inhibitors of matriptase and matriptase-2, in
which positions P4, P3 and P2 were varied using unnatural amino acids as a strategy to improve the pharmacokinetic
properties as well as the selectivity of matriptase-2 over matriptase. The structures and activity data are outlined
in Tables 8–11. Table 8 shows the inhibitory data of P4 structural modifications to the Y-Y-V-R-Kbt scaffold
(compound 72). S4 pocket modifications attempted to improve the proteolytic stability of the inhibitors, replacing
Tyr of 72 with desamino (homo Tyr) compound 73 has improved plasma stability, results reported in original
article [94]. Moreover, (H)Y-Y-V-R (compound 73) not only has improved potency compared with reference
compound 72 (Ki 2.6 nM) but has elevated matriptase-2 (Ki 2 nM) selectivity being 17-fold more active over
matriptase (Ki 34 nM). When the P4 Tyr residue was replaced by the bulky group dessamino-adamantane (74)
the potency decreases for matriptase-2 but not matriptase. Further SAR suggests that replacing Tyr with (H)Phe
(compound 76) slightly improves the potency for matriptase-2 (Ki 1.74 nM) compared with compound 72. In
future science group
10.4155/fmc-2018-0446
Review
Damalanka & Janetka
Table 7. Biological activity data of tetrapeptides for matriptase and matriptase-2.
50, 57–71
Compound
P4 -P3 -P2 -P1
Matriptase Ki (nM)
Matriptase-2 Ki (nM)
50
R-Q-A-R
0.011
3.30
[116]
Ref.
57
R-Q-P-R
0.061
7.80
[116]
58
R-Q-Y-R
0.18
0.74
[116]
59
R-Q-F-R
0.23
1.40
[116]
60
R-Y-A-R
0.14
6.30
[116]
61
R-R-A-R
0.72
15.40
[116]
62
L-Q-A-R
0.15
13.47
[116]
63
S-Q-A-R
0.092
34.80
[116]
64
W-R-E-R
14.67
46.78
[116]
65
R-N-P-R
0.91
47.77
[116]
66
K-N-A-R
0.45
24.84
[116]
67
W-C-Y-R
7.98
1.03
[116]
68
Y-Y-V-R
31.54
2.56
[116]
69
L-W-W-R
3.22
1.98
[116]
70
R-L-S-R
1.40
2.48
[116]
71
Y-K-A-R
0.89
20.79
[116]
contrast, the potency decreases against matriptase (Ki 43 nM) so the selectivity for matriptase-2 is effectively
doubled for 76 compared with 72 (25-fold vs. 12-fold).
In another set of analogs (Table 9), they explored P3 modifications with different amino acid residues to further
investigate the specificity of S3 pocket based on the reference compounds 72 and 76. Compound 77, having a
replacement of Tyr with Phe results in lowered potency on both proteases matriptase (Ki 63 nM) and matriptase-2
(Ki 9 nM), indicating the importance of hydroxyl group at position P3 on both the proteases. Replacement of
Tyr with a chloro substituent in the P3 position is tolerated for matriptase but gives a twofold drop in activity
for matriptase-2 (Ki 5 nM). To further explore the S3 specificity, changes were implemented on P3 position of
compound 76. Replacement of Tyr with a 4-thiazolyl group leads to slightly improved inhibition of matriptase-2
(Ki 1.2 nM) but reduced inhibition of matriptase (Ki 12 nM). Introduction of a benzyl group to phenol (80) and
replacement of (H)Phe (81) yields decreased activity for matriptase ∼twofold (Ki 71 and 65 nM vs. 43 nM for
76) and matriptase-2 (Ki 3.5 and 2.1 nM vs. 1.74 nM for 76). These SAR data reveal the importance of Tyr or a
hydroxyl group in the S3 subpocket [96].
