U.S. patent application number 12/381572 was filed with the patent office on 2009-11-19 for ai-2 compounds and analogs based on salmonella typhimurium lsrb structure.
This patent application is currently assigned to The Trustees of Princeton University. Invention is credited to Bonnie L. Bassler, Shawn R. Campagna, Frederick M. Hughson, Stephen T. Miller, Martin F. Semmelhack, Michiko E. Taga, Karina B. Xavier.
Application Number | 20090286873 12/381572 |
Document ID | / |
Family ID | 35150540 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090286873 |
Kind Code |
A1 |
Miller; Stephen T. ; et
al. |
November 19, 2009 |
AI-2 compounds and analogs based on Salmonella typhimurium LsrB
structure
Abstract
This invention relates to crystals comprising apo-LsrB and
holo-LsrB. The structure of holo-LsrB identifies a
tetrahydroxytetrahydrofuran derived from
4,5-dihydroxy-2,3-pentanedione (DPD) as the active autoinducer-2
(AI-2) molecule in Salmonella typhimurium. The X-ray
crystallographic data can be used in a drug discovery method.
Additionally the invention provides AI-2 analogs based on this
discovery as well as pharmaceutical compositions containing those
analogs.
Inventors: |
Miller; Stephen T.;
(Swarthmore, PA) ; Xavier; Karina B.; (Princeton,
NJ) ; Taga; Michiko E.; (Somerville, MA) ;
Campagna; Shawn R.; (Hamilton, NJ) ; Semmelhack;
Martin F.; (Princeton, NJ) ; Bassler; Bonnie L.;
(Princeton, NJ) ; Hughson; Frederick M.;
(Princeton, NJ) |
Correspondence
Address: |
MATHEWS, SHEPHERD, MCKAY, & BRUNEAU, P.A.
29 THANET ROAD, SUITE 201
PRINCETON
NJ
08540
US
|
Assignee: |
The Trustees of Princeton
University
|
Family ID: |
35150540 |
Appl. No.: |
12/381572 |
Filed: |
March 13, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11228707 |
Sep 16, 2005 |
7547726 |
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12381572 |
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11104681 |
Apr 12, 2005 |
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11228707 |
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60561659 |
Apr 12, 2004 |
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Current U.S.
Class: |
514/473 ;
549/476 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 33/04 20180101; Y02A 50/30 20180101; Y02A 50/47 20180101; Y02A
50/483 20180101; C07K 2299/00 20130101; Y02A 50/481 20180101; C07H
7/02 20130101; C07H 3/02 20130101; Y02A 50/471 20180101; Y02A
50/473 20180101; C07K 14/255 20130101 |
Class at
Publication: |
514/473 ;
549/476 |
International
Class: |
A61K 31/341 20060101
A61K031/341; C07D 307/04 20060101 C07D307/04; A61P 33/04 20060101
A61P033/04 |
Goverment Interests
GOVERNMENT INTERESTS
[0002] This invention was funded in part through a grant from the
National Institutes of Health. Therefore, the federal government
has certain rights in this invention.
Claims
1-3. (canceled)
4. A compound represented by formula I or formula J: ##STR00023##
wherein Y is hydrogen or hydroxyl; and R is lower alkyl; or a
pharmaceutically acceptable salt thereof.
5. (canceled)
6. A compound represented by formula K or formula L: ##STR00024##
wherein Y is hydrogen or hydroxyl; X and Z are independently
hydrogen, lower alkyl or hydroxyl; and R is lower alkyl; or a
pharmaceutically acceptable salt thereof.
7-9. (canceled)
10. A pharmaceutical composition comprising one or more compounds
of claim 4 in admixture with a pharmaceutically acceptable
carrier.
11. A pharmaceutical composition comprising one or more compounds
of claim 6 in admixture with a pharmaceutically acceptable
carrier.
14-39. (canceled)
Description
[0001] This application is a continuation-in-part of U.S. Ser. No.
11/104,681, filed Apr. 12, 2005, which claims benefit under 35
U.S.C. .sctn. 119(e) of U.S. Provisional Ser. No. 60/561,659, filed
Apr. 12, 2004, each of which is incorporated herein by reference in
its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to crystals comprising apo-LsrB and
holo-LsrB. The structure of holo-LsrB identifies a
tetrahydroxytetrahydrofuran derived from
4,5-dihydroxy-2,3-pentanedione (DPD) as the active autoinducer-2
(AI-2) molecule in Salmonella typhimurium. The X-ray
crystallographic data can be used in a drug discovery method.
Additionally the invention provides AI-2 analogs based on this
discovery as well as pharmaceutical compositions containing those
analogs.
BACKGROUND OF THE INVENTION
[0004] Many bacteria modulate their behavior in response to
cell-cell communication in a process termed quorum sensing
(Bassler, 2002). Intercellular communication is accomplished
through the production, release, and detection of small signaling
molecules called autoinducers. Typically, Gram-negative bacteria
use acylated homoserine lactones as autoinducers, whereas
Gram-positive bacteria use modified oligopeptides. In its simplest
form, quorum sensing consists of the accumulation of high
autoinducer concentrations at high bacterial population densities.
The bacteria respond with a population-wide alteration of gene
expression, allowing the community to coordinate behavior in a
manner akin to cells in a multicellular organism. Quorum sensing
provides a mechanism for the collective regulation of processes
including biofilm formation and virulence in Pseudomonas aeruginosa
and Vibrio cholerae, antibiotic production in Photorhabdus
luminescences, and light production in Vibrio harveyi (Miller et
al., 2001). In general, different bacterial species produce and
respond to chemically distinct autoinducers, restricting quorum
sensing to intraspecies communication.
[0005] Genetic and biochemical studies of quorum sensing in the
marine bacterium V. harveyi led to the identification of a novel
autoinducer used to control bioluminescence (Bassler et al., 1994,
1997; Chen et al., 2002; Schauder et al., 2001; Surette et al.,
1999). This autoinducer signal, termed AI-2, is unusual in that it
is produced by a large number of bacterial species in addition to
V. harveyi. Furthermore, AI-2-responsive genes have been identified
in a variety of bacteria (Xavier et al., 2003). Consequently, AI-2
has been proposed to serve as a "universal" quorum-sensing signal
that enables interspecies communication (Schauder et al.,
2001).
[0006] The enzyme LuxS, which has been identified in more than 55
Gram-negative and Gram-positive bacterial species, is responsible
for AI-2 biosynthesis (Surette et al., 1999; Xavier et al., 2003).
AI-2 signals are derived from S-adenosylmethionine (SAM), whose
consumption as a methyl donor yields S-adenosylhomocysteine (SAH)
(FIG. 1A). SAH is metabolized to adenine and S-ribosylhomocysteine
(SRH) (Cornell et al., 1998). SRH is the substrate for LuxS (Lewis
et al., 2001; Schauder et al., 2001), which cleaves it to generate
homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD, FIG. 1A).
[0007] The products of the LuxS reaction strongly stimulate light
production in V. harveyi (Meijler et al., 2004; Schauder et al.,
2001; Zhao et al., 2003). One of these products, homocysteine, has
no autoinducer activity. The other product, DPD, is expected to
cyclize spontaneously to form two epimeric furanoses, (2R,4S)- and
(2S,4S)-2,4-dihydroxy-2-methyldihydrofuran-3-one (R- and S-DHMF,
respectively; FIG. 1B). Hydration of R- and S-DHMF would give rise
to (2R,4S)- and
(2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R- and
S-THMF, respectively; FIG. 1B).
[0008] Because DPD exists in equilibrium with other chemical
species in solution (this work and Meijler et al., 2004),
identifying the form that is active in AI-2 signaling in V. harveyi
proved difficult. Trapping V. harveyi AI-2 in its receptor LuxP
greatly facilitated its identification. X-ray crystallography
allowed direct visualization at 1.5 .ANG. resolution of the ligand
bound to LuxP (Chen et al., 2002), establishing that the signal
molecule is S-THMF-borate (FIG. 1B). Formation of this molecule
from DPD can be explained by a simple mechanism. Since borate
reacts readily with adjacent hydroxyl groups on furanosyl rings
(Loomis et al., 1992), it is chemically reasonable that
S-THMF-borate forms spontaneously by addition of borate, which is
abundant (ca. 0.4 mM) in marine environments, to S-THMF (FIG. 1B).
Consistent with this scheme, chemically synthesized DPD induces
bioluminescence in V. harveyi, but only in the presence of boric
acid (Meijler et al., 2004). S-THMF-borate is unrelated to
previously characterized autoinducers and is highly unusual in
containing boron, an element rarely observed in biological
molecules.
[0009] The presence of boron in the LuxP ligand raised the question
of whether S-THMF-borate is the sole bacterial signaling molecule
derived from DPD. Hence, this question was addressed by determining
whether other bacteria that respond to AI-2 signals recognize
S-THMF-borate or whether, instead, they recognize different
derivatives of DPD. In the latter case, the use of S-THMF-borate as
a signaling molecule might be confined, for example, to bacteria
such as marine vibrios that live in relatively high-borate
environments. The identification of LsrB as an AI-2 binding protein
in S. typhimurium and Escherichia coli (Taga et al., 2001, 2003)
provided a starting point for characterizing the spectrum of AI-2
signal molecules.
[0010] S. typhimurium carries the LuxS enzyme and synthesizes DPD.
Genetic analysis has identified a set of lsr (LuxS-regulated) genes
whose expression is controlled by the LuxS-generated AI-2 signaling
molecule (Taga et al., 2001). The Lsr proteins appear to function
in the binding, internalization, and metabolism of the AI-2 signal
(Taga et al., 2001, 2003). LsrB, as suggested by its homology to
periplasmic sugar binding proteins, binds the AI-2 signal directly.
Other genes in the lsr operon encode LsrA, LsrC, and LsrD. These
proteins form an ABC transporter complex, homologous to the ribose
transporter, that internalizes the signal molecule. Internalized
AI-2 is subsequently processed by additional lsr operon encoded
enzymes (Taga et al., 2003). Thus, one consequence of activating
the lsr operon at high cell density is that S. typhimurium clears
AI-2 signaling activity from its environment. This might represent
a strategy for terminating AI-2 signaling or for interfering with
AI-2 signaling by other species (Taga and Bassler, 2003).
