Combinatorial tuning of peptidic drug candidates: High-affinity matriptase inhibitors through incremental structure-guided optimization

Article abstract of DOI:10.1039/c3ob27469a

Herein we report a convenient strategy for the development of novel, highly-potent peptidic inhibitors of the trypsin-like serine protease matriptase based on the monocyclic variant of the sunflower trypsin inihibitor-1 (SFTI-1[1,14]). We screened SFTI-1[1,14] variants possessing incremental modifications of the parent peptide for beneficial binding properties. This compound library comprising 6 peptides and 16 triazole-containing peptidomimetics was established via structure-guided rational design and synthesized using a divergent strategy employing copper-click chemistry. The most favorable amino acid substitutions were combined in one framework yielding potent SFTI-1-derived matriptase inhibitor-1 (SDMI-1) and the truncated dodecapeptide variant (SDMI-2) with single-digit nanomolar inhibition constants. In silico studies indicated that the improved matriptase affinity compared to the parent peptide is caused by the successful establishment of additional favorable proton donor-acceptor interactions between basic inhibitor side chains and acidic residues on the surface of the target enzyme. SDMI-1 and 2 are potent inhibitors of the pharmaceutically relevant protease matriptase at a near physiological pH and, thus, may find applications in therapy or diagnostics.

Full text of DOI:10.1039/c3ob27469a

Organic &
Biomolecular Chemistry
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Combinatorial tuning of peptidic drug candidates:
high-affinity matriptase inhibitors through incremental
structure-guided optimization†
Cite this: Org. Biomol. Chem., 2013, 11,
1848
Heiko Fittler, Olga Avrutina, Bernhard Glotzbach, Martin Empting* and
Harald Kolmar*
Herein we report a convenient strategy for the development of novel, highly-potent peptidic inhibitors
of the trypsin-like serine protease matriptase based on the monocyclic variant of the sunflower trypsin
inihibitor-1 (SFTI-1[1,14]). We screened SFTI-1[1,14] variants possessing incremental modifications of the
parent peptide for beneficial binding properties. This compound library comprising 6 peptides and 16
triazole-containing peptidomimetics was established via structure-guided rational design and synthesized
using a divergent strategy employing “copper-click” chemistry. The most favorable amino acid substi-
tutions were combined in one framework yielding potent SFTI-1-derived matriptase inhibitor-1 (SDMI-1)
and the truncated dodecapeptide variant (SDMI-2) with single-digit nanomolar inhibition constants.
In silico studies indicated that the improved matriptase affinity compared to the parent peptide is caused by
the successful establishment of additional favorable proton donor–acceptor interactions between basic
inhibitor side chains and acidic residues on the surface of the target enzyme. SDMI-1 and 2 are potent
inhibitors of the pharmaceutically relevant protease matriptase at a near physiological pH and, thus, may
find applications in therapy or diagnostics.
Received 19th September 2012,
Accepted 8th January 2013
DOI: 10.1039/c3ob27469a
www.rsc.org/obc
generated from inactive precursors through endoproteolytic
cleavage by specific TTSPs.4 Hence, they play crucial
Introduction
Trypsin is one of the most prominent digestive enzymes roles in the cellular development and maintenance of
ubiquitously found in the small intestine of vertebrates.1 Its homeostasis.3
intriguing molecular framework includes the famous triad
A well-studied example of a membrane-anchored trypsin-
Asp-His-Ser as a core feature implementing its proteolytic like serine protease with pharmaceutical relevance is
activity.2 This prototypic architecture and the ability to cleave matriptase.8–18 It is widely expressed on the surface of epi-
peptide bonds after basic residues constitute the structural thelial cells in healthy tissue where its proteolytic activity is
and functional groundwork of a whole class of biocatalysts precisely regulated by natural protease inhibitors like the hep-
referred to as trypsin-like serine proteases.3 Members of this atocyte growth factor activator inhibitor-1 and 2 (HAI-1,
enzyme family are involved in diverse biological processes and HAI-2).10,13 However, dysregulation of this physiological inhibi-
occur in soluble form or as membrane-anchored entities.4 tor–protease balance is believed to facilitate pathological pro-
Type II transmembrane serine proteases (TTSP), for instance, cesses. Indeed, a number of studies associate matriptase
are bound to the cell surface via the N-terminus and have been overexpression with the development and progression of
characterized as important mediators of the pericellular epithelial tumors, as well as osteoarthritis and
procession and activation of various effector molecules.3–7 atherosclerosis.14–18 Furthermore, Napp et al. observed pro-
Active forms of peptide hormones, growth and differentiation nounced in vivo matriptase activity in a murine orthotopic pan-
factors, receptors, enzymes, and adhesion molecules are creatic tumor model and showed that the administration of
active-site inhibitors significantly reduces proteolysis of the
substrate analyte.11 Hence, potent and selective matriptase
Clemens-Schöpf Institute of Organic Chemistry and Biochemistry, Technische
inhibitors are of great therapeutic importance, and their deve-
lopment is a challenging task. To date, a number of small syn-
thetic organic compounds as well as large antibody fragments
Universität Darmstadt, Petersenstr. 22, 64287 Darmstadt, Germany.
