Changing the selectivity profile–from substrate analog inhibitors of thrombin and factor Xa to potent matriptase inhibitors

Article abstract of DOI:10.3109/14756366.2016.1172574

The type II transmembrane serine protease matriptase is a potential target for anticancer therapy and might be involved in cartilage degradation in osteoarthritis or inflammatory skin disorders. Starting from previously described nonspecific thrombin and factor Xa inhibitors we have prepared new noncovalent substrate-analogs with superior potency against matriptase. The most suitable compound 35 (H-d-hTyr-Ala-4-amidinobenzylamide) binds to matriptase with an inhibition constant of 26 nM and has more than 10-fold reduced activity against thrombin and factor Xa. The crystal structure of inhibitor 35 was determined in the surrogate protease trypsin, the obtained complex was used to model the binding mode of inhibitor 35 in the active site of matriptase. The methylene insertion in d-hTyr and d-hPhe increases the flexibility of the P3 side chain compared to their d-Phe analogs, which enables an improved binding of these inhibitors in the well-defined S3/4 pocket of matriptase. Inhibitor 35 can be used for further biochemical studies with matriptase.

Full text of DOI:10.3109/14756366.2016.1172574

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Changing the selectivity profile – from substrate
analog inhibitors of thrombin and factor Xa to
potent matriptase inhibitors
Alexander Maiwald, Maya Hammami, Sebastian Wagner, Andreas Heine,
Gerhard Klebe & Torsten Steinmetzer
To cite this article: Alexander Maiwald, Maya Hammami, Sebastian Wagner, Andreas Heine,
Gerhard Klebe & Torsten Steinmetzer (2016): Changing the selectivity profile – from substrate
analog inhibitors of thrombin and factor Xa to potent matriptase inhibitors, Journal of Enzyme
Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2016.1172574
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1172574
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Date: 13 May 2016, At: 00:56

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–9
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/14756366.2016.1172574
!
RESEARCH ARTICLE
Changing the selectivity profile – from substrate analog inhibitors of
thrombin and factor Xa to potent matriptase inhibitors
Alexander Maiwald*, Maya Hammami*, Sebastian Wagner, Andreas Heine, Gerhard Klebe, and Torsten Steinmetzer
Department of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University, Marburg, Germany
Abstract
Keywords
The type II transmembrane serine protease matriptase is a potential target for anticancer
therapy and might be involved in cartilage degradation in osteoarthritis or inflammatory skin
disorders. Starting from previously described nonspecific thrombin and factor Xa inhibitors we
have prepared new noncovalent substrate-analogs with superior potency against matriptase.
The most suitable compound 35 (H-D-hTyr-Ala-4-amidinobenzylamide) binds to matriptase
with an inhibition constant of 26 nM and has more than 10-fold reduced activity against
thrombin and factor Xa. The crystal structure of inhibitor 35 was determined in the surrogate
protease trypsin, the obtained complex was used to model the binding mode of inhibitor 35 in
the active site of matriptase. The methylene insertion in D-hTyr and D-hPhe increases the
flexibility of the P3 side chain compared to their D-Phe analogs, which enables an improved
binding of these inhibitors in the well-defined S3/4 pocket of matriptase. Inhibitor 35 can be
used for further biochemical studies with matriptase.
Benzamidines, matriptase, protease inhibition,
trypsin, X-ray structure
History
Received 5 February 2016
Revised 25 March 2016
Accepted 28 March 2016
Published online 6 May 2016
Introduction
promotes extracellular proteolysis by generating the broad
spectrum protease plasmin, which is an activator of MMPs, of
certain growth hormones, and is involved in the degradation of
extracellular matrix proteins (ECM). The components of the
plasminogen activation system are upregulated in several tumor
entities and promote tumor invasion and metastasis. In addition to
some other proteases including hepatocyte growth factor activator
(HGFA), matriptase efficiently converts pro-hepatocyte growth
factor/scattering factor (pro-HGF/SF) to its active form10. HGF/
SF has high affinity to the receptor tyrosine kinase c-Met, thereby
inducing signaling pathways, which promote tumorigenesis and
invasive growth12,13. The G-protein coupled protease-activated
receptor 2 (PAR2) is an additional candidate substrate of
matriptase, which is localized on the extracellular surface9.
PAR2 mediates cell adhesion, cell motility and inflammation and
has been suggested to stimulate metastasis14. Beside these
functions in cancer, a matriptase-catalyzed PAR2 activation also
leads to an increased collagenase expression contributing to
enhanced cartilage degradation in osteoarthritis15. Moreover, a
strong increase in matriptase expression was observed in inflam-
matory skin disorders16. Recent findings suggest that the host
protease matriptase might also be involved in the activation of
certain H1 and H9 influenza virus hemagglutinins, which is
Matriptase is one of the best characterized members among the 17
conserved type II transmembrane serine proteases (TTSPs) found
in human and mice1. Like all other TTSPs matriptase possesses a
complex modular structure containing an extracellular C-terminal
trypsin-like serine protease domain. It is widely expressed in
epithelial tissues and mediates various pleiotropic effects in
development, cell–cell adhesion and tissue homeostasis2.