Further modifications to the P2 position of the reference compound 76 include replacement of Val with
diphenylalanine (compound 82) which displayed a significant decrease in potency for both the proteases (Table 10)
matriptase and matriptase-2 (Ki 1380 and 340 nM). This result is explained through the steric clash of His665 in
the S2 subpocket [94]. A loss of potency from 2 to 20-fold for matriptase-2 was observed from replacement of Val
with heterocyclic groups or aromatic groups. For matriptase, the activity increased when the Val was replaced by
Tyr and Trp with Ki values of 5.6 and 7 nM (compounds 86 and 89 vs. compound 72).
To improve the selectivity on matriptase-2, modifications on compound 81 in the P2 position were attempted
with branched aliphatic amino acids, shown in Table 11 (compounds 90-100). Replacement of Val by Leu had no
effect on selectivity, but the activity decreased twofold for both matriptase and matriptase-2 (Ki 120 and 5.2 nM
vs. 65 and 2.1 nM compound 81). The selectivity of matriptase-2 improved 58-fold upon replacement with Ile
(compound 92) but the potency diminished fourfold for matriptase and twofold for matriptase-2. Fine-tuning of
side chain stereochemistry, replacement of Val by D-Val (compound 90), D-Ile (compound 93) and D-allo-Ile
(compound 95) led to decreased potency. Thus, D-amino acids are not suited in the S2 subpocket for matriptase
10.4155/fmc-2018-0446
Future Med. Chem. (Epub ahead of print)
future science group
Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
Review
Table 8. Biological activity data of various P4 residue peptidomimetic inhibitors of matriptase and matriptase-2.
72–76
Compound
P4 residue
Structure
Matriptase Ki (nM)
Matriptase-2 Ki (nM)
Ref.
72
Tyr
32
2.6
[94]
73
(H)Tyr
34
2
[94]
74
(H)Adamantane
39
6
[94]
75
(H)Leucine
28
6
[94]
76
(H)Phe
43
1.74
[94]
and matriptase-2. Replacement of Val by linear alkyl side chains such as Abu (compound 96) and Nva (compound
97) increased inhibition by 3-fold for matriptase but had no effect on matriptase-2. The bulkier t-Bu group
drastically decreased the potency for matriptase and matriptase-2 (Ki 15000 and 560 nM, respectively), defining
the steric limits in the S2 subpocket. Introduction of Thr and allo-Thr in the P2 position increased preference for
matriptase-2 over matriptase 64-fold. The selectivity of matriptase-2 was explained the H-bond interaction between
water, the hydroxyl group of Thr/allo-Thr and His665 in the S2 sub-pocket of matriptase-2. This SAR indicates
that the binding pocket tolerates a certain degree of steric hindrance, sustains its asymmetry and matriptase-2
prefers branched, lipophilic residues to ascertain selectivity over matriptase [94].
future science group
10.4155/fmc-2018-0446
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Damalanka & Janetka
Table 9. Biological activity data of various P4-P3 residue peptidomimetic inhibitors of matriptase and matriptase-2.
72, 76, 77–81
Compound
P4 residue
Matriptase Ki (nM)
Matriptase-2 Ki (nM)
Ref.
72
Tyr
32
2.6
[94]
77
Tyr
63
9
[94]
78
Tyr
34
5
[94]
76
(H)Phe
43
1.74
[94]
79
(H)Phe
12
1.2
[94]
80
(H)Phe
71
3.5
[94]
81
(H)Phe
65
2.1
[94]
10.4155/fmc-2018-0446
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Future Med. Chem. (Epub ahead of print)
future science group
Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
Review
Table 10. Biological activity data of various P4-P3-P2 residue peptidomimetic inhibitors of matriptase and matriptase-2.
82–89
Compound
P4-P3 residues
Matriptase Ki (nM)
Matriptase-2 Ki (nM)
Ref.
