Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-09
2001-02-13
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C514S002600, C514S04400A
Reexamination Certificate
active
06187541
ABSTRACT:
RELATED APPLICATIONS
This application claims priority to Martin et al.,
STAPHYLOCOCCUS AUREUS ANTIBACTERIAL TARGET GENES
, United States Provisional Application No. 60/003,798, filed Sep. 15, 1995, now abandoned, and to Benton et al.,
STAPHYLOCOCCUS AUREUS ANTIBACTERIAL TARGET GENES
, U.S. Provisional Application No. 60/009,102, filed Dec. 22, 1995, now abandoned, which are incorporated herein by reference including drawings.
BACKGROUND
This invention relates to the field of antibacterial treatments and to targets for antibacterial agents. In particular, it relates to genes essential for survival of a bacterial strain in vitro or in vivo.
The following background information is not admitted to be prior art to the pending claims, but is provided only to aid the understanding of the reader.
Despite the development of numerous antibacterial agents, bacterial infections continue as a major, and currently increasing, medical problem. Prior to the 1980s, bacterial infections in developed countries could be readily treated with available antibiotics. However, during the 1980s and 1990s, antibiotic resistant bacterial strains emerged and have become a major therapeutic problem. There are, in fact, strains resistant to essentially all of the commonly used antibacterial agents, which have been observed in the clinical setting, notably including strains of
Staphylococcus aureus
. The consequences of the increase in resistant strains include higher morbidity and mortality, longer patient hospitalization, and an increase in treatment costs. (B. Murray, 1994
, New Engl. J. Med
. 330:1229-1230.) Therefore, there is a pressing need for the development of new antibacterial agents which are not significantly affected by the existing bacterial resistance mechanisms.
Such development of new antibacterial agents can proceed by a variety of methods, but generally fall into at least two categories. The first is the traditional approach of screening for antibacterial agents without concern for the specific target.
The second approach involves the identification of new targets, and the subsequent screening of compounds to find antibacterial agents affecting those targets. Such screening can involve any of a variety of methods, including screening for inhibitors of the expression of a gene, or of the product of a gene, or of a pathway requiring that product. However, generally the actual target is a protein, the inhibition of which prevents the growth or pathogenesis of the bacterium. Such protein targets can be identified by identifying genes encoding proteins essential for bacterial growth.
SUMMARY
Each pathogenic bacterial species expresses a number of different genes which are essential for growth of the bacteria in vitro or in vivo in an infection, and which are useful targets for antibacterial agents. This invention provides an approach to the identification of those genes, and the use of those genes, and bacterial strains expressing mutant forms of those genes, in the identification, characterization, and evaluation of targets of antibacterial agents. It further provides the use of those genes and mutant strains in screening for antibacterial agents active against the genes, including against the corresponding products and pathways. Such active compounds can be developed into antibacterial agents. Thus, this invention also provides methods of treating bacterial infections in mammals by administering an antibacterial agent active against such a gene, and the pharmaceutical compositions effective for such treatment.
For the
Staphylococcus aureus
essential genes identified in this invention, the essential nature of the genes was determined by the isolation of growth conditional mutants of
Staphylococcus aureus
, in this case temperature sensitive mutants (ts mutants). Each gene was then identified by isolating recombinant bacteria derived from the growth conditional mutant strains, which would grow under non-permissive conditions but which were not revertants. These recombinant bacteria contained DNA inserts derived from the normal (i.e., wild-type)
S. aureus
chromosome which encoded non-mutant products which replaced the function of the products of the mutated genes. The fact that a clone having such a recombinant insert can complement the mutant gene product under non-permissive conditions implies that the insert contains essentially a complete gene, since it produces functional product.
The Staphylococcal genes described herein have either been completely sequenced or have been partially sequenced in a manner which essentially provides the complete gene by uniquely identifying the coding sequence in question, and providing sufficient guidance to obtain the complete sequence and equivalent clones. For example, in some cases, sequences have been provided which can be used to construct PCR primers for amplification of the gene from a genomic sequence or from a cloning vector, e.g., a plasmid. The primers can be transcribed from DNA templates, or preferably synthesized by standard techniques. The PCR process using such primers provides specific amplification of the corresponding gene. Therefore, the complete gene sequence is obtainable by using the sequences provided.
In a first aspect, this invention provides a method of treating a bacterial infection in a mammal by administering a compound which is active against a bacterial gene selected from the group of genes corresponding to SEQ ID NO. 1-105. Each of these genes has been identified as an essential gene by the isolation of growth conditional mutant strains, and the complementation in recombinant strains of each of the mutated genes under non-permissive conditions, by expression from artificially-inserted DNA sequences carrying genes identified by the specified sequences of SEQ ID NO. 1-105. In particular embodiments of this method, the infection involves a bacterial strain expressing a gene corresponding to one of the specified sequences, or a homologous gene. Such homologous genes provide equivalent biological function in other bacterial species. Also in a preferred embodiment, the compound has a structure described by the general structure below:
in which
R, R
1
, R
2
, and R
3
are independently H, alkyl (C
1
-C
5
), or halogen;
R
4
is H, alkyl (C
1
-C
5
) halogen, SH, or S-alkyl (C
1
-C
3
);
R
5
is H, alkyl (C
1
-C
5
), or aryl (C
6
-C
10
);
R
6
is CH2NH2, alkyl (C1-C4), 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, or aryl (C
6
-C
10
);
or
R
5
and R
6
together are —C(R
7
)═C(R
8
)—C(R
9
)═C(R
10
)—, —N═C(R
8
)—C(R
9
)═C(R
10
)—, —C(R
7
)═N—C(R
9
)═C (R
10
)—, —C(R
7
)═C(R
8
)—N═C(R
10
)—, or —C(R
7
)═C(R
8
)—C(R
9
)═N—;
in which R
7
, R
8
, R
9
, and R
10
are independently H, alkyl (C
1
-C
5
), halogen, fluoroalkyl (C
1
-C5);
or
R
7
and R
8
together are —CH═CH—CH═CH—.
The term “alkyl” refers to a branched or unbranched aliphatic hydrocarbon group, e.g., methyl, ethyl, n-propyl, iso-propyl, and tert-butyl. Preferably the group includes from 1 to 5 carbon atoms and is unsubstituted, but alternativly may optionally be substituted with functional groups which are commonly attached to such chains, e.g., hydroxyl, fluoro, chloro, aryl, nitro, amino, amido, and the like.
The term “halogen” refers to a substituent which is fluorine, chlorine, bromine, or iodine. Preferably the substituent is fluorine.
The term “pyridyl” refers to a group from pyridine, generally having the formula C
5
H
4
N, forming a heterocyclic ring, which may optionally be substituted with groups commonly attached to such rings.
The term furyl refers to a heterocyclic group, having the formula C
4
H
3
O, which may be either the alpha or beta isomer. The ring may optionally be substituted with groups commonly attached to such rings.
The term “thienyl refers to a group from thiophen, generally having a formula C
4
H
3
S
The term “aryl” refers to an aromatic hydrocarbon group which includes a ring structure in which the electrons are delocalized. Commonly, aryl groups contain
Benton Bret
Lee Ving J.
Malouin Francois
Martin Patrick K.
Schmid Molly B.
Lyon & Lyon LLP
Marschel Ardin H.
Microcide Pharmaceuticals, Inc.
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