Substrate specificity and screening of the integral membrane protease Pla

Article abstract of DOI:10.1016/j.bmcl.2007.09.104

This paper reports a study to find small peptide substrates for the important virulence factor of Yersinia pestis, plasminogen activator, Pla. The method used to find small substrates for this protease is reported along with studies examining the ability of these peptides to inhibit activity of the enzyme. Through the use of parallel synthesis and positional scanning, small tripeptides were identified that are viable substrates for the protease.

Full text of DOI:10.1016/j.bmcl.2007.09.104

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 427–431
Substrate specificity and screening of the integral
membrane protease Pla
Anton Agarkov,a Sadhana Chauhan,b Pedro J. Lory,a
Scott R. Gilbertsona,* and Vladimir L. Motinb,*
aChemical Biology Program, Department of Pharmacology and Toxicology, The University of Texas Medical Branch,
301 University Boulevard, Galveston, TX 77555-0650, USA
bDepartment of Pathology and Department of Microbiology & Immunology, The University of Texas Medical Branch,
301 University Boulevard, Galveston, TX 77555-0609, USA
Received 4 August 2007; revised 28 September 2007; accepted 28 September 2007
Available online 12 October 2007
Abstract—This paper reports a study to find small peptide substrates for the important virulence factor of Yersinia pestis, plasmin-
ogen activator, Pla. The method used to find small substrates for this protease is reported along with studies examining the ability of
these peptides to inhibit activity of the enzyme. Through the use of parallel synthesis and positional scanning, small tripeptides were
identified that are viable substrates for the protease.
Ó 2007 Elsevier Ltd. All rights reserved.
Yersinia pestis, the agent of plague, has been recognized
as one of the most devastating, epidemic-causing bacte-
ria experienced by mankind.1 Typically, Y. pestis is
transmitted to humans via a bite from a flea that had
previously fed on an infected rodent. From the initial
site of infection, bacteria disseminate to the draining
lymph node, causing swelling of this lymph node to
form a bubo (bubonic plague).
Moreover, it was shown that Pla allows Y. pestis to rep-
licate rapidly in the airways, thus playing the essential
role in causing pneumonic plague.6 The invasive proper-
ties of Pla, which is a cell surface-located protease, are
likely due to the ability of this enzyme to induce fibrino-
lysis and degrade extracellular matrix and basement
membranes.7,8 The activation of plasminogen to plasmin
while degrading the plasmin inhibitor a2-antiplasmin by
Pla is the mechanism used by Y. pestis to progress from
the peripheral sites into the circulation and systemic
infection.9,10 This paper reports work to identify inhibi-
tors of Pla through parallel synthesis of small peptidic
substrates for the enzyme.
Y. pestis contains a unique, 9.5-kb plasmid pPCP that
expresses plague plasminogen activator (Pla), which is
responsible for fibrinolytic and coagulase activities.2
Pla expression is associated with the marked ability of
Y. pestis to colonize the vicera and thus cause lethal
infection upon administration by peripheral (i.e. intra-
dermal (id), subcutaneous (sc), or intraperitoneal) routes
of infection.3 The importance of Pla for plague patho-
genesis was verified with isogenic Pla mutants of epi-
demic Y. pestis strains KIM and CO92, which showed
an up to 106-fold reduced virulence by the sc routes.3,4
Recently, it was confirmed that bubonic plague, but
not septicemic, depends on Pla to facilitate the rapid dis-
semination of bacteria from the site of id infection.5
One of the classic approaches for the development of
protease inhibitors has been to determine the substrate
specificity of the protease of interest and then use that
information for the design of inhibitors.11 Such an ap-
proach is particularly useful with membrane bound pro-
teases, such as Pla, where there is limited structural
information. An approach was undertaken where
ensembles of peptides were screened for their ability to
act as substrates for Pla. Knowledge of the small peptide
substrates accepted by the enzyme can often serve as a
starting point for the development of enzyme inhibitors.
Keywords: Yersinia pestis; Plasminogen activator; Protease substrate;
Peptide inhibitor; Positional scan; Parallel synthesis.
The original approach undertaken was to synthesize, on
solid support, ensembles of fluorogenic peptides based
*
Corresponding authors. Tel.: +1 409 772 9703; fax: +1 409 772 9700
(S.R.G.); e-mail addresses: srgilber@utmb.edu; vlmotin@utmb.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.104