[0011] The structure of LsrB, both unliganded and in complex with
its DPD-derived ligand was determined. Like other periplasmic
binding proteins, LsrB undergoes a significant conformational
change upon ligand binding. Most strikingly, the LsrB ligand
differs from the LuxP ligand and lacks boron. Thus, two different
bacterial AI-2 receptors bind chemically distinct derivatives of
DPD. These findings mean that the earlier use of the term "AI-2" to
refer exclusively to S-THMF-borate is not accurate. Instead, the
AI-2 response in different bacterial species can be triggered by at
least two different derivatives of the LuxS product, DPD.
SUMMARY OF THE INVENTION
[0012] The present invention is directed to crystals comprising
apo-LsrB or holo-LsrB, i.e., an LsrB-ligand complex. The crystals
diffract X-rays to a resolution of greater than. 5.0 .ANG., and
preferably to a resolution greater than 1.5 .ANG. or 1.3 .ANG.. In
accordance with the discoveries of the invention, the ligand
comprises an autoinducer-2 (AI-2) molecule which comprises a furan
moiety. In one embodiment the ligand is R-THMF as having the
chemical formula:
##STR00001##
[0013] In accordance with the invention, another embodiment relates
to methods of using the crystal structures from the crystals of the
invention to identify whether a ligand binds to LsrB. This method
involves obtaining the atomic coordinates for at least a selected
portion of LsrB and using those atomic coordinates to computer
model the identification of and/or docking of potential ligands
that can bind to the selected portion of LsrB. The selected
portion, preferably includes the R-THMF binding site, and more
preferably, includes one or more amino acid residues selected from
the group consisting of Lys35, Asp116, Asp166, Gln 167, Pro220 and
Ala 222.
[0014] In a further aspect of the invention, the potential ligand
is tested for AI-2 antagonist or agonist activity by obtaining a
sample of the potential ligand, contacting a prokaryotic cell with
the sample under conditions to assess whether the ligand can bind
to LsrB and/or affect the quorum sensing activity of the cells
exposed to the potential ligand. Those ligands identified by these
methods, and pharmaceutical compositions containing those ligands,
are contemplated as part of the instant invention.
[0015] Another aspect of the invention provides pharmaceutical
compositions with a compound having the chemical formula:
##STR00002##
in admixture with a pharmaceutically acceptable carrier. Such
compositions are useful for treating bacterial infections when
administered for a time and in an amount that is therapeutically
effective to treat the bacterial infection. The above compound is
(2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran, and also
referred to herein as R-THMF.
[0016] Another aspect of the invention relates to AI-2
antagonists/agonists designed to bind to LsrB, LuxP and/or LuxQ, or
their counterparts from any bacterial species, based on the
formation pathways for the AI-2 signaling molecules recognized by
V. harveyi (upper branch) and S. typhimurium shown in FIG. 1B.
These analogs are based on both the R and S stereoisomers at the 2
position of the furan ring.
[0017] The set of analogs based upon the S stereoisomer are the
Series A-H compounds. The Series A-H compounds are specifically
represented by the following formulas,
[0018] for Series A and B by
##STR00003##
wherein X is O, NH, S, CH.sub.2, CFH or CF.sub.2; Y is hydrogen,
hydroxyl, methyl or amino; Z is hydroxyl or amino; and R is lower
alkyl, aryl or alkenyl; with the proviso that when X is O in
formula A, then simultaneously R cannot be methyl, Y cannot be
hydroxyl and Z cannot be hydroxyl; or a pharmaceutically-acceptable
salt thereof;
[0019] for Series C and D by
##STR00004##
wherein W is hydroxyl or amino; X is O, NH, S, CH.sub.2, CFH or
CF.sub.2; Y is hydrogen, hydroxyl, methyl or amino; Z is hydroxyl
or amino; and R.sub.1 and R.sub.2 are independently lower alkyl,
aryl or alkenyl; or a pharmaceutically-acceptable salt thereof;
[0020] for Series E and F by
##STR00005##
wherein W is hydroxyl or amino; X is O, NH, S, CH.sub.2, CFH or
CF.sub.2; Y is hydrogen, hydroxyl, methyl or amino; Z is hydroxyl
or amino; and R.sub.1 and R.sub.2 are independently lower alkyl,
aryl or alkenyl; or a pharmaceutically-acceptable salt thereof;
and
[0021] for Series G and H by
##STR00006##
wherein X is O, NH, S, CH.sub.2, CFH or CF.sub.2; Y is hydrogen,
hydroxyl, methyl or amino; Z is hydroxyl or amino; and R is lower
alkyl, aryl or alkenyl; with the proviso that when X is O in
formula G, then simultaneously R cannot be methyl, Y cannot be
hydroxyl and Z cannot be hydroxyl; or a pharmaceutically-acceptable
salt thereof.
[0022] For the Series A and B compounds, X is preferably CFH or
CF.sub.2.
[0023] The set of analogs based upon the R stereoisomer are the
Series I-P compounds. The Series I-P compounds are specifically
represented by the following formulas,
[0024] for Series I and J by
##STR00007##
wherein X is O, NH, S, CH.sub.2, CFH or CF.sub.2; Y is hydrogen,
hydroxyl, methyl or amino; Z is hydroxyl or amino; and R is lower
alkyl, aryl or alkenyl; with the proviso that when X is O in
formula A, then simultaneously R cannot be methyl, Y cannot be
hydroxyl and Z cannot be hydroxyl; or a pharmaceutically-acceptable
salt thereof;
[0025] for Series K and L by
##STR00008##
wherein W is hydroxyl or amino; X is O, NH, S, CH.sub.2, CFH or
CF.sub.2; Y is hydrogen, hydroxyl, methyl or amino; Z is hydroxyl
or amino; and R.sub.1 and R.sub.2 are independently lower alkyl,
aryl or alkenyl; or a pharmaceutically-acceptable salt thereof;
[0026] for Series M and N by
##STR00009##
wherein W is hydroxyl or amino; X is O, NH, S, CH.sub.2, CFH or
CF.sub.2; Y is hydrogen, hydroxyl, methyl or amino; Z is hydroxyl
or amino; and R.sub.1 and R.sub.2 are independently lower alkyl,
aryl or alkenyl; or a pharmaceutically-acceptable salt thereof;
and
[0027] for Series 0 and P by
##STR00010##
wherein X is O, NH, S, CH.sub.2, CFH or CF.sub.2; Y is hydrogen,
hydroxyl, methyl or amino; Z is hydroxyl or amino; and R is lower
alkyl, aryl or alkenyl; with the proviso that when X is O in
formula O, then simultaneously R cannot be methyl, Y cannot be
hydroxyl and Z cannot be hydroxyl; or a pharmaceutically-acceptable
salt thereof.
[0028] For the Series I and J compounds, X is preferably CFH or
CF.sub.2.
[0029] The invention also includes pharmaceutical compositions
comprising one or more of the foregoing compounds in admixture with
a pharmaceutically acceptable carrier.
[0030] A still further aspect of the invention is directed to
methods of regulating the activity of an autoinducer-2 (AI-2)
receptor by contacting the AI-2 receptor with an AI-2 analog for a
time and in an amount sufficient to regulate said activity, wherein
said AI-2 analog is a compound represented by any one of Series A
to Series P. These compounds can be used to regulate activity of
the AI-2 receptors LsrB, LuxP and/or LuxQ, or their analogs (i.e.,
counterparts) from any bacterial species. Preferably the receptors
are found on a bacterial cell, including bacteria in warm blooded
hosts. The regulated activity includes any regulated by quorum
sensing such as bacterial cell growth, siderophore expression,
bacterial virulence, biofilm formation exopolysaccharide production
in bacterial cells and bacterial colony morphology.
[0031] Yet another aspect of the invention provides a method for
treating a subject infected with a pathogenic bacteria by
administering a therapeutically-effective amount of a
pharmaceutical composition containing at least one of the Series
A-Series P compounds to a subject for a time and in an amount
sufficient to inhibit AI-2 activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 illustrates the chemistry of AI-2 Signaling
Molecules. FIG. 1A shows the metabolic pathway leading to DPD, the
key product of the enzyme LuxS. FIG. 1B illustrates the formation
pathways for the AI-2 signaling molecules recognized by V. harveyi
(upper branch) and S. typhimurium (lower branch). S-THMF-borate
binds to the V. harveyi receptor LuxP (Chen et al., 2002), whereas
R-THMF binds to the S. typhimurium receptor LsrB. Previously,
S-DHMF and S-THMF-borate were referred to as pro-AI-2 and AI-2,
respectively (Chen et al., 2002). IUPAC carbohydrate nomenclature
is provided in Example 1.
[0033] FIG. 2 provides ribbon and space filling models for the
structure of Apo- and Holo-LsrB. FIG. 2A shows an overview of S.
typhimurium apo-LsrB. The ribbon diagram is colored in rainbow
order from N- to C terminus. FIG. 2B shows a comparison of apo-
(red, left side) and holo-LsrB (orange, right side) that reveals
domain closure upon ligand binding. FIG. 2C shows the structure of
holo-LsrB with R-THMF. The Fo-Fc electron density map, contoured at
5.sigma., was calculated without ligand built into the binding site
and with waters in and around the binding site deleted. FIG. 3D
provides a stereoview of R-THMF in Fo-Fe electron density contoured
at 3.sigma.. FIG. 3E provides a stereoview of R-THMF in Fo-Fc
density contoured at 10.sigma.. Phase bias was avoided as
described.
