The development of first Staphylococcus aureus SplB protease inhibitors: Phosphonic analogues of glutamine

Article abstract of DOI:10.1016/j.bmcl.2012.07.011

Produced by Staphylococcus aureus, SplB belongs to the chymotrypsin-like serine protease family. Since the biological role of SplB protease is unknown, the design and application of its specific inhibitors may help to reveal the function of this enzyme. Until now no SplB inhibitors have been reported. Herein, we present the design and synthesis of novel α-aminophosphonic analogues of glutamine, as well as their peptidyl derivatives. The inhibitory effects of these compounds towards the newly discovered SplB serine protease from S. aureus are characterized. We have also investigated the influence of aromatic ester substituents on inhibitory potency towards SplB. One of the compounds - Cbz-Glu-Leu-Gln^P(OC₆H₄-4-O-CH ₃)₂ - displayed an apparent second-order inhibition rate value of 1400 M^-1 s^-1.

Full text of DOI:10.1016/j.bmcl.2012.07.011

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5574–5578
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The development of first Staphylococcus aureus SplB protease inhibitors:
Phosphonic analogues of glutamine
a
a
a
b,c
b
_
´
Ewa Burchacka , Maciej Walczak, M. , Marcin Sienczyk , Grzegorz Dubin , Michał Zdzalik ,
Jan Potempa b, Józef Oleksyszyn a,
Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspian´skiego 27, 50-370 Wrocław, Poland
b Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland
c Malopolska Centre of Biotechnology, Gronostajowa 7a, 30-387 Krakow, Poland
⇑
a
_
a r t i c l e i n f o
a b s t r a c t
Article history:
Produced by Staphylococcus aureus, SplB belongs to the chymotrypsin-like serine protease family. Since
the biological role of SplB protease is unknown, the design and application of its specific inhibitors
may help to reveal the function of this enzyme. Until now no SplB inhibitors have been reported. Herein,
Received 24 May 2012
Revised 29 June 2012
Accepted 4 July 2012
Available online 13 July 2012
we present the design and synthesis of novel a-aminophosphonic analogues of glutamine, as well as their
peptidyl derivatives. The inhibitory effects of these compounds towards the newly discovered SplB serine
protease from S. aureus are characterized. We have also investigated the influence of aromatic ester sub-
stituents on inhibitory potency towards SplB. One of the compounds—Cbz-Glu-Leu-GlnP(OC6H4-4-O-
Keywords:
SplB protease
Staphylococcus aureus
CH3)2—displayed an apparent second-order inhibition rate value of 1400 Mꢀ1 sꢀ1
.
Ó 2012 Elsevier Ltd. All rights reserved.
a-Aminoalkylphosphonates
Serine protease inhibitors
Staphylococcus aureus is a Gram-positive spherical bacterium
that forms microscopic clusters resembling grapes.1 This pathogen
is characterized by a worldwide spread and is responsible for many
types of diseases including pneumonia, meningitis, osteomyelitis,
endocarditis, toxic shock syndrome (TSS), bacteremia, and sepsis.2
At the same time, S. aureus asymptomatically colonizes the skin
and mucous membranes of more than 30% of the human popula-
tion, as well as skin and mucous membranes of animals.3 Due to
the increasing number of antibiotic-resistant Staphylococcus aureus
strains, new strategies to treat staphylococcal infections need to be
developed. One possible approach focuses on the application of
synthetic inhibitors that block key bacterial proteases and may
thus possibly overcome S. aureus infection.
Currently, genomes of several dozen different Staphylococcus
aureus strains are available in publically accessible databases.4
Analysis of this data demonstrates that some of the mobile genetic
elements, including the pathogenicity islands (PAIs), encode sev-
eral enterotoxins and other virulence factors. Among them a clus-
ter of up to six genes organized into one operon encoding serine
protease-like (Spl) proteins has been discovered.5,6 The sequences
of Spl proteins reveal their high homology to the S. aureus V8 pro-
tease and epidermolytic toxins.5,7 This structural similarity to
known staphylococcal virulence factors, clustering of the spl genes
with genes encoding superantigens, and their location on PAI
suggest that Spl proteases may contribute to S. aureus pathogenic-
ity.8 The first report on Spl proteins was provided by Reineck et al.9
They discovered a high level of antibodies directed against the SplC
protein produced by S. aureus in patients with endocarditis.