428
A. Agarkov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 427–431
on loop 5 of Pla, a region of the enzyme that is known to
undergo self cleavage,10 and then screen those libraries
for fluorescence (Fig. 1). Six-residue peptides, overlap-
ping by 1 amino acid, were synthesized with the DAB-
CYL/EDANS quencher/fluorophor system chosen for
this work.12 In theory, peptides in the mixture that are
substrates for Pla would be hydrolyzed resulting in the
separation of the quencher (DABCYL) from the fluoro-
phor (EDANS) and fluorescence of the bead. The tar-
geted libraries synthesized on the PEGA1900 support
resulted in no fluorescence when submitted to the puri-
fied enzyme. Additionally, targeted libraries based on
the region of plasminogen that is known to be cleaved
by Pla were also screened, resulting in no fluorescence.
Some potential reasons for the lack of a signal are: the
enzyme and the solid support the peptides were attached
to may not be compatible in terms of porosity or hydro-
phobicity; the enzyme may not accept small substrates
or substrates that do not have a particular secondary
structure.
peated for positions 3, 4, and 5 and then the final introduc-
tion of DABCYL. Each peptide mixture was cleaved from
the support and then tested in a reaction with Pla to deter-
mine which amino acid in the first position resulted in the
hydrolysis. Fluorescence was detected using a plate reader
at excitation and emission wavelengths of 360 and
460 nm, respectively. The amino acid in position 1 that re-
sulted in fluorescence was then held constant and the sec-
ond position is scanned in the same manner as the first.
This process is then repeated for the third, fourth, and
fifth positions. (It should be noted that while the original
paper reports isokinetic ratios for Boc-protected amino
acids and usage of 10 equiv, we used Fmoc-based strategy
and 5 equiv during coupling.) Despite the limitation of a
positional scan approach, the method provided substrates
for Pla enzyme, with a sequence Arg-Arg-Ile-Asn-Arg
being selected as the ‘best’. The substrate based on this se-
quence was resynthesized on Sieber amide resin to elimi-
nate influence of Gly/Ala, and the resulting DABCYL-
Arg-Arg-Ile-Asn-Arg-Glu(EDANS)-NH2 was confirmed
to be a fluorogenic substrate for Pla.
To determine if the enzyme would accept small pep-
tide substrates in general, mixtures of ‘all possible’
combinations of trimers, tetramers, pentamers, and
hexamers were generated in solution and incubated
with the enzyme. These mixtures were generated using
a modified version of the isokinetic approach of
Houghton13,14 and consisted of all the natural amino
acids, excluding cysteine and tryptophan. Upon treat-
ment of Pla with a given volume of each peptide mix-
ture, the solutions of pentamers and hexamers were
found to fluoresce. This indicated that the problem
with the initial solid supported screen was probably
due to incompatibility of the enzyme with the support.
Rather than attempting to remedy this problem, an
effort was undertaken to identify active peptides in
solution. The positive results with pentamers and
hexamers, versus the lack of fluorescence with trimers
and tetramers, were explained through an assumption
that Pla needs at least 5 amino acids to recognize its
substrate. This assumption was later proven to be
incorrect and will be discussed later.
Once a small peptide substrate was identified, the Pla
activity was measured in a fluorimetric assay using sub-
strate DABCYL-Arg-Arg-Ile-Asn-Arg-Glu(EDANS)-
NH2. In Figure 2, increase in fluorescence is given as a
function of time for both purified recombinant Pla and
an isogenic pair of Y. pestis strains either expressing or
not the Pla protease on the cell surface. Both purified
and cell-associated Pla enzymes cleaved the substrate
in a time-dependent manner, reaching a plateau in
about 60 min. The Pla-negative mutant showed low
background fluorescence indicating substrate specificity
toward Pla. At high concentrations of the fluorogenic
peptide, inhibition was observed with both purified Pla
as well as the enzyme expressed on the surface of Y. pestis
(Fig. 3). The exact origin of this inhibition is not known
but it was confirmed by LC–MS analysis of the reaction
mixtures.
In addition to cleavage of the flurogenic substrate, a
functional assay was performed. Pla activates plasmino-
gen to plasmin by limited proteolysis. The inhibitory
activity of the substrate was tested by examining its abil-
ity to inhibit the conversion of plasminogen to plasmin
in a fibrin plate assay.16 Upon plasminogen’s conversion
to plasmin, the resulting proteolytic dissolution of fibrin
is seen as a clear liquid spot on an opalescent fibrin film.
Pre-incubation of purified Pla with the substrate, DAB-
CYL-Arg-Arg-Ile-Asn-Arg-Glu(EDANS)-NH2, followed
by spotting the samples on the fibrin film resulted in a
concentration-dependent inhibition of Pla-mediated
fibrinolysis (Fig. 4). It was also observed that the fluoro-
genic substrate can inhibit Pla expressed by Y. pestis
cells in a similar manner, but did not prevent the action
of mammalian plasminogen activator urokinase (data
not shown).
Having established a ‘minimal’ length of the substrate, a
positional scan approach, similar to the work of Hough-
ten, was used to identify individual peptides (Scheme
1).15 In this approach 1 amino acid position is held con-
stant while the others are varied. The Fmoc-Glu(E-
DANS)-Gly-Wang or Fmoc-Glu(EDANS)-Ala-Wang
resin was divided into 20 equal portions, and each portion
was coupled to an individual amino acid followed by cou-
pling with an isokinetic mixture of 18 amino acids. This
provides each vessel with a mixture of dimers with the
known amino acid at position 1 and the other 18 amino
acids represented at position 2. This process was then re-
Following hydrolysis of the fluorogenic substrate, the
reaction products were identified by LC–MS analysis.
The site of hydrolysis was at a basic site, in this case
between the two arginines. This observation correlates
with the substrate specificity of the omptin family of
Figure 1. Loop 5 sequence was analyzed as overlapping six-residue
peptides.