E-mail: M.Empting@Biochemie-TUD.de, Kolmar@Biochemie-TUD.de
†Electronic supplementary information (ESI) available: Tabulated data of ESI-MS
exhibiting single-digit nanomolar to subnanomolar inhibition
constants have been reported.19–21 Very recently, Marsault
analysis, RP-HPLC chromatograms, and plotted kinetic data of enzyme inhi-
bition assays for all compounds. See DOI: 10.1039/c3ob27469a
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and coworkers have demonstrated the applicability of peptido- elastase, cathepsin G, β-tryptase, proteinase K or kallikrein-
mimetics bearing a benzothiazole-based “serine trap” for the related peptidase.27–31 Moreover, unlike a majority of peptidic
development of very potent matriptase inhibitors.21 Reactive molecules, both bicyclic and monocyclic variants of SFTI-1
carbonyl functionality at the P1 position allowed for the esta- demonstrate
blishment of a transient covalent linkage between the ligand (35–76 h).30,32 Additionally, a latent inhibitory activity against
and the active-site serine.21,25,26
matriptase in the nanomolar range (Ki = 1–150 nM) has been
a
sufficient stability in blood serum
In this study, we present a convenient strategy for the reported for the bicyclic peptide at the pH optimum of the
development of non-covalent high-affinity matriptase inhibi- TTSP (pH ∼ 8.5–9).22,33–36 Very recently, we showed that this
tors starting from a naturally occurring peptidic framework moderate potency is essentially decreased in an environment
comprising a canonical protease inhibitor loop (Fig. 1). We with near physiological parameters (Ki = 1.1 μM at pH 7.6).22
used the rigid and well-defined tetradecapeptide framework of This is of particular importance for potential in vivo appli-
the sunflower trypsin inhibitor-1 (SFTI-1) as a starting point cations. A therapeutic/diagnostic agent must possess high
for structure-guided lead compound optimization. This mole- activity under homeostasis or – in the case of tumor as well as
cular scaffold possesses a canonical (substrate-mimicking) inflammation-induced hypoxia – more acidic conditions.37–41
loop for anti-proteolytic activity and has already been success-
Using a structure-guided incremental optimization strategy
fully used for the generation of inhibitors of chymotrypsin, we were able to generate SFTI-1 derivatives with single-digit
Fig. 1 Predicted structure of the SFTI-1[1,14] (1)–matriptase complex generated via molecular dynamics simulations followed by a two-step docking procedure.22
Blue: nitrogen, green: sulfur, red: oxygen, yellow: carbon, hydrogen is omitted for clarity. Reported X-ray/NMR structures 3P8F and 1JBN were used as initial coordi-
nates.12,23 (A) Molecular surface of the catalytic domain of matriptase in complex with 1 (sticks). Color gradients (red to blue) indicate electrostatic surface potentials
(negative to positive) at pH 7.4 calculated via the Particle Mesh Ewald method with a maximum value of 500 kJ mol−1 24
(B) Enlarged depiction of the active site.
.
Regions concealing parts of the ligand are shown transparently. Bound inhibitor 1 is highlighted by a black outline. (C) Sequence and schematic representation of
SFTI-1[1,14] (1). (D) Depiction of the active site indicating side chains of matriptase residues (cyan sticks, black outline) which have been considered within the pre-
sented approach for the structure-guided optimization of the SFTI-1 framework (sticks, black outline).
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nanomolar Ki as well as an improved trypsin/matriptase selec- the methyl ester group yielding the corresponding cystine-
tivity profile.
bridged products 5 and 6.
Unfortunately, none of the position 7 variants 1–8 showed
an improved matriptase affinity over SFTI-1[1,14] in enzyme
inhibition assays (Table 1). Thus, we focused our further experi-
ments on the optimization of positions 10 and 12.
Results and discussion
In our previous study, we reported that the monocyclic variant
SFTI-1[1,14] possesses a slightly improved inhibition constant
compared to the bicyclic wild type.22 Thus, the open-chain
derivative represents a promising starting point for the design
of novel matriptase inhibitors. A combination of molecular
dynamics simulation and docking experiments yielded an
in silico SFTI-1[1,14]-matriptase complex, very similar to the
reported wild-type inhibitor-protease X-ray structure
(Fig. 1).12,22
Using these structural data and considering suggestions
made by Yuan et al.,12 we designed a minimal compound
library 1–22 suitable for an initial search for incremental
enhancements of the lead structure (step 1, Fig. 2). In a second
step we investigated whether a combination of the individual
beneficial modifications of the SFTI-1 framework results in an
additive effect (step 2). Finally, we truncated the most promis-
ing variant to study the influence of the C-terminal residues
on matriptase affinity and selectivity (step 3).
In the ligand–receptor complex, the side chain of residue
10 of SFTI-1[1,14] is oriented towards a surface area of matrip-
tase with a pronounced negative polarization (Fig. 1). The
β-carboxylic group of the Asp96 contributes significantly to
this environment. Yuan et al. suggested installing short basic
side chain functionalities with low degrees of conformational
freedom like aminoalanine or aminohomoalanine at position
10 of the inhibitor to establish favorable electrostatic inter-
actions.12 Nevertheless, initial molecular modeling implied
that a linker of about four to five methylene units is needed to
position a basic functionality in proximity to the β-carboxylic
group of Asp96 in its native conformation present in the
crystal structure (PDB-ID: 3P8F). Thus, we designed SFTI-1
[1,14] derivatives 9–16 possessing planar and rigid triazolyl
linkers of appropriate length to investigate the possibility for
new favorable interactions between the ligand and the receptor
(Fig. 2A).
Initially, synthetic routes for 9–16 were devised as modifi-
cations of the approach used for SFTI-1[1,14] variants 1–8.