Matriptase is required for normal epithelial development and
postnatal survival, hence, knockout mice die within 48 h of birth
due to severe dehydration caused by impaired epidermal barrier
function3. Individuals harboring a missense mutation leading to a
Gly!Arg change in residue 216 of the protease domain
(chymotrypsin numbering), which results in strongly reduced
matriptase activity, suffer from ichthyosis with hypotricosis4,5
.
Furthermore, matriptase mRNA and protein are downregulated in
inflamed colonic tissues from Crohn’s disease and ulcerative
colitis patients. The loss of matriptase leads to leaky tight
junctions within the gut and reduced transepithelial resistance
promoting intestinal inflammation6,7
.
Beside these important functions in normal physiological
processes matriptase can activate numerous substrates involved in
cancer8 and other diseases. For instance, matriptase efficiently
cleaves soluble single-chain pro-urokinase type plasminogen
activator (pro-uPA), as well as receptor-bound pro-uPA9–11. uPA
essential for virus propagation17,18
.
Therefore, matriptase emerged as a potential drug target,
especially for the treatment of epithelial tumors. First successful
proof of concepts studies with prostate cancer models in mice
have been performed with the arginal-derived matriptase inhibitor
CVS-398319, and with the 3-amidinophenylalanine derivative CJ-
173720 (Figure 1). Moreover, CJ-1737 and related inhibitors
reduced the pro-HGF/SF driven phosphorylation of the HGF
receptor/c-Met and the overall cellular invasiveness of the human
*These authors contributed equally to this work.
Address for correspondence: Torsten Steinmetzer, Department of
Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University,
Marbacher Weg 6, Marburg D-35032, Germany. E-mail: steinmetzer@
uni-marburg.de

2
A. Maiwald et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
HOOC
O
Figure 1. Matriptase inhibitors used in tumor
models in mice and various cell culture
assays.
O
O
H
O
O
H
N
N
H2N
N
S
N
H3C
N
H
H
H
O
N
O O
O
H
NH2
NH
NH2
HN
NH2
HN
HN
NH2
CJ-1737 (Ki = 3.8 nM)
CVS-3983 (Ki = 3.3 nM)
HN
HN
NH2
O
H
O
CH3
O
O
N
H
H
S
N
N
N
N
H2N
N
O O
H
Cl
Cl
NH2
O
S
O
NH2
NH
NH2
NH2
HN
HN
IN-1 (Ki = 0.011 nM)
MI-432 (Ki = 2.0 nM)
pancreatic adenocarcinoma cell line AsPC-121. Even stronger 5 mm, 100 A, 32 ꢀ 250 mm, Macherey-Nagel, Du¨ren, Germany)
effects on pro-HGF/SF induced c-Met activation on primary by a linear gradient with the same solvents as described above at a
mammary carcinoma cells and three other human breast cancer flow rate of 20 ml/min. All final inhibitors were obtained as TFA-
cell lines were described for the ketobenzothiazole inhibitor salts after lyophilization. The molecular mass of the synthesized
IN-122 (Figure 1), meanwhile various highly potent ketone- compounds was determined using a QTrap 2000 ESI spectrometer
derived inhibitors have been described23,24. The invasiveness and (Applied Biosystems, now Life Technologies, Carlsbad, CA). The
migration of various tumor cell lines could be also inhibited by 1H- and 13C-NMR spectra were recorded on a Jeol JNM-GX-400
˚
other benzamidine-derived matriptase inhibitors25,26
.
at 400 and 100 MHz (Jeol Inc., Peabody, MA) and are referenced
During the search for inhibitors of matriptase-2, a related to internal solvent signals.
TTSP, we have previously observed a significant inhibitory
potency of the substrate analog inhibitor BAPA (benzylsulfonyl- Synthesis
D-Arg-Pro-4-amidinobenzylamide, compound 5 in Table 2)
The most selective inhibitor 35 was synthesized as described in
Scheme 1.
against matriptase (Ki ¼ 55 nM)27. Its C-terminal arginine
mimetic 4-amidinobenzylamide (4-Amba) is an excellent anchor
for all trypsin-like serine proteases and can be produced in large
quantities28, but it does not contribute to any selectivity within
H-Ala-4-Amba ꢁ 2 TFA (1)
this family. It was initially described during the development of Boc-Ala-OH (333 mg, 1.76 mmol) and 4-amidinobenzylamine ꢁ 2
the thrombin inhibitor melagatran/ximelagatran29 and was later HCl35 (391 mg, 1.76 mmol) were suspended in 10 ml DMF. After
used for the design of potent uPA, factor Xa, factor VIIa, and cooling to 0ꢂ C the mixture was treated with 1.155 g (2.61 mmol)
plasmin inhibitors30–33
.