A. Agarkov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 427–431
429
Scheme 1. The highlighted amino acids are amino acids that resulted in preferred cleavage of the substrate. Ultimately, based on qualitative
differences in the rate of cleavage between different active sequences, we selected DABCYL-Arg-Arg-Ile-Asn-Arg-Glu(EDANS)-NH2 as the substrate
candidate for Pla.
proteases which have a preference to cleave between two
basic amino acids.17
not inhibit the activation of plasminogen at the concen-
tration in the range of 100–350 lM but was still a sub-
strate for the enzyme. Every truncated version, except
the dipeptide DABCYL-Arg-Arg-OH, was found to be
a substrate for Pla (Table 1). Apparently EDANS plays
a role in increasing inhibitory activity. We are in the
process of examining the origin of this effect through
the incorporation of molecules with structures similar
to EDANS. This effect was observed when EDANS
was attached by either the side chain or main chain car-
boxyl of the peptide (1 and 6, Table 1). Synthesis of the
Truncated versions of the initial six-residue substrate
were examined to probe the length of peptide necessary,
as well as to examine the role of the fluorogenic and
quencher molecules. Versions of the hexapeptide sub-
strate with and without the EDANS group were evalu-
ated as substrates and as inhibitors in the fibrinolytic
assay. A derivative of the peptide without the fluoro-
phore EDANS but with the quencher DABCYL did