While variants 10, 12, 14, and 16 were readily accessible via
Step 1: tuning affinity of singular residues (increments)
Trypsin and matriptase share a very similar substrate spec- this strategy (Fig. 2C), compounds 9, 11, 13, and 15 could not
trum. Thus, we rationalized that substitutions within the cano- be synthesized in this manner. An attempt to assemble the
nical region of SFTI-1[1,14] (Fig. 1C) would be detrimental Aza10 analog of peptide-resin 25 on the solid support resulted
towards improved affinity. However, we included the P2′ posi- in an undefined mixture of side products (data not shown).
tion (Ile7) as a site for side-chain replacements within our Nevertheless, we were able to synthesize compounds 9, 11, 12,
molecular design (Fig. 2A). Histidines 40 and 143 of matrip- and 15 via the resin-bound pentapeptide intermediate 26 as
tase are in close proximity to this residue, therefore they might depicted in Fig. 2D.
provide the possibility for favorable hydrogen bonding inter-
actions with the ligand (Fig. 1D).
Interestingly, a significant increase of matriptase affinity
over SFTI-1[1,14] was observed for four of the eight triazolyl-
To reduce the synthetic expense and to cover an adequate containing peptidomimetics 9–16 (Table 1).
structural and functional space, we set up a divergent synthetic
The most pronounced enhancement within the set of
procedure. Azide-bearing peptidic scaffolds 23 and 24 were inhibitors 9, 10, 13, and 15 was detected for the aminomethyl-
assembled on the solid support using commercially available functionalized 1,2,3-triazole 9. The difference of the free ener-
building blocks Fmoc-L-azidoalanine (Fmoc-Aza-OH) and gies of binding/dissociation ΔBG between compound 9 and
Fmoc-L-azidohomoalanine (Fmoc-Aha-OH) (Fig. 2B). On-resin SFTI-1[1,14] was calculated as 3.9 kJ mol−1. Thus, additional
copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) with favorable electrostatic inhibitor–enzyme interactions were suc-
different alkyne components allowed for the facile installation cessfully established by furnishing the peptide with a basic
of an amine, a carboxylic acid, the corresponding methyl ester functionality at position 10. However, if length of the triazolyl
and a phenyl functionality at position 7. This combinatorial linker is increased by only one methylene unit (10), the attrac-
approach is quite similar to the “tethered fragment” strategy tive contribution is significantly reduced. Furthermore, the
previously described by Burke Jr. and coworkers.42,43 Acidolytic installation of an acidic carboxy group at this position (11, 12)
cleavage from the solid support, oxidative macrocyclization is detrimental for matriptase inhibition. Noteworthy, a signifi-
and chromatographic isolation gave SFTI-1[1,14] derivatives cant increase of inhibitor potency compared to SFTI-1[1,14]
1–8 (Fig. 2C). Interestingly, both free carboxylic acids 3 and 4 is also observed for the formally uncharged methyl ester
as well as corresponding methyl esters 5 and 6 were selectively derivative 13 although not as pronounced as in the case of the
accessible from the respective open-chain methyl ester precur- amino-functionalized variant 9.
sor peptides through the choice of macrocyclization con-
Additionally, we investigated the impact of basic canonical
ditions. The methyl carboxylate was hydrolyzed during amino acids, lysine and arginine, at position 10 (17, 18) on
disulfide bond formation at pH 8.4 giving free acids 3 and 4. matriptase affinity. Surprisingly, compound 18 equipped with
Setting the pH to 7.7, however, allowed for the preservation of a flexible aliphatic linker demonstrated the highest potency of
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Fig. 2 Design and synthesis of a small compound library comprising SFTI-1[1,14] derivatives 1–22. (A) Collection of triazolyl-containing peptides 1–16 as well as
variants 17–22 with singular substitutions at positions 10 and 12 using canonical amino acids lysine, arginine, alanine, valine, isoleucine, or histidine. (B) Synthesis of
precursors 23, 24, 25, and 26 as well as inhibitors 17–22. (C) and (D) Schematic depictions of synthetic routes yielding triazolyl-containing peptides 1–16. Con-
ditions: (a) microwave-assisted Fmoc-SPPS; (b) acidolytic cleavage from the solid support using TFA–H2O–anisole–TES (47 : 1 : 1 : 1, v : v : v : v) and dithiothreitol (DTT),
(c) air-mediated oxidative macrocyclization in 100 mM (NH4)2CO3 aq. (pH = 8.4) at 1 mg peptide per mL; (d) on-resin CuAAC using given alkyne component (5 eq.),
CuSO4·5H2O (20 mol%), sodium ascorbate (20 mol%), and N,N-diisopropylethylamine (DIEA, 8 eq.) in DMF at ambient temperature (overnight) (e) air-mediated oxi-
dative macrocyclization in 100 mM (NH4)2CO3 aq. (pH = 7.7) at 1 mg peptide per mL. All compounds 1–22 were isolated through preparative reversed-phase HPLC.
