Autoinducer compounds

Details Autoinducer compounds Autoinducer compounds

1998-06-18

2002-01-08

Ketter, James (Department: 1636)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C514S473000, C514S507000, C514S513000, C549S321000

Reexamination Certificate

active

06337347

ABSTRACT:

TECHNICAL FIELD
The present invention relates to autoinducer compounds which enhance gene expression in a wide variety of microorganisms. The present invention further relates to therapeutic compositions and therapeutic methods wherein, for example, gene expression within microorganisms is regulated.
BACKGROUND ART
Before 1981, microbiologists generally assumed that bacteria lacked the requirement and the capability of producing cell-cell signaling molecules. In 1981, by Eberhard, et al. Biochemistry, 20, 2444-2499, 1981, showed that the bacterium
Photobacterium fischeri
produces a compound 3-oxo-N-(tetrahydro-2-oxo-3-furanyl) hexanamide, also known as vibrio (photobacterium) autoinducer (VAI), which is associated with bacterial luminescence under conditions of high cell density. The cell membrane of
P. fischeri
was shown to be permeable to VAI by Kaplan and Greenberg in 1985 (J. Bacteriol., 163, 1210-1214, 1985). At low bacterial cell densities in broth medium, VAI passively diffuses out of the cells along a concentration gradient, where it accumulates in the surrounding medium. At high cell densities, the concentration of VAI outside the cells is equivalent to the concentration of VAI inside the cells. Under such conditions VAI was shown to initiate transcription of luminescence genes. Using such a system, bacteria are able to monitor their own population density and regulate the activity of specific genes at the population level.
For several years it was presumed that the autoinducer involved in bacterial luminescence was unique to the few bacteria that produce light in the marine environment. Then, in 1992, the terrestrial bacterium
Erwinia carotovora
was shown to use an autoinducer system to regulate the production of the &bgr;-lactam antibiotic carbapenem (Bainton, et al., Biochem J., 288, 297-1004, 1992b). The molecule found to be responsible for autoinduction of carbapenem was shown to be an acylated homoserine lactone (HSL), a member of the same class of molecules responsible for autoinduction in bioluminescence. This finding led to a general search for HSLs in a wide range of bacteria. To affect the search, a bioluminescence sensor system was developed and used to screen for HSL production in the spent supernatant liquids of a number of bacterial cultures. Many different organisms were shown by the screening to produce HSLs. These included:
Pseudomonas aeruginosa, Serratia marcescens, Erwinia herbicola, Citrobacter freundii, Enterobacter agglomerans
and
Proteus mirabilis
(Brainton, et al., Gene. 116, 87-91, 1992a; Swift, et al., Mol. Microbiol., 10, 511-520, 1993). More recently, the list has grown to include Erwinia stewartii (Beck, J. Bacteriol, 177, 5000-5008, 1993),
Yersinia enterocolitica
(Throup, et al., Mol. Microbiol., 17, 345-356, 1995),
Agrobacterium tumefaciens
(Zhang, et al., Nature, 362, 446-448, 1993),
Chromobacterium violaceum
(Winston, et al., Proc. Natl. Acad. Sci., USA, 92, 9427-9431, 1995),
Rhizobium leguminosarium
(Schripsema, et al., J. Bacteriol, 178, 366-371 1996 and others. Today it is generally assumed that all enteric bacteria, and the gram negative bacteria generally, are capable of cell density regulation using HSL autoinducers.
In 1993 Gambello, et al. Infect. Immun., 61, 1880-1184, (1993) showed that the &agr;-HSL product of the LasI gene of
Pseudomonas aeruginosa
controls the production of exotoxin A, and of other virulence factors, in a cell density dependent manner. Since that time, the production of a large number of Pseudomonas virulence factors have been shown to be controlled by &agr;-HSL compounds produced by the LasI and RhlI regulatory systems (Ochsner, et al., Proc. Natl. Acad. Sci., USA 92, 6424-6428, 1995; Winson, et al., supra; Latifi, et al., 1995), in a manner reminiscent of the Lux system. Latifi, et al. Mol. Microbiol, 21, 1173-1146, (1996) have also shown that many stationary phase properties of
P. aeruginosa
, including those controlled by the stationary phase sigma factor (RpoS), are under the hierarchical control of the LasI and RhlI cell-cell signaling systems.
In all cases, homoserine lactone autoinducers are known to bind to a DNA binding protein homologous to LuxR in
Photobacterium fischeri
, causing a conformational change in the protein initiating transcriptional activation. This process couples the expression of specific genes to bacterial cell density (Latifi, et al. supra, 1996). Regulation of this type has been called ‘quorum sensing’ because it suggests the requirement for a ‘quorate’ population of bacterial cells before activation of the target genes (Fuqua, et al., J. Bacteriol., 176, 269-275, 1994b). Expression of certain of these ‘virulence factors’ has been correlated with bacterial cell density (Finley and Falkow, Microbiol. Rev. 53, 210-230, 1989).
In
P. aeruginosa
, quorum sensing has been shown to be involved in the regulation of a large number of exoproducts including elastase, alkaline protease, LasA protease, hemolysin, cyanide, pyocyanin and rhamnolipid (Gambello, et al., supra; Latifi, et al., supra; Winson, et al., supra; Ochsner, et al., 1995). Most of these exoproducts are synthesized and exported maximally as
P. aeruginosa
enters stationary phase.
The concept of cell signalling and quorum sensing has been studied in the art. See for example U.S. Pat. No. 5,591,872, to Pearson et al; Passador et al, Journal of Bacteriology, pages 5990-6000, October, 1996; PCT W092/18614 and U.S. Pat. No. 5,593,827.
Given the importance of these signalling molecules in the regulation of diverse metabolic functions, there exists a need for new autoinducer compounds which regulate gene expression in bacteria.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide novel autoinducer compounds and compositions comprising said novel compounds.
A further object of the invention is to provide novel methods for regulation, i.e., inhibition, enhancement, dispersion, etc., by administration of the compounds of the present invention.
Additional objects and advantages of the present invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from the practice of the invention. The objects and advantages of the invention may be realized and obtained as particularly pointed out in the appended claims.
According to the present invention, the foregoing and other objects are achieved in part by compounds of the following formulae:
wherein in the above formulae R
1
-R
2
, are selected from H, C
1
-C
4
alkyl group (preferably CH
3
), OH, NH
2
, SH or a halogen such as fluorine, chlorine bromine or iodine;
R
22
and R
23
are selected from S, O, and N—R,
R
24
-R
28
, are H or a halogen, and
X, X
1
and X
2
are selected from O, S, H
2
or any combination of H plus one halogen or two halogens when one or more R groups is substituted.
A further object of the present invention is to provide methods for regulating gene expression with a microorganism, which method comprises adding an inventive compound to a microorganism culture to cause expressing of a selected gene that would not otherwise be expressed.
Additional objects and advantages of the present invention will become readily apparent to those having ordinary skill in this art from the following detailed description, wherein only the preferred embodiment of the invention is shown and described, simply by way of illustration of the best mode contemplated for carrying out the present invention. As will be realized, the present invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
DESCRIPTION OF THE INVENTION
The present invention relates to autoinducer compounds of the formulae:
wherein in the above formulae R
1
-R
21
, are selected from H, C
1
-C
4
alkyl

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