The first potent diphenyl phosphonate KLK4 inhibitors with unexpected binding kinetics

Article abstract of DOI:10.1039/c5md00288e

KLK4 is a serine protease from the kallikrein family that is involved in cancer progression. The diphenyl phosphonate warhead is intended to bind irreversibly with serine proteases, but unexpectedly, very potent KLK4 diphenyl phosphonate inhibitors were d

Full text of DOI:10.1039/c5md00288e

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The first potent diphenyl phosphonate KLK4
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DOI: 10.1039/C5MD00288E
inhibitors with unexpected binding kinetics
Cite this: DOI: 10.1039/x0xx00000x
Jeroen Van Soom,°a Giuliana Cuzzucoli Crucitti,°a,b Rafaela Gladysz,a Pieter Van
der Veken,a Roberto Di Santo,b Ingmar Stuyver,a Victoria Buck,c Anne-Marie
Lambeir,d Viktor Magdolen,c Jurgen Joossensa and Koen Augustyns*a
Received 00th January 2012,
Accepted 00th January 2012
KLK4 is a serine protease from the kallikrein family that is involved in cancer progression.
The diphenyl phosphonate warhead is intended to bind irreversibly with serine proteases, but
unexpectedly, very potent KLK4 diphenyl phosphonate inhibitors were discovered with
reversible inhibition kinetics.
DOI: 10.1039/x0xx00000x
www.rsc.org/
examples are a uPA inhibitor reported by Joossens et al.
(compound 8b, kapp = 1x106 M-1s-1)21, an elastase inhibitor by
Winiarski et al. (compound 61, kapp = 2x106 M-1s-1)34, a
Introduction
Human tissue kallikrein 4 (KLK4) is a trypsin-like serine
protease of 24.1 kDa which belongs to a 15-member family of
closely related peptidases (kallikrein-related peptidases, or
KLKs). KLK4 is expressed as a preproprotein of 254 amino
acid (aa) residues, converted to a 228 aa proprotein, and
activated to a 224 aa mature proteinase by a metalloprotease.1
Strikingly, KLK4 is imperative for tooth development,
particularly in the formation of enamel, which also depends on
MMP-20.2,3 In adults, KLK4 is mainly expressed in prostate
epithelial cells. Even though its physiological role in the
prostate is not extensively studied, it was recently demonstrated
that KLK4 is up-regulated in prostate cancer4,5 and is related to
a poor prognosis.6 Like other KLKs, KLK4 has extracellular
hydrolytic activity, such as activating and/or degrading matrix
proteins,7 cancer associated proteins,8 proteases,9 and signalling
receptors.10 In both patho- and physiological conditions, KLK4
expression is regulated by steroid hormones in a tissue-specific
manner. The androgens influence its expression in prostate and
breast cancer cells,11,12 whereas estrogen causes expression in
endometrial and ovarian cancer cells.13 Notably, KLK4 has the
capacity to activate the urokinase-type plasminogen activator
(pro-uPA), as well as to modulate the activity of its receptor
(uPAR), both of which play a significant role in metastatic
cancers.10,14 All these findings suggest that KLK4 can find
application both as cancer biomarker,15,16 and therapeutic
target.8,12
subtilisin inhibitor by Pietrusewicz et al. (compound 43, kapp
=
1x105 M-1s-1)35, a GluC inhibitor by Burcharcka et al.
(compound 8a, kapp = 8x103 M-1s-1)36, a DPP IV inhibitor by
Belyaev et al. (compound 11e, kapp = 2x103 M-1s-1)31, and a
DPP8 inhibitor by Van der Veken et al. (compound 2e, kapp
=
4x103 M-1s-1)33. Here we describe a series of diphenyl
phosphonate compounds, as the first small molecule inhibitors
of KLK4 (Table 1). KLK4 belongs to the S1 protease family
with a preference for cleaving amide substrates following Arg >
Lys > Tyr at the P1 positions.37,38 Traditionally a peptidic tail is
added to that position to obtain more potent and selective
diphenyl phosphonate inhibitors. Nevertheless we published the
design and evaluation of very potent and selective non-peptidic
diphenyl phosphonate uPA inhibitors21 lacking a peptidic tail.
We designed these small compounds around the preferred P1
position since it drove the diphenyl phosphonate warhead most
optimally in the direction of the catalytically active serine,
followed by the formation of a covalent bond between the
phosphorous atom of the warhead and the serine alcohol of the
enzyme. The binding mechanism of the diphenyl phosphonate
warhead is explained in Figure 1. Based on the same rationale
we made a selection of Arg, Lys and Tyr diphenyl phosphonate
mimetics and evaluated them for KLK4 inhibition.20-22, 39-40, 42
This study will show that screening of a focussed library of
compounds designed for irreversible inhibition can generate
surprising results. Specific attention should be given to the
kinetic binding mechanism, even if the IC50 values show strong
binding to the target. Because selectivity issues are generally
observed with protease inhibitors, we decided to test the
compounds against a broader protease panel.
To date, a number of natural inhibitors (serine protease
inhibitors, or serpins) with physiological implication in KLK4
activity regulation are known, but only a small group of non-
natural inhibitors has been discovered..17 Among these we can
find peptides like sunflower trypsin inhibitor and its
analogues.18 Currently, no small-molecules are known as KLK4
inhibitors. Since its predominant role in the development and
Results and discussions
19
progression of prostate cancer
and chemoresistance in
ovarian cancer,11 KLK4 emerges as a suitable target for the
development of antitumor agents.
Compounds 1a-f,h and 2a-b were synthesized as previously
21, 24,
described (see Scheme 1 and 2, Supporting Information).
25, 39, 41
Compounds 1a-i and 2a-b were tested in enzymatic
Several diphenyl phosphonate compounds have been identified
as potent, irreversible serine protease inhibitors.20-36 Interesting
assays to determine their ability to inhibit KLK4 as well as 8
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 1