SFTI-1[1,14] variants 1–22 listed in Table 1. Due to the inherent corresponding to an increase of ΔBG by 6.5 kJ mol−1 resulted
degrees of conformational freedom of the respective amino from a singular amino acid substitution (18).
acid side chains, a pronounced entropic penalty has been
Finally, we investigated the impact of modifications at posi-
expected for compounds 17 and 18.12 Indeed, ε-amino-functio- tion 12 of the SFTI-1[1,14] framework on matriptase affinity.
nalized derivative 17 was slightly less potent than inhibitor Yuan et al. described a pronounced undesirable entropic effect
9 possessing a more rigid and restrained linker motif of on Phe12 of SFTI-1 due to conformational restraints caused by
comparable length. However, a double-digit nanomolar Ki Phe97 and Phe99 side chains forming the S4 subsite of the
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Table 1 Determined inhibition constants of compounds 1–22 against matrip-
tase at pH 7.6 and calculated differences in free energies of binding/dissociation
compared to SFTI-1[1,14]
c
Relative
activityb
ΔBG(x) − ΔBG(SFTI−1[1,14])
/
Entry
Kia/nM
703 87d
(kJ mol−1
)
SFTI-1[1,14]
1
7.0
5.4
19.8
3.4
2.3
15.4
4.8
0
Pos. 7e
1
2
3
4
5
6
7
8
4892 663
3822 494
13 886 1711
2380 291
1629 200
10 857 1349
3342 414
1252 155
148 19
−4.8 0.5
−4.2 0.4
−7.4 0.4
−3.0 0.4
−2.1 0.4
−6.8 0.4
−3.9 0.4
−1.4 0.4
3.9 0.4
1.8
Pos. 10e
9
0.21
0.73
65.8
11.5
0.30
3.2
0.79
6.8
0.33
0.072
2.3
10
11
12
13
14
15
16
17
18
19
20
21
22
513 66
0.8 0.4
46 287 5728
8096 1011
208 27
2224 277
556 70
4750 585
232 29
50.6 6.5
1614 206
580 72
−10.4 0.4
−6.1 0.4
3.0 0.4
−2.9 0.4
0.6 0.4
−4.7 0.4
2.7 0.4
6.5 0.4
−2.1 0.4
0.5 0.4
Fig. 3 Structures of triazolyl-containing SFTI-1[1,14] derivative 42, SFTI-1
derived matriptase inhibitor-1 (SDMI-1) 43 and SFTI-1 derived matriptase inhibi-
tor-2 (SDMI-2) 44.
Pos. 12e
0.83
0.45
0.29
319 40
206 27
2.0 0.4
3.0 0.5
Inhibitors 42 and 43 demonstrated a further increased
matriptase affinity compared to SFTI-1[1,14] derivatives 9, 18,
and 22 (Table 2). Our calculations suggest that the observed
improvement in ΔBG relies predominantly on an additive
effect. The determined individual favorable contributions of
compounds 18 and 22 sum up to 9.6 0.6 kJ mol−1, whereas,
the difference of free energies of binding/dissociation between
43 and SFTI-1[1,14] was calculated as 10.3 0.4 kJ mol−1. The
difference between these two values could be accounted for a
synergistic behavior.44 However, the observed effect is negli-
gible considering the respective estimated uncertainties. In
the case of inhibitor 42 even an unfavorable non-additive
portion was detected for the installed modifications as the cal-
culated increase of ΔBG was smaller than the sum of the incre-
mental improvements (Table 2).
a Determined as described in the Experimental section. b Relative
activity given as the ratio Ki of the corresponding compound/Ki of
SFTI-1[1,14]. c ΔBG(x) refers to the free energy of binding/dissociation of
compound x. ΔBG were calculated from respective Ki using ΔBG =
−RTlnKi.22,44 Errors of the given differences ΔBG(x) − ΔBG(SFTI−1[1,14])
were calculated by propagation of errors. d As published before.22 e The
modified position of the SFTI-1[1,14] framework is given.
enzyme (Fig. 1D).12 As a consequence, substitution of Phe12 by
non-natural amino acids possessing larger aromatic side
chains has a detrimental effect on bioactivity.34 Thus, we
decided to replace the native phenylalanine residue by smaller
aliphatic, hydrophobic, as well as heteroaromatic amino acids
alanine, valine, isoleucine, and histidine (Fig. 2A). Respective
compounds 19–22 were synthesized through routine micro-
wave-assisted Fmoc-SPPS followed by oxidative macrocycliz-
ation and chromatographic isolation (see ESI†).
Nevertheless, tetradecapeptide 43 which we refer to as the
SFTI-1-derived matriptase inhibitor-1 (SDMI-1) possesses a low
inhibition constant of 11 nM near the physiological pH.
Furthermore, in the enzyme assay conducted near the pH
optimum of matriptase (pH = 8.5) a single-digit nanomolar Ki
(1.1 nM) was observed.
Indeed, we observed a beneficial effect of Phe12Ile (21) as
well as Phe12His (22) substitutions on matriptase inhibition
in our in vitro assays. In particular, the imidazole functionality
at position 12 (22) facilitated an improved interaction between
the SFTI-1 framework and the surface of the enzyme of about
Step 3: truncation of most promising variant
3 kJ mol−1
.
In a very recent study we performed a structural analysis of
several monocyclic triazole-bridged SFTI-1 derivatives
in silico.22 The yielded results indicated a pronounced influ-
Step 2: combination of beneficial increment modifications
Encouraged by the enhanced matriptase affinities observed for ence of the terminal regions on matriptase affinity. An entropic
compounds 9, 18 and 22, we combined the singular modifi- penalty caused by the fixation of the Phe12 residue of the
cations of position 10 (9, 18) with the described Phe12His sub- parent bicyclic peptide through aromatic side chains of the
stitution (22). The resulting constructs 42 and 43 (Fig. 3) were enzyme has already been described and addressed in the pre-
synthesized either via microwave-assisted Fmoc-SPPS (42) or by sented work.12 However, additional conformational constraints
using a strategy analogous to that described for compounds of residues Pro13 and Asp14 forming the secondary loop may
9–16 (42) (compare Fig. 2D).
cause unfavourable entropic contributions upon binding to
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Table 2 Determined inhibition constants of compounds 42–44 against matriptase at pH 7.6 (8.5), calculated differences in free energies of binding/dissociation
compared to SFTI-1[1,14] as well as trypsin affinity and selectivity at pH 7.6a
Relative
activity
ΔBG(x) − ΔBG(SFTI−1[1,14])
/
Sum of increment
Entry
Matriptase Kib/nM
(kJ mol−1
)
contributionsc/kJ mol−1
Trypsin Ki/nM
Selectivityd
SFTI-1[1,14]
42
43 (SDMI-1)
44 (SDMI-2)
703 87e
89.5 11.1
11.0 1.4 (1.1 0.3)
6.2 1.2 (2.3 0.8)
1
0
n.a.