[0034] FIG. 3 illustrates the ligand binding sites for LsrB and
LuxP. FIG. 3A shows a comparison of R-THMF and the LsrB binding
site (left) with S-THMF-borate and the LuxP binding site (right;
Chen et al., 2002). FIGS. 3B and 3C show stereoviews of the LsrB
and LuxP ligand binding sites, respectively, in orientations
matched by aligning the overall protein structures. FIG. 3D
provides a structure-based sequence alignment of LsrB and LuxP
(Shindyalov et al., 1998), with the sequence of LsrB being SEQ ID
NO. 1 and the sequence of LuxP being SEQ ID NO. 2. Residues that
form specific interactions (hydrogen bonds or salt bridges) with
the ligand are indicated with filled triangles.
[0035] FIG. 4 depicts .sup.11B-NM spectra showing that the LsrB
ligand lacks boron but, upon release, can form a borated
derivative. FIG. 4A shows the .sup.11B-NMR spectra of holo-LuxP
which were collected before (top) or after (middle) the addition of
5 mM boric acid. The holo-LuxP ligand was then released by thermal
denaturation; its spectrum is shown in the bottom panel. FIG. 4B
shows the .sup.11B-NMR spectra of holo-LsrB (prepared as a GST
fusion protein as described in the Experimental Procedures) which
were collected before (top) or after (middle) the addition of 5 mM
boric acid. The holo-LsrB ligand was then released by thermal
denaturation; its spectrum is shown in the bottom panel.
[0036] FIG. 5 graphically illustrates that boric acid enhances AI-2
signaling in V. harveyi and inhibits AI-2 signaling in S.
typhimurium. FIG. 5A is a bar graph showing light produced by the
V. harveyi strain MM32 (LuxN.sup.-, LuxS.sup.-) assayed following
the addition of water (no ligand), in vitro synthesized DPD, LsrB
ligand, or LuxP ligand. Light units were measured following 8 hr of
growth in borate-depleted AB medium (black bars) or in the same
medium plus 5 mM boric acid (white bars). Ligand concentrations
were approximately 0.2 nM, within the linear range of the assay. In
borate-depleted medium, approximately 5000 times more ligand was
required to produce a measurable increase in light production.
Error bars represent the standard deviations for four independent
cultures. FIG. 5B shows the expression of the lsr operon in S.
typhimurium strain MET844 (LuxS.sup.-) assayed following the
addition of NMR buffer (no ligand), in vitro synthesized DPD, LsrB
ligand, or LuxP ligand. .beta.-galactosidase activities were
measured after 4 hr of growth in borate-depleted LB medium (black
bars) or in the same medium plus 5 mM boric acid (white bars).
Ligand concentrations were approximately 10 .mu.M for DPD and LsrB
ligand and 4 .mu.M for LuxP ligand. A lower concentration was used
for the LuxP ligand because the solubility of holo-LuxP limits the
attainable concentration of the released ligand. Error bars
represent the standard deviations for two independent cultures.
DETAILED DESCRIPTION OF THE INVENTION
Crystallography and Rational Ligand Design
[0037] The present invention provides a crystal comprising LsrB
without any ligand bound. This form is also referred to herein as
apo-LsrB. In this context, those skilled in the art will understand
that the term "crystal" refers to an ordered arrangement of atoms,
the crystal having an overall size and quality sufficient for the
elucidation of the atomic arrangement by X-ray crystallography.
Preferably, the crystal diffracts X-rays to a resolution of greater
than about 5.0 Angstroms (.ANG.), more preferably greater than
about 2.5 .ANG., even more preferably greater than about 1.5 or 1.3
.ANG.. A resolution "greater than" a particular value means a
resolution that numerically exceeds the recited value. For example,
in X-ray crystallography, a resolution of 2.8 .ANG. is greater than
a resolution of 5.0 .ANG.. Crystals comprising LsrB are preferably
prepared by the methods described in the Examples below. The atomic
coordinates for LsrB are preferably determined by X-ray
crystallography of a crystal comprising LsrB, preferably by the
methods described in the Examples below but can be determined using
other methods known in the crystallographic art. A set of atomic
coordinates for the apo-LsrB crystal has been deposited in the
Protein Data Bank under accession codes 1TM2.
[0038] Another crystal of the invention comprises LsrB and a
ligand. That ligand has been identified as containing a furan
moiety. As used herein, the term "ligand" refers to a molecule or
ion that binds to LsrB. Preferably, binding between the ligand and
LsrB occurs at an LsrB binding site, which a region of LsrB that
interacts with the ligand to produce an LsrB-ligand complex in
which the ligand binds relatively tightly to LsrB. Such strong
binding may be produced, for example, when the shapes of the
binding site and ligand are mutually compatible (e.g., "lock and
key"), and/or when at least some of the ligand atoms are attracted
to at least some of the LsrB atoms in the vicinity of the binding
site by intermolecular forces, e.g., dipole-dipole interactions,
Van der Waals attractions, hydrogen-bonding, etc. A set of atomic
coordinates for the holo-LsrB crystal has been deposited in the
Protein Data Bank under accession codes 1TJY. The binding site for
the LsrB-ligand complex is shown schematically in FIG. 3A, left
panel.
[0039] Binding sites have significant utility in fields such as
drug discovery. The association of natural ligands with the binding
sites of their corresponding proteins, enzymes or receptors is the
basis of many biological mechanisms of action. Similarly, many
drugs exert their biological effects through association with the
binding sites of proteins, enzymes, and receptors. Such
associations may occur with all or any parts of the binding site.
An understanding of such associations enables the design of drugs
having more favorable associations with their target proteins,
enzymes or receptors, and thus, improved biological effects.
Therefore, this information is valuable in designing potential
inhibitors of the binding sites of biologically important
targets.
[0040] For example, the holo-LsrB structure can be used to
computationally dock compounds into the binding pocket. Compounds
with high affinity may block transport via LsrB, whether or not the
bound conformation closely resembles holo-LsrB. In another example,
the apoLsrB structure, or the two domains of the holo-LsrB
structure separately, can be used to search for compounds that bind
in the interdomain interface. Such compounds do not necessarily
need to bind to the exact same site as R-THMF and could prevent
LsrB from adopting the holo-LsrB conformation, thereby preventing
it from interacting functionally with the LsrC/D transporter. In
either case, the crystal structures are used to carry out virtual
screening. Potential "hits" can then be tested in quorum sensing
assays. The high resolution structures of holo- and apo-LsrB will
aid in such rationale design and search for LsrB ligands
[0041] Hence, the atomic coordinates of the apo-LsrB and holo-LsrB
can be used to identify whether a ligand binds to LsrB, and thus
may be used for a variety of purposes, such as drug discovery. A
preferred method comprises obtaining the atomic coordinates in the
crystal of at least a selected portion of LsrB. Preferably, the
selected portion comprises the ligand binding site. More
preferably, the selected portion includes the amino acid residue
found at the ligand binding site including residues Gln167, Asp116,
Pro220, Ala 222, Lys35 and Asp166. Lys35, Asp116 and Asp166 are
involved in hydrogen bonding with the R-THMF. Additionally,
hydrophobic residues near the methyl group of R-THMF include Phe41
and Leu265.
[0042] The atomic coordinates are preferably used to model the
selected portion. Such modeling is preferably accomplished by
storing crystallographic information about the selected portion on
a computer and then using the computer to translate the atomic
coordinates into the three-dimensional structure of the selected
portion of LsrB. Computers and software suitable for carrying out
these functions are commercially available. Computer packages
include Sybyl version 6.8 from Tripos, Inc. and MacroModel version
8.0 from Schrodinger Software. A potential ligand is then
identified, and the likelihood of binding between the ligand and
LsrrB is determined by docking the potential ligand to the selected
portion of holo-LsrB. Such docking preferably involves
computationally evaluating the ligand for its ability to bind with
LsrB, preferably using the commercially available computational
packages described above. Ligands that bind with LsrB are potential
drug candidates. The LsrB structure encoded by the crystallographic
data may be displayed in a graphical three-dimensional
representation on a computer screen. This allows visual inspection
of the structure, as well as visual inspection of the structure's
association with the ligand. Preferably, a computer is used for the
identifying of the potential ligand or the docking of the potential
ligand to the binding site, or both. A general review of
computation docking methods is found in Perola et al. (2004) and
Kellenberger et al. (2004).
[0043] After docking (preferably by the computational methods
described above) indicates that a particular ligand has the
potential to bind to LsrB, the interaction of the indicated ligand
is preferably examined by obtaining a sample of the potential
ligand and testing that ligand for activity. Preferably, the
compounds are tested in quorum-sensing assays using prokaryotic
cells, e.g., bacteria, to determine whether and to what extent the
ligand affects quorum sensing.
AI-2 Analogs
[0044] The final biosynthetic product in the AI-2 signaling pathway
is DPD. This molecule can cyclize to give two furanoketones, S-DHMF
and R-DHMF as shown in FIG. 1B. Each of these can add water
(hydrate) leading to S-THMF and R-THMF, also shown in FIG. 1B.
Addition of borate to S-THMF produces the AI-2 signaling molecule
for V. harveyi, S-THMF borate, which acts through binding to the
periplasmic protein, LuxP. The hydrated version R-THMF is the
member of this set which is active in Salmonella, binding to the
sugar transport protein, LsrB. The hydration reaction and boron
complexation are spontaneous for these molecules under
physiological conditions. DPD and these isomers are in rapid
equilibrium and are relatively unstable.
[0045] Accordingly, another aspect of the present invention
provides a series of stable compounds that exhibit
antagonist/agonist activity for AI-2. These compounds are also
referred to herein as AI-2 analogs. The discovery that LsrB binds
R-THMF provides a new mechanism of bacterial control.
[0046] These compounds were designed to satisfy three criteria. The
compounds of the invention were designed to be (1) chemically
stable, (2) capable of spontaneous, favorable hydration in the case
of analogs S-DHMF and R-DHMF, and (3) to optimize binding to the
receptor proteins via matching of the shape and positioning of
functional groups. These compounds of the present invention are
analogs of the monocyclic forms of DPD and hydrated DPD.
[0047] Compounds designated herein as Series A and B represent
agonists/antagonists which are direct analogs of monocyclic
structure S-DHMF. A preferred set of these compounds have one or
two fluoride substituents on the carbon at C-1 and C-5. The
electron-withdrawing effect of the fluoride favors hydration of the
carbonyl group at C-3 and mimics the natural signals, S-DHMF and
R-DHMF. The compounds represented in Series A and B are stable
toward ring opening when X is CH.sub.2, CFH or CF.sub.2 and give
static, cyclic structures.
