Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1999-12-29
2003-12-02
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S007200, C435S009000, C435S006120, C435S183000, C435S243000, C530S350000, C536S024320, C536S024100
Reexamination Certificate
active
06656703
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for identifying inhibitors of fatty acid biosynthesis in bacteria.
BACKGROUND OF THE INVENTION
Fatty acid biosynthesis (FAB) is necessary for the production of bacterial cell walls, and therefore is essential for the survival of bacteria (Magnuson et al., 1993, Microbiol. Rev. 57:522-542). The fatty acid synthase system in
E. coli
is the archetypal type II fatty acid synthase system. Multiple enzymes are involved in fatty acid biosynthesis, and genes encoding the enzymes fabH, fabD, fabG, acpP, and fabF are clustered together on the
E. coli
chromosome. Clusters of FAB genes have also been found in
Bacillus subtilis, Haemophilus influenza
Rd,
Vibrio harveyi
, and
Rhodobacier capsulatus
. Examples of FAB genes in
B. subtilis
include fabD, yjaX and yhjB (encoding synthase III), fabG, ywpB, yjbW, yjaY, ylpC, fabG, and acpA. The ylpC, fabG, and acpA genes are contained within a single operon that is controlled by the PylpC promoter.
Using genetic and biochemical methods, the targets and mechanisms of action of several inhibitors of the elongation part of the FAB pathway have been identified (FIG.
1
). The FAB inhibitor cerulenin inhibits &bgr;-ketoacyl-ACP synthase, and the inhibitor thiolactomycin inhibits acetoacyl ACP synthase (Omura, 1981, Meth. Enzymol. 72:520-532; Moche et al., 1999, J. Biol. Chem. 274:6031-6034; and Jackowski et al., 1989, J. Biol. Chem. 264:7624-7629). The FAB inhibitors isoniazid, triclosan, and diazaborine inhibit enoyl-acyl carrier protein reductase (Quemard et al., 1995, Biochem. 34:8235-8241; McMurray et al., 1998, Nature 394:531-532; Heath et al., 1998, J. Biol. Chem. 273:30316-30320; Heath et al., 1999, J. Biol. Chem. 274:11110-11114; Levy et al., 1999, Nature 398:383-384; and DeBoer et al., 1999, Mol. Microbiol. 31:443-450). In
Pseudomonas aeruginosa
, triclosan has been shown to inhibit the enoyl-acyl carrier protein reductase (FabI), which converts trans-2-enoyl-ACP to acyl-ACP (Hoang and Schweizer, supra). The FAB pathway provides the acyl groups for production of acylated homoserine lactones (HSLs). HSLs are the signaling molecules involved in quorum sensing, i.e., bacterial cell-to-cell signaling, in a wide variety of bacteria. In pathogenic bacteria, such as Pseudomonas, quorum sensing is a mechanism for regulating the expression of virulence factors (Hastings and Greenberg, 1999, J. Bacteriol. 181:2667-2668).
SUMMARY OF THE INVENTION
The invention is based upon the discovery that the activity of promoters of certain genes is increased in the presence of compounds that inhibit
B. subtilis
FAB. Thus, compounds that inhibit FAB can be identified by their ability to increase the activity of the
B. subtilis
PyhfB and PylpC promoters. Various promoters can be used in the invention, provided that the activity of the promoter is upregulated by a FAB inhibitor, such as cerulenin or triclosan. FAB inhibitors that slow the growth of, or kill, bacteria are candidate antibacterial agents that can be used in methods of treating bacterial infections. The invention thus provides a rapid and convenient method for identifying (i) compounds that inhibit FAB and which can subsequently be derivatized to produce antibacterial agents, as well as (ii) compounds that inhibit FAB and which are antibacterial agents. Because the FAB pathway is involved in the synthesis of HSLs, the invention also provides a method for identifying compounds that inhibit HSL synthesis. Such compounds can be used to inhibit bacterial virulence. If desired, such inhibitors of HSL synthesis can be further derivatized using standard medicinal chemistry techniques to produce inhibitors of virulence having increased potency.