82
(H)Phe-Tyr-
1380
340
[94]
83
Tyr-Tyr
48
56
[94]
84
Tyr-Tyr
12
5
[94]
85
Tyr-Tyr
20
13
[94]
86
Tyr-Tyr
7
1.1
[94]
87
Tyr-Tyr
44
5.3
[94]
88
Tyr-Tyr
19
6
[94]
future science group
R
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Damalanka & Janetka
Table 10. Biological activity data of various P4-P3-P2 residue peptidomimetic inhibitors of matriptase and matriptase-2
(cont.).
82–89
Compound
P4-P3 residues
89
Tyr-Tyr
R
Matriptase Ki (nM)
Matriptase-2 Ki (nM)
Ref.
5.6
3.1
[94]
Substrate-based peptide inhibitors of HGFA, hepsin & matriptase
Janetka and co-workers have developed small molecular weight peptidyl inhibitors based on the substrates pro-HGF
and pro-MSP, as summarized in Table 12 [32,41]. Compounds 101 and 102 are tetrapeptides with the electrophilic
ketone group ketothiazole (kt) as the serine-trapping warhead. These inhibitors were shown to decrease the catalytic
processing of pro-HGF and pro-MSP by HGFA and phosphorylation of MET in MDA-MB-231 invasive breast
cancer cells [41]. Compound 101 (Ac-KQLRkt) was designed and synthesized by capping acetyl group at Nterminal P4 Lys of pro-HGF substrate and installing the ketothiazole at the P1-P1 reactive site [32]. Compound
101 displayed strong inhibition activity against all three proteases HGFA (Ki 53 nM), matriptase (Ki 0.92 nM) and
hepsin (Ki 0.22 nM). However, compound 102, Ac-SKLR-kt (based on pro-MSP) peptide had twofold decreased
inhibitory activity for HGFA, and 63-fold for matriptase, and fivefold for hepsin. All these tetrapeptide analogs
showed stronger inhibitory activity against matriptase and hepsin over HGFA. The data in the Table 12 indicates
that variation of amino acids from P4-P2 regions significantly affected the potencies against all three proteases.
Replacement of Lys with Trp in P4 of Ac-KQLR-kt (101) had no effect on potency (101b) in HGFA and hepsin
but 34-fold decrease in matriptase, while replacement of Ser of Ac-SKLR-kt (102) with Trp in P4 (102b) leads to
the increase in activity twofold in HGFA, sevenfold in matriptase and twofold in hepsin. However, replacing Lys
of (101) with Ser (101f) the activity decreases in all three proteases threefold (HGFA), tenfold (matriptase) and
twofold (hepsin). Substitution of Arg (102h) in P4 (102) the potency enhanced in all three proteases. This data
reveals that the HGFA prefers larger side chains in the S4 pocket [32]. The P3 amino acid in pro-HGF peptides
basic side chains are preferred for HGFA which enhances the activity with no effect on hepsin while the potency
decreases in matriptase (101g and 101h). However, since the residue in 101h is the largest sidechain of Trp, it also
might suggest that these large group cannot be accommodated in matriptase and HGFA. Interestingly, when the
P3 Lys in pro-MSP analog 102 is changed to an Arg (102e) the inhibition of all three protease is improved with Ki
values 24, 5.8 and 0.68 nM of HGFA, matriptase and hepsin, respectively. The SAR for 101 and 102 and analogs
thereof demonstrate that HGFA prefers either Arg or Trp in the S4 and S3 pockets, P2 prefers small hydrophobic
amino acid side chains [32,81]. Several analogs have been reported to abrogate the processing of the protein substrates
pro-HGF and pro-MSP with similar potency to HAI-1.
This same group further reported on the rational drug design of α-ketobenzothiazole (kbt) and substituted kbt
inhibitors 103–121 as exemplified in Table 12. The compounds were evaluated for their biochemical inhibition
of HGFA, matriptase and hepsin using a kinetic enzyme inhibition assay which employs a fluorescent peptide
substrate [41]. A computational model of compound 101 with HGFA (Figure 3) taught that the thiazole group
occupied in the S1 pocket and reveals that the S3 pocket is largely accessible through substitution on the thiazole
10.4155/fmc-2018-0446
Future Med. Chem. (Epub ahead of print)
future science group
Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
Review
Table 11. Biological activity data of various P2 residue peptidomimetic inhibitors of matriptase and matriptase-2.