[0048] The compounds of Series A are represented by formula A and
the compounds of Series B are represented by formula B in the
structures shown below:
##STR00011## [0049] wherein [0050] X is O, NH, S, CH.sub.2, CFH or
CF.sub.2; [0051] Y is hydrogen, hydroxyl, methyl or amino; [0052] Z
is hydroxyl or amino; and [0053] R is lower alkyl, aryl or alkenyl;
[0054] with the proviso that when X is O in formula A, then
simultaneously R cannot be methyl, Y cannot be hydroxyl and Z
cannot be hydroxyl.
[0055] Compounds designated herein as Series C and D represent
agonists/antagonists which have substituents positioned to mimic
closely the hydrated form S-THMF. In particular, the
stereoconfiguration at position C-2 parallels the arrangement in
S-THMF. Lacking a carbonyl group at C-3, these compounds are
generally stable with respect to hydroxy-keto exchange and loss of
water, and are capable of spontaneously binding borate to produce
analogs of S-THMF-borate.
[0056] The compounds of Series C are represented by formula C and
the compounds of Series D are represented by formula D in the
structures shown below:
##STR00012##
[0057] wherein [0058] W is hydroxyl or amino; [0059] X is O, NH, S,
CH.sub.2, CFH or CF.sub.2; [0060] Y is hydrogen, hydroxyl, methyl
or amino; [0061] Z is hydroxyl or amino; and [0062] R.sub.1 and
R.sub.2 are independently lower alkyl, aryl or alkenyl.
[0063] Compounds designated herein as Series E and F are
stereoisomers of those in Series C and D, also generally stable,
but cannot complex with borate at C-2/C-3. However, borate binding
is possible at C-3/C-4 when Y is OH or NH.sub.2.
[0064] The compounds of Series E are represented by formula E and
the compounds of Series F are represented by formula F in the
structures shown below:
##STR00013##
[0065] wherein [0066] W is hydroxyl or amino; [0067] X is O, NH, S,
CH.sub.2, CFH or CF.sub.2; [0068] Y is hydrogen, hydroxyl, methyl
or amino; [0069] Z is hydroxyl or amino; and [0070] R.sub.1 and
R.sub.2 are independently lower alkyl, aryl or alkenyl.
[0071] Compounds designated herein as Series G and H are the
hydrated analogs of the Series A and B compounds and spontaneously
dehydrate to be in equilibrium with the isomers with a carbonyl
group at C-3. In Series G, those compounds with X being O, NH, and
S can equilibrate through the same processes as represented in FIG.
1: reversible hydration at C-3 and reversible ring opening at
C-1/C-2. In series H, ring opening is possible only for the
compounds having X be O, NH, or S and simultaneously having Y be OH
or NH.sub.2.
[0072] The compounds of Series G are represented by formula G and
the compounds of Series H are represented by formula H in the
structures shown below:
##STR00014## [0073] wherein [0074] X is O, NH, S, CH.sub.2, CFH or
CF.sub.2; [0075] Y is hydrogen, hydroxyl, methyl or amino; [0076] Z
is hydroxyl or amino; and [0077] R is lower alkyl aryl or alkenyl;
[0078] with the proviso that when X is O in formula G, then
simultaneously R cannot be methyl, Y cannot be hydroxyl and Z
cannot be hydroxyl.
[0079] Compounds designated herein as Series I and J represent
agonists/antagonists which are direct analogs of monocyclic
structure R-DHMF. A preferred set of these compounds have one or
two fluoride substituents on the carbon at C-1 and C-5. The
electron-withdrawing effect of the fluoride favors hydration of the
carbonyl group at C-3 and mimics the natural signals, S-DHMF and
R-DHMF. The compounds represented in Series I and J are stable
toward ring opening when X is CH.sub.2, CFH or CF.sub.2 and give
static, cyclic structures.
[0080] The compounds of Series I are represented by formula I and
the compounds of Series J are represented by formula J in the
structures shown below:
##STR00015## [0081] wherein [0082] X is O, NH, S, CH.sub.2, CFH or
CF.sub.2; [0083] Y is hydrogen, hydroxyl, methyl or amino; [0084] Z
is hydroxyl or amino; and [0085] R is lower alkyl, aryl or alkenyl;
[0086] with the proviso that when X is O in formula I, then
simultaneously R cannot be methyl, Y cannot be hydroxyl and Z
cannot be hydroxyl.
[0087] Compounds designated herein as Series K and L represent
agonists/antagonists which have substituents positioned to mimic
closely the hydrated form R-THMF. In particular, the
stereoconfiguration at position C-2 parallels the arrangement in
R-THMF. Lacking a carbonyl group at C-3, these compounds are
generally stable with respect to hydroxy-keto exchange and loss of
water but cannot complex with borate.
[0088] The compounds of Series K are represented by formula K and
the compounds of Series L are represented by formula L in the
structures shown below:
##STR00016##
[0089] wherein [0090] W is hydroxyl or amino; [0091] X is O, NH, S,
CH.sub.2, CFH or CF.sub.2; [0092] Y is hydrogen, hydroxyl, methyl
or amino; [0093] Z is hydroxyl or amino; and [0094] R.sub.1 and
R.sub.2 are independently lower alkyl, aryl or alkenyl.
[0095] Compounds designated herein as Series M and N are
stereoisomers of those in Series K and L, also generally stable,
and are capable of spontaneously binding borate at C-2/C-3 to
produce R-THMF-borate analogs. Borate binding is also possible at
C-3/C-4 when Y is OH or NH.sub.2.
[0096] The compounds of Series M are represented by formula M and
the compounds of Series N are represented by formula N in the
structures shown below:
##STR00017##
[0097] wherein [0098] W is hydroxyl or amino; [0099] X is O, NH, S,
CH.sub.2, CFH or CF.sub.2; [0100] Y is hydrogen, hydroxyl, methyl
or amino; [0101] Z is hydroxyl or amino; and [0102] R.sub.1 and
R.sub.2 are independently lower alkyl, aryl or alkenyl.
[0103] Compounds designated herein as Series O and P are the
hydrated analogs of the Series I and J compounds and spontaneously
dehydrate to be in equilibrium with the isomers with a carbonyl
group at C-3. In Series O, those compounds with X being O, NH, and
S can equilibrate through the same processes as represented in FIG.
1: reversible hydration at C-3 and reversible ring opening at
C-1/C-2. In series P, ring opening is possible only for the
compounds having X be O, NH, or S and simultaneously having Y be OH
or NH.sub.2.
[0104] The compounds of Series 0 are represented by formula 0 and
the compounds of Series P are represented by formula P in the
structures shown below:
##STR00018## [0105] wherein [0106] X is O, NH, S, CH.sub.2, CFH or
CF.sub.2; [0107] Y is hydrogen, hydroxyl, methyl or amino; [0108] Z
is hydroxyl or amino; and [0109] R is lower alkyl aryl or alkenyl;
[0110] with the proviso that when X is O in formula O, then
simultaneously R cannot be methyl, Y cannot be hydroxyl and Z
cannot be hydroxyl.
[0111] As used herein, "lower alkyl" means both branched- and
straight-chain, saturated aliphatic hydrocarbon groups having 1 to
6 carbon atoms. Lower alkyl groups include, but are not limited to,
for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl and
the like.
[0112] As used herein, "alkenyl" means hydrocarbon chains of either
a straight or branched configuration and one or more unsaturated
carbon-carbon bonds, such as ethenyl, propenyl, and the like. Such
alkenyl groups have 2 to 6 carbon atoms.
[0113] As used herein, "aryl" includes "aryl" and "substituted
aryl." Thus "aryl" of this invention means any stable 6- to
14-membered monocyclic, bicyclic or tricyclic ring, containing at
least one aromatic carbon ring, for example, phenyl, naphthyl,
indanyl, tetrahydronaphthyl (tetralinyl) and the like. The presence
of substitution on the aryl group is optional, but when present,
the substituents can be halo, alkyl, alkoxy, hydroxyl, amino,
cyano, nitro, trifluoromethyl, acylamino or carbamoyl.
[0114] As used herein, "stable compound" or "stable structure"
means a compound that is sufficiently robust to survive isolation
to a useful degree of purity from a reaction mixture, and
formulation into an efficacious therapeutic agent. When it is clear
from the context, preferred stable compounds are those which are
chemically stable and do not readily isomerize in accordance with
the pathways shown in FIG. 1B.
[0115] As those of skill in the art appreciate, the actual chemical
stability of each compound will, however, vary depending on the
particular substituents and their positions relative to one
another. Methods to measure chemical stability are known to those
of skill in the art. Certain AI-2 analogs of the invention, while
sufficiently stable for isolation and formulation as therapeutic
agents, however, may undergo isomerization and ring opening. Such
compounds remain within the scope of stable compounds suitable for
uses as AI-2 analogs. Hence, the invention contemplates use of
isolated isomers, mixtures of isomers, isolated stereoisomers and
racemic mixtures of stereoisomers as therapeutic agents. Those AI-2
analogs expected to isomerize and undergo ring opening are those
compounds where X is O, NH or S and the X position in the ring is
adjacent to a carbon atom with an OH or NH.sub.2 group. For
example, such analogs include compounds of formula A with X being
O, NH or S at the C1 position and the C2 position having Z as OH or
NH.sub.2 as well as compounds of formula B, having X be O, NH or S
at the C5 position while the C4 position has Y as OH or NH.sub.2.
Similar combinations and positioning of substituents exist for the
series C-P compounds.
[0116] As used herein, "pharmaceutically acceptable salts" refer to
derivatives of the disclosed compounds that are modified by making
acid or base salts. Examples include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues and the like.
Pharmaceutically acceptable salts include, but are not limited to,
hydrohalides, sulfates, methosulfates, methanesulfates,
toluenesulfonates, nitrates, phosphates, maleates, acetates,
lactates and the like.