Although the existence of Spl proteases was demonstrated more
than a decade ago, their biological function is still unclear.
The SplB protease belongs to a chymotrypsin-like serine prote-
ase family.7,8,10 The crystal structure of SplB protease reveals the
presence of the Ser157, His39 and Asp77 catalytic triad in the ac-
tive site of the protein.8 Substrate mapping of SplB specificity using
positional scanning synthetic combinatorial libraries revealed a
narrow P1 residue selectivity towards Asp, Asn or Gln accommo-
dated by the protease S1 binding pocket.7 Interestingly, the side
chain of the glutamic acid residue was not accepted at this posi-
tion. Application of the CLiPS (cellular library of peptide substrates)
method revealed Trp-Glu-Leu-Gln (P4-P1) as the optimal sequence
recognized by the SplB protease.10
Since the role of the SplB protease for bacterial growth and the
progression of infection is unknown, the application of specific and
potent inhibitors may help to discover its function in vivo. More-
over, SplB inhibitors will allow the detailed characterization of
the protease binding site (specificity determinants) by solving
the crystal structure of the enzyme-inhibitor complex. Such infor-
mation may be further used to determine potential natural sub-
strates of SplB in the host organism.
An effective SplB inhibitor should be highly specific and stable
under experimental conditions. One of the choices are the peptidyl
⇑
Corresponding author. Tel.: +48 71 320 4027; fax: +48 71 320 2427.
E-mail address: jozef.oleksyszyn@pwr.wroc.pl (J. Oleksyszyn).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.011

E. Burchacka et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5574–5578
5575
derivatives of
a-aminophosphonate diaryl esters. They are highly
binding pocket, but also extended interactions within the S2 and
S3 pockets.11–13,17
specific towards the target serine protease and do not react
with acetylcholinesterase, cysteine proteases, or threonine prote-
ases of proteasome, and do not inhibit metallo and aspartyl prote-
ases.11–13 The design of such inactivators of serine proteases is
straightforward—the replacement of the C-terminal amino acid of
the substrate of best fit by a structurally corresponding diaryl
aminophosphonate analogue leads to generation of a potent inhib-
itor.13,14 The best substrate is the one with the highest kcat/KM sec-
ond order hydrolysis rate of the peptide bond, where the Km value
reflects only initial noncovalent binding of the substrate in the
ground state. The initial step of inhibitor binding, the formation
of a noncovalent reversible complex, is very likely similar to the
binding of the substrate. In the next step, an irreversible reaction
at the active site catalytic serine side chain nucleophile occurs.
One of the aryl ester groups leaves the phosphorus phosphonate
atom through nucleophilic substitution with a formation of the tri-
gonal bipyramid transition state, in which the leaving group occu-
pies the apical position in relation to the attacking serine
hydroxylate. The resulting mixed diester undergoes hydrolysis
(in a process called ‘complex aging’) and a final tetrahedral mono-
ester (phosphonate-O-serine) complex is formed. Such a complex
resembles the transition state tetrahedral intermediate observed
during peptide bond hydrolysis by serine proteases. Therefore, a
complex of phosphonate-based inhibitors with serine protease is
extremely stable, with a half-life of hydrolysis in the range of a
few hours to a few days.12,13 The second order inhibition rate con-
stant is the function of the initial noncovalent binding (Ki) and the
rate of the irreversible step (k2). Although its relation to Ki is evi-
dent and substrate mapping provides sufficient data for the inhib-
itors design, the influence of k2 is more complex and depends on
phosphorus atom electrophilicity. The Ki of phosphonate type
inhibitors are not identical to the substrate KM due to the fact that
they are structurally different. The irreversible step (k2) represents
the energy of activation for the phosphonylation reaction of serine-
Oꢀ or stabilization of TS (transition state) for such a reaction—
trigonal bipyramid. Such stabilization is provided by interaction
with enzyme and by the electrophilicity of the phosphorus atom
of the phosphonate molecule. The phosphorus atom of the inhibi-
tor should possess some basic electrophilicity, since phosphonic
dialkyl esters are devoid of reactivity with serine proteases.11–13
The introduction of some electrowithdrawing substituents within
the phenyl ester ring, such as NO2, SO2CH3, COOR, significantly in-
creases the electrophilic character of the phosphorus leading to ex-
tremely potent irreversible inhibitors of serine proteases.15,16
However, increased electrophilicity results in decreased stability
and higher susceptibility to water hydrolysis, which excludes them
from practical application.