90–100
Compound
Residues
Matriptase Ki (nM)
Matriptase-2 Ki (nM)
Ref.
90
D-Val
240
7
[94]
91
L-Leu
120
5.2
[94]
92
L-Ile
230
4
[94]
93
D-Ile
150
5
[94]
94
L-allo-Ile
51
2.6
[94]
95
D-allo-Ile
12000
690
[94]
96
Abu
21
1.6
[94]
97
Nva
26
2.3
[94]
future science group
R
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Damalanka & Janetka
Table 11. Biological activity data of various P2 residue peptidomimetic inhibitors of matriptase and matriptase-2 (cont.).
90–100
Compound
Residues
R
Matriptase Ki (nM)
Matriptase-2 Ki (nM)
Ref.
98
(t-Bu)Gly
15000
560
[94]
99
L-Thr
120
5.3
[94]
100
L-allo-Thr
200
3.1
[94]
P2
P3’
P4
P1’
P1
P2’
P3
Figure 3.
10.4155/fmc-2018-0446
Computational model of compound 101 with HGFA [41].
Future Med. Chem. (Epub ahead of print)
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Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
Review
Table 12. Biological activity data of peptidyl analogs against HGFA, matriptase and hepsin.
101–102
103–108
HGFA
Matriptase
Ref.
Structure
R
Ki (nM)
Ki (nM)
Ki (nM)
101 (101 pro-HGF)
A
Ac-K-Q-L-
53
0.92
0.22
[32]
101a
A
Ac-K-H-L-
96
22
0.41
[32]
101b
A
Ac-W-Q-L-
65
32
0.21
[32]
101c
A
Ac-K-Q-F-
58
0.69
0.58
[32]
101d
A
Ac-K-F-L-
80
15
2.1
[32]
101e
A
Ac-R-Q-L-
60
0.32
0.28
[32]
101f
A
Ac-S-Q-L-
182
9.2
0.34
[32]
101g
A
Ac-K-R-L-
12
1.1
0.57
[32]
101h
A
Ac-W-R-L-
21
5.5
0.21
[32]
102 (102 pro-MSP)
A
Ac-S-K-L-
81
58
1.2
[32]
102a
A
Ac-S-H-L-
332
104
0.6
[32]
102b
A
Ac-W-K-L-
56
8.6
0.55
[32]
102c
A
Ac-S-K-F
57
3.0
8.5
[32]
102d
A
Ac-N-K-L-
79
12.0
1.4
[32]
102e
A
Ac-S-R-L-
24
5.8
0.68
[32]
102f
A
Ac-T-K-L-
103
8.7
0.61
[32]
102g
A
Ac-S-W-L-
63
69
1.2
[32]
102h
A
Ac-R-K-L-
17
0.83
0.47
[32]
103
B
Ac-K-Q-L
30
0.54
0.09
[41]
104
B
Ac-S-K-L-
51.8
3.7
0.07
[41]
105
B
W-R-L-
11
2.3
0.17
[41]
106
B
Ac-F-L-F-
75
4
1.4
[41]
107
B
Ac-K-R-L-
7.5
1.2
0.11
[41]
108
B
Ac-W-L-F-
125
7.7
0.35
[41]
109
C
Ac-K-Q-L-
-Leu-NH2
12.4
5.2
0.43
[41]
110
C
Ac-S-K-L-
-Leu-NH2
16.7
8.2
0.22
[41]
111
C
Ac-K-R-L-
-Leu-NH2
6.3
9
0.23
[41]
112
C
Ac-S-K-L-
COOH
139
54.3
0.16
[41]
113
C
Ac-K-R-L-
6.4
0.95
0.52
[41]
114
C
Ac-K-R-L-
6.5
1.4
0.64
[41]
115
C
Ac-K-R-L-
6.1
7.6
0.08
[41]
116
C
Ac-K-R-L-
3.7
2.5
0.67
[41]
117
C
Ac-S-K-L-
4-NH-piperidine
7.2
1.3
0.27
[41]
118
C
Ac-S-K-L-
Trp-NH2
12.1
4.1
0.25
[41]
119
C
Ac-S-K-L-
-Phe-NH2
22.9
5.7
0.21
[41]
future science group
R1
109–121
Compound
Hepsin
10.4155/fmc-2018-0446
Review
Damalanka & Janetka
Table 12. Biological activity data of peptidyl analogs against HGFA, matriptase and hepsin (cont.).