BOP and 1.36 ml (7.83 mmol) DIPEA. After stirring overnight at
Because of the poor selectivity profile of BAPA, which is a room temperature, the solvent was removed in vacuo, and the
significant stronger inhibitor of the clotting proteases thrombin Boc-protected intermediate was purified by preparative HPLC.
and factor Xa (fXa) with inhibition constants of 3.5 and 2.5 nM, The product containing fractions (HPLC: tR ¼ 23.89 min, start at 1%
respectively34, we tried to develop new substrate-analog inhibitors solvent B) were combined and evaporated. The remaining residue
with improved selectivity for matriptase. The results of this work was treated with 10 ml trifluoroacetic acid and stirred for one hour
are summarized in the current paper.
at room temperature. The solvent was evaporated and the product
was lyophilized from water providing a white solid (yield: 285 mg,
0.64 mmol, 36%, HPLC: tR ¼ 8.88 min, start at 1% solvent B, MS
calcd m/z 220.13, m/z found 221.2 (M + H)+. 1H NHMR (400MHz,
DMSO-d6) 1.39 (d, J ¼ 7.20 Hz, 3H), 3.91 (s, 1H), 4.45 (d,
J ¼ 6.00Hz, 2H), 7.49 (d, J ¼ 8.00 Hz, 2H), 7.79 (d, J ¼ 8.00 Hz,
2H), 8.10 (s, 3H), 8,98 (t, J ¼ 6.00Hz, 1H), 9,26 (s, 1H).
Materials and methods
Reagents for synthesis, including protected standard amino acids,
coupling reagents and solvents were obtained from Bachem,
Fluka, Acros, Merck or Aldrich.
Analytical HPLC experiments were performed on a Shimadzu
˚
LC-10A system (column: Nucleodur C18, 5 mm, 100 A,
Boc-D-hTyr-OH (2)
4.6 ꢀ 250 mm, Machery-Nagel, Du¨ren, Germany) with a linear
gradient of acetonitrile (solvent B) and water (solvent A) both 111 mg (0.4 mmol) of H-D-hTyr-OHꢁ HBr (Iris Biotech GmbH,
containing 0.1% TFA at a flow rate of 1 ml/min (1% increase Marktredwitz, Germany) was dissolved in 4 ml dioxane, 2 ml
solvent B per min, starting at 1% solvent B), detection at 220 nm. water and 1 ml 1 N NaOH. The mixture was cooled to 0ꢂ C and
The final inhibitors were purified to more than 95% using treated with 100 mg (0.46 mmol) Boc2O. After 10 min the ice bath
preparative HPLC (pumps: Varian PrepStar Model 218 gradient was removed and the mixture was stirred at room temperature for
system, detector: ProStar Model 320 with detection at 220 nm, 2 h. The solvent was removed in vacuo and the remaining residue
fraction collector: Varian Model 701; column: C8, Nucleodur, was dissolved in a mixture of 5% KHSO4 and ethyl acetate. The

DOI: 10.3109/14756366.2016.1172574
Substrate analog matriptase inhibitors
3
NH
NH
Scheme 1. Synthesis of inhibitor 35.
Conditions and reagents (a) 1 equiv Boc-
Ala-OH, 1 equiv BOP, 3 equiv DIPEA; (b)
TFA, 1 h room temperature; (c) 1.15 equiv
Boc2O in dioxane and water, pH ꢄ 8.5
adjusted with 1 N NaOH; (d) 1 equiv BOP, 3
equiv DIPEA; (e) TFA, 1 h, room tempera-
ture, preparative HPLC.
2
H N
2
H-D-hTyr-OH · HBr
2 HCl
a,b
c
O
H
N
Boc
OH
O
H N
2
N
H
NH
2
2
1
NH
2 TFA
OH
d,e
NH
NH
2
H
N
O
O
H N
2
N
H
35
2 TFA
OH
organic layer was washed 3 ꢀ with 5% KHSO4 and 3 ꢀ with 8.69 (m, 1H), 8,76 (m, 1H), 9.21 (s, 3), 9.26 (s 1H 13C NMR
saturated aqueous NaCl. The organic phase was dried over (100 MHz, DMSO-d6) 19.0, 30.1, 34.0, 42.3, 49.1, 52.8, 115.8,
Na2SO4, and the solvent was removed in vacuo to afford a 127.1, 127.8, 128.7, 129.5, 131.1, 146.3, 156.2, 166.1, 168.7,
colorless viscous oil (yield: 95 mg, 0.322 mmol, 80.5%, HPLC 172.4).
start at 1% solvent B: tR ¼ 40.32 min, MS calcd m/z 295.14, m/z
Racemic P3 homoamino acids, which were not commercially
found 294.25 (MꢃH)ꢃ. 1H NHMR (400 MHz, DMSO-d6) 1.39 (s, available (Figure 2), were prepared from appropriately substituted
9H), 1.83 (m, 2 h), 2.50 (m, 2 h), 3.82 (m, 1H), 6.65 phenylethyl chlorides or bromides by reaction with ethyl
(d, J ¼ 8.40 Hz, 2H), 6.96 (d, J ¼ 8.40 Hz, 2H), 7.21 (m, 1H) acetamidocyanoacetate in presence of potassium carbonate and
9.11 (s, 1 H), 12.40 (s, 1H).
potassium iodide, as described previously36. The synthesis of the
inhibitors with these residues was performed as shown in
Scheme 1 for the D-hTyr inhibitor. The final diastereomeric
inhibitors could be separated by preparative reversed phase
HPLC. Substrate analog inhibitors of trypsin-like serine proteases
normally prefer D-configurated P3-residues. Therefore, the com-
pounds with stronger inhibitory potency were assigned to contain
the P3 residue in D-configuration, whereas their analogs with
lower potency possess a L-configurated P3 amino acid.