0.21 0.03e
9.2 2.1
11.2 2.0
38.0 11.1
0.0003
0.1
1.0
0.13
0.016
0.009
5.1 0.4
10.3 0.4
11.7 0.6
6.9 0.6
9.6 0.6
n.a.
6.1
a Experiments were performed as described in the footnote of Table 1 and in the Experimental section. b Values in brackets correspond to Ki
determined at pH 8.5. c Errors were calculated by propagation of errors. d Calculated as the ratio of Ki against matriptase (pH 7.6)/Ki against
trypsin (pH 7.6). e As published before.22
matriptase. According to our calculations interaction with
trypsin is not affected in this manner. As a consequence, the
truncation of the two C-terminal residues might have a bene-
ficial effect on matriptase inhibition. Additionally, we ration-
alized that removing the negative charge of the terminal
carboxy group by introduction of a C-terminal amide might
enhance the overall charge complementarity between the
target enzyme and the inhibitor.
Inspired by these considerations, we synthesized the mono-
cyclic dodecapeptide 44 as described in the Experimental
section (Fig. 3). Indeed, we observed an additional minor
improvement of inhibitor potency resulting in a Ki of 6.2
1.2 nM. This SFTI-1-derived matriptase inhibitor-2 (SDMI-2)
exhibits a 2.3
0.8 nM inhibition constant at pH 8.5 and
shows an improved selectivity towards matriptase over trypsin
(6-fold). To our knowledge, 43 and 44 are to date the most
potent Bowmann–Birk matriptase inhibitors described.
Finally, we modeled the inhibitor–protease complex for
SDMI-2 (44) based on the in silico coordinates of SFTI-1[1,14]
and matriptase described above (see Fig. 1).22 First, the amino
acid exchanges Ile10Arg and Phe12His were introduced into
the parent model. Then, residues Pro13 and Asp14 were
replaced by a C-terminal amide. Optimization of side-chain
geometry and subsequent energy minimization yielded a struc-
ture quite similar to the corresponding SFTI-1 co-crystal (3P8F,
Fig. 4A). Nevertheless, additional favorable interactions
between 44 and matriptase were observed (Fig. 4B). The side
chain of Asp96 was reoriented to form a bidentate hydrogen
bond with the guanidinyl functionality of Arg10. Furthermore,
the possibility for
a proton donor–acceptor interaction
between the backbone carbonyl oxygen of Asp96 and the ε2-
nitrogen of His12 was detected. However, an X-ray structure of
the SDMI-2–matriptase complex is needed to undoubtedly
prove whether the predicted in silico coordinates are valid.
Yuan et al. suggested to use short basic residues like amino-
Fig. 4 (A) Predicted structure of SFTI-1 derived matriptase inhibitor-2 (SDMI-2)
44 (thick colored sticks) upon binding to matriptase (surface). The SFTI-1-matrip-
tase X-ray structure (3P8F) is shown as thin white sticks and the calculated
peptide backbone root mean square deviation (RMSD) is given for both com-
plexes. The angle of view for the picture below is indicated. (B) Close-up of the
homoalanine at position 10 to establish an effective electrostatic predicted inhibitor 44–enzyme complex. Important matriptase residues are
depicted as transparent white sticks with black outlines and putative hydrogen
interaction with Asp96 while attenuating entropic penalties
bond interactions are shown as dashed yellow lines. Blue: nitrogen, green:
arising from extended flexible linkers.12 However, it remains to
sulfur, red: oxygen, yellow: carbon, hydrogen is omitted for clarity. Color gradi-
be tested whether such a short side chain is sufficient to posi-
ents (red to blue) indicate electrostatic surface potentials (negative to positive)
tion a basic functionality in proximity to the carboxylic group
of Asp96.
at pH 7.6 calculated via the Particle Mesh Ewald method with a maximum value
of 500 kJ mol−1 24
.
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Analytical and semi-preparative RP-HPLC were performed
on a Varian 920-LC system and a Varian 940-LC system,
Conclusion
In this study we present a convenient strategy for the develop- respectively. A Phenomenex Hypersil 5u BDS C18 LC column
ment of novel, highly potent inhibitors of matriptase based on (150 × 4.6 mm, 5 μm, 130 Å) and a Phenomenex Luna 5u C18
the SFTI-1 framework. Using a divergent synthetic route we LC column (250 × 12.20 mm, 5 μm, 100 Å) were used as
screened 22 compounds for beneficial modifications of the stationary phases. The eluent system consisted of eluent A
parent peptide. Combination of the most favorable amino acid (0.1% aq. TFA) and eluent B (90% aq. acetonitrile containing
substitutions and subsequent C-terminal truncation yielded 0.1% TFA).