101–102
103–108
109–121
Matriptase
Ref.
Compound
Structure
R
R1
HGFA
Hepsin
Ki (nM)
Ki (nM)
Ki (nM)
120
C
Ac-S-K-L-
-NH-benzyl
13.4
0.8
0.14
[41]
121
C
Ac-S-K-L-
4-NH-pyridine
16
0.98
0.29
[41]
ring [41]. It was discovered that when the thiazole group of analog 101 was replaced with benzothiazole 103, it
increased the potency against all three proteases HGFA, matriptase and hepsin with Ki values 30, 0.54 and 0.09 nM,
respectively. Moreover, substitution of the kbt on the reference analog 96 with a valine amide as in compound 109,
elevated the potency of HGFA by 4-fold (Ki 12.4 nM) but lowered the activity of matriptase >tenfold (Ki 5.2 nM)
and hepsin >threefold (Ki 0.43 nM). Parallel results were obtained via the modification of the pro-MSP truncated
substrate (102). These results might be expounded by the position of amino acids in S3 pocket by docking, where
HGFA contains a His (H451) that is less sterically hindered, matriptase contains Asp (D60) and hepsin has an Asn
(N209).
Aromatic amino acid substitution of the kbt tetrapeptides at both P2 and P4 sites (compounds 106 and 108)
decreased the potency of all three proteases compared with compound 103 and 104, suggesting that hepsin does
not prefer aromatic side chains in the P2 and P4 sites and SAR data reveal that preference of HGFA as well.
Comparing the results of 103, 107 with 104, 106 and 108 clearly provides strong evidence that matriptase strongly
prefers basic side chain at the S4 pocket.
In the SKLR and KRLR sub library of compounds, substitution was varied at the R1 position of the ketobenzothiazole (compounds 112-121). The HGFA potency of these analogs systematically increased but matriptase
and hepsin activity were only slightly affected in most cases. For example, benzyl analogs 115 and 116 with a
carboxylic acid at the meta and para position, both significantly increased the potency toward HGFA (Ki 6.1 and
3.7 nM), hepsin (Ki 0.08 and 0.67 nM) but actually decreased the inhibition of matriptase (Ki 7.6 and 2.5 nM).
The piperidine analog 117 was shown to be a promising and potent ‘triplex’ inhibitor of HGFA, matriptase and
hepsin with Ki values 7.2, 1.3 and 0.27 nM, respectively. Triplex inhibitors of HGFA, matriptase and hepsin,
such as compounds 103 and 110 have been demonstrated to block the tumor-associated fibroblast scattering of
invasive DU145 prostate tumor cells by inhibiting the activation of pro-HGF to HGF [32]. In addition, multiple
analogs from Table 12 have been shown to be effective in reversing the resistance to EGFR inhibitors gefitinib
and cetuximab in lung cancer cells [117–120]. These initial results from the kbt inhibitors in anticancer studies in
vitro are very promising but further testing in vitro and more importantly in animal models of cancer along with
optimization of lead compounds will be required to develop these triplex inhibitors of the TTSPs matriptase and
hepsin and related protease HGFA as clinical candidate drugs to treat cancer patients.
