Towards quorum-quenching catalytic antibodies

Article abstract of DOI:10.1039/b819819e

The development of a novel method to attenuate bacterial virulence is reported, which is based upon the use of designed transition-state analogues to select human catalytic antibodies capable of degrading bacterial quorum-sensing molecules. The Royal Soci

Full text of DOI:10.1039/b819819e

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Towards quorum-quenching catalytic antibodiesw
Prashant B. Kapadnis,a Evan Hall,b Madeleine Ramstedt,a Warren R. J. D. Galloway,a
Martin Welchb and David R. Spring*a
Received (in Cambridge, UK) 6th November 2008, Accepted 28th November 2008
First published as an Advance Article on the web 11th December 2008
DOI: 10.1039/b819819e
The development of a novel method to attenuate bacterial
virulence is reported, which is based upon the use of designed
transition-state analogues to select human catalytic antibodies
capable of degrading bacterial quorum-sensing molecules.
and the synthesis of AHL mimics that block natural signals.14
Nature is known to have evolved quorum-quenching enzymes
that are capable of hydrolyzing both the amide and lactone
moieties of AHL signalling molecules.15 For example, a class
of enzymes known as paraoxanases has been identified in
several mammals, which are capable of inactivating OdDHL
and thus attenuating P. aeruginosa quorum sensing in cell
cultures and in vivo.16 Recently, the concept of quorum
quenching using antibody catalysis17 has been introduced.18
This approach uses small molecules as haptens to elicit anti-
bodies capable of catalyzing AHL hydrolysis and thus inhibit
quorum sensing. A haptenic small molecule that closely
resembles the transition state of a reaction should elicit anti-
bodies that act as catalysts of the reaction.19 However,
previous reports on the application of antibody catalysis in
quorum quenching18 have employed haptens that were not
specifically designed to be structural mimics of the transition
state for AHL hydrolysis. Consequently, antibodies procured
to such haptens catalyzed AHL hydrolysis with only very
moderate levels of activity. Herein we report on the synthesis
of sulfones deliberately designed to resemble the transition-
state structure for AHL-ring hydrolysis (Scheme 1).
Antibiotic drugs have played an essential role in the global
increase in life expectancy and quality of life that has occurred
over the last century.1 However, the emergence and increasing
prevalence of multi-drug-resistant bacterial strains are eroding
such gains.1,2 Existing antibiotics generally inhibit bacterial
cellular processes that are essential for microbial survival.2,3
An inherent problem with this approach is that it creates a
selection pressure for drug-resistant mutations.4,5 Bacterial
antivirulence therapies seek to avoid the development of
treatment-induced resistance. In this context, bacterial quorum-
sensing systems offer an attractive target.6
Quorum sensing is the intercellular signalling mechanism,
mediated by small molecules, which many bacteria use to
co-ordinate gene expression with population density.7 Quorum
sensing is used by many bacterial pathogens to regulate virulence;
however, it is not essential for survival.8 Thus, disruption of
quorum sensing (so-called ‘quorum quenching’) should attenuate
pathogenicity without imposing the same selection for resistance,
compared to existing antibiotic treatments.9
N-acylated-L-homoserine lactones (AHLs) are used by
many Gram-negative bacteria for intercellular communica-
tion.10 For example, Pseudomonas aeruginosa utilizes BHL
and OdDHL (Fig. 1) to regulate virulence.10
Scheme 1 Catalytic hydrolysis of AHL (active in quorum sensing) to
products (inactive in quorum sensing) via the predicted transition state
of the rate-determining step. Note that the transition-state structure is
only representative and is highly simplified.
Fig. 1 Examples of AHL small molecules employed by Gram-negative
bacteria in quorum sensing. BHL = N-butyryl-L-homoserine lactone;
HHL = N-hexanoyl-L-homoserine lactone; OdDHL = N-(3-oxo-
dodecanyl)-L-homoserine lactone.
Our studies began with the design of a suitable transition-
state analogue for the rate-determining step of lactone ring
hydrolysis in AHL molecules. Hydrolysis proceeds via
approach of water or hydroxide towards the lactone carbonyl,
forming a pseudo-tetrahedral transition-state structure,20 with
a degree of negative charge delocalized over the two exo
carbon–oxygen bonds (1, Scheme 1). We identified sulfone 2
as a stable transition-state mimic (Fig. 2). Many stable
electronic surrogates21 of transition states for amide and ester
hydrolysis have been investigated;22 however, to the best of
our knowledge, the use of a sulfone has yet to be exploited.23
Three sulfones were targeted for synthesis: transition-state
analogues for BHL (2a), OdDHL (2b) and biotinoylated
HHL (2c, Fig. 2).24
Several methods have been employed previously to block
AHL-dependent quorum sensing,11 including enzymatic
degradation of AHL,12 AHL sequestration by antibodies13
a Department of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, UK CB2 1EW. E-mail: drspring@ch.cam.ac.uk