SFTI-1 derived matriptase inhibitors-1 and 2 (SDMI-1 and 2)
ESI mass spectra were recorded with a Shimadzu
with inhibition constants in the low nanomolar to single-digit LCMS-2020 using a Phenomenex Jupiter 5u C4 LC column
nanomolar range. The structure-guided design of the initial (50 × 1 mm, 5 μm, 300 Å) as well as a binary eluent system
peptide/peptidomimetic library as well as the shortening of consisting of eluent A (0.1% aq. formic acid, LC-MS grade) and
the amino acid sequence were inspired by in silico experiments eluent B (100% acetonitrile containing 0.1% formic acid,
which were used as an idea generator towards beneficial modi- LC-MS grade).
fications of the wild-type compound.12,22 Thus, an inhibitor
NMR spectra were recorded in DMSO-d6 on a Bruker DRX
possessing only twelve residues as well as an inverted trypsin– 500 instrument (500 MHz) using a Shigemi 5 mm symmetrical
matriptase selectivity in favor of the latter enzyme was NMR microtube (magnetic susceptibility matched to DMSO).
developed.
Peptides were synthesized using a Liberty 12-channel auto-
Although the applied synthetic strategy using “copper-click” mated peptide synthesizer on a Discover SPS microwave
chemistry was applicable for the assembly of the majority of peptide synthesizer platform (CEM) following the Fmoc
the triazolyl-containing peptidomimetics 1–16, it was incom- strategy.
patible with the use of non-natural amino acid azidoalanine at
position 10 (compounds 9, 11, 13, and 15). In our previous
works, we did not observe significant limitations using Fmoc-
Chemistry
Aza-OH as a building block for microwave assisted Fmoc-
General procedures of peptide synthesis. All peptides
SPPS.45 However, the unexpected restricted applicability of the bearing a C-terminal carboxy acid were assembled on a Tenta-
unprotected azide functionality under the described con- Gel S AC resin (Rapp Polymere) or a 2-chlorotrityl chloride
ditions was circumvented through a modified synthetic route.
resin (Iris Biotech) preloaded with Fmoc-L-Asp(tBu). Corres-
Novel compounds 43 and 44 are potent inhibitors of ponding peptide amides were assembled on an AmphiSpheres
matriptase in a pH range near the physiological one and, thus, 40 RAM resin (Agilent).
may find applications in therapy or diagnostics. Notably, the
Loading of 2-chlorotrityl chloride resin was performed
activity of these SFTI-1 derivatives composed exclusively of manually as follows. A solution of 103 mg Fmoc-L-Asp(tBu)-OH
natural amino acids is not in the range of recently reported (0.25 mmol) and 34 μL N,N-diisopropylethylamine (DIEA,
peptidomimetics bearing reactive carbonyl functionalities.21 1 mmol) in a minimal amount of dichloromethane (DCM) was
Since only two residues have been furnished with new side- added to the resin. The resulting mixture was shaken for 2 h at
chain functionalities, the SFTI-1 framework provides further ambient temperature. The solution was removed by filtration
potential for the establishment of unique attractive inter- and the loaded resin was washed with DCM–methanol–DIEA
actions with matriptase and, therefore, improvement of affinity (17 : 2 : 1; 3×), DCM (3×), dimethylformamide (DMF, 3×) and
and selectivity. Additionally, our ongoing studies show that 43 DCM (3×).
and 44 can serve as versatile platforms for conjugation with
Canonical amino acids were attached by double or triple
reporter molecules without sacrificing bioactivity. This strategy coupling using 4 equiv. of the corresponding amino acid,
would allow for in vivo imaging experiments using for example 3.9 equiv. of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronoium
PET/SPECT or NIRF methodologies.46–49
hexafluorophosphate (HBTU) and 8 equiv. of DIEA, or, in the
Nevertheless, further selectivity tests using an extended set case of cysteine, 3–4 equiv. of 2,4,6-trimethylpyridine (colli-
of relevant trypsin-like proteases as well as characterization of dine). Arginine and cysteine were coupled using a two-step
these two peptidic inhibitors in vivo are issues that have to be microwave program: 1. RT, 0 W, 25 min; 2. 75 °C, 25 W,
addressed in the future.
0.5 min (Arg) and 1. RT, 0 W, 2 min; 2. 50 °C, 25 W, 4 min
(Cys), respectively. All other amino acids were coupled using a
standard microwave program: 75 °C, 21 W, 5 min.
Non-natural azide-bearing building blocks Fmoc-L-Aza-OH
and Fmoc-L-Aha-OH were attached via double coupling using
2 equiv. of the corresponding amino acid, 1.9 equiv. HATU, and
4 equiv. DIEA and a two-step microwave program (1. 60 °C, 30 W,
Experimental
General
All chemicals and solvents were purchased from Bachem, Iris 45 min, 2. 75 °C, 20 W, 5 min).
Biotech, Novabiochem, Sigma-Aldrich, Rapp Polymere, Roth or
Varian (Agilent).