Byun and coworkers reported an excellent review on the recent development of hepsin inhibitors [121]. The same
group also reported the peptidyl inhibitors of hepsin and matriptase with configurational modification in the Arg
Cα of Ac-KQLR-kt (Compound 101) [32] and truncated peptides, the activity data shown the hepsin affinity of
(R)-isomer is similar to that of the (S)-isomer, indicating that the epimerization/racemization of Arg residue did
not make significant difference on hepsin inhibition [93].
Pant and et al. reported the novel hepsin inhibitors with nanomolar activity, in the cell-based assay one of
their leading molecule inhibit the dual oncogenic functions of hepsin and also inhibited MET activation and loss
of cellular function [122]. Vasioukhin and co-workers reported the development of a novel, nontoxic and orally
bioavailable small molecule hepsin inhibitors, HepIn1-25 [55]. Out of these 25, HepIn-13 (Figure 4) inhibits hepsin
10.4155/fmc-2018-0446
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Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
Review
Br
HN
Figure 4.
NH
Small molecule hepsin inhibitor.
H2N
NH
O
N
H
HN
O
Figure 5. CVS-3983 small molecule inhibitor for
matriptase.
O
O
NH
H
N
O
OH
O
N
H
NH2
and blocks prostate cancer metastasis. In the double transgenic line, LPB-Tag/PB-Hepsin mice, which develops
bone metastasis [55], HepIn-13 reduced metastasis to the lungs with 42% of control group vs. 20% of treatment
group [55].
In a xenograft model of orthotropic prostate cancer, in which LnCap-34 cells display hepsin-overexpressing
capability to invade and development lymph growth metastasis, the HAI-1 protein-based hepsin inhibitor, a
mono PEGylated form of the Kunitz domain-1 (KD1-PEG) suppressed contralateral prostate invasion and growth
metastasis by 70% and 43%, respectively [122].
CVS-3983 (Figure 5), a small molecule matriptase inhibitor developed by the Carvos international, was found
to significantly suppress the growth of androgen independent human prostate tumor xenografts [123]. MCOTII, a cyclic microprotein of the squash Momordica cochinchinensis trypsin-inhibitor family, which is potent for
matriptase and blocks pro-HGF activation in cell-free and cell culture models, selectively inhibit invasion of
matriptase expression in prostate cancer cells [124].
Conclusion
In the past 10 years, a large number of small molecule benzamidine and substrate-based peptidomimetic inhibitors
of TTSPs (matriptase, hepsin and matriptase-2) in addition to the closely related soluble plasma protease HGF-A
have been identified through rational design. Peptidomimetics and some of the benzamide analogs were identified as
potent inhibitors for HGFA, matriptase, hepsin and matriptase-2 with IC50 and Ki values at single-digit nanomolar
concentrations. These inhibitors thus achieve the incredible potency at levels appropriate and mostly required
for modern drugs, but the challenges of selectivity, metabolic stability, safety and oral administration still exist.
Furthermore, TTSP and HGFA inhibitors have been demonstrated to have strong anticancer properties in vitro in
multiple phenotypic cancer cell assays. More advanced studies, such as in vivo therapeutic efficacy in relevant animal
models of cancer are now necessary to further develop these exciting lead compounds for clinical use. Increasing
evidence suggests that TTSPs matriptase, hepsin and HGFA play a key role in the clinical resistance to anticancer
therapy as well as tumor progression/metastasis and for matriptase-2 in iron overload diseases.
Future perspective
TTSPs exemplify a large class of promising druggable targets for therapeutic intervention especially in cancer,
however the development small molecule inhibitors have been sparse and mainly from academic groups, thus their
further development as marketed drugs has been stunted. Currently, no specific TTSP inhibitors are in clinical
trials and to our knowledge are not being actively pursued by the pharma industry. However, based on the existing
research and availability of lead chemical inhibitors of several chemical series, it is evident that the small molecule
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Damalanka & Janetka
inhibitors can potently block the in vitro oncogenic cell signaling in cancer. Future studies will need to assess the
in vivo pharmacokinetics (PK) and efficacy of these currently available lead compounds in relevant animal models.