[0117] Pharmaceutically-acceptable salts of the compounds of the
invention can be prepared by reacting the free acid or base forms
of these compounds with a stoichiometric or greater amount of the
appropriate base or acid in water or in an organic solvent, or in a
mixture of the two; generally, nonaqueous media like ether, ethyl
acetate, ethanol, isopropanol, or acetonitrile are preferred. The
salts of the invention can also be prepared by ion exchange, for
example. Lists of suitable salts are found in Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton,
Pa., 1985, p. 1418, the disclosure of which is hereby incorporated
by reference in its entirety.
Synthesis of the AI-2 Analogs
[0118] A general procedure for preparation of the fluorine
containing analogs (Series A, B, I and J compounds) is illustrated
with the preparation of 5 as shown below in Scheme 1. Hydroxylation
of the cyclopentenone 1 with standard methods followed by
protection of the secondary hydroxyl group gives 2. Then
fluorination with one of several fluorinating agents gives 3.
Deprotection and oxidation gives the ketone 4. Standard
alpha-hydroxylation conditions produce 5. Examples of fluorinating
agents can be found, for example, in Chaddick, et al., (2001). For
the general synthesis method, see: Singh et al. (2002).
##STR00019##
[0119] Examples of structures in the Series C and D are synthesized
by a general method involving standard cis-hydroxylation of an
alkene:
##STR00020##
[0120] Examples of structures in the Series E, F, M and N are
synthesized by a general method involving standard
cis-hydroxylation of an alkene:
##STR00021##
[0121] Examples of structures in the Series G, H, O and P are
prepared by the addition of water to the corresponding ketones with
acid catalysis:
##STR00022##
[0122] The compounds of the invention can be synthesized using the
above methods or by methods known to those of skill in the art. The
methods outlined above can be improved by one skilled in the art
by, for instance, changing the temperature, duration, stoichiometry
or other parameters of the reactions. Any such changes are intended
to fall within the scope of this invention.
Uses of the AI-2 Analogs
[0123] "Autoinducer-2 analog" or "AI-2 analog" means any compound
of the Series A-Series P compounds. Such compounds may act to
inhibit AI-2 activity or to induce or enhance AI-2 activity. "AI-2
inhibition" refers to compounds that interfere with the ability of
the AI-2 moiety in a particular species to be detected, recognized,
or bound by its receptor, to act as a signal for luminescence,
bacterial growth, or pathogenesis, or any other activity controlled
by quorum sensing and includes molecules that degrade, sequester or
bind to AI-2, and the compounds act to inhibit or reduce the
activity of AI-2 to any degree. Such inhibition can be partial or
complete; "AI-2 activation" is similar except that the compounds
act to enhance or stimulate the activity of AI-2 to any degree.
[0124] Another embodiment of the invention provides a method of
regulating the activity of an autoinducer-2 (AI-2) receptor which
comprises contacting said AI-2 receptor with an AI-2 analog for a
time and in an amount sufficient to regulate said activity, wherein
said AI-2 analog is any one of the Series A-Series P compounds.
This method can be used for any bacterial species and thus can be
used with the AI-2 receptor is LsrB, LuxP or LuxQ or the equivalent
receptor from the bacterial species in question. The methods can be
conducted in vitro or in vivo, on cells or with extracts.
Regulation of activity can be assessed by any convenient
measurements means, such as assays for the level of AI-2, DPD
consumption or bioluminescence assays. These methods are well known
to those of skill in the art and some are described below in the
Examples. Preferably, AI-2 activity is regulated by the AI-2
analogs of the inventions when the AI-2 receptor is found on a
bacterial cell, as distinguished from the receptor being in an
extract or cell lysate.
[0125] The regulated activities include any associated with or
regulated in response to quorum sensing and can be regulated both
positively or negatively, i.e., the compounds can activate or
inhibit AI-2 activity. Examples of activities that can be regulated
include, but are not limited to, bacterial cell growth, siderophore
expression, bacterial virulence, biofilm formation
exopolysaccharide production in bacterial cells and bacterial
colony morphology. In the case of siderophore expression, the
activity can preferably be inhibition of siderophore expression.
For exopolysaccharide production, the activity includes rugose
polysaccharide production. With bacterial colony morphology, the
activity is smooth colony morphology formation.
[0126] This method can be used to regulate the AI-2 receptor when
the bacterial cell is found in a warm blooded host. Warm-blooded
hosts includes domesticated animals (including pets and livestock),
humans, rodents, primates and other mammals.
[0127] In accordance with the invention, the AI-2 receptor is
preferably on or from a bacterial cell of one of the following
species: V. harveyi, V. cholerae, V parahaemolyticus, V.
alginolyticus, Pseudomonas phosphoreum, Yersinia enterocolitica, E.
coli, S. typhimurium, S. typhi, Haemophilus influenzae,
Helicobacter pylori, Bacillus subtilis, Borrelia burgfdorferi,
Neisseria meningitidis, Neisseria gonorrhoeae, Yersinia pestis,
Campylobacter jejuni, Deinococcus radiodurans, Mycobacterium
tuberculosis, Enterococcus faecalis, Streptococcus pneumoniae,
Streptococcus pyogenes and Staphylococcus aureus.
[0128] The invention further provides methods of inhibiting the
infectivity of a pathogenic organism as well as therapeutic
compositions containing the AI-2 analogs of the present invention.
The methods comprise administering to a subject a therapeutically
effective amount of a pharmaceutical composition that inhibits the
activity of naturally-occurring AI-2.
[0129] When used therapeutically, the Series A to P compounds of
the invention are administered in a "therapeutically-effective
amount. Such an amount refers to that amount necessary to
administer to a host to inhibit or activate the pathways regulated
by quorum sensing, including, but not limited, to virulence gene
expression, biofilm formation, production of antibiotic, to
modulate bioluminescence, to inhibit siderophore production, to
inhibit exopolysaccharide and/or to modulating the mammalian
inflammatory response and particularly for ameliorating or reducing
inflammation in inflammatory diseases and conditions associated
with production of IL-1 and IL-6. Those compounds which act as
inhibitors of AI-2 induced responses are also therapeutically
useful as antibiotics. Methods of determining
therapeutically-effective amounts are well known.
Pharmaceutical Preparations
[0130] The Series A to P compounds of the invention can be
formulated as pharmaceutical compositions comprising one or more of
those molecules together with a pharmaceutically acceptable
carrier. Pharmaceutically acceptable carriers can be sterile
liquids, such as water and oils, including those of petroleum,
animal, vegetable or synthetic origin, such as peanut oil, soybean
oil, mineral oil, sesame oil and the like. Water is a preferred
carrier when the pharmaceutical composition is administered
intravenously. Saline solutions and aqueous dextrose and glycerol
solutions can also be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical carriers are
described in Gennaro et al., (1995). In addition to the
pharmacologically active agent, the compositions can contain
suitable pharmaceutically acceptable carriers comprising excipients
and auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically for delivery
to the site of action. Suitable formulations for parenteral
administration include aqueous solutions of the active compounds in
water-soluble form, for example, water-soluble salts. In addition,
suspensions of the active compounds, as appropriate in oily
injection suspensions may be administered. Suitable lipophilic
solvents or vehicles include fatty oils, for example, sesame oil or
synthetic fatty acid esters, for example, ethyl oleate or
triglycerides. Aqueous injection suspensions can contain substances
which increase the viscosity of the suspension include, for
example, sodium carboxymethyl cellulose, sorbitol, and dextran.
Optionally, the suspension can also contain stabilizers. Liposomes
can also be used to encapsulate the agent for delivery into the
cell.
[0131] The pharmaceutical formulation for systemic administration
according to the invention can be formulated for enteral,
parenteral or topical administration. Indeed, all three types of
formulations can be used simultaneously to achieve systemic
administration of the active ingredient.
[0132] Suitable formulations for oral administration include hard
or soft gelatin capsules, pills, tablets, including coated tablets,
elixirs, suspensions, syrups or inhalations and controlled release
forms thereof.
[0133] The Series A to P compounds of the invention can also be
incorporated into pharmaceutical compositions which allow for the
sustained delivery of those compounds to a mammal for a period of
several days, to at least several weeks, to a month or more. Such
formulations are described in U.S. Pat. Nos. 5,968,895 and
6,180,608 B1.
[0134] For topical administration, any common topical formation
such as a solution, suspension, gel, ointment or salve and the like
can be employed. Preparation of such topical formulations are well
described in the art of pharmaceutical formulations as exemplified,
for example, by Remington's Pharmaceutical Sciences. For topical
application, the Series A to P compounds of the invention can also
be administered as a powder or spray, particularly in aerosol form.
The active ingredient can be administered in pharmaceutical
compositions adapted for systemic administration. As is known, if a
drug is to be administered systemically, it can be confected as a
powder, pill, tablet or the like or as a syrup or elixir for oral
administration. For intravenous, intraperitoneal or intra-lesional
administration, the active ingredient will be prepared as a
solution or suspension capable of being administered by injection.
In certain cases, it may be useful to formulate the active
ingredient in suppository form or as an extended release
formulation for deposit under the skin or intramuscular injection.
In a one embodiment, quorum sensing regulators can be administered
by inhalation. For inhalation therapy the compound can be in a
solution useful for administration by metered dose inhalers or in a
form suitable for a dry powder inhaler.
[0135] It will be appreciated by those skilled in the art that
various omissions, additions and modifications may be made to the
invention described above without departing from the scope of the
invention, and all such modifications and changes are intended to
fall within the scope of the invention, as defined by the appended
claims. All references, patents, patent applications or other
documents cited are herein incorporated by reference in their
entirety.