Fmoc deprotection was achieved in two steps by reaction
with 20% piperidine in DMF at 75 °C, 42 W for 0.5 min (initial
1854 | Org. Biomol. Chem., 2013, 11, 1848–1857
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Paper
deprotection) followed by a second deprotection step with 20% copper(II) sulfate pentahydrate (CuSO4·5H2O), 1 eq. sodium
piperidine in DMF at 75 °C, 42 W for 3 min. ascorbate (NaAsc) and 8 eq. DIEA in 5 mL argon-flushed DMF
Cleavage of peptides from the solid support and removal of was added to the peptide resin and shaken at ambient temp-
side-chain protecting groups were achieved via acidolysis using erature overnight. Then, the solution was removed by filtration
a standard cleavage cocktail consisting of trifluoroacetic acid and the peptide resin was washed with methanol (3×), 0.5%
(TFA)–H2O–anisole–triethylsilane (TES) (47 : 1 : 1 : 1, v : v : v : v) sodium diethyldithiocarbamate in DMF (w/v, 3×), DMF (3×)
and DTT to suppress unwanted oxidation. The resulting reac- and DCM (3×) yielding intermediates 36–38.
tion mixture was shaken for 3 h at RT followed by precipitation
and subsequent washing (3×) with methyl tertiary butyl ether described procedure. The remaining N-terminal residues were
(MTBE) to yield crude linear peptides. attached to each triazolyl-containing peptide resin yielding
Air-mediated oxidative macrocyclization of the crude pep- intermediates 39–41. After Fmoc deprotection, acidolytic clea-
tides was conducted in 100 mM (NH4)2CO3 aq. (1 mg mL−1
vage from the solid support, ether precipitation, and oxidative
The Fmoc protecting group was removed according to the
,
pH = 8.4) and monitored by analytical RP-HPLC. After disulfide formation, macrocyclic peptides were isolated via
complete conversion (1–7 days) the solvent was removed by semi-preparative RP-HPLC. This gave compounds 9, 11, 13,
freeze-drying to yield the crude peptides. To suppress and 15 as white solids in the following yields. 9: 16.8 mg
unwanted saponification of the methyl ester of compounds 5, (21.2%); 11: 6.6 mg (8.2%); 13: 4.1 mg (5.1%); 15: 4.2 mg
6, 13, and 14, a 100 mM (NH4)2CO3 aq. buffer (pH 7.7) was (5.1%). For analytical data, please refer to the ESI.†
used (Fig. 2).
Triazolyl-containing peptide 42. Inhibitor 42 was syn-
Triazolyl-containing peptides 1–8, 10, 12, 14, and 16. Azide- thesized following a strategy similar to that described for
bearing peptide resins 23, 24, and 25 were assembled on a compounds 9, 11, 13, and 15. First, peptide-resin intermediate
TentaGel S AC-Asp(t-Bu) Fmoc resin (loading: 0.21 mmol g−1
)
Fmoc-Aza-Cys(Trt)-His(Trt)-Pro-Asp(tBu)-resin was assembled
according to the described procedures on a 0.25 mmol scale, on a 2-chlorotrityl chloride resin preloaded with Fmoc-Asp
washed (3× with DCM and 3× with ether) and dried in an (tBu) (loading: 0.23 mmol g−1) on a 0.05 mmol scale. Then, a
exsiccator.
solution of 39 mg N-Boc-propargylamine (0.25 mmol), 14 mg
On-support copper(I)-catalyzed azide–alkyne cycloaddition CuSO4·5H2O (0.05 mmol), 10 mg NaAsc (0.05 mmol)
(CuAAC) was conducted with 0.05 mmol of intermediates 23, and 70 μL DIEA (0.4 mmol) in 5 mL argon-flushed DMF
24, and 25 and the respective alkyne component (propargyl- was added to the peptide resin and shaken at ambient
amine, methyl propiolate, or phenylacetylene) according to the temperature overnight. The solution was removed by filtration
scheme laid out in Fig. 2C. A solution of 5 equiv. alkyne, and the peptide resin was washed with methanol (3×), 0.5%
1 equiv. copper(II) sulfate pentahydrate (CuSO4·5H2O), 1 equiv. sodium diethyldithiocarbamate in DMF (w/v, 3×), DMF (3×)
sodium ascorbate (NaAsc) and 8 equiv. DIEA in 5 mL argon- and DCM (3×).
flushed DMF was added to the peptide resin and shaken at
The Fmoc protecting group was removed according to the
ambient temperature overnight. Then, the solution was described procedure. The remaining N-terminal residues were
removed by filtration and the peptide resin was washed with attached to the triazolyl-containing peptide resin. After Fmoc
methanol (3×), 0.5% sodium diethyldithiocarbamate in DMF deprotection, acidolytic cleavage from the solid support, ether
(w/v, 3×), DMF (3×) and DCM (3×) yielding intermediates precipitation, and oxidative disulfide formation, macrocyclic
27–35.
peptide 42 was isolated via semi-preparative RP-HPLC. This
After Fmoc deprotection, acidolytic cleavage from the solid gave 0.9 mg (1.2%) of pure 42 as a white solid. For analytical
support, ether precipitation, and oxidative disulfide formation, data, please refer to the ESI.†
macrocyclic peptides were isolated via semi-preparative
Peptides 17–22, and 43. Linear precursors were synthesized
RP-HPLC. This gave compounds 1–8, 10, 12, 14, and 16 as on a 2-chlorotrityl chloride resin preloaded with Fmoc-Asp-
white solids in the following yields. 1: 13.5 mg (17%); 2: (tBu) (loading: 0.23 mmol g−1) on a 0.05 mmol scale.