H-D-hTyr-Ala-4-Amba ꢁ 2 TFA (35)
H-Ala-4-Amba ꢁ 2 TFA (111 mg, 0.25 mmol) and Boc-D-hTyr-OH
(74 mg, 0.25 mmol) were suspended in 5 mL of DMF. The
mixture was cooled to 0ꢂ C and treated with 110 mg (0.25 mmol)
BOP and 130 mL (0.75 mmol) DIPEA. After stirring overnight at
room temperature, the solvent was removed in vacuo. The
remaining residue was treated with 5 ml trifluoroacetic acid and
stirred for one hour at room temperature. After precipitation in
ether and centrifugation, the obtained solid was purified by
preparative HPLC. The product containing fractions were
combined, concentrated and lyophilized from water providing a
white solid (yield: 32 mg, 0.05 mmol, 20%, HPLC:
tR ¼ 17.33 min, start at 1% solvent B, MS calcd m/z 397.48,
m/z found 398.21(M + H)+ 1H NHMR (400 MHz, DMSO-d6) 1.29
For the synthesis of the benzylsulfonyl protected inhibitors
shown in Table 2, the appropriate benzylsulfonyl-protected P3
amino acids were used instead of the Boc derivatives. The
analytical data of all inhibitors are provided as Supplementary
material available online.
Determination of inhibition constants
(d, J ¼ 6.16 Hz, 3H), 1.94 (m, 2H), 3.92 (m, 1H), 4.40 (m, 3H), The Ki values for the inhibition of the catalytic domain of
6.69 (d, J ¼ 8.80 Hz, 2H), 6.96 (d, J ¼ 8.40 Hz, 2H), 7.46 matriptase (23 pM in assay), which was prepared as described
(d, J ¼ 8.80 Hz, 2H), 7.77 (d, J ¼ 8.40 Hz, 2H), 8.18 (s, 3H), previously20, and factor Xa (97 pM in assay, purchased from

4
A. Maiwald et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
O
O
O
O
O
H N
H N
H N
H N
H N
2
2
2
2
2
OH
OH
OH
OH
OH
Cl
Cl
Cl
OMe
Cl
Cl
D/L-hPhe(4-OMe)
HPLC: 23.60 min
MS calc.: 209.25
found: 210.12
D/L-hPhe(2-Cl)
HPLC: 27.17 min
MS calc.: 213.06
found: 214.15
D/L-hPhe(3-Cl)
HPLC: 28.35 min
MS calc.: 213.06
found: 214.05
D/L-hPhe(2,4-Cl )
D/L-hPhe(4-Cl)
HPLC: 29.37 min
MS calc.: 213.06
found: 214.09
2
HPLC: 33.60 min
MS calc.: 247.02
found: 248.02
Figure 2. Analytical data and abbreviations of prepared racemic homo amino-acids. Their determined mass corresponds to the (M + H)+ ion, the HPLC
analysis started at 1% solvent B.
Enzyme Research South Bend, Indiana, USA) were determined
with the substrate methylsulfonyl-D-Arg-Pro-Arg-AMC (prepared
in house37, for matriptase: KM ¼ 6 mM, used concentrations in
assay 14, 7 and 3.5 mM; for FXa: KM ¼ 28 mM, used concentra-
tions in assay 50, 25 and 12.5 mM), while Tos-Gly-Pro-Arg-AMC
(KM ¼ 5.4 mM, used concentrations in assay 10, 5 and 2.5 mM)
was used for bovine thrombin prepared according to Walsmann38
(31 pM in assay). The measurements were performed in a
Fluoroscan microplate reader (ꢀex ¼ 355 and ꢀem ¼ 460 nm;
Thermo Fisher Scientific, Vantaa, Finland) with 100 ml buffer
(50 mM Tris, 154 mM NaCl, pH 8.0), 20 ml substrate solution
(substrate dissolved in water) and 20 ml enzyme39. The Ki values
were obtained from Dixon plots40 and are the average of at least
two measurements.
Table 1. Data collection and refinement statistics for the trypsin/inhibitor
35 complex.
PDB-code
4MTB
(A) Data collection and processing
˚
Wavelength (A)
0.91841
P3121
Space group
Unit cell parameters
˚
a, b, c (A)
55.0, 55.0, 108.1
90.0, 90.0, 120.0
a, b, g (ꢂ)
Matthews coefficient (A /Da)
3
˚
2.0
39.2
1
Solvent content (%)
Molecules in asymetric unit
(B) Diffraction dataa
˚
Resolution range (A)
40–1.22 (1.24–1.22)
54536
4.8 (42.2)
Unique reflections
R (I) sym (%)
Completeness
Redundancy
I/s (I)
Crystal structure analysis
95.5 (93.8)
5.0 (4.0)
29.7 (2.7)
Cocrystallization of trypsin with inhibitor 35: Bovine beta-
Trypsin (Sigma, # T8003) was dissolved in a solution of 10 mM
CaCl2 at 20 mg/ml for 30 minutes on ice. The inhibitor 35 was
dissolved in water at 2.5 mg/ml. The crystallisation buffer
contained 0.1 M imidazole, 0.1 M (NH4)2SO4, 20% PEG
8000, 0.1% sodium azide at pH 8. In the next step, 50 mL of the
trypsin solution was mixed with 10 mL of the inhibitor solution
and filled up to 100 mL with water. This new solution was mixed
1:1 with the crystallization buffer and 5 mL of this solution were
placed in the center of a cover slip. Crystallization was carried
out at 16 ꢂC by the hanging-drop method. The wells of the
crystallization trays were filled with 500 mL of the crystallization
buffer. Subsequently, the cover slips were placed over the wells
and sealed. Crystals of good diffracting quality could be
obtained after 20 to 30 days.