Herein, we present the first synthetic procedure for the genera-
tion of 1-aminophosphonate diaryl esters—phosphonic analogues
of glutamine and their peptidyl derivatives; structures which were
based on the optimal SplB substrate sequence (Glu-Leu-Gln/P3-P2-
P1). In addition to our synthetic approach, we determined the
inhibitory activity of these novel compounds against the SplB pro-
tease, including simple Cbz-protected phosphonates as well as
their peptidyl derivatives.
The rate of SplB protease (100 nM, expressed and purified as
previously described8) inhibition was measured in 0.1 M Tris–
HCl, 0.01% Triton X-100 buffer (pH 7.6) at 37 °C. The inhibition of
chymotrypsin (3 nM, Sigma–Aldrich, Poznan, Poland) and subtili-
sin (5 nM, Sigma–Aldrich, Poznan, Poland) was me assured in
0.1 M HEPES, 0.5 M NaCl, 0.03% Triton X-100 buffer (pH 7.5) at
37 °C. The enzymes were assayed using fluorogenic substrates:
Ac-Trp-Glu-Leu-Gln-ACC (10
lM, SplB, Ex. 355 nm, Em. 460 nm)
and Suc-Ala-Ala-Pro-Phe-AMC (5
lM, chymotrypsin and subtilisin,
Ex. 350 nm, Em. 460, Calbiochem, Merck, Warszawa, Poland). The
calculated Michaelis constant (KM) values for SplB, chymotrypsin
and subtilisin were 135 lM, 70 lM, and 60 lM, respectively.
The kinetics of SplB protease inhibition was determined by
addition of the enzyme into the solution of the substrate and inhib-
itor tested ([E]0 << [I]0) according to the following mechanism:
k2
E þ I ꢀ EI ! Ei
ð1Þ
Ki
where Ki is the reversible enzyme-inhibitor complex (EI) dissocia-
tion constant, and k2 is the rate of irreversible complex (Ei) forma-
tion. The observed rate of inhibition was determined by the
progress curve method using the following (Eq. 2):
lnð½Pꢁ1 ꢀ ½PꢁtÞ ¼ ln½Pꢁ1 ꢀ kobs
t
ð2Þ
where [P]1 is the product concentration at the end of the reaction,
[P]t is the product concentration at time t and kobs is a pseudo-first-
order rate constant.18 Control curves in the absence of the inhibitor
were linear at the evaluated conditions. For derivatives showing an
inhibitory activity of less than 50% at 200
lM concentration we pre-
sumed a k2/Ki value <10 Mꢀ1 sꢀ1. For inhibitors displaying a mini-
mum 50% activity at 200 lM concentration kinetic parameters
(kobs, Ki and k2/Ki) were examined according to the (Eq. 3)18
:
k2
Ki
kobs
½Iꢁ
¼
ð3Þ
½Sꢁ
½Iꢁ
1 þ K þ K
m
i
All measurements were performed using a Spectra Max Gemini
XPS spectrofluorometer (Molecular Devices, USA) at 37 °C.
The synthesis of phosphonic diaryl ester analogues of
glutamine (Scheme 1) started with the preparation of a 4-oxo-
N-tritylbutanamide. Briefly, the succinic anhydride (1) was
suspended in methanol and was refluxed for 3 h until a single spot
appeared on TLC.19 After the reaction was completed the volatile
products were evaporated. The resulting mono-methyl hydrogen
succinate (2) was used directly in the next step without further
purification. Coupling of triphenylmethylamine with mono-methyl
hydrogen succinate was performed in acetonitrile using HBTU as
the coupling agent in the presence of triethylamine leading to
the methyl 4-oxo-4-(tritylamino)butanoate (3). The reduction of
an ester group of 3 was achieved by the application of lithium
borohydride leading to 4-hydroxy-N-tritylbutanamide (4). Further
oxidation of 4-hydroxy-N-tritylbutyryloamid was performed
under Swern conditions leading to 4-oxo-N-tritylbutanamide (5).
