Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1998-10-07
2002-07-09
Raymond, Richard L. (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
C514S211110, C514S261100, C514S262100, C514S272000, C514S274000, C540S468000, C540S552000, C544S276000, C544S277000, C544S312000, C544S321000
Reexamination Certificate
active
06417180
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to DNA polymerases and antimicrobial compounds.
BACKGROUND OF THE INVENTION
Gram-positive eubacteria include a number of human pathogens, including Staphylococcus aureus, responsible for many human nosocomial soft-tissue infections. Like other common eubacteria, Gram-positive eubacteria absolutely require DNA polymerase III for their growth and replication.
Discovered in 1972, eubacterial DNA polymerase III (pol III) is the major polymerase enzyme involved in DNA replication and is therefore essential for cell division. Two classes of pol IIIs are known.
The Gram-positive pol IIIs are so-named because they were first discovered in the Gram-positive eubacterium Bacillus subtilis. Later it was recognized that Gram-positive pol IIIs are encoded by the polC gene. The polC gene product is generally a polypeptide which is about 1430-1460 amino acids in length, and which integrates both an 3′-5′ exonuclease site and a polymerase site. The Gram-positive pol IIIs are uniquely sensitive to inhibitory dGTP analogs of the so-called “HPUra” type (Brown,
Proc. Natl. Acad. Sci. USA,
67:1454, 1970).
Gram-negative pol IIIs are so-named because they were first discovered in the Gram-negative bacterium
Escherichia coli.
The Gram-negative pol IIIs are encoded by the dnaE gene, are typically 1155-1165 amino acids in length, contain only the polymerase site, and are completely insensitive to HPUra-like compounds.
The genomes of Gram-negative eubacteria apparently contain dnaE but not polC. The genomes of Gram-positive eubacteria and mycoplasmas contain both polC and dnaE. The dnaE gene product is required for replication of the Gram-negative bacterial genome, while the polC gene product is required for replication of the Gram-positive and mycoplasmal bacterial genomes. The function of the dnaE gene product in Gram-positive bacteria and mycoplasma is unclear.
SUMMARY OF THE INVENTION
The invention is based on the discovery that the DNA polymerase III of Gram-positive eubacteria and mycoplasmas contain a zinc finger domain adjacent to the polymerase active site, and that the integrity of the zinc finger is required for polymerase activity.
Accordingly, the invention features methods of identifying compounds that inhibit infections by Gram-positive eubacteria and mycoplasmas and the new antimicrobial compounds themselves.
In general, the invention features a compound for inhibiting Gram-positive eubacterial or mycoplasmal infection. The compound includes a zinc finger-reactive moiety, a linker, and a Gram-positive eubacterial or mycoplasmal DNA polymerase III active site-binding moiety connected to the zinc finger-reactive moiety via the linker. The compound can have the formula:
A—(L—B)
m
where B is a zinc finger-reactive moiety, L is a linker, and A is a polymerase III active site-binding moiety. Examples of A include:
in which each of R
1
and R
2
, independently, is hydrogen, C
1-3
alkyl, C
1-3
haloalkyl, or —L—B; each of R
3
and R
4
, independently, is hydrogen, C
1-3
alkyl, halo, C
1-3
haloalkyl, or —L—B; m is 1 or 2; and n is 0, 1, or 2; provided that at least one of R
1
, R
2
, R
3
, and R
4
, is —L—B. The invention also includes salts of the compounds of the invention. L can be a direct bond or a C
1-18
alkylene chain. The alkylene chain optionally containing 1 to 5 ether groups, thioether groups, amine groups, ester groups, thioester groups, or amide groups. B can contain an azodi(bis)urea group, an aromatic or aliphatic disulfide group, an aromatic or aliphatic nitroso group, a thiosulfonate group, or a thiazolidone group.
Examples of B include:
where each of R
a
and R
b
, independently, is hydrogen, C
1-6
alkyl, phenyl, C
1-6
hydroxyalkyl, C
1-6
haloalkyl, amine, or —L—A; and p is 1, 2, 3, or 4; provided that either one of R
a
and R
b
is —L—A, and R
a
and R
b
are not —L—A simultaneously. The invention also includes a salt of any of the above compounds.
