Inhibitor profiling of the Pseudomonas aeruginosa virulence factor LasB using N-alpha mercaptoamide template-based inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.08.099

We report on the synthesis and biological evaluation of a focussed library of N-alpha mercaptoamide containing dipeptides as inhibitors of the zinc metallopeptidase Pseudomonas aeruginosa elastase (LasB, EC 3.4.24.26). The aim of the study was to derive a

Full text of DOI:10.1016/j.bmcl.2009.08.099

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6230–6232
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitor profiling of the Pseudomonas aeruginosa virulence factor LasB using
N-alpha mercaptoamide template-based inhibitors
George R. Cathcart a, Brendan F. Gilmore a, Brett Greer b, Pat Harriott b, Brian Walker a,
*
a School of Pharmacy, Queen’s University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK
b Department of Biological Sciences, Medical Biology Centre, Queen’s University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We report on the synthesis and biological evaluation of a focussed library of N-alpha mercaptoamide con-
taining dipeptides as inhibitors of the zinc metallopeptidase Pseudomonas aeruginosa elastase (LasB, EC
3.4.24.26). The aim of the study was to derive an inhibitor profile for LasB with regard to mapping the
S01 binding site of the enzyme. Consequently, a focussed library of 160 members has been synthesised,
using standard Fmoc-solid phase methods (on a Rink-amide resin), in which a subset of amino acids
including examples of those with basic (Lys, Arg), aromatic (Phe, Trp), large aliphatic (Val, Leu) and acidic
(Asp, Glu) side-chains populated the P02 position of the inhibitor sequence and all 20 natural amino acids
were incorporated, in turn, at the P01 position. The study has revealed a preference for aromatic and/or
large aliphatic amino acids at P01 and a distinct bias against acidic residues at P02. Ten inhibitor sequences
were discovered that exhibited sub to low micromolar Ki values.
Received 25 June 2009
Revised 27 August 2009
Accepted 28 August 2009
Available online 10 September 2009
Keywords:
LasB
Pseudolysin
Pseudomonas elastase
Biofilm
Ó 2009 Elsevier Ltd. All rights reserved.
Mercaptoamide
Protease inhibitor
Pseudomonas elastase, also known as Pseudolysin or LasB, is a
metalloprotease, which has long been recognised as a key viru-
lence factor produced by the bacterium Pseudomonas aeruginosa.1
The secreted protease degrades a broad-range of host tissue pro-
teins, and key biomolecules involved in innate immunity such as
the immunoglobulins, complement factors and cytokines.2
In addition, LasB acts within the bacterial cell, as a key regulator
in the generation of the secreted polysaccharides that constitute
the bacterial biofilm. In this ‘defensive’ mode of growth, the bacte-
rial cells are resistant to biocides and to the host immune re-
sponse.3 Growth as a biofilm is strongly correlated with chronic
infection, and the increased resistance make such infections diffi-
cult to eradicate.4 Resistance to anti-microbials is up to 1000-fold
greater within the biofilm, and the biofilm exopolysaccharide pre-
vents the immune system from generating a response to the bacte-
rial cell.5 LasB thus plays a key role in the pathogenesis of
Pseudomonal infection, by mediating both the defence and viru-
lence of this opportunistic pathogen.6
hoped that such a ‘second-generation’ class of antimicrobials
might not place a strong selection pressure on the bacteria for
emergence of resistant strains.8 This strategy has shown some
promise recently, and so inhibition of LasB represents a rational
target for attenuation of Pseudomonal virulence in a variety of
pathologies of Pseudomonal infection.9
Against this background, we have embarked upon a comprehen-
sive structure-function study aimed at achieving inhibitor profiling
of LasB. The present work reports on the initial ‘mapping’ of the S10
binding site of the enzyme using N-mercaptoamide dipeptides.
The generalised structure of these inhibitors is shown in Figure 1,
along with the structures of previously reported compounds.
We have previously reported on the solid phase synthesis of a
series of N-alpha mercaptoamide-containing dipeptide amides as
inhibitors of matrix metalloproteases and we have employed this
methodology in the present study.10 The synthetic method is out-
lined in Figure 2 and includes the incorporation of S-trityl pro-
tected mercaptoacetic acid into the range of dipeptide target
sequences, constructed using standard Fmoc-solid phase synthesis
protocols, on a Rink amide resin, and employing an Advanced
Chemtech 396 parallel automated peptide synthesiser. The synthe-
sis of the target inhibitor templates proceeded without difficulty
except in those sequences containing very hydrophobic amino
acids. In these instances, the syntheses were repeated using a
CEM LibertyTM microwave assisted peptide synthesiser.11 The iden-
tity and purity of all peptide products were confirmed using re-
versed phase HPLC and electrospray mass spectrometry.
Inhibitors of this virulence factor have previously been demon-
strated to protect host corneal tissue from the destruction that
characterises Pseudomonal eye infection, and anti-LasB antibody
immunization reduces the virulence of infection.7 Inhibition of
bacterial virulence factors has gained momentum recently, as an
antimicrobial strategy that is non-destructive to the bacteria. It is
* Corresponding author. Tel.: +44 (0)2890 97 2117.
E-mail address: brian.walker@qub.ac.uk (B. Walker).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.099