(C) Refinement
Resolution range
Reflections used in refinement
(work/free)
35.715–1.22
54489/2763
Final R values for all reflections
(work/free) (%)
Protein residues
Calcium ions
Inhibitor atoms
13.5/16.4
223
1
29
336
Water molecules
RMSD from ideality
˚
Bond length (A)
0.009
1.390
Bond angles (ꢂ)
Ramachandran plot (PROCHECK)
Residues in preferred regions (%)
Residues in allowed regions (%)
Outliers (%)
Mean B-factor (A )
Protein
97.0
3.0
0.0
Data collection and processing: Obtained crystals were frozen
at 77 K for data collection. The data set was collected at 100 K at
synchrotron beamline 14.2 at the BESSY using MARMOSAIC
225 mm CCD detector. Data processing and scaling were
performed using the HKL2000 package41.
2
˚
11.9
11.1
9.2
Ligand
Calcium
Imidazole
Water molecules
18.9
24.3
Structure determination and refinement: The coordinates of
bovine trypsin (PDB: 2ZFS)42, were used for molecular replace-
ment with Phaser from the CCP4 program package43. For initial
rigid body refinement of the protein molecule, followed by
repeated cycles of maximum likelihood energy minimization
simulated annealing and B-factor refinement the program
PHENIX44 was used. A randomly chosen 5% of all data were
used for the calculation of Rfree and were not used in the
refinement. Amino acid side chains were fit into s-weighted 2Fo
– Fc and Fo – Fc electron density maps using Coot45. After the
first refinement cycle, water molecules and subsequently ions and
ligands were located in the electron density and added to the
aNumbers in brackets are for the highest resolution shell.
model. Restraints were applied to bond lengths and angles,
planarity of aromatic rings and van der Waals contacts. Multiple
side-chain conformations were built in case an appropriate
electron density was observed and maintained during the refine-
ment, and if the minor populated side chain showed at least 20%
occupancy. The final model was validated using PHENIX own
validation options or ADIT. Data collection, unit cell parameters

DOI: 10.3109/14756366.2016.1172574
Substrate analog matriptase inhibitors
5
and refinement statistics are given in Table 1. The naming of the screened with matriptase, thrombin and fXa. In a first series, the
protein amino acids was done according to Bode et al.46. P3 position was modified, whereas proline was maintained as P2
Coordinates and structure factors have been deposited in the residue, while in a second one the D-arginine in P3 position was
Protein Data Bank with the accession code 4MTB.
kept constant and the P2 residue was modified (Table 2).
Although a few analogs, e.g. inhibitors 3–6, revealed a
significant matriptase affinity with inhibition constants550 nM,
all compounds with proline in P2 position showed a considerably
stronger thrombin inhibition and most substances also possess a
Results
Modeled complex of BAPA in matriptase
Due to the limited amount of matriptase we could not determine high fXa affinity. Interestingly, a ꢄ25-fold improved matriptase
its crystal structure in complex with substrate analog inhibitors so inhibition was found for the D-homophenylalanine (D-hPhe)
far. For initial modeling of their binding mode in matriptase we derivative 3 over its D-Phe analog 11. A relatively high potency
used the recently described crystal structure of a trypsin mutant in was also determined for inhibitors containing basic P3 side
complex with benzylsulfonyl-D-Arg-Gly-4-Amba, the P2 glycine chains, which are probably involved in cation-ꢁ-interactions to
analog of BAPA 5 (3pmj.pdb)47. The complex was superimposed matriptase residues Trp215 and Phe99.
by a Ca-atom fit with the previously determined crystal structure
of matriptase (2gv6.pdb)20, all water molecules were deleted. constant P3 residue, which was available from previous work on
After replacement of the P2 glycine with proline, the bound inhibitors of the human airway trypsin-like protease (HAT)49,51
inhibitor was energy-minimized using the program MOE48, In comparison to the P2 Pro inhibitor 5, a 1.5-fold stronger
whereas the coordinates of matriptase were kept constant. matriptase inhibition was observed for the Ala-analog 15. As
Therefore, we screened a second series 15–22 with D-Arg as
.