Example 1
Experimental Procedures
A. LsrB Production
[0136] S. typhimurium LsrB without its amino-terminal signal
peptide (residues 1-26) was cloned into plasmid pGEX4TI for
expression as a glutathione-S-transferase (GST) fusion protein in
E. coli strain BL21. Protein expression was induced by the addition
of 0.1 mM isopropyl .beta.-D-thiogalactopyranoside for 6 hr prior
to harvesting the bacteria. The GST-LsrB fusion protein was
purified by glutathione agarose affinity chromatography. The GST
tag was removed by thrombin digestion, leaving two additional
residues at the N terminus (GlySer) of LsrB. The protein was
further purified by hydrophobic affinity chromatography (Phenyl
Superose; Pharmacia) and size-exclusion chromatography (Superdex
200; Pharmacia). LsrB (>95% pure) was concentrated for
crystallization experiments to 8 mg/ml in 20 mM Tris-HCl (pH 8.0),
150 mM NaCl, and 1 mM dithiothreitol. Selenomethionyl protein was
overexpressed in E. coli B834. Cells were grown in M9 medium as
described in Doublie' (Doublie', 1997) with selenomethionine at 0.3
mM. Purification was the same as for the native protein.
B. Crystallization and Diffraction Data Collection
[0137] Both apo-LsrB and holo-LsrB crystallized by the hanging drop
method in 0.1 M Tris-HCl (pH 8.5), 22%-25% PEG 4000 (w/v) in space
group P2.sub.12.sub.12.sub.1. The apo-LsrB crystals initially
obtained (a=38.0, b=74.0, c=116.2) were used to seed
crystallization in 0.1 M Tris-HCl, pH 8.5, 18%-24% PEG 4000.
Crystals were cryoprotected by brief soaks in 0.1 M Tris-HCl, pH
8.5, 20% PEG 4000, 16% (v/v) glycerol and flash frozen in liquid
nitrogen. Native crystals diffracted to 1.9 .ANG. and data were
collected at 100 K using an R-AXIS-IV image plate detector mounted
on a Rigaku 200HB generator. Selenomethionine LsrB crystals were
grown and frozen in the same conditions as native crystals.
Selenomethionine crystals diffracted to 2.1 .ANG. resolution at
NSLS beam line X25, where MAD data were collected using an ADSC
Q315 CCD detector. Holo-LsrB crystals (a=37.8, b=76.6, c=109.7)
were prepared by addition of approximately 0.25 mM in vitro LuxS
reaction product (Schauder et al., 2001) to the native LsrB
crystallization conditions, giving a DPD:LsrB molar ratio of
slightly over 1:1. Crystals diffracted to 1.3 .ANG. resolution at
NSLS beam line X25. To test the possibility that LsrB can bind a
borated adduct of DPD, crystals of LsrB were grown as above with
both .about.0.25 mM in vitro LuxS reaction product and 0.5 or 5 mM
boric acid. Crystals grown under these conditions were isomorphous
with native crystals. Data were collected at NSLS beam line X25
where crystals diffracted to 1.3 .ANG. for 0.5 mM boric acid and
2.0 .ANG. for 5 mM boric acid. In all cases, data were processed
using the HKL package (Otwinowski and Minor, 1998).
C. Structure Determination and Refinement
[0138] Positions of the selenium atoms were determined using SOLVE
(Terwilliger et. al., 1999) with subsequent density modification
and initial automatic model building by RESOLVE (Terwilliger,
2002). The automatically generated partial model was used as a
starting point for model building using the program O (Jones et
al., 1991). The apo-LsrB structure was refined using native data to
1.9 .ANG. and water molecules added with the program CNS (Brunger
et al., 1998). The final model contains all LsrB residues present
in the protein (27-340) plus 348 ordered water molecules and has
good geometry (Table 1) with only one residue (Asp116) outside of
the allowed regions of the Ramachandran plot (see below).
[0139] The structure of holo-LsrB was solved via molecular
replacement using CNS, treating the two domains of apo-LsrB as
separate objects in the search. The model was built in 0 and
refined with CNS and CCP4 (CCP4, 1994) to 1.3 .ANG. resolution.
Asp116 again lies in a disallowed region of the Ramachandran plot,
with both backbone and side chain conformations identical to those
observed in apoLsrB. Its proper positioning is nevertheless
unambiguous in both the 1.9 .ANG. apo-LsrB and 1.3 .ANG. holo-LsrB
electron density maps. The ligand present in the holo-LsrB crystals
was not built until the R.sub.cryst and R.sub.free, had dropped to
0.18 and 0.20, respectively, and water molecules had been included
in the model (but not in the ligand binding site). The electron
density in the binding site was well ordered and clearly
interpretable and was modeled as
(2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF). The
ligand was refined using CNS, with parameter files generated by the
HIC-Up server (Kleywegt et al., 1998). Given the high resolution of
the data, the geometric terms were relaxed during later cycles of
refinement. Omitting all waters and examining a simulated annealing
omit map revealed 11 side chains with multiple conformations. The
final model contains 399 water molecules and the two heterologous
N-terminal residues (Gly-Ser) remaining after removal of the GST
tag. Molecular images were prepared using PyMOL (DeLano, 2002).
[0140] The structures of the holo-LsrB complex crystallized in the
presence of 0.5 and 5 mM boric acid were determined by molecular
replacement using holo-LsrB with the ligand omitted via the program
EPMR (Kissinger et al., 1999). The structures were partially
refined to R.sub.free values of 24.3 (data to 1.3 .ANG.) and 24.0
(data to 2.0 .ANG.), respectively, by which point it was clear that
the ligand was identical to that present in the fully refined
holo-LsrB complex.
D. .sup.11B-NMR
[0141] Holo-LuxP was purified as described previously (Chen et al.,
2002), exchanged into NMR buffer (20 mM potassium phosphate [pH
7.5], 150 mM NaCl, 1 mM dithiothreitol) using a small gel
filtration column (PD10; Amersham Biosciences), and concentrated to
200 .mu.M. To prepare holo-LsrB, GST-LsrB was incubated overnight
with an approximately equimolar amount of in vitro LuxS reaction
product (Schauder et al., 2001). Unbound ligand was removed by
immobilizing the protein on glutathione agarose beads and washing
extensively with NMR buffer. Finally, the fusion protein was eluted
using NMR buffer plus 10 mM glutathione and concentrated to
approximately 1 mM. .sup.11B NMR spectra were collected on each
sample before and after addition of boric acid to a final
concentration of 5 mM. Then, each sample was heated 3 min at
70.degree. C. to release the ligand from the protein, the denatured
protein was pelleted, and spectra were collected for the
ligand-containing supernatants. All .sup.11B NMR spectra were
collected at 4.degree. C. using a Varian Unity/INOVA spectrometer
at 128.4 Mhz equipped with a 8 mm tunable X/.sub.1H probe (Nalorac)
and were referenced to BF.sub.3O(Et)2. 180,000 scans were averaged
for each spectrum with a 0.25 s recycle time using an approximately
30.degree. flip-angle pulse.
E. Bacterial Strains and Growth Conditions
[0142] V. harveyi strain MM32 (luxN::Cm, luxS::Tn5Kan) was used for
bioluminescence assays. This strain was constructed by introducing
luxS::Tn5Kan onto the chromosome of strain JAF305 (luxN::Cm)
(Bassler et al., 1993; Freeman et al., 1999). S. typhimurium strain
MET844 (rpsL, putRA::Kan-lsr-lacZYA, .DELTA.lsrFGE::Cm, luxS::TPOP)
was used for lsr-lacZ assays (Taga et al., 2003). V. harveyi was
grown in borate-depleted autoinducer bioassay (AB) medium
(Greenberg et al., 1979), and S. typhimurium was grown in
borate-depleted Luria-Bertani (LB) medium. To remove borate, the
media were filtered through a borate anion-specific resin,
Amberlite IRA743 (Sigma-Aldrich). Specifically, 500 ml of medium
was passed three times through 30 ml of resin and the column was
regenerated between each passage according to a method described
previously (Bennett et al., 1999). Following filtration, the pH of
the medium was adjusted using KOH made with borate-depleted water.
For all experiments involving borate-depleted reagents, only
plastic supplies were used. To test the effect of boron on the
bioluminescence and lsr-lacZ assays, boric acid was added to the
borate-depleted media to a final concentration of 5 mM. As
expected, the addition of boric acid (pK.sub.a=9.2) did not affect
the pH of the media. The presence or absence of boric acid had no
effect on the growth of either organism.
F. AI-2 Bioassays
[0143] V. harveyi MM32 was grown 14 hr in borate-depleted AB at
30.degree. C. with aeration and subsequently diluted 1:5,000 into
fresh borate-depleted AB medium in the presence or absence of 5 mM
boric acid. 10% autoinducer samples (v/v) were added to the diluted
cells and light production was measured hourly in a Wallac Model
1450 Microbeta Plus liquid scintillation counter. In the presence
of 5 mM borate, addition of 0.1-1 nM (final concentration) DPD to
the MM32 reporter strain induced a linear response in light
production following 6-8 hr incubation. Bioluminescence is reported
as the light produced by the cells divided by the background
obtained in medium alone.
[0144] AI-2-dependent induction of the lsr operon in S. typhimurium
was measured by determining the .beta.-galactosidase activity of
the lsr-lacZ promoter fusion in S. typhimurium strain MET844.
Overnight cultures were grown in borate-depleted LB medium at
37.degree. C. with aeration and were diluted 1:100 into fresh
borate-depleted LB medium in the presence or absence of 5 mM boric
acid. To the diluted cells (900 .mu.l), 10% (v/v) autoinducer
samples were added (100 .mu.l), and cells were grown for 4 hr. Cell
lysates were prepared and .beta.-galactosidase activity was
measured as described previously (Taga et al., 2003).
.beta.-galactosidase units are defined as [(OD.sub.420 min.sup.-1 X
dilution factor)/OD.sub.600].
[0145] Ligands were released from LsrB and LuxP as described above.
All ligand concentrations were estimated by .sup.11B-NMR in NMR
buffer supplemented with 5 mM boric acid; the area of the boric
acid peak served as an internal concentration standard.