Accordingly, the invention features a method for determining whether a test compound is an inhibitor of bacterial FAB. The method includes: (i) contacting a bacterial cell with a test compound, wherein the bacterial cell contains (a) a promoter (e.g., PyhfB or PylpC), the activity of which is increased in the presence of a compound that inhibits FAB, operably linked to (b) a reporter gene; and (ii) measuring activity of the promoter, wherein an increase in activity, relative to the level of activity of the promoter in the absence of the test compound, indicates that the test compound is an inhibitor of bacterial FAB.
The invention also includes a method for determining whether a test compound is an antibacterial agent, the method comprising: (i) contacting a bacterial cell with a test compound, wherein the bacterial cell contains (a) a promoter (e.g., PyhfB or PylpC), the activity of which is increased in the presence of a compound that inhibits FAB, operably linked to (b) a reporter gene; (ii) measuring activity of the promoter, wherein an increase in activity, relative to the level of activity of the promoter in the absence of the test compound, indicates that the test compound is an inhibitor of FAB; and (iii) determining whether the compound is an antibacterial agent by determining whether the compound kills, or slows the growth of, bacteria. Optionally, the test compound may be further assayed in a biochemical assay (e.g., in an extract of the cell) to determine which step in the pathway is inhibited, and to confirm that the test compound inhibits fatty acid biosynthesis. For example, inhibition of fatty acid biosynthesis can be detected as inhibition of incorporation of acetate into fatty acids or phospholipids. Conventional methods can be used to measure inhibition of incorporation of acetate.
An increase in activity of the promoter can be measured, for example, by measuring expression of a reporter gene that is operably linked to the promoter, such as a lacZ cat, gus, a green fluorescent protein gene, or a luciferase gene. Other suitable reporter genes are well known in the art and can be used in the invention. If desired, the activity of the promoter can be measured by measuring binding of antibodies to a product of the reporter gene (e.g., a protein encoded by the reporter gene), with an increase in the level of bound antibodies reflecting an increase in activity of the promoter. Alternatively, activity can be measured by measuring the level of mRNA transcribed from the reporter gene, with an increase in the mRNA level reflecting an increase in promoter activity.
The invention also provides methods of preparing (i) an inhibitor of fatty acid biosynthesis and/or (ii) an antibacterial agent. The methods include: screening multiple test compounds by the methods described above; identifying candidate compounds that upregulate promoter activity; isolating one or more lead compounds from the candidate compounds; identifying and selecting a lead compound that inhibits fatty acid biosynthesis or bacterial growth; and formulating the selected lead compound as an inhibitor of fatty acid biosynthesis or as an antibacterial agent. A “lead compound” is a test compound that increases promoter activity by at least 3 times the standard deviation, plus the mean. If desired, lead compounds can subsequently be derivatized using conventional medicinal chemistry methods, as described herein.
Similarly, the invention features methods for preparing (i) an inhibitor of FAB or (ii) an antibacterial agent. The methods include screening multiple test compounds by the methods described above; identifying candidate compounds that upregulate promoter activity; isolating one or more lead compounds from the candidate compounds; derivatizing the lead compound(s), thereby producing a derivative of the lead compound; identifying derivatives that inhibit FAB or bacterial growth; and formulating the derivative as an inhibitor of FAB or as an antibacterial agent (e.g., by admixture with a pharmaceutically acceptable carrier). Inhibitors of FAB and antibacterial agents prepared by such methods also are included within the invention. Such compounds can be used in methods for inhibiting bacterial fatty acid biosynthesis or growth of bacteria in an organism having a bacterial infection.
The invention also provides a method for identi
Murphy Christopher
Youngman Philip
Fish & Richardson P.C.
Millennium Pharamaceuticals, Inc.
Shahnan-Shah Khatol S
Swartz Rodney P
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