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other related trypsin-like serine proteases and
hydrolase.
1
serine The diphenyl phosphonate derivatives are known as irreversible
inhibitors of serine proteases, hence, we performed kinetic
Our compound evaluation approach started with the evaluation studies to determine the inhibition mechanismVieowfArtsiceleleOcntleinde
DOI: 10.1039/C5MD00288E
of enzyme inhibition at 3 inhibitor concentrations (250 µM, 2.5 compounds with significant KLK4 inhibition at concentrations
µM and 25 nM), followed by the IC50 and progress curve lower than 2.5 µM. Based on the obtained progress curves we
determinations for compounds showing more than 50% determined the type of binding kinetics. (Figure 2A) For the
inhibition at 2.5 µM. Corresponding IC50 values were calculated most interesting compounds we confirmed the type of
Figure 1. Binding mechanism of diphenyl phosphonates. The warhead is directed towards the active serine followed by a
pentacoordinate transition state. After release of one phenol a covalent bond is formed. The second phenol can be released during a so
called aging process by the influence of a water molecule
using dose-response curves and are reported in Table 1. The 4 inhibition by a dilution experiment. (Figure 2B). Unexpectedly,
Parameter Logistic nonlinear regression model was used for the progress curve of 1e shows a reversible binding type for
curve-fitting analysis (Grafit.v7 (erithacus.com)). This model KLK4. (Figure 2) In these experiments, enzyme and inhibitors
does not make any assumptions about the interaction and were incubated at 37°C upon addition of the substrate (Boc-
provides 4 parameters: a minimum asymptote, a maximum Val-Pro-Arg-AMC). The conversion of the substrate to the
asymptote, the Hill slope describing the steepness of the curve, fluorescent aminomethyl-coumarin is monitored and the results
and the inflection point at a concentration defined as the IC50. are expressed as relative fluorescence unit (RFU) against time.
All available curves and equations can be found in the A typical irreversible binder shows progress curves which show
supplementary information. Most of the tested compounds final velocities equal to zero and these curves are described by
exhibited excellent potency in inhibiting KLK4. In fact, the following equation: vt = voe-kobs*t. However, in the
compounds 1b, 1e-h and 2a-b showed IC50 values in the sub- reversible inhibition curve, (1e) the steady-state rate is different
micromolar/nanomolar concentration range (IC50 range 0.003- from zero since the enzyme is not completely blocked and
0.250 µM).
conversion of the substrate continues after the time dependent
The most potent KLK4 inhibitor within this series was inhibitor binding step. This curve is characterised by three
compound 1e, which showed an IC50 value of 3.4 nM. We phases: an initial phase that extrapolates to time =0 with slope
recently published compounds 2a and 2b which were generated v0, final phase with a slope equal to vs, and an exponential
during the validation of a MSAS approach to obtain uPA phase that connects the two linear phases with a pseudo-first