Compound 5 was used as the starting material for the synthesis
of all 1-aminoalkylphosphonate diaryl esters presented in this
study.
Although the influence of the electrowithdrawing character on
the increased overall inhibition could be clearly noticed,15,16 the
electrodonating substituents could, in some cases, induce the per-
fect fitting of the phosphoryl oxygen to the oxyanion hole and in-
crease the electrophilicity of the phosphorous atom by a ‘steric’
and not ‘electronic’ effect. In another words, the substituent on
the phenyl ester ring could stabilize TS for the phosphonylation
reaction and increase k2/Ki despite its electrodonating character.
Distinguishing the effect of each of these two factors is difficult
and its understanding requires further studies.
Additionally, when the chemical stability of the 1-amin-
oalkylphosphonate diaryl esters and their outstanding selectivity
of action, together with the stability of enzyme-inhibitor complex,
are taken into consideration, it is clear that these compounds rep-
resent a perfect tool to study the role of the SplB protease in vivo.
In order to effectively inactivate a target protease, several types
of short distance interactions are required that include not only the
optimal structural fit of the P1 residue of the inhibitor into the S1

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E. Burchacka et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5574–5578
O
O
H
N
O
O
O
HO
MeOH
reflux
Trt-NH2
HBTU, Et3N, MeCN
O
O
O
O
1
2
3
LiBH4, THF, EtOH
H
H
H
N
Swern ox.
N
O
OH
O
O
5
4
Scheme 1. General strategy of 4-oxo-N-tritylbutanamide synthesis.
N-benzyloxycarbonyl
were obtained as racemic mixtures of diastereoisomers in the
-amidoalkylation reaction of triaryl phosphite with benzyl carba-
a
-aminoalkylphosphonate diaryl esters
structure of obtained SplB inhibitors was based on the protease
substrate recognition pattern.7
a
We began our search for potential SplB inhibitors by screening
the collection of different diaryl esters of phosphonic analogues of
amino acids, including AlaP, LeuP, and PheP. None of the tested
compounds influenced the activity of SplB at a concentration below
mate and 4-oxo-N-tritylbutanamide in dry dichloromethane using
cooper triflate as the catalyst (Scheme 2).20 The Trt-protective
group in resulting compounds was removed using a mixture of
trifluoroacetic acid and triisopropylsilane (TIPS) (95:5, v/v).
The synthesis of target phosphonic glutamine peptidyl deriva-
tives started with the removal of the benzyloxycarbonyl protective
group of Cbz-(Trt)GlnP(OAr)2 by hydrogenolysis on Pd/C. The ami-
no acid coupling steps were performed in acetonitrile using HBTU
as the coupling agent in the presence of triethylamine. The ob-
tained derivatives were purified on Silica Gel. In order to evaluate
the inhibitory properties of the obtained dipeptides their Trt group
was first removed by TFA:TIPS (95:5, v/v) solution and the result-
ing compounds were purified by HPLC prior to SplB inhibition
studies.
200 l
M. Moreover, tripeptide Cbz-Glu-Leu-GluP(OPh)2, which dif-
fers from the optimal SplB substrate only at the P1 residue, was also
completely inactive against SplB [Skorenski, M.; unpublished re-
sults]. These observations are in agreement with the absolute sub-
strate specificity observed for the SplB protease. Additionally, none
of the synthesized simple Cbz-protected phosphonic analogues of
glutamine (7a–d) displayed any inhibitory activity against SplB
protease at concentrations below 200 lM. As expected, 7a–d were
also inactive against chymotrypsin. Weak subtilisin inhibition was
observed for derivative 7b (k2/Ki = 33 Mꢀ1 sꢀ1). The lack of
inhibitory activity against SplB observed for simple Cbz-protected
derivatives may be the consequence of weak hydrogen bond
network formation between the protease and the inhibitor
(Table 1).