G. IUPAC Nomenclature
[0146] The IUPAC carbohydrate nomenclature for the structures in
FIG. 1B is as follows: DPD, L-glycero-1-dehydro-penta-2,3-diulose;
S-DHMF, .alpha.-L-glycero-1-dehydro-penta-2,3-diulo-2,5-furanose;
S-THMF,
.alpha.-L-glycero-1-dehydro-3-hydro-penta-2,3-diulo-2,5-furanose;
S-TH F-borate,
.alpha.-L-glycero-1-dehydro-3-hydro-penta-2,3-diulo-2,5-furanos-
yl-2,3-cyclic borate; R-DHMF,
.beta.-L-glycero-1-dehydro-penta-2,3-diulo-2,5-furanose; R-THMF,
.beta.-L-glycero-1-dehydro-3-hydro-penta-2,3-diulo-2,5-furanose.
H. Accession Numbers
[0147] Atomic coordinates for apo-LsrB and holo-LsrB have been
deposited in the Protein Data Bank under accession codes 1TM2 and
1TJY, respectively.
Example 2
Structure of LsrB
[0148] The structure of S. typhimurium LsrB was determined to 2.1
.ANG. resolution using multiwavelength anomalous diffraction (MAD)
phasing and subsequently refined to 1.9 .ANG. resolution (Table 1).
Despite low sequence identity (11%), LsrB exhibits the same fold as
the V. harveyi AI-2 signaling receptor LuxP (Chen et al., 2002),
with a three-stranded hinge connecting two similar .alpha./.beta.
domains (FIG. 2A). LsrB also has strong structural homology with
several other sugar binding proteins including E. coli ribose
binding protein (RBP) and S. typhimurium galactose binding protein,
as well as repressors such as E. coli purine nucleotide synthesis
repressor and trehalose repressor (Hars et al., 1998; Mowbray et
al., 1992; Mowbray et al., 1983; Schumacher et al., 1994). In
periplasmic binding proteins, including LuxP, the ligand binding
site is near the hinge between the two domains. In the crystal
structure of LsrB, the domains are in an open conformation similar
to, though less pronounced than, the open conformations observed in
unliganded RBP (Bjorkman and Mowbray, 1998), leaving the putative
binding site exposed to solvent (FIG. 2B). While several
well-ordered water molecules are visible in this region, no density
corresponding to an autoinducer molecule can be identified. This
structure is apo-LsrB.
TABLE-US-00001 TABLE 1 Phasing and Refinement Statistics Apo SeMet
Holo Native Peak Inflection Remote Native Wavelength (.ANG.) 1.5418
0.9789 0.9793 0.9500 1.1000 Resolution (.ANG.) 1.9 2.1 2.1 2.1 1.3
Unique reflections 26,175 18,993 18,917 18,650 77,033 R.sub.sym %
(outer shell) 4.3 (17) 7.2 (20) 6.4 (22) 7.0 (25) 8.2 (28) I/_I
(outer shell) 14.4 (4.7) 10.0 (4.9) 11.5 (4.0) 10.5 (3.7) 9.7 (3.4)
Complete (%) 98.3 99.8 98.7 97.5 97.0 Anomalous Phasing at 2.1
.ANG. Heavy atom sites 6 Overall FOM 0.63 Refinement Apo Holo
Resolution (.ANG.) 60-1.9 63-1.3 R.sub.cryst/R.sub.free 0.191/0.227
0.156/0.172 Rms deviation Bond length (.ANG.) 0.005 0.006 Bond
angle (.degree.) 1.30 1.26 Dihedrals (.degree.) 22.74 22.63
Improper (.degree.) 0.85 0.88 Average B factor Protein 18.55 9.52
Ligand -- 13.14 Water 32.07 22.55 All atoms 20.26 11.24 R.sub.sym =
.SIGMA..sub.h.SIGMA..sub.i|I.sub.i(h) - <
I(h)>|/.SIGMA..sub.h.SIGMA..sub.iI.sub.i(h), where I.sub.i(h) is
the ith measurement of h and <I(h)>is the mean of all
measurements of I(h) for reflection h. R.sub.free is R.sub.cryst
calculated with only the test set (5%) of reflections. FOM, figure
of merit.
Example 3
Structure of the LsrB:Ligand Complex
[0149] To identify the LsrB ligand, LsrB was crystallized in the
presence of DPD and the other products generated by incubating SAH
with recombinant Pfs and LuxS enzymes as previously described
(Schauder et al., 2001) (FIG. 1A). The structure was determined by
molecular replacement and refined to 1.3 .ANG. resolution (Table
1). In this structure, the domains of LsrB have closed around the
binding site, rotating shut about the hinge region by 21.degree.
relative to one another (FIG. 2B) (Hayward et al., 2002).
Nonprotein electron density is prominent between the two domains
(FIG. 2C) in a location analogous to the ligand binding sites of
LuxP and other periplasmic binding proteins. As detailed below,
this electron density is consistent with R-THMF, a DPD derivative
not previously known to be biologically active (FIGS. 2D and
2E).
[0150] The LsrB ligand R-THMF differs from the V. harveyi LuxP
ligand S-THMF-borate in two respects (FIG. 3A). First, no borate is
present. Second, the stereochemistry of the LsrB ligand appears to
be opposite to that of the LuxP ligand at position 2, the anomeric
center (see FIG. 1B). This stereochemical assignment is supported
by the crystallographic data. Specifically, examination of a 1.3
.ANG. resolution F.sub.o-F.sub.c map at high contour level shows
stronger electron density in the position modeled as a hydroxyl
group (FIG. 2E). Indeed, the oxygens in the ligand all display
stronger electron density in the F.sub.o-F.sub.c map than the
carbons (FIG. 2E). (To eliminate phase bias, the map was calculated
using a model in which the ligand had never been present and from
which any water molecules in or around the ligand binding site had
been removed.) In addition, the proposed stereochemistry places the
methyl group in a hydrophobic environment (the nearest residues are
Phe41 and Leu265, 3.6 and 3.9 .ANG. away, respectively), while the
hydroxyl group is situated near polar atoms, the closest of which
is the backbone oxygen of Pro220 (3.3 .ANG. away). A possibility
remains that the S stereoisomer, or a mixture of the R and S
stereoisomers, is present in the crystal structure, and their
chemical interconvertability prevents us from testing their
biological activity individually. Nonetheless, both the electron
density and the placement of polar and hydrophobic residues within
the binding site support the identification of the LsrB ligand as
R-THMF (FIG. 3A).
Example 4
Comparison of Ligand Binding by LsrB and LuxP
[0151] While LsrB and LuxP share the same fold, their binding sites
are distinctive and appear to be designed to accommodate different
ligands (FIGS. 3A-3D). Key residues involved in hydrogen bonding
between LuxP and S-THMF-borate are not conserved in LsrB. Gln77,
Ser79, and Thr266, polar residues in the LuxP binding site that
hydrogen bond with S-THMF-borate, are replaced in LsrB by the
nonpolar residues Val39, Gly40, and Ala222, respectively. LuxP
residues Arg215 and Arg310, each of which makes multiple hydrogen
bonds with S-THMF-borate, are replaced in LsrB by Trp170 and
Trp266, respectively, whose ring nitrogens are too distant from
R-THMF to participate in hydrogen bonding. LuxP Trp82, whose ring
nitrogen hydrogen bonds with S-THMF-borate, corresponds to Phe42 in
LsrB.
[0152] Perhaps the most striking difference between the LsrB and
LuxP ligand binding sites is that they differ in net charge (FIG.
3A). The binding site in LsrB has three charged residues, Lys35,
Asp116, and Asp166. Lys35 and Asp166 are positioned to form a salt
bridge, neutralizing their respective charges, while still
contributing to ligand binding. Asp116 does not have a salt bridge
partner, leaving a net negative charge in the binding pocket. This
contrasts with the binding pocket in LuxP, which contains two
positively charged residues (Arg215 and Arg310) that stabilize the
negative charge on S-THMF-borate. The negative charge in the LsrB
binding pocket makes it unlikely that S-THMF-borate would bind to
LsrB.
[0153] The furanosyl rings of the two DPD derivatives are oriented
differently in the LsrB and LuxP binding pockets (compare FIGS. 3B
and 3C). The furanosyl ring in LsrB occupies roughly the same
position as the boratering in LuxP and the ribose ring in RBP.
Barring a large structural rearrangement, there does not appear to
be sufficient room in the LsrB binding pocket to accommodate
S-THMF-borate. Overall, because of hydrogen bonding, electrostatic,
and steric differences, LsrB and LuxP have binding sites that
accommodate chemically distinct signaling molecules derived from
the same precursor, DPD. Consistent with this proposal, LsrB
crystallized in the presence of both DPD and boric acid (0.5 or 5
mM) displayed electron density (at 1.3 and 2.0 .ANG., respectively)
in the ligand binding pocket indistinguishable from the ligand
density in holo-LsrB. Thus, even with the addition of high levels
of boric acid, the analyzed crystals of S. typhimurium LsrB have
never been observed to bind the borated form of AI-2 responsible
for quorum sensing in V. harveyi.
Example 5
LsrB and LuxP Bind Different Ligands in Solution
[0154] .sup.11B-NMR was used to establish that LsrB and LuxP bind
specifically to different ligands in solution (FIG. 4). Consistent
with previous results (Chen et al., 2002), holo-LuxP displays a
single boron peak at 6.1 ppm, indicating the presence of the bound
S-THMF-borate (FIG. 4A, top trace). This peak was unaffected by the
addition of 5 mM boric acid, although the boric acid itself gives
rise to a large peak at 18.8 ppm (FIG. 4A, middle trace). (Borate,
with a pK.sub.a of 9.2, is present almost entirely as undissociated
boric acid at physiological pH.) After heating the holo-LuxP/boric
acid sample to denature LuxP and release the ligand, and removing
the denatured protein by centrifugation, a new peak appeared at 5.8
ppm (FIG. 4A, bottom trace). This new peak likely corresponds to
THMF-borate, the small change in chemical shift reflecting the
altered chemical environment of the released ligand. A small peak
at 9.5 ppm may arise from molecules in which two five-membered
furanosyl rings are crosslinked by a single borate; such compounds
have characteristic .sup.11B NMR chemical shifts of 6.9-11.1 ppm
(van den Berg et al., 1994).