inhibitors.42 Interestingly they also show some KLK4 order rate constant of kobs. All equations used for fitting the
inhibition, although the alpha amino substitutent is not present. progress curves are described in Lambeir et al.43 The progress
This emphasizes the strength of the P1 side chain as a directing curve of 1e is consistent with a slow reversible type of
group for the diphenyl phosphonate interaction with the inhibition. Fitting the progress curve of this reaction gives a
catalytic serine of the protease.
value of kobs = koff + kon[I] and thus allows to estimate koff when
In particular, all the active compounds are characterized by the kon and [I] are known. In the experiment of Figure 2B the rate
presence of a guanidine or amidine function, as expected from of dissociation is comparable with the rate of binding at the
the substrate selectivity of the enzyme. As mentioned before, lowest inhibitor concentrations used, so there is no need to
KLK4 is able to recognize and cleave the peptide bonds of the hypothesize that a covalent adduct is formed and subsequently
substrate predominantly after an arginine residue, however it hydrolyzed over the course of hours.
also cleaves after lysine or tyrosine at the P1 position of a We have performed progress curves and dilution experiments
substrate. Compounds characterized by aniline and phenol for all compounds showing more than 50% inhibition at 2.5
moieties in the P1 position (1c, 1d, 1i) did not display potent µM. (see supplementary data). Compounds 1b, 1e, 1f, 1g, 1h,
KLK4 inhibition. The most potent KLK4 inhibitor of this series 2a and 2b all show a reversible type of binding. This study
(1e) also shows a nice selectivity. The selectivity ratio (IC50 suggests that in the case of KLK4 inhibition, the presence of the
enzyme/IC50 KLK4) is about 70 for KLK2 and KLK1 and is diphenyl phosphonate warhead combined with its optimal P1
more than 300 for the other enzymes.
fragment is not sufficient to guarantee an irreversible binding
2 | J. Name., 2012, 00, 1-3
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Table 1 Biological evaluation of KLK4 inhibitors 1a-i and 2a-b
View Article Online
DOI: 10.1039/C5MD00288E
IC50 (µM)
tPA
KLK4
Type of
binding
Cpd
R1
R2
KLK4
>2.5
KLK2
>2.5
KLK1
>10
KLK8
>10
uPA
Thrombin
>2.5†
Plasmin
>250†
FXa
AChE
>10
†
>250†
>2.5†
>250†
ND
Rev
ND
1a
NH-
Cbz
†
1b
0.84**†
44†
0.8*†
30†
>10
/
>10†
>10
0.04
>10
>2.5
>2.5
>10
0.82
>10
0.125
>10
>10
>10
NH-
Cbz
¥
>10
>10
32
>10
1c
NH-
Cbz
†
ND
Rev
Rev
>10
>10
0.24*
>10
>10
19.80
62
>10
/
1d
NH-
Cbz
¥
0.22*
0.53*
2.70†
250†
250†
4.90†
3.60†
>10†
0.0034
0.012
1.16
>10
>20
1e
NH-
Cbz
†
0.17*
0.79**
5
0.007**†
12†
2.40*†
3†
1f
NH-
Cbz
†
Rev
Rev
1g
>2.5†
>2.5†
0.196*†
>2.5†
6.20†
>2.5†
13.7†
0.041
0.015
>2.5
>2.5
1.37
>10
>20
NH-
Cbz
†
1h
0.72*
0.90**
68†
17†
NH-
Cbz
1i€
ND
>10
>2.5
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.086**
>2.5
NH-
Cbz
§
2a
2.44**
1.44*
1.40*§
H
Rev
0.25
§
3.02**
0.17*
0.0079**§
H
Rev
0.08
>2.5
>10
>10
>10
>10
>10
2b
ND: not determined because IC50 above 2.5 µM; Rev: Reversible binding; *Reversible binding, **Irreversible binding. Standard deviation
available in supplementory information. Synthesis or datapoints also reported in † ref 21, ¥ ref 25, €ref 39, § ref. 42,

