Synthesis and molecular modeling provide insight into a pseudomonas aeruginosa quorum sensing conundrum

Article abstract of DOI:10.1021/ja111138y

The triphenyl amide/ester 12 was originally reported to be a potent mimic of the natural 3-oxo-dodecanoyl homoserine lactone quorum sensing molecule in Pseudomonas aeruginosa. However, explicit synthesis/chemical characterization was lacking, and a later

Full text of DOI:10.1021/ja111138y

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Synthesis and Molecular Modeling Provide Insight into a
Pseudomonas aeruginosa Quorum Sensing Conundrum
Joseph S. Zakhari, Isao Kinoyama, Anjali K. Struss, Prasanna Pullanikat, Colin A. Lowery, Matthew Lardy, and
Kim D. Janda*
The Skaggs Institute for Chemical Biology, Departments of Chemistry and Immunology, and Worm Institute for Research and Medicine
(WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
S Supporting Information
b
12 (termed TP-1P), was structurally unrelated to 3OC12-HSL
ABSTRACT: The triphenyl amide/ester 12 was originally
reported to be a potent mimic of the natural 3-oxo-dodeca-
noyl homoserine lactone quorum sensing molecule in
Pseudomonas aeruginosa. However, explicit synthesis/che-
mical characterization was lacking, and a later report provid-
ing protein crystallographic data inferred 12 to be incorrect,
with 9 now being the surmised structure. Because of these
inconsistencies and our interest in quorum sensing mole-
cules utilized by Gram-negative bacteria, we found it neces-
sary to synthesize 9 and 12 to test for agonistic activity in a P.
aeruginosa reporter assay. Despite distinct regiochemical
differences, both 9 and 12 were found to have comparable
EC50 values. To reconcile these unanticipated findings,
modeling studies were conducted, and both compounds
were revealed to have comparable properties for binding to
the LasR receptor.
but had an activity greater than that of the natural ligand (EC50
=
14 vs 140 nM).7 However, the chemical identity of this novel
activator was never explicitly confirmed and was subsequently
proven incorrect in an X-ray crystallography study of LasR by
Zou and Nair. According to the electron density maps of TP-1
bound to LasR, Zou and Nair proposed that the chlorine atom and
the nitro group were reversed in the actual structure of TP-1, 9
(herein referredtoasTP-1R).8 Thus, the proposed potency ofTP-
1P, coupled with the quandary of its chemical structure, provided
an impetus to synthesize both previous and revised compounds to
be fully characterized and tested in a P. aeruginosa QS reporter
assay system. Herein, we report these findings and the unex-
pected activity of both compounds.
Synthesis of both TP-1R and TP-1P began from commercially
available 3,5-dibromosalicylaldehyde 1, which was protected as
the MOM ether 2 (Scheme 1). Reduction of the aldehyde using
NaBH4, followed by conversion of alcohol 3 to the mesylate,
afforded compound 4. At this point, Gabriel synthesis was
invoked to provide primary amine 6, which serves as the common
intermediate for both TP-1R and TP-1P. Toward TP-1R, amide
7 was formed in the presence of 2-nitrobenzoic acid, EDC, and
HOBt, followed by cleavage of the MOM ether to achieve phenol
8. Finally, esterification of 8 with 2-chlorobenzoic acid yielded
TP-1R (9).
The synthesis of TP-1P from 6 follows the same sequence with
the following modifications: amide formation was performed in
the presence of 2-chlorobenzoic acid to afford 10, followed by
MOM deprotection and ester formation in the presence of
2-nitrobenzoic acid to provide TP-1P (12).