[0155] For comparison, an identical set of experiments was carried
out using GST-LsrB preincubated with the same in vitro DPD
synthesis reaction products used for crystallization of holo-LsrB;
unbound ligand was chromatographically removed. In this case, no
boron peak was observed in .sup.11B-NMR spectra (FIG. 4B, top
trace). Addition of 5 mM boric acid had no effect on the NMR
spectrum (FIG. 4B, middle trace). In a separate experiment,
simultaneous incubation of unliganded GST-LsrB with both 5 mM
borate and the in vitro DPD synthesis reaction products gave
identical results. Thus, within the detection limits of this
experiment, LsrB does not bind a borated derivative of DPD.
Strikingly, however, thermal release of the bound LsrB ligand into
5 mM boric acid (FIG. 4B, bottom trace) led to the appearance of a
peak at 5.8 ppm, exactly as observed upon release of the bound LuxP
ligand. This result is consistent with the chemical scheme in FIG.
1B according to which, upon release into excess boric acid, THMF
would be converted spontaneously into THMF-borate. Taken together,
the .sup.11B-NMR results are in agreement with the hypothesis,
based on crystallographic evidence, that LsrB binds an unborated
ligand. Furthermore, they indicate directly that the LsrB ligand,
once released, can be converted into a borated form. This property
may underlie the ability of AI-2 activity secreted by S.
typhimurium to stimulate light production in V. harveyi (Bassler et
al., 1997; Surette et al., 1999).
Example 6
V. harveyi and S. typhimurium AI-2 Bioassays
[0156] The crystallographic and .sup.11B NMR results imply that V.
harveyi and S. typhimurium recognize different derivatives of DPD,
one that contains boron and one that does not. That this
distinction was not previously recognized may stem, in part, from
the ability of the molecules to interconvert, as indicated by
chemical considerations, earlier functional studies, and the NMR
results (Bassler et al., 1997; Chen et al., 2002; Meijler et al.,
2004; Schauder et al., 2001; Surette et al., 1999) (FIG. 4). To
test these ideas further, V. harveyi and S. typhimurium bioassays
were used to examine whether AI-2 signaling molecules released from
the two receptors are in equilibrium with one another. Furthermore,
the position of this equilibrium, and thus the signaling activity,
can be influenced by the presence of boric acid. The model (FIG.
1B) predicts that boric acid should enhance AI-2 signaling in V.
harveyi but inhibit AI-2 signaling in S. typhimurium.
[0157] To directly examine the influence of borate on
AI-2-dependent signaling in V. harveyi and S. typhimurium,
borate-depleted medium was prepared. AI-2 responses
(bioluminescence in V. harveyi, lsr operon induction in S.
typhimurium) were measured both with and without added boric acid.
In this experiment, V. harveyi strain, MM32 was used because this
strain lacks the LuxS enzyme needed to biosynthesize DPD and thus
produces no endogenous AI-2 signal. This strain also has the AI-1
pathway inactivated. Bioluminescence was measured following
addition of enzymatically synthesized DPD or, alternatively, ligand
released from either LsrB or LuxP.
[0158] No light was produced when the V. harveyi reporter strain
was exposed to DPD or to the released LsrB ligand in
borate-depleted medium (FIG. 5A, black bars). The released LuxP
ligand also failed to stimulate light production. The inability of
the released V. harveyi ligand to stimulate V. harveyi light
production suggests that, in borate-depleted medium, S-THMF-borate
dissociates into THMF and boric acid (see FIG. 1B). In all cases, a
large increase in ligand-stimulated light production occurred when
boric acid (5 mM) was added to the borate-depleted medium (FIG. 5A,
white bars). The ability of both DPD and the released S.
typhimurium ligand to stimulate light production in the presence of
boric acid confirms that DPD and THMF are in equilibrium with one
another and that THMF, once borated, is active in V. harveyi AI-2
signaling (Meijler et al., 2004).
[0159] AI-2 induction of the lsr operon of S. typhimurium can be
monitored by measuring .beta.-galactosidase activity in strain
MET844 (lsr-lacZ, luxS.sup.-). FIG. 5B shows that, even in
borate-depleted medium, addition of DPD or the ligand released from
either LsrB or LuxP induces lsr expression (black bars). Note that
approximately 2.5-fold less LuxP ligand was used in this experiment
compared to DPD and the LsrB ligand. Strikingly, whereas boric acid
strongly enhances the ability of DPD or either receptor's ligand to
activate the V. harveyi assay (FIG. 5A), it inhibits the ability of
the same molecules to activate the S. typhimurium assay (FIG.
5B).
[0160] The results demonstrate that borate is required for the
AI-2-response in V. harveyi but inhibits the AI-2 response in S.
typhimurium. These findings are consistent with the model shown in
FIG. 1B, which posits that DPD, R-THMF, and S-THMF-borate are in
equilibrium with one another. This equilibrium, as expected, can be
shifted toward borated forms by the addition of boric acid, and
toward unborated forms by borate depletion. Hence, in accordance
with the crystallographic results, it appears that R-THMF is the
active species for S. typhimurium AI-2 signaling whereas
S-THMF-borate is the active species for V. harveyi AI-2
signaling.
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Sequence CWU 1
1
21306PRTSalmonella typhimurium 1Ala Glu Arg Ile Ala Phe Ile Pro Lys
Leu Val Gly Val Gly Phe Phe1 5 10 15Thr Ser Gly Gly Asn Gly Ala Gln
Glu Ala Gly Lys Ala Leu Gly Ile 20 25 30Asp Val Thr Tyr Asp Gly Pro
Thr Glu Pro Ser Val Ser Gly Gln Val 35 40 45Gln Leu Val Asn Asn Phe
Val Asn Gln Gly Tyr Asp Ala Ile Ile Val 50 55 60Ser Ala Val Ser Pro
Asp Gly Leu Cys Pro Ala Leu Lys Arg Ala Met65 70 75 80Gln Arg Gly
Val Lys Ile Leu Thr Trp Asp Ser Asp Thr Lys Pro Glu 85 90 95Lys Arg
Ser Tyr Tyr Ile Asn Gln Gly Thr Pro Lys Gln Leu Gly Ser 100 105
110Met Leu Val Glu Met Ala Ala His Gln Val Asp Lys Glu Lys Ala Lys
115 120 125Val Ala Phe Phe Tyr Ser Ser Pro Thr Val Thr Asp Gln Asn
Gln Trp 130 135 140Val Lys Glu Ala Lys Ala Lys Ile Ser Gln Glu His
Pro Gly Trp Glu145 150 155 160Ile Val Thr Thr Gln Phe Gly Tyr Asn
Asp Ala Thr Lys Ser Leu Gln 165 170 175Thr Ala Glu Gly Ile Ile Lys
Ala Tyr Pro Asp Leu Asp Ala Ile Ile 180 185 190Ala Pro Asp Ala Asn
Ala Leu Pro Ala Ala Ala Gln Ala Ala Glu Asn 195 200 205Leu Lys Arg
Asn Asn Leu Ala Ile Val Gly Phe Ser Thr Pro Asn Val 210 215 220Met
Arg Pro Tyr Asn Gln Arg Gly Thr Val Lys Glu Phe Gly Leu Trp225 230
235 240Asp Val Val Gln Gln Gly Lys Ile Ser Val Tyr Val Ala Asn Ala
Leu 245 250 255Leu Lys Asn Met Pro Met Asn Val Gly Asp Ser Leu Asp
Ile Pro Gly 260 265 270Ile Gly Lys Val Thr Val Ser Pro Asn Ser Glu
Gln Gly Tyr His Tyr 275 280 285Glu Ala Lys Gly Asn Gly Ile Val Leu
Leu Pro Glu Arg Val Ile Phe 290 295 300Asn Lys3052279PRTVibrio
harveyi 2Pro Ile Lys Ile Ser Val Val Tyr Pro Gly Gln Gln Val Ser
Asp Tyr1 5 10 15Trp Val Arg Asn Ile Ala Ser Phe Glu Lys Arg Leu Tyr
Lys Leu Asn 20 25 30Ile Asn Tyr Gln Leu Asn Gln Val Phe Thr Arg Pro
Asn Ala Asp Ile 35 40 45Lys Gln Gln Ser Leu Ser Leu Met Glu Ala Leu
Lys Ser Lys Ser Asp 50 55 60Tyr Leu Ile Phe Thr Leu Asp Thr Thr Arg
His Arg Lys Phe Val Glu65 70 75 80His Val Leu Asp Ser Thr Asn Thr
Lys Leu Ile Leu Gln Asn Ile Thr 85 90 95Thr Pro Val Arg Glu Trp Asp
Lys His Gln Pro Phe Leu Tyr Val Gly 100 105 110Phe Asp His Ala Glu
Gly Ser Arg Glu Leu Ala Thr Glu Phe Gly Lys 115 120 125Phe Phe Pro
Lys His Thr Tyr Tyr Ser Val Leu Tyr Phe Ser Glu Gly 130 135 140Tyr
Ile Ser Asp Val Arg Gly Asp Thr Phe Ile His Gln Val Asn Arg145 150
155 160Asp Asn Asn Phe Glu Leu Gln Ser Ala Tyr Tyr Thr Lys Ala Thr
Lys 165 170 175Gln Ser Gly Tyr Asp Ala Ala Lys Ala Ser Leu Ala Lys
His Pro Asp 180 185 190Val Asp Phe Ile Tyr Ala Cys Ser Thr Asp Val
Ala Leu Gly Ala Val 195 200 205Asp Ala Leu Ala Glu Leu Gly Arg Glu
Asp Ile Met Ile Asn Gly Trp 210 215 220Gly Gly Gly Ser Ala Glu Leu
Asp Ala Ile Gln Lys Gly Asp Leu Asp225 230 235 240Ile Thr Val Met
Arg Met Asn Asp Asp Thr Gly Ile Ala Met Ala Glu 245 250 255Ala Ile
Lys Trp Asp Leu Glu Asp Lys Pro Val Pro Thr Val Tyr Ser 260 265
270Gly Asp Phe Glu Ile Val Thr 275
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