14.9 mg (18.6%); 3: 9.3 mg (11.6%); 4: 6.3 mg (7.8%); 5: 2.4 mg
After acidolytic cleavage from the solid support, ether
(3%); 6: 2 mg (2.5%); 7: 0.5 mg (0.6%); 8: 5.1 mg (6.2%); 10: precipitation, and oxidative disulfide formation, macrocyclic
4.6 mg (5.8%); 12: 4 mg (5%); 14: 4 mg (4.9%); 16: 2 mg peptides were isolated via semi-preparative RP-HPLC. This
(2.4%). For analytical data, please refer to the ESI.†
gave compounds 17–22 and 43 as white solids in the following
Triazolyl-containing peptides 9, 11, 13, and 15. Azide- yields. 17: 3 mg (3.9%); 18: 6.2 mg (7.9%); 19: 17.4 mg (23.9%);
bearing peptide resin 26 was assembled on a TentaGel S 20: 11.2 mg (15.1%); 21: 23.2 mg (31%); 22: 9.2 mg (12.1%);
AC-Asp(t-Bu) Fmoc resin (loading: 0.21 mmol g−1) according to 43: 0.9 mg (1.2%). For analytical data, please refer to the ESI.†
the described procedures on a 0.25 mmol scale, washed (3×
with DCM and 3× with ether) and dried in an exsiccator.
Inhibitor 44. The linear precursor of 44 was synthesized on
an AmphiSpheres 40 RAM resin (0.44 mmol g−1) on a
On-support copper(I)-catalyzed azide–alkyne cycloaddition 0.1 mmol scale. After acidolytic cleavage from the solid
(CuAAC) was conducted with 0.05 mmol of intermediates 26 support, ether precipitation, and oxidative disulfide formation,
and the respective alkyne component (N-Boc-propargylamine, the macrocyclic peptide was isolated via semi-preparative
methyl propiolate, or phenylacetylene) according to the RP-HPLC. This gave 10.5 mg (7.8%) of 44 as a white solid. For
scheme laid out in Fig. 2D. A solution of 5 eq. alkyne, 1 eq. analytical data, please refer to the ESI.†
This journal is © The Royal Society of Chemistry 2013
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Paper
Organic & Biomolecular Chemistry
In vitro assays
variant. Residues Pro13 and Asp14 were deleted and the
C-terminal amide functionality was installed. Then, Arg10 and
His12 side-chain geometries were optimized using a rotamer
library and steepest decent minimization.53,54 A global energy
minimization with an increased scaling factor for non-bonded
interactions (1.2 instead of 1.0) was performed. Finally, a
second energy minimization was performed with a reset
scaling factor for non-bonded interactions (1.0) to yield the
SDMI-2–matriptase complex depicted in Fig. 4.
Enzymes. Recombinant production, autocatalytic activation
and purification of matriptase were conducted as previously
described.22 Bovine trypsin was purchased from Sigma-
Aldrich. Active-site titration and determination of Michaelis–
Menten constants KM for the chromogenic substrate Boc-QAR-
pNA were performed as reported earlier.22
Data collection and analysis. All experiments were per-
formed in triplicate. Active-site titrated matriptase (final con-
centration: [E] = 0.9 nM) or trypsin (final concentration: [E] =
0.5 nM) were incubated with the respective inhibitor in
different concentrations [I] at pH 7.6 or 8.5 for 30 min. Then, Acknowledgements
the chromogenic substrate Boc-QAR-pNA (final concentration:
This work was supported by the Deutsche Forschungs-
gemeinschaft through grant Ko 1390/9-1, by BMBF, and LOE-
WE-Soft Control.
[S] = 250 μM) was added. The residual proteolytic activity (ν/ν0)
was determined by monitoring the absorption of the corres-
ponding samples in 96-well plates (NUNC, round bottom,
clear) at 405 nm in 60 s intervals over 30 min at RT using the
Tecan GENios microplate reader. Slopes of the initial reaction
velocities (steady-state) were calculated and normalized to the References
data of the uninhibited enzymatic hydrolysis of Boc-QAR-pNA.
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were fitted either by eqn (1) or in the case of tight-binding
inhibition (trypsin: 42; matriptase: 43 at pH 8.5, 44 at pH 7.6
and 8.5) by eqn (2) through non-linear regression.
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ꢀ
ꢁꢁ1
ν
½Iꢀ
¼
1 þ
ð1Þ
ν0
IC50
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢂ
ꢃ
ꢂ
ꢃ
2
½Eꢀ þ ½Iꢀ þ Kiapp
ꢁ
½Eꢀ þ ½Iꢀ þ Kiapp ꢁ4½Eꢀ½Iꢀ
ν
¼ 1 ꢁ
ð2Þ
6 J. D. Hooper, J. A. Clements, J. P. Quigley and T. M. Antalis,
J. Biol. Chem., 2001, 276, 857–860.
ν0
2½Eꢀ
Substrate-independent inhibition constants Ki were calcu-
lated from respective IC50 values or apparent (substrate-depen-
dent) inhibition constants Kai pp using eqn (3) and (4),
respectively.
7 S. Netzel-Arnett, J. D. Hooper, R. Szabo, E. L. Madison,
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ꢀ
ꢁꢁ1
ꢁꢁ1
½Sꢀ
Ki ¼ IC50 1 þ
ð3Þ
ð4Þ
KM
ꢀ
½Sꢀ
Ki ¼ Kapp 1 þ
i
KM
Errors were calculated through propagation of errors
(compare Tischler et al.).50
In silico procedures
General. All in silico experiments were performed on an
Intel® Core™ i7–2600 workstation with 8 virtual cores using 12 C. Yuan, L. Q. Chen, E. J. Meehan, N. Daly, D. J. Craik,
the YASARA structure package (YASARA Biosciences) and the M. D. Huang and J. C. Ngo, BMC Struct. Biol., 2011, 11.
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M. S. Lee, M. D. Johnson, R. B. Dickson and C. Y. Lin,
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matriptase complex (PDB ID: 3P8F) has been recently
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T. Murdock, B. Burke, B. S. Nielsen, J. S. Gutkind and
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