430
A. Agarkov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 427–431
Figure 2. Kinetic of cleavage of identified fluorescent substrate by Pla.
The activity was measured with purified recombinant Pla, Y. pestis
cells expressing Pla and Y. pestis cells lacking Pla gene.
Figure 4. Fibrinolytic assay of Pla activity at various substrate
concentrations. The substrate is taken in 2-fold dilutions. Spots 4
and 6 represent the samples with no substrate added. Spots 1, 2, 3, and
5 have different substrate concentrations. The substrate was dissolved
either in DMSO or in water.
Table 1. Peptide sequences tested as substrates and functional
inhibitors
Compounda Sequence
Sb Ic
1
2
3
4
5
6
7
8
DABCYL-RRINR-Glu(EDANS)-NH2
+
+
+
+
À
+
+
+
+
À
À
À
À
+
+
+
DABCYL-RRINR-OH
DABCYL-RRIN-OH
DABCYL-RRI-OH
DABCYL-RR-OH
DABCYL-RRINR-Glu-EDANS
DABCYL-RRIN-EDANS
DABCYL-RRI-EDANS
a Compounds 2–5 and DABCYL-Arg-OH have been synthesized and
used as LC–MS standards to determine cleavage fragments. Based on
retention time and mass detection, DABCYL-Arg-OH was one of
products in all cleavage mixtures except 5.
Figure 3. Fluorimetric assay of Pla activity at various substrate
concentrations. The activity was measured at the end point of the
reaction with purified recombinant Pla (a) and Y. pestis cells expressing
Pla on their surface (b).
b Substrate, cleavage was determined by LC-MS.
c Inhibitor, the inhibitory activity of the peptides was determined using
functional assay on fibrin plates.
hexapeptide omitting DABCYL also resulted in the loss
of the inhibitory activity, indicating that both fluoro-
phore and quencher play a role in the inhibition of
Pla. As a control, both free EDANS and DABCYL
were examined and found not to inhibit the enzyme.
As shown in Table 1 the length of the peptide can be re-
duced to a trimer DABCYL-Arg-Arg-Ile-EDANS and
maintain activity.
amount of the mixtures there is significantly more of
each individual trimer than each pentamer (up to
182-fold). Consequently under the conditions of the ini-
tial assay, substrate inhibition was observed with the tri-
mer and tetramer mixtures. However, when discrete
tetra- and tripeptides were synthesized and tested, they
displayed substrate and inhibitory properties similar to
those of the lead hexamer substrate.
Previously, in the case of mixtures of peptides, it was
found that only hexamer and pentamer mixtures re-
sulted in fluorescence. Since our initial experiments were
conducted with the same amount of total peptide re-
sulted from the isokinetic approach, the difference be-
tween the experiment discussed here and the results
with mixtures are likely due to the fact that in a given
Since a tripeptide DABCYL-Arg-Arg-Ile-EDANS was
found to be a substrate similar to the hexapeptide DAB-
CYL-Arg-Arg-Ile-Asn-Arg-Glu(EDANS)-NH2, its optimi-
zation was undertaken. A focused library of tripeptide

A. Agarkov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 427–431
431
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.09.104.
References and notes
Figure 5. Profiling the tripeptide DABCYL-Arg-Arg-X-EDANS
library. The X represents one of the 20 amino acid residues. The y
axis is end point fluorescent signal after 2 h of incubation of the
substrate with purified Pla. The x axis provides the spatial address of
the amino acid as represented by the one-letter code. Each substrate
was examined at concentration of ꢀ35 lmol.
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Acknowledgments
This work was supported by the Region VI ‘Western’
RCE (NIH award 1-U54-AI-057156) grant, an award
from the Sealy Center for Vaccine Development of the
University of Texas Medical Branch, and the Robert
A. Welch Foundation. We thank Dr. Robert R. Bru-
baker for providing the strains of Y. pestis and helpful
discussions.