The obtained model reveals the expected overall binding mode expected, this replacement strongly diminished the inhibition of
known from many structures of various trypsin-like serine thrombin, whereas the affinity against fXa was slightly enhanced.
proteases in complex with substrate analog inhibitors, containing A similar matriptase inhibition was found for the P2 Arg inhibitor
a P3 residue in D-configuration (Figure 3). The D-Arg side-chain 16, all other P2 modifications reduced the potency.
is oriented towards the distal S3/4 binding pocket above Trp215
From previous work we know that the potency of this inhibitor
and might be involved in cation-ꢁ interactions. However, its side- type against fXa strongly depends on the presence of a suitable P4
chain guanidino group is not involved in any specific hydrogen group. Elimination of the N-terminal sulfonyl group of Bzls-
bonds with matriptase. In contrast, all known interactions of the DSer(tBu)-Gly-4-Amba (Ki ¼14 nM) resulted in a &1000-fold
benzamidine group in the S1 pocket and between the inhibitor reduced fXa affinity (Ki ¼16 mM), whereas the affinity against
backbone and the matriptase residues Ser214, Gly216 and Gly219 thrombin was only 6-fold reduced52. Moreover, in previous work
were found. The model suggested that it should be possible to
Table 2. Inhibition of matriptase, thrombin and fXa by benzylsulfonyl-
protected inhibitors available from previous work27,49,50
replace the P3 D-arginine and P2 proline by other residues to
improve affinity of the inhibitors.
.
Determination of inhibition constants
NH
Initially, benzylsulfonyl protected inhibitors containing D-con-
figurated P3 residues available from previous studies27,49,50 were
NH
2
H
N
P3
S
P2
O
O
Ki (mM)
No.
P3
P2
Matriptase
Thrombin
fXa
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
D-hPhe
D-hArg
D-Arga
D-Lys
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Ala
Arg
0.013
0.03
0.033
0.04
0.073
0.2
0.001
0.0012
0.004
0.001
0.001
0.0004
0.003
0.00012
0.0016
0.0013
0.005
0.004
0.075
0.18
0.029
0.068
0.5
0.037
0.66
0.31
0.014
0.0027
0.0029
0.052
0.0022
0.017
0.15
0.035
0.077
0.012
0.83
D/L-hAla(2Pyr)b
D-Lys(Cbz)
D-Phe(4-Am)c
D-Chaa,d
D-Phe
0.3
0.33*
0.34
0.77
2.76
5.91
0.02
0.023
0.065
0.12
0.2
D-Val
D-Ala
Gly
D-Arg
D-Arg
D-Arg
D-Arg
D-Arg
5.17
0.0014
0.015
0.0021
0.037
0.02
0.011
0.021
0.015
Abue
Nvaf
Ser
D-Arg
D-Arg
D-Arg
Leu
Val
Phe
0.46
0.57
0.24
Figure 3. Modeled binding mode of BAPA (inhibitor 5) in matriptase.
The coordinates of matriptase were taken from the pdb entry 2gv6. The
protease is shown with its solvent-accessible transparent surface in gray.
The inhibitor is displayed as stick model with carbon atoms in white,
nitrogen in blue, oxygen in red, and sulfur in yellow. Selected residues of
matriptase forming the S3/4 pocket (Trp215 at the bottom, Phe99 on the
right, Gln175 on the left and Phe97 on the top), as well as residues
involved in polar contacts to the inhibitor (Asp189, Ser190, Gly216, and
Gly219) are labeled and shown as sticks with carbons in green. All
colored figures were prepared using PyMOL v0.98 (DeLano Scientific,
San Carlos, CA).
aThe matriptase inhibition by compounds 5 and 10 has been previously
published27
.
bRacemic D/L-homo-2(pyridyl)alanine.
D-4-amidinophenylalanine.
c
d
D-cyclohexylalanine.
ea-aminobutyric acid.
fNorvaline.

6
A. Maiwald et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Table 3. Inhibition of matriptase, thrombin and factor Xa by inhibitors
lacking a P4 residue.
are very potent inhibitors of bovine trypsin with Ki values of
0.81 nM and 3.7 nM, respectively. Since we had insufficient
amounts of matriptase for structure determination, inhibitor 35
was crystallized in the surrogate protease trypsin (Figure 4A). All
typical contacts of the P1 benzamidine within the S1 pocket and
from the backbone of the inhibitor to trypsin residues Ser214 and
Gly216 have been found and are identical as previously observed
in crystal structures of substrate-analog inhibitors with throm-
bin50, fXa52 or uPA30. The P2 carbonyl binds to the side-chain
amide of Gln192 and two water molecules, one of them mediates
a contact to the free P3 amino group. The D-hTyr backbone
interacts with Gly216, and the P3 side chain fits well into the
distal S3/4 pocket. The hydroxyl group forms a close contact to
the carbonyl of the Gln175 side-chain amide and is surrounded by
a complex water network. Only two of them, which directly bind
to the inhibitor, are shown in Figure 4(A), one of them enables
water-mediated contacts to the main chain carbonyls of Thr98 and
Gln175.
NH
NH
2
H
N
P
2
P
2
Ki (mM)
No.