Before biological evaluation, the solubility and stability of TP-1P
and TP-1R were measured to ensure optimal conditions
for the cell-based QS assays. This is particularly important due to
the presence of a seemingly labile o-nitro ester in TP-1P. Never-
theless, in agreement with previous reports,7 TP-1P and TP-1R
were stable in MES and TRIS buffer systems, with half-lives of
59.91 and 607.5 h at pH 8 (Supporting Information, Figure S1-S4
and Table S1). Furthermore, TP-1P and TP-1R were found to have
a maximum solubility of 5 μM in 10% DMSO and a minimum
tested solubility of 3.12 nM in 0.16% DMSO, thus ensuring the
integrity of each compound under the assay conditions.
uorum sensing (QS) is the process in which bacteria
Q
communicate intercellularly through chemical signals
termed autoinducers.1 QS, a population-density-dependent phe-
nomenon, allows bacterial populations to regulate a variety of
physiological processes such as bioluminescence, antibiotic bio-
synthesis, biofilm differentiation, and production of virulence
factors. Pseudomonas aeruginosa, an opportunistic pathogen, is
found in many immunocompromised patients suffering from
diseases such as cystic fibrosis, AIDS, burns, or neutropenic
cancer.2 P. aeruginosa, like many other Gram-negative bacteria,
regulates QS through the exchange of acyl homoserine lactone-
based autoinducers. One of the major autoinducers of P.
aeruginosa, 3-oxo-dodecanoyl homoserine lactone (3OC12-
HSL), along with its cognate receptor LasR, has been the subject
of intense investigation.3 Accordingly, and due to the dire effects
of P. aeruginosa on human health, a myriad of approaches to the
modulation of QS have been examined,4 including the construc-
tion of structural analogues of 3OC12-HSL to act as QS agonists
and antagonists.5
Recently, a library of 200 000 compounds was screened by
Greenberg and co-workers in the hopes of discovering potent
inhibitors or activators of the LasR-dependent QS pathway.6
Both activators and inhibitors of QS were uncovered, and,
excitingly, one compound exhibited more potent QS activation
than the natural 3OC12-HSL signal. This compound, triphenyl
A reporter strain of P. aeruginosa based on the luxCDABE gene
cassette was used to test agonistic activity of TP-1P and TP-1R
Received: December 10, 2010
Published: February 24, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of TP-1R (top)a and TP-1P (bottom)b
a Conditions: (a) MOMCl, K2CO3, DMF, rt, 16 h, 93%; (b) NaBH4,
MeOH, THF, rt, 14 h, 96%; (c) MsCl, Et3N, CH2Cl2, rt, 3 h, 65%; (d)
potassium phthalimide, DMF, rt, 19 h, 80%; (e) H2NNH2-H2O,
EtOH, reflux, 17 h, 85%; (f) (2-NO2)PhCO2H, EDC-HCl, HOBt,
DMF, rt, 19 h, quant.; (g) TFA, CH2Cl2, 0 °C to rt, 3 h, 61%; (h) (2-
Cl)PhCO2H, EDC-HCl, DMAP, THF, rt, 13 h, 50%. b Conditions: (a)
(2-Cl)PhCO2H, EDC-HCl, HOBt, rt, 19 h, 66%; (b) TFA, CH2Cl2, 0
°C to rt, 3 h, quant.; (c) (2-NO2)PhCO2H, EDC-HCl, DMAP, THF,
rt, 13 h, 52%.
Table 1. Biological Activity and Binding Energies of TP-1R,
TP-1P, Tp scaffold, and 3OC12-HSL
Figure 1. Simulations from the 3IX4 structure show the amide in TP-
1R forming hydrogen bonds with Asp73 and Tyr56 (A), whereas with
TP-1P, Tyr56 has moved away and no longer interacts with TP-1P. In
TP-1P, the amide is rotated compared to that in TP-1R and makes
hydrogen bonds with Asp73 and Ser129 (B). In both simulations, Asp73
is caged through interactions with Tyr64 and two interactions
with Thr75.
compound
EC50 (nM)
ΔG (kcal/mol)a
9, TP-1R
12, TP-1P
13
28.3 ( 4.3
42.8 ( 4.0
>10 000
-11.5
-11.7
ndb
3OC12-HSL
30.9 ( 1.3
ndb
a Represents relative free energy of binding between ligand and LasR.
requirements of binding, computational studies were initiated
to model the TP ligands into the LasR binding domain. Studies of
TP-1P and TP-1R were simulated using Gromacs v4.0.7 to
determine the necessity of the nitro and chloro substituents.