The low metabolic stability of the most potent series of peptide substrate-based inhibitors will also have to be
addressed in compound optimization. Positive data to this and efficacy in animal models, will almost certainly and
significantly raise the interest level for furthering more significant work on inhibitor and drug development efforts.
With the very recent interest in developing small molecule inhibitors of the TTSPs within the last 5–10 years,
it is thought that interest will grow to realize the high potential of these inhibitors as promising new anticancer
drugs. This novel approach should be attractive to the biotechnology and pharmaceutical industries in light of
the lack of patient responses and rapid resistance to targeted therapies like kinase inhibitors, which have been
developed over the last 20–30 years of coordinated work. Targeting the proteases HGFA, matriptase and hepsin
with small molecule inhibitors has been recently shown to both prevent and overcome the clinical resistance to
several different kinase inhibitors. This observation and data combined with the obvious application as stand-alone
nonkinase kinase pathway inhibitors will likely serve as a foundation for the near future medicinal chemistry efforts
and clinical applications using TTSP inhibitors in cancer and other diseases. Importantly, this therapeutic strategy
in cancer is predicted to be effective in abrogating tumor and disease progression, including metastasis, which is
the biggest obstacle to prolonging the life of cancer patients.
Executive summary
Background
Type II transmembrane serine protease family
• Type II transmembrane serine protease (TTSP) plays vital roles in biological processes including epithelial
differentiation, homeostasis, iron metabolism, hearing and blood pressure regulation.
• HGF plays a key role cancer disease progression; matriptase, hepsin and HGFA activate the HGF.
• Matriptase is overexpressed in a broad range of epithelial tumors including breast, prostate, colon, uterus, skin
and cervix; which is first discovered in breast cancer cell lines and highly expressed in human breast carcinoma
cells.
• Hepsin is primarily expressed in the liver but also commonly in human kidney, prostate cancer, renal cell
carcinomas, ovarian and breast.
• HGFA is not a TTSP but located in the plasma, it belongs to the S1 trypsin-like serine proteases. HGFA has been
extensively investigated in breast cancer, but also found in AML and multiple myeloma.
• Matriptase-2 plays a pivotal role in the regulation of iron homeostasis in mice and humans, it is mainly restricted
to liver.
• Matriptase, hepsin, HGFA and matriptase-2 are potential therapeutic targets for the treatment and prevention of
metastatic cancer and iron overload diseases, respectively.
• We have provided an overview on the discovery of small molecule and substrate-based peptidomimetic inhibitors
of the TTSPs matriptase, hepsin and matriptase-2.
Synthetic inhibitors of hepsin, HGFA, matriptase & matriptase-2
• A limited number of research groups have reported small molecule and peptidomimetic inhibitors as new drugs
for cancer and iron overload diseases.
• We have summarized the different chemical series of benzguanidine, benzamidine, benzamidine sulfonamide,
benzimidamide, bisbenzamidine, substrate-based peptide inhibitors and their activity against hepsin, HGFA,
matriptase and matriptase-2.
Future perspective
• TTSPs are promising targets for the therapeutic development of novel drugs which we expect lead inhibitors to
enter clinical evaluation within the next 5–10 years.
Financial & competing interests disclosure
JW Janetka is a cofounder and shareholder of ProteXase Therapeutics and who is an inventor on a patent application covering
inhibitors of HGFA, matriptase and hepsin. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript
apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
Review
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•
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10.4155/fmc-2018-0446
Future Med. Chem. (Epub ahead of print)
future science group
Recent progress on inhibitors of the TTSPs, hepsin, matriptase & matriptase-2
Review
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Important article for the identification of subpocket selectivity using docking studies for matriptase and matriptase-2.
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••
Good review article of the recent development of hepsin inhibitors.
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10.4155/fmc-2018-0446