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DOI: 10.1039/C5MD00288E
A
B
Figure 2. A: Progress curve of compound 1e in the presence of KLK4. Each curve represents a different concentration of compound 1e ((a) 0 µM,
(b) 3 nM, (c) 6 nM, (d) 13 nM, (e) 25 nM, (f) 50 nM, (g) 100 nM, (h) 200 nM). The curves represent a reversible binding mechanism with slow type
binding characteristics. B: This graph represents the results of a dilution experiment ( (a) No inhibitor, (b) in the presence of 1e). Compound 1e
was incubated at 300 nM with an enzyme concentration 100 times higher than within the IC50 assay. After 15 minutes the mixture was 100 times
dilutes with buffer. If compound 1e was an irreversible inhibitor the enzyme activity would be completely inhibited also after dilution (no increase
represent a good starting point to develop selective KLK4
inhibitors. Irreversibility is a desirable characteristic for
diphenyl phosphonate inhibitors and a necessary prerequisite to
develop activity-based probes to validate KLK4 as a target
and/or biomarker in cancer. Further results in this area will be
reported in due course.
mode of the inhibitor to the enzyme. In the case of the here
reported KLK4 inhibitors it can be hypothesised that upon
interaction of the compounds with enzyme’s P1 pocket, the
phosphonate esters may not be properly positioned for a
nucleophilic attack by the serine residue. We can state that the
reason should not be found in the chemical properties such as
stability or lower reactivity of the warhead, because compounds
1b, 1f, 1g, 1h, 2a and 2b show irreversible inhibition for uPA,
KLK8 or KLK2. Detailed information can be found in Table 1
and the supplementary information. Compound 1g is a
promising hit candidate but shows strong inhibition of
thrombin. This could cause side effects related to interference
with the coagulation cascade, which needs to be avoided.
Nevertheless, also for thrombin the determination of the
binding characteristics is important. Reversible or irreversible
inhibition of thrombin could lead to a different interpretation of
the safety risk. We conclude that the ideal P1 substitution is not
yet found to obtain potent, selective and irreversible KLK4
inhibitors. Alternatively, additional modifications such as
modifications of the alpha amino substituents or replacements
of the phenyl esters may be necessary to obtain selective
irreversible KLK4 binders. Experiments are currently in
progress to identify improved compounds which will be
presented later in combination with their biological results
Acknowledgements
This work was supported by the Special Fund for Research of
the University of Antwerp (BOF-UA), the Industrial Research
Fund of the University of Antwerp (IOF-UA), the Agency for
Innovation by Science and Technology (IWT) with a Strategic
Basic Research (SBO) grant (ChemProTools) and an
Innovation Mandate (Jurgen Joossens), the Hercules
Foundation Flanders with medium-scale research infrastructure
grants, the Research Foundation Flanders (FWO) with a
research project (G013513N) and by the German Research
Society (DFG, MA 1236/8-1). The Laboratories of Medicinal
Chemistry and Medical Biochemistry belong to the Department
of Pharmaceutical Sciences and are partners of the Antwerp
Drug Discovery Network (www.ADDN.be). The excellent
technical assistance of S. Lyssens and Sabine Creutzburg is
greatly appreciated. We use the academic license of Instant
JChem from ChemAxon to manage our compound data.
Notes and references
a Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences,
University of Antwerp (UA), Universiteitsplein 1, B-2610 Antwerp,
Belgium.
Conclusions
b
Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-
For the first time, we report potent small molecule KLK4
inhibitors. A set of compounds from a focused library designed
for irreversible inhibition was tested in a KLK4 inhibition
assay. From this preliminary study, five compounds (1d-h)
emerged as good inhibitors with IC50 values in the range of sub-
micromolar/nanomolar concentrations. Compound 1e is a
single digit nanomolar inhibitor of KLK4 with at least a 70-fold
Fondazione Cenci Bolognetti, “Sapienza” Università di Roma, P.le Aldo
Moro 5, Roma I-00185, Italy.
c Klinische Forschergruppe der Frauenklinik, Klinikum rechts der Isar der
TU München, Ismaninger Strasse 22, 81675 München, Germany.
d
Medical Biochemistry, Department of Pharmaceutical Sciences,
University of Antwerp (UA), Universiteitsplein 1, B-2610 Antwerp,
Belgium.
selectivity in
a panel of highly homologous proteases.
Surprisingly 1e showed reversible binding kinetics. This study
demonstrates that the type of inhibition should always be
carefully investigated, even if potent IC50 values are obtained
with compounds designed to be irreversible. These data
° Jeroen Van Soom and Giuliana Cuzzucoli Crucitti are co-first authors.

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DOI: 10.1039/C5MD00288E
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