P3
P2
Matriptase
Thrombin
fXa
2.58
1.32
0.45
0.74
1.87
1.21
1.04
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
D-hPhe
D-hPhe(4-Cl)
D-hPhe(2-Cl)
D-hPhe(2,4-Cl2)
D-hPhe(4-OMe)
D-hPhe(3-Cl)
hPhe(3-Cl)
hPhe(4-OMe)
hPhe(2,4-Cl2)
hPhe(2-Cl)
hPhe(4-Cl)
hPhe
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
0.06
0.11
0.14
0.19
0.37
0.46
5.28
7.69
11.6
12.0
15.0
19.7
0.026
0.044
0.046
0.11
0.15
0.16
0.28
3.02
3.94
4.17
6.32
9.97
0.072
0.014
0.013
0.15
0.012
0.032
0.65
0.74
1.13
1.21
0.37
7.61
0.30
0.73
0.007
0.08
0.15
0.07
0.18
0.57
0.57
4.26
2.12
2.69
89.4
31.6
24.8
39.2
66.4
0.57
1.79
Superposition of the complex with the crystal structure of
matriptase (2gv6.pdb) suggests a very similar binding mode,
all characteristic interactions were maintained (Figure 4B).
The positively charged amidine group binds to the side chains
of matriptase residues Asp189, Ser190 and to the carbonyl
D-hTyr
D-hPhe
D-hPhe(4-Cl)
D-hPhe(2-Cl)
D-hPhe(2,4-Cl2)
D-hPhe(3-Cl)
D-hPhe(4-OMe)
hPhe(4-Cl)
hPhe(2,4-Cl2)
hPhe(4-OMe)
hPhe(2-Cl)
2.30 oxygen of Gly219. Additional H-bonds exist between the P1
0.19
0.25
1.33
6.87
33.9
12.5
46.71
10.58
10.44
amide NH and the carbonyl oxygen of Ser214 and between
the backbone of D-hTyr with the carbonyl oxygen and NH of
Gly216. Moreover, the insertion of an additional methylene
group increases the flexibility of D-hTyr, which enables a
deeper binding of the P3 side chain into the distal S3/4 pocket
of matriptase. This might explain the increased affinity of the
D-hPhe and D-hTyr inhibitors compared to the less flexible D-
Phe or D-Cha-analogs.
hPhe(3-Cl)
Discussion
we found an enhanced matriptase inhibition with biphenyl-3-
sulfonyl-substituted derivatives of 3-amidinophenylalanine. The
most potent derivatives of this series were substituted by chlorine
atoms (e.g., see inhibitor MI-432 in Figure 1) or methoxy groups
at their N-terminal phenyl ring, which occupies a similar position
in the S3/4 pocket of matriptase as known for the D-configurated
P3 side chain of substrate-analog inhibitors. Based on these
findings and due to the high potency of compound 3, new
substrate analog inhibitors without a P4 benzylsulfonyl group in
combination with chloro- or methoxy-substituted D-hPhe analogs
in P3 position have been prepared. All these inhibitors were
synthesized with Pro or Ala as P2 residue (Table 3). Comparison
of compound 3 with compound 23 revealed that the deletion of
the Bzls-group reduced the matriptase inhibition only by a factor
of five, whereas the potency against fXa was &200-fold
decreased. Further replacement of P2 Pro by Ala provided
compound 36, the first substrate-analog inhibitor with superior
affinity for matriptase compared to thrombin and fXa. However,
all chloro- or methoxy-substituted D-hPhe analogs showed
reduced matriptase affinity, whereas in few cases the inhibition
of thrombin and fXa was slightly enhanced. Interestingly,
incorporation of the commercially available D-hTyr provided
inhibitor 35, which possesses an improved affinity against all
three proteases and maintains the selectivity profile as found for
compound 36. As expected, a poor inhibitory potency was found
for all inhibitors containing L-configurated P3 amino acids.
Starting with a screening of available benzylsulfonyl-protected
inhibitors we identified D-hPhe and Ala as most suitable P3 and
P2 residues for substrate-analog matriptase inhibitors. The
replacement of P2 Pro by Ala is a simple method to achieve a
significant reduction in thrombin affinity, whereas it leads to a
slightly better matriptase and fXa inhibition. A P2 Ala is also
found in the P4-P4’ segment (RQAR#VVGG) of the autocatalytic
cleavage site of matriptase and it can be well accommodated in
the S2 pocket below the side chains of Phe99 and His57. The
RQAR sequence served recently for the design of highly potent
arginyl-ketone derived matriptase inhibitors23. For comparison we
have also incorporated the natural P4-P2 substrate segment in our
series, the resulting inhibitor H-Arg-Gln-Ala-4-Amba possesses a
ca. 5-fold reduced potency against matriptase (Ki ¼ 0.12 mM)
compared with inhibitor 35, although it has a nearly 100-fold and
therefore more pronounced selectivity over thrombin
(Ki ¼ 14.7 mM) and fXa (Ki ¼ 14.5 mM). Otherwise, L-configu-
rated peptides can suffer from rapid degradation in vivo, whereas
the incorporation of D-amino acids often contributes to an
improved stability.