b Department of Biochemistry, University of Cambridge, Cambridge,
UK
w Electronic supplementary information (ESI) available: Full experi-
mental details and analytical data for all novel compounds. See DOI:
10.1039/b819819e
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monitored using isothermal titration calorimetry (ITC). To the
best of our knowledge, this is the first reported use of ITC to
measure AHL degradation. Using ITC we obtained kcat
and KM(HHL) values for MBP–AiiA (in the absence of 2a) of
0.027 sÀ1 and 0.49 mM, respectively (Fig. 3). These kcat and
KM values are lower than those reported for AiiA by previous
workers,29d,e perhaps reflecting the fact that our ITC titrations
were carried out using much lower AHL concentrations
(sub-millimolar) than previously, or the improved assay tech-
nique. Crucially, as the concentration of 2a present was
increased, KM(HHL) values increased without any significant
effect upon kcat, which suggested that 2a was acting as a potent
competitive inhibitor of AiiA with a Ki of 3 mM. It should be
noted that the transition-state analogues 2a–c are not
themselves biologically active as AHL mimetics (they did not
display either AHL agonist or antagonist activity in vivo
in responsive organisms (Serratia marcescens, Pseudomonas
aeruginosa and Erwinia carotovora) at concentrations
below 1 mM).
Fig. 2 Target AHL hydrolysis transition-state analogues (2).
The successful formation of the amide bond in 2 was anticipated
to be the key reaction in their synthesis, since the obvious
disconnection gives an unstable amino-sulfone.25 Therefore, to
avoid the free amine, traceless Staudinger ligation methodology
was investigated.26 2-Azido-tetrahydrothiophene 3 was readily
synthesized from tetrahydrothiophene according to the method
of Still et al.27 Oxidation to the corresponding sulfone using
potassium permanganate supported on manganese dioxide28
provided the desired azido-sulfone 4 (Scheme 2).
Scheme 2 Synthesis of azido-sulfone 4.
Phosphine coupling partners 5a–c were synthesized in two
steps from o-iodophenol (see ESIw). Reaction with azide 4
generated the amide products 2a and 2b in good yields
(Scheme 3). The phosphines and azide gave aza-ylide inter-
mediates that were trapped intramolecularly by the phenolic
esters, thereby avoiding formation of the unstable amine.
Biotin-tagged derivative 2c was synthesized by a similar route
involving biotin coupling as the final step (Scheme 3).
Fig. 3 Competitive inhibition of AiiA activity by 2a. (A) Raw ITC
output showing that 2a inhibits AiiA-dependent hydrolysis of HHL;
(B) Lineweaver–Burk plot showing that 2a is a potent competitive
inhibitor. [S] = HHL (substrate) concentration.
The fact that 2a acts as competitive inhibitor of AiiA implies
that compounds of the general form 2 are indeed good
transition-state mimics for AHL lactone ring degradation
and should therefore act as haptens for the selection of anti-
body catalysts for this hydrolysis process.
In summary, we have reported the design and synthesis of
predicted transition-state mimics for AHL lactone hydrolysis
and demonstrated their ability to act as competitive inhibitors of
the AHL hydrolase AiiA. Initial proof-of-principle work indi-
cates that these mimics, once immobilized, can be used to select
for binders from a human antibody phage display library.30 The
ability of these transition-state binders to degrade bacterial
quorum-sensing molecules is currently being investigated. To
the best of our knowledge, this work represents the first report
aiming to use deliberately designed transition-state analogues to
biopan for catalytic antibodies capable of degrading AHL
molecules. If proved successful it may lead to the development
of a new method for the treatment of bacterial infections.
We gratefully acknowledge financial support from the
EPSRC, BBSRC, MRC, Swedish Research Council and the
Jawaharlal Nehru Memorial, Cambridge Commonwealth and
Newman Trusts. Also, we thank Walter Fast (University of
Texas, USA) and Jung-Kee Lee (Korean Research Institute
for Bioscience and Biotechnology, Korea) for generously
providing expression constructs of AiiA.
Scheme 3 Summary of analogue synthesis: (i) azide 4, DMF, 70 1C,
then H2O; (ii) TFA, CH2Cl2; (iii) Biotin, EDCÁHCl, NEt3, DMAP,
CH2Cl2.
In order to determine if the sulfone (2) was an ‘accurate’
mimic of the transition state for AHL ring hydrolysis, we
purified a known AHL lactonase, AiiA.29 AiiA was purified as
a maltose binding protein (MBP) fusion. The ability of 2a to
inhibit MBP–AiiA-dependent hydrolysis of HHL in vitro was
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30 Previous reports on antibody-catalyzed quorum quenching (see
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library derived from a cloned human immune repertoire allows the
generation of human catalytic antibodies, which are more suitable
for human therapeutic applications than monoclonal antibodies of
animal origin (see H. Dorsam, M. Braunagel, C. Kleist, D. Moynet
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540 | Chem. Commun., 2009, 538–540