G. R. Cathcart et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6230–6232
6231
Figure 1. Comparison of chemical structures of previously reported thiol-based inhibitors of LasB, with the dipeptide templates of the present study.
Figure 2. Reaction scheme for synthesis of focussed library of N-alpha mercaptoamide dipeptides. Reagents: (I) 20% piperidine/DMF (v/v); (II) Fmoc-NHCHR2–
CO2H + HATU + DIPEA (threefold molar excess over resin loading); (III) repeat step I; (IV) Fmoc-NHCHR1–CO2H + HATU + DIPEA (threefold molar excess over resin loading);
(V) repeat step I; (VI) S-trityl thioglycolic acid + HATU + DIPEA (threefold molar excess over resin loading); VII, TFA/TIPS/H2O (95:2.5:2.5% v/v).
Table 1
Each peptide was screened for inhibitory activity against LasB,
Ki Values (
1000
lM) for inhibitor library. ‘NI’ (no inhibition) has been stated for values over
obtained from Elastin Products Company, using a microtitre-based
fluorimetric assay. Hydrolysis of the fluorogenic substrate Amin-
obenzoyl-Ala-Gly-Leu-Ala-p-Nitro-Benzyl-Amide by LasB was car-
ried out in buffer containing 0.05 M Tris–HCl, 2.5 mM CaCl2, 1%
dimethylformamide, pH 7.2. The change in fluorescence was mon-
itored using a BMG FLUOstar OPTIMA fluorescence microplate
reader. Those peptides that exhibited inhibitory activity were then
investigated further over an extended concentration range in order
to determine the Ki for each. Figure 3 shows a typical dose–re-
sponse relationship for SH–CH2–CO–Y–V–NH2, as an example. This
figure clearly demonstrates the competitive nature of the inhibi-
tion, while Table 1 summarises the kinetic data that was generated
in this initial inhibitor screening.
l
M. Values in grey identify a general trend for low Ki values in inhibitors
containing P01 Trp and Tyr residues
P20
Ki (l
M)
Basic
Aromatic
Large aliphatic
Acidic
Lys
Arg
Phe
Trp
Val
Leu
Asp
Glu
P10
His
Arg
Lys
Ile
332
135
433
190
76
(NI)
(NI)
(NI)
(NI)
(NI)
623
25
21
18
47
306
(NI)
123
1.3
645
53
(NI)
650
971
142
(NI)
587
38
(NI)
(NI)
(NI)
(NI)
(NI)
(NI)
91
224
126
(NI)
146
113
1.1
125
(NI)
366
206
300
49
(NI)
555
1.8
11
(NI)
4.1
Phe
Leu
Trp
Ala
Met
Pro
Cys
Asn
Val
Gly
Ser
Gln
Tyr
Asp
Glu
Thr
14
10
3.7
153
3.9
766
274
289
22
451
(NI)
380
8.5
491
553
(NI)
115
6.6
56
646
280
69
641
444
217
3.0
(NI)
867
(NI)
131
37
72
51
75
(NI)
6.5
395
204
562
108
70
(NI)
122
(NI)
91
51
98
21
316
7.0
(NI)
(NI)
(NI)
(NI)
(NI)
(NI)
(NI)
(NI)
(NI)
27
It can be appreciated from Table 1, that a number of inhibitor
sequences have been identified in the present study that exhibit
Ki values in the low micromolar range with SH–CH2–CO–Y–V–
(NI)
246
(NI)
508
69
138
510
540
33
157
161
180
10
457
229
937
0.77
(NI)
730
377
(NI)
(NI)
(NI)
(NI)
(NI)
(NI)
(NI)
5.5
14
551
(NI)
616
(NI)
146
225
207
704
65
118
(NI)
(NI)
(NI)
(NI)
(NI)
(NI)
(NI)
(NI)
NH2 being the most potent sequence identified with a Ki value of
0.77 M. This value is directly comparable to those reported before
l
for LasB inhibitor templates that have been derived from studies of
substrate sequences.12 These published studies revealed a prefer-
ence for aromatic or large aliphatic residues in the S01 and S20 pock-
ets of the LasB active site.
This preference for aromatic/hydrophobic residues was also
recapitulated in this present study, although an aromatic residue
Figure 3. Typical progress curves for hydrolysis of aminobenzoyl-Ala-Gly-Leu-Ala-
p-nitro-benzyl-amide by LasB in the presence of a range of concentrations of SH–
CH2–CO–Y–V–NH2.

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G. R. Cathcart et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6230–6232
The same general preference also exists for the P02 residue, with
P02 Phe-, Leu-, and Val-based compounds occupying seven out of
the top 10 inhibitors. This confirms the known preference for aro-
matic or large aliphatic residues in the S02 pocket, indicating that
this region of the active site is also predominantly hydrophobic.
The acidic residues Asp and Glu performed poorly in the P02 posi-
tion, while the basic residues produced moderate inhibitors. How-
ever, no single amino acid residue occupying the P02 position of the
inhibitors produced active compounds in every instance, suggest-
ing that the P01 position is the driving determinant for recognition.
Acknowledgements
This work was supported, in part, by the award of a postgradu-
ate studentship to G.R.C. from the Department of Employment and
Learning (D.E.L.) Northern Ireland.
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