The affinity against fXa could be significantly reduced by
deletion of the P4 benzylsulfonyl group. In case of fXa it binds
in a shallow subpocket above the Cys220-Cys191 disulfide-
bridge, where its methylene group comes in close contact with
one of the phenyl-ring carbons of the P1 4-Amba residue
˚
(distance 3.7 A), thus stabilizing the compact horseshoe-like
inhibitor conformation52. Although the binding mode of the
benzylsulfonyl group in thrombin is very similar53 and probably
also comparable in matriptase (see model in Figure 3), its
Crystal structure of inhibitor 35 in trypsin and model in
complex with matriptase
Although we could reduce the potency against thrombin and fXa elimination has a less pronounced influence on the inhibition of
it was found that the D-hTyr inhibitor 35 and its D-hPhe analog 36 these two enzymes.

DOI: 10.3109/14756366.2016.1172574
Substrate analog matriptase inhibitors
7
Figure 4. Compound 35 in complex with trypsin and matriptase: (A) Crystal structure of compound 35, shown as sticks (carbon in green, nitrogen in
blue, oxygen in red, and water molecules as red spheres) in complex with bovine trypsin presented with its backbone in beige (4MTB.pdb). Selected
trypsin residues are shown as sticks with light pink carbon atoms; (B) Model of compound 35 in the active site of matriptase (shown with gray surface
and backbone as cartoon in green), obtained by superimposition of the 35/trypsin complex with the crystal structure of matriptase (2GV6), followed by
an energy minimization of the D-homotyrosine side-chain using the program MOE48. All other inhibitor atoms and the matriptase residues were fixed,
water molecules were deleted. A pdb file of the modeled matriptase/inhibitor 35 complex is provided as Supplementary material for download.
two chlorine atoms (Ki ¼ 110 nM)54, we expected a beneficial
effect after incorporation of chloro-substituted D-hPhe analogs.
However, all of these analogs, including the p-methoxy-deriva-
tives 27 and 41, provided less potent matriptase inhibitors. A
superimposition of inhibitor 35 and MI-432 reveals that the
D-hTyr phenyl ring penetrates much deeper into the S3/4 pocket.
It occupies a slightly different position compared to the dichloro-
substituted phenyl ring of the 3-amidinophenylalanine-derived
inhibitor, which makes a closer contact to the phenyl ring of
Phe99 (Figure 5). Moreover, the D-hTyr ring is more vertically ori-
ented against Trp215, whereas the terminal dichloro-substituted
phenyl ring of inhibitor MI-432 adopts a rather parallel orienta-
tion above the indole and vertically points against Phe99. These
differences may explain why we could not achieve any improve-
ment after incorporation of chloro- or methoxy-substituted
D-hPhe analogs. So far, D-hTyr is the preferred residue in P3
position of this matriptase inhibitor type.
Conclusion
Figure 5. Model of matriptase in complex with inhibitor MI-432 (sticks
with white carbon atoms, oxygen in red, nitrogen in blue, chlorine in
Starting from highly potent benzylsulfonyl protected substrate
green, structure shown in Figure 1) and the D-hTyr-derivative 35 (sticks
analog inhibitors of thrombin and fXa we could modulate their
with light pink carbons). The superpositions of both inhibitors reveals that
selectivity profile and obtained inhibitors with superior matriptase
affinity. This was achieved by a combination of three modifica-
tions: (i) the elimination of the P4 benzylsulfonyl group, (ii)
incorporation of D-hPhe analogs in P3 position, and (iii)
replacement of the P3 Pro by Ala. However, we have to admit
that the inhibitors 35 and 36 are still stronger trypsin inhibitors,
although trypsin is mainly located in the intestinal tract and
should not come in direct contact with injectable inhibitors.
Presently, we cannot exclude that these compounds may also bind
to other trypsin-like serine proteases, which were not tested so far.
Moreover, the presence of two basic groups at the P3 and P1
residues makes it very unlikely that the unprotected inhibitors
lacking the P4 moiety could be orally available. Nevertheless, the
new inhibitors 35 and 36 are easily accessible and stable
compounds and may serve as suitable tools for further biochem-
ical studies with matriptase.
their terminal phenyl rings occupy different positions in the distal S3/4
binding pocket. The matriptase residues Phe97, Phe99, Gln175, and
Trp215, which shape the S3/4 pocket, and Asp189 are shown as sticks
with green carbon atoms.
The improved potency of the D-hPhe and D-hTyr inhibitors
against matriptase resembles a trend, which has been previously
observed during the development of substrate-analog fXa inhibi-
tors31. In that case also the shorter P3 D-Phe provides less active
inhibitors. The improved potency can be explained based on the
crystal structure of inhibitor 35 in complex with trypsin, assuming
a similar binding mode in fXa and matriptase. Due to the
methylene insertion, the side chains of the P3 homoamino acids
find better access to the well-defined S3/4 binding sites of fXa
and matriptase. In trypsin, the D-hTyr enables additional water
mediated contacts to carbonyl groups at the end of this pocket,
although this results in only ca. 2–4-fold enhanced potency of
inhibitor 35 compared to its D-hPhe analog 36 for all enzymes.
Based on the approximately 50-fold improved potency of
inhibitor MI-432 (Figure 1) compared to its analog lacking the
Acknowledgements
The authors thank the beamline support staff at BESSY for their advice
during data collection.

8
A. Maiwald et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
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Declaration of interest
The authors report no conflicts of interest.
The authors acknowledge a travel grant from the Helmholtz Zentrum
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Supplementary material available online