In another aspect, the invention includes a method of inhibiting the polymerase activity of a zinc finger-containing DNA polymerase (e.g., a Gram-positive eubacterial DNA polymerase III or a mycoplasmal DNA polymerase III, such as the
Bacillus subtilis
DNA polymerase III) by contacting the DNA polymerase with a compound (e.g., a compound of the invention) under conditions sufficient for the compound to remove or interacts with a zinc ion bound to a zinc finger in the DNA polymerase.
The invention also includes a method of decreasing the rate of cell division of a bacterium containing a zinc finger-containing DNA polymerase (e.g., a Gram-positive eubacterial DNA polymerase III or a mycoplasmal DNA polymerase III, such as the
Bacillus subtilis
DNA polymerase III) by exposing the bacterium to a compound (e.g., a compound of the invention) under conditions sufficient for the compound to enter the bacterium and interact with a zinc ion bound to a zinc finger in the DNA polymerase.
In yet another aspect, the invention includes a method for testing whether a compound decreases the rate of cell division of a bacterium (e.g., a Gram-positive eubacterium or a mycoplasma) containing a zinc finger-containing DNA polymerase by exposing a bacterium containing a zinc finger-containing DNA polymerase to the compound under conditions sufficient for the compound to enter the bacterium; and determining whether a zinc ion (e.g., a
65
Zn ion) is bound to a zinc finger of the DNA polymerase, where binding of a zinc ion to the zinc finger in the absence of the compound but not in the presence of the compound indicates that the compound decreases the rate of cell division of the bacterium. The zinc finger-containing DNA polymerase can be at least 70% homologous or identical to SEQ ID NO:1 and comprises the sequence:
Z—X
2
—Cys—X
15-27
—Cys—X
2
—Cys (SEQ ID NO:5)
where Z is His or, Cys, X
2
is any two consecutive amino acids, and X
15-27
is any 15 to 27 consecutive amino acids. For example, the zinc finger-containing DNA polymerase can include SEQ ID NO:2. In other embodiments, the zinc finger-containing DNA polymerase comprises the sequence:
CyS—X
2
—Cys—X
19-21
—Cys—X
2
—Cys (SEQ ID NO:6)
where X
2
is any two consecutive amino acids, and X
19-21
is any 19 to 21 consecutive amino acids.
The invention also includes a method for testing whether a compound inhibits a zinc finger-containing DNA polymerase by providing a mixture that includes a polypeptide including a zinc finger of a zinc finger-containing DNA polymerase; mixing the compound with the mixture under conditions sufficient to allow the compound to contact the zinc finger; and determining whether a zinc ion is bound to the zinc finger, where binding of the zinc ion to the zinc finger in the absence of the compound but not in the presence of the compound indicates that the compound inhibits the DNA polymerase. In some embodiments, the mixture includes a cell containing the polypeptide.
In a different aspect, the invention includes a method of determining whether a compound inhibits a zinc finger-containing DNA polymerase by providing a mixture that includes a bacterium containing a zinc finger-containing DNA polymerase; mixing the compound with the mixture under conditions sufficient to allow the compound to contact the DNA polymerase within the bacterium, the compound including a group that interacts with zinc in a zinc finger; and measuring polymerase activity of the DNA polymerase in the presence of the compound, where a polymerase activity in the presence of the compound less than the polymerase activity in the absence of the compound indicates that the compound inhibits the DNA polymerase.
In still another aspect, the invention includes a method of treating a mammal susceptible to or having an undesirable Gram-positive eubacterial or mycoplasmal infection by administering to the mammal an amount of a compound (e.g., a compound of the invention) sufficient to interact with zinc in a zinc finger-containing DNA polymerase within a bacterium such that the polymerase activity of the DNA polymerase is
Barnes Marjorie H.
Brown Neal C.
Wright George E.
Fish & Richardson P.C.
McKenzie Thomas
Raymond Richard L.
University of Massachusetts
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