Several previous studies have demonstrated that the amino acid
sequence of the most potent peptidyl inhibitor usually corresponds
to the sequence of the best substrates.12,15,21 Therefore, the
O
NH
O
NH2
H
H
O
O
N
O
OAr
OAr
OAr
OAr
Cbz-NH2, P(OAr)3
TFA:TIPS/95:5
O
N
H
P
O
N
H
P
O
O
O
Cu(CF3SO3)2, DCM
5
6a-d
7a-d
Ar
=
a
b
c
O
O
OH
O
NH2
O
O
H
N
OAr
OAr
d
O
N
N
H
P
H
O
O
13a-d
Scheme 2. Synthesis of analogues of phosphonic glutamine.

E. Burchacka et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5574–5578
5577
Table 1
Activity of phosphonic analogues of glutamine (Cbz-GlnP(OAr)2) against SplB, chymotrypsin and subtilisin
O
NH2
O
R
O
O
O
N
H
P
O
R
Compound
R
SplB
Chymotrypsin
Subtilisin
k2/Ki (Mꢀ1 sꢀ1
<10
Ki (
l
M)
k2/Ki (Mꢀ1 sꢀ1
)
Ki (
l
M)
k2/Ki (Mꢀ1 sꢀ1
)
Ki (
l
M)
)
7a
7b
7c
7d
H
>200
>200
>200
>200
<10
<10
<10
<10
>200
>200
>200
>200
<10
<10
<10
<10
>200
111 3.2
>200
4-O-Methyl
4-t-Butyl
4-Ethyl
33
<10
<10
>200
Table 2
Activity of dipeptidyl derivatives of phosphonic glutamine against SplB, chymotrypsin and subtilisin
O
NH2
R
O
H
N
O
O
O
N
H
P
O
O
R
Compound
R
SplB
Chymotrypsin
Subtilisin
Ki (
lM)
k2/Ki (Mꢀ1 sꢀ1
)
Ki (l
M)
k2/Ki (Mꢀ1 sꢀ1
)
Ki (l
M)
k2/Ki (Mꢀ1 sꢀ1
)
10a
10b
10c
10d
H
>200
>200
>200
>200
<10
<10
<10
<10
>200
>200
>200
>200
<10
<10
<10
<10
>200
35 1.5
>200
<10
57
<10
<10
4-O-Methyl
4-t-Butyl
4-Ethyl
>200
We have further tested the corresponding dipeptides (10a–d)
but these also have not displayed inhibitory activity against SplB
inhibitor found in this study (Table 3). It is possible that some addi-
tional interactions may occur during 13b binding to the enzyme
such as hydrogen bond formation with the oxygen atom of the
methoxy group leading to increased inhibitory potency.
at concentrations lower than 200
agreement with previously reported data showing that the
Cbz-protected dipeptidyl -aminoalkylphosphonate diaryl esters
lM. This observation is in close
a
In terms of inhibitor specificity, all phosphonic tripeptides ob-
tained were completely inactive against chymotrypsin. However,
compounds 13b and 13d inhibited subtilisin almost three times
more potently than they inhibited SplB. An absolute selectivity of
action against tested serine proteases was displayed only by com-
pound 13a which inhibited SplB and not chymotrypsin or subtili-
sin, however at a cost of potency of SplB inhibition. Compound
13a showed the highest binding affinity to the SplB protease
among all the obtained derivatives (Ki/KM ratio of 0.031) however
13b, the most potent inhibitor, displayed higher Ki value of
display poor inhibitory potency against serine proteases in com-
parison to simple Cbz-protected phosphonates.12,13,22 For example,
N-terminally Boc-protected dipeptidyl derivatives of
a-amin-
oalkylphosphonate diaryl esters showed only moderate activity
against subtilisin.16 A similar observation is made in the current
study, as 10b displayed only weak inhibition of subtilisin with a
k2/Ki value of 57 Mꢀ1 sꢀ1 (insignificant increase over 7b) (Table 2).