Simulations of the 3IX4 complex8 did not show any highly
occupied hydrogen bonds between TP-1R and LasR. The most
consistent interactions were between the amide proton of TP-1R
and Asp73 and the amide carbonyl of TP-1R and Tyr56, but
these interactions were transient, as Asp73 also hydrogen bonds
with Thr75 and Tyr64 throughout the simulation and Tyr56 also
hydrogen bonds with Ser129 (Figure 1). The exchange of the
nitro and chloro groups in TP-1R and TP-1P does not affect the
occupancy of either the hydrogen bond with Asp73 or that with
Tyr56 in TP-1R. The only significant difference in the simula-
tions was that, with TP-1P, the amide carbonyl occasionally
forms a hydrogen bond with Ser129, which is unseen with TP-
b Not determined.
compared to that of the natural autoinducer 3OC12-HSL.9 TP-
1P, TP-1R, and 3OC12-HSL all demonstrated similarly potent
agonistic activity of LasR-dependent signaling (Table 1). Inter-
estingly, maximal luminescence was nearly equal for TP-1R and
3OC12-HSL, but TP-1P induced only 50% luminescence rela-
tive to the former compounds (SI, Figure S5). Nevertheless, the
finding of similar EC50 values between the two triphenyl
compounds was unforeseen, in light of the perceived effect of
the regiochemical change of the chloro and nitro positioning in
the two agonists. This was even more puzzling considering that
the LasR receptor protein has evolved to bind a vastly dissimilar
ligand in 3OC12-HSL.
Seeking to delineate the similar QS activity of TP-1P and TP-
1R on a molecular level and to elucidate the structural
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COMMUNICATION
1R. Pleasingly, truncated Alchemical FEP simulations (Table 1)
show only a small difference in the relative binding energy ΔΔG
= 0.2 kcal/mol between TP-1R and TP-1P.
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Due to the high degree of shape similarity of TP-1R and TP-
1P, both compounds’ ability to make hydrogen bonds through
their amide moiety to nearby residues, and the low ΔΔG of
binding between the two molecules, it is not surprising from our
simulations that they are both of similar potency. The positioning
of the nitro and chloro groups within the binding pocket of LasR
with no consistent hydrogen-bonding or electrostatic interac-
tions with nearby residues sheds light on the similar activity
observed with TP-1P and TP-1R. To investigate the effect of the
scaffold alone, 2-(benzamidomethyl)phenyl benzoate (13) was
synthesized in one step from 2-hydroxybenzyl amine and benzoic
acid and tested in the P. aeruginosa reporter assay. This com-
pound failed to give any QS activation at concentrations up to 10
μM (Table 1). Thus, while the regiochemical interchange of the
nitro and chloro substituents does not seem paramount for
activity, the presence of an electronegative substituent appears
to be critical for LasR binding, dimerization, and ultimately gene
expression.
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In summary, through chemical synthesis, we have firmly estab-
lished the chemical identities of TP-1R/TP-1P, and through model-
ing, we have ascertained the minimal necessary chemical architecture
for LasR activation. Lastly, we highlight the potential synthetic
interchangeable pieces found within the 2-(benzamidomethyl)phenyl
benzoate. The ester and amide units could readily serve as a viable
grounding for the diversity and development of additional agonists
and antagonists against LasR-dependent QS in P. aeruginosa.
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures,
b
spectral data, and biological protocols. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
kdjanda@scripps.edu
’ ACKNOWLEDGMENT
We gratefully acknowledge the NIH (AI077644) for support
and Prof. Michael Surette (University of Calgary) for providing
the luminescent PAO-JP2 P. aeruginosa strain.
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