Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-03-03
2003-03-18
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007400, C435S018000, C435S019000, C435S024000, C435S006120, C435S032000, C435S007320, C435S069100, C435S069800, C435S183000, C435S173300, C435S173300, C435S243000, C536S024300, C536S024320
Reexamination Certificate
active
06534278
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to methods for identifying compounds that kill bacteria or inhibit bacterial growth. The invention also relates to methods for identifying compounds that can be used to treat infections (e.g., bacterial infections in organisms such as mammals).
Bacterial cell wall peptidoglycan biosynthesis is a multistep process (see FIG.
1
). Although there is some variation between bacterial species, each step in the respective synthetic pathways is essential for the growth of the bacteria. Inhibition of any step can be lethal, and each step is therefore a potential target against which new antibacterial drugs are sought. Inhibitors are already known for some steps in the biosynthetic pathway; however, bacteria have developed resistance to many of these inhibitors, thus necessitating continued searching for new antibacterial agents.
One mode of defense that gram positive bacteria use to resist a certain class of antibacterial agents (i.e., the &bgr;-lactams, which inhibit peptidoglycan formation) is to produce an enzyme called &bgr;-lactamase. Production of &bgr;-lactamase is induced in some bacterial strains by the presence of &bgr;-lactams in the cell. &bgr;-Lactamase reacts with &bgr;-lactam drugs (e.g., penicillin or cephalosporin), rendering the drugs inactive. Certain species of gram negative bacteria such as Enterobacter (e.g.,
E. cloacae, E. kobei, E. agglomerans
, or
E. flavus
) and
Citrobacter freundii
also produce &bgr;-lactamase, in response to the build-up of cell wall degradation products, not just in the presence of &bgr;-lactams per se. Because bacterial cell walls are continuously degraded and reassembled throughout the life cycle of a bacterium, the build-up of degradation products can be due to inhibition of at least one step in the peptidoglycan biosynthetic pathway.
SUMMARY OF THE INVENTION
The invention features new assays based on the discovery that induction of the &bgr;-lactamase gene can be used to identify compounds that kill bacteria (i.e., bacteriocidal activity) or inhibit bacterial growth (i.e., bacteriostatic activity), and thus to treat bacterial infections (i.e., to reduce symptoms of existing infections and to prevent infections) in organisms such as mammals. The &bgr;-lactamase can be encoded, for example, by a &bgr;-lactamase gene normally carried by a bacterial host, or inserted into a host, e.g., a heterologous host. The new methods are highly efficient and sensitive, and can be used, for example, for high throughput screening of libraries of potential inhibitors.
In one embodiment, the invention features a method for identifying a candidate compound (e.g., a single compound or a member of a library of potential inhibitors) that inhibits bacterial growth. The method includes the steps of contacting bacteria with the candidate compound to form a reaction mixture, and then assaying the reaction mixture for induction of &bgr;-lactamase, which indicates inhibition of bacterial growth.
The assaying step can, for example, include measuring the optical absorbance (e.g., optical density (OD)) of the reaction mixture (e.g., to detect the absorbance of &bgr;-lactamase at 490 nm); detecting the binding of antibodies to &bgr;-lactamase; or probing for &bgr;-lactamase mRNA.
In this context, a “candidate compound” is any compound not previously known to inhibit “bacterial growth,” which includes proliferation of bacteria, budding, cell division, endospore formation, and other forms of reproduction. “Inhibitors of bacterial growth” include both compounds that prevent growth of bacteria (i.e., bacteriostatic compounds) and compounds that kill bacteria (i.e., bacteriocidal compounds).
The invention also features a method for identifying an inhibitor of cell wall biosynthesis. The method includes the steps of contacting bacteria with a candidate compound to form a reaction mixture; and assaying the reaction mixture for induction of &bgr;-lactamase, wherein induction of &bgr;-lactamase indicates that the candidate compound is an inhibitor of cell wall biosynthesis.
In another embodiment, the invention features a method for identifying a candidate compound that can be used to treat infection in an organism by a bacteria. The method includes the steps of contacting the bacteria with the candidate compound to form a reaction mixture, and then assaying the reaction mixture for induction of &bgr;-lactamase, which indicates that the candidate compound can be used to treat bacterial infection.
Organisms that can be treated include mammals (e.g., humans, non-human primates, horses, cows, pigs, sheep, goats, dogs, and cats); non-mammalian animals (e.g., chickens or frogs); other eukaryotes (e.g., plants); and prokaryotes.
The invention also features a method for identifying a candidate compound that inhibits bacterial growth. The method includes the steps of providing bacteria carrying a gene that encodes &bgr;-lactamase; incubating the bacteria with the candidate compound under conditions that enable cell wall biosynthesis to form a reaction mixture; and assaying for induction of &bgr;-lactamase, which indicates that the candidate compound is an inhibitor of bacterial growth.
A bacteria “carrying a gene” is a bacteria that contains a plasmid, cosmid, vector, or other nucleic acid molecule that includes the gene. The gene can be incorporated into a chromosome (e.g., integrated into a bacterial chromosome) or can be extrachromosomal, but still within the bacterial cell. The gene can be from the same bacterial species as the host, e.g., preexisting in the host, or from a different species (i.e., heterologous). The gene can be a &bgr;-lactamase gene from a bacterial species selected from the group of genera consisting of Citrobacter, Enterobacter, Serratia, Pseudomonas, and Proteus. For example, the gene can be, ampC from
Citrobacter freundii
. The gene can also include a reporter gene such as lacZ or luc. The reporter gene can be fused to the &bgr;-lactamase gene or otherwise under the control of the same regulators as &bgr;-lactamase.
The method can also include the steps of obtaining a cell extract containing enzymes, cofactors, and carrier molecules necessary for a particular step or steps of cell wall biosynthesis; supplying a substrate for the step or steps; incubating the candidate compound with the cell extract and the substrate under conditions that enable the step or steps to proceed to form an incubation mixture; and assaying the incubation mixture for the substrate and the product produced in the step or steps. The production of an amount of product less than that normally produced in the step or steps relative to the amount of substrate indicates the presence of an inhibitor of the step or steps.
In addition, the invention features a method for identifying an inhibitor of a particular step or steps of cell wall biosynthesis. The method includes the steps of providing bacteria carrying a gene that encodes &bgr;-lactamase; incubating the bacteria with a candidate compound under conditions that enable cell wall biosynthesis to form a reaction mixture; assaying the reaction mixture for induction of &bgr;-lactamase to identify an inhibitor of cell wall biosynthesis; obtaining a cell extract containing. enzymes, cofactors, and carrier molecules necessary for the particular step or steps; supplying a substrate for the step or steps; incubating the inhibitors with the cell extract and the substrate under conditions that enable the step or steps to proceed; and assaying the incubation mixture (e.g., by chromatography) for the substrate and the product normally produced in the step or steps. The production of an amount of product less than that normally produced in the step or steps relative to the amount of substrate indicates the presence of an inhibitor of the step or steps.
The cell extract can be a whole cell, a cell membrane preparation, or a cytoplasmic extract, for example. The substrate can be detectably labeled (e.g., with a fluorescent tag, an isotopic label, or biotin).
The particular step of cell wall biosynthe
Fish & Richardson P.C.
Graser Jennifer E.
Millennium Pharmaceuticals Inc.
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