Activity is significantly increased in phosphonic tripeptides
(13a–d) compared to the amino acid analogue (7a–d) and dipeptide
(10a–d) series. This is likely explained by the fact that the peptide
chain of the inhibitor first directs the molecule in the proper bind-
ing conformation and then the active site nucleophile attacks the
phosphorus atom of the inhibitor leading to formation of the en-
zyme-inhibitor complex. The peptidyl phosphonic glutamine deriv-
ative Cbz-Glu-Leu-GlnP(OC6H4)2 (13a) displayed a k2/Ki value of
500 Mꢀ1 sꢀ1 toward SplB protease. Introduction of electrodonating
groups at the para position of the phenyl ester ring such as ethyl
(13d) or t-butyl (13c) resulted in decreased inhibitory properties
with k2/Ki values of 180 Mꢀ1 sꢀ1 and 15 Mꢀ1 sꢀ1, respectively. De-
spite the fact that isosteric ethyl and methoxy substituents at para
position are electrodonating, the highest potency towards the SplB
protease was observed for Cbz-Glu-Leu-GlnP(OC6H4-4-O-CH3)2
16 lM and it is highly likely the 4-O-methyl substituent provides
additional interaction that stabilizes TS of the reaction.
In order to evaluate the role of the Cbz group in the inhibitory
properties the activity of H-Glu-Leu-GlnP(OPh)2 and H-Glu-Leu-
GlnP(OC6H4-4-CH2CH3)2 towards the SplB protease was measured.
Both derivatives were found to be less potent as compared to the
Cbz-protected parent compounds 13a and 13d displaying the
apparent second-order inhibition rate values <10 Mꢀ1 sꢀ1. This
could in part be explained by the formation of some additional
bond networks inside the S4 binding pocket with the Cbz group
of the inhibitor. These findings are consistent with previous reports
showing that peptides devoid of the N-protective function (either
Cbz or Boc group) are less potent inhibitors when compared to pro-
tected peptides.23,24
(13b, k2/Ki = 1400 Mꢀ1 sꢀ1
) which was the most potent SplB

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E. Burchacka et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5574–5578
Table 3
Activity of tripeptidyl derivatives of phosphonic glutamine against SplB, chymotrypsin and subtilisin
O
OH
O
NH2
O
O
R
R
H
N
O
O
O
N
H
N
H
P
O
O
Compound
R
SplB
Chymotrypsin
Subtilisin
k2/Ki (Mꢀ1 sꢀ1
)
Ki (l
M)
k2/Ki (Mꢀ1 sꢀ1
)
Ki (l
M)
k2/Ki (Mꢀ1 sꢀ1
)
Ki (lM)
13a
13b
13c
13d
H
4.2 0.4
16 0.9
145 3.1
12 1.1
500
1400
15
>200
>200
>200
>200
<10
<10
<10
<10
>200
<10
4100
48
4-O-Methyl
4-t-Butyl
4-Ethyl
8.5 0.7
49 2.2
113 2.3
180
440
In conclusion, we have provided the first ever example of the
synthesis of phosphonic diaryl ester glutamine analogues and their
peptidyl derivatives. Despite the lack of selectivity of the obtained
compounds with highest potency between SplB and subtilisin (13b
and 13d), a highly specific SplB protease inhibitor was identified
(13a). As such, our preliminary data opens the door to further opti-
mization and the development of potent SplB protease inhibitors.
Moreover, all of the synthesized analogues of glutamine presented
in this study were obtained as diastereoisomer mixtures. Since
only one diastereoisomer reacts with the protease active site, true
inhibitory potency can be at least twofold greater than the potency
of the diastereoisomer mixture.21 It is worth noting that this is the
first report on SplB protease inhibitor design ever described in the
literature. Additionally, further investigation on obtaining the crys-
tal structure of SplB protease complexed with inhibitor 13d is cur-
rently underway and will be published separately in due course.
References and notes
1. Eijkman, C. Zbl Bakt Parasitenk Infektionskr. 1901, 29, 841.
2. Michaell, R. D.; Roberts, B. Clin. Microbiol. Rev. 2010, 3, 616.
3. van Belkum, A.; Verkaik, N. J.; de Vogel, C. P.; Boelens, H. A.; Verveer, J.;
Nouwen, J. L.; Verbrugh, H. A.; Wertheim, H. F. J. Infect. Dis. 1820, 2009,
199.
4. Lindsay, J. A.; Holden, M. T. Trends Microbiol Assess. 2004, 12, 378.
5. Reed, S. B.; Wesson, C. A.; Liou, L. E.; Trumble, W. R.; Schlievert, P. M.; Bohach,
G. A.; Bayles, K. W. Infect. Immun. 2001, 69, 1521.
6. Kuroda, M.; Ohta, T.; Uchiyama, I.; Baba, T.; Youzawa, H.; Kobayashi, I. J. Mol.
Biol. 2001, 357, 1225.
7. Popowicz, G. M.; Dubin, G.; Stec-Niemczyk, J.; Czarny, A.; Dubin, A.; Potempa,
J.; Holak, T. A. J. Mol. Biol. 2006, 358, 270.
8. Dubin, G.; Stec-Niemczyk, J.; Kisielewska, M.; Pustelny, K.; Popowicz, G.; Bista,
M.; Kantyka, T.; Boulware, K.; Stennicke, H. R.; Czarny, A.; Phopaisarn, M.;
Doughtry, P. S.; Thogerson, I. B.; Enghlid, J. J.; Thornberry, N.; Dubin, A.;
Potempa, J. J. Mol. Biol. 2008, 379, 343.
9. Reineck, K.; Renneberg, J.; Diamant, M.; Gutschik, E.; Bendtzen, K. Biochim.
Biophys. Acta 1997, 1350, 128.
10. Stec Niemczyk, J.; Pustelny, K.; Kisielewska, M.; Bista, M.; Boulware, K.;
Stennicke, H. R.; Thogersen, I. B.; Daughtry, P. S.; Enghild, J. J.; Baczynski, K.;
Popowicz, G.; Dubin, A.; Potempa, J.; Dubin, G. Biochem. J. 2009, 419, 555.
11. Oleksyszyn, J.; Powers, J. C. Biochem. Biophys. Res. Commun. 1989, 161, 143.
12. Oleksyszyn, J.; Powers, J. C. Biochemistry 1991, 30, 485.
13. Oleksyszyn, J.; Powers, J. C. Methods Enzymol. 1994, 244, 423.
14. Sienczyk, M.; Oleksyszyn, J. Curr. Med. Chem. 2009, 16, 1673.
15. Sienczyk, M.; Winiarski, Ł.; Kasperkiewicz, P.; Psurski, M.; Wietrzyk, J.;
Oleksyszyn, J. Bioorg. Med. Chem. Lett. 2011, 21, 1310.
16. Pietrusewicz, E.; Sienczyk, M.; Oleksyszyn, J. J. Enzyme Inhib. 2009, 24,
1229.
17. Schecker, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157.
18. Knight, C. G. In Proteinase Inibitors; Barett, A. J., Salvesen, G., Eds.; Elsevier:
Amsterdam, 1986; pp 23–51.
19. Bronwen, M.; Wheatley, M.; Keay, B. A. J. Org. Chem. 2007, 72, 7253.
20. Jossens, J.; Van der Veken, P.; Surpateanu, G.; Lambeir, A. N.; El-Sayed, I.; Ali, O.
M.; Augustyns, K.; Haemers, A. J. Med. Chem. 2006, 49, 5785.
21. Winiarski, L.; Oleksyszyn, J.; Sienczyk, M. J Med Chem. in press.
22. Bode, W.; Mayer, J.; Powers, J. C. Biochemistry 1951, 1989, 28.
23. Oleksyszyn, J.; Boduszek, B.; Kam, C. M.; Powers, J. C. J. Med. Chem. 1994, 37,
226.
Acknowledgments
This work was supported by grants N N401 596340 (to J.O.) and
N N301 032834 (to G.D.) from the Polish Ministry of Science and
Higher Education. E.P. acknowledges a fellowship co-financed by
the European Union within the European Social Fund. M.S.
acknowledges Wroclaw University of Technology Statute Funds
(S10156/Z0313). The authors would like to thank Dr Keri Csenc-
sits-Smith (University of Texas Health Science Center at Houston,
TX, USA) for critical reading of the manuscript and Dr Marcin Drag
(Wroclaw University of Technology, Wroclaw, Poland) for his kind
gift of the SplB protease fluorogenic substrate.
Supplementary data
24. Joossens, J.; Ali, O. M.; El-Sayed, I.; Surpateanu, G.; Van der Veken, P.; Lambeir,
A. M.; Setyono-Han, B.; Foekens, J. A.; Schneider, A.; Schmalix, W.; Haemers, A.;
Augustyns, K. J. Med. Chem. 2007, 50, 6638.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012. 07.011.