Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
1999-03-25
2003-01-07
Brusca, John S. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C435S006120, C435S007310, C435S007320, C435S007210, C435S091100, C435S091400, C536S023740
Reexamination Certificate
active
06505126
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for identifying fungal-derived nucleic acids and polypeptides that are useful as molecular targets for diagnosis and treatment of pathological conditions, as well as the isolated nucleic acids and polypeptides themselves. The invention also relates to compositions and methods for the diagnosis, prevention, and amelioration of pathological conditions resulting from fungal infection.
BACKGROUND OF THE INVENTION
Development of effective methods and compositions for the prevention and the treatment of fungal infections is a critical goal of the pharmaceutical and agricultural industries. The major fungal animal pathogens in North America are Candida species and Aspergillus species, as well as
Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis
, and
Cryptococcus neoformans
(Medically Important Fungi, Second Edition, Davise H. Larone, Ed., American Society for Microbiology, Washington, D.C.).
Fungal infections are a significant and growing health problem, especially in immunocompromised patients. Although several classes of antifungal agents are currently marketed, including compounds such as flucytosine, azoles, allylamines, and amphotericin, all of these agents are limited either by lack of fungicidal activity, toxicity, or development of resistance.
For example, patients suffering from AIDS may contract aspergillosis and endemic infections such as coccidiodomycoses and histoplasmoses. Cancer and transplant patients suffer high morbidity and mortality from opportunistic fungal infections such as those caused by Aspergillus and Fusarium. Furthermore, the development of fungal and yeast strains that are resistant to the above compounds will necessitate the development of antifungals with new mechanisms of action. There is also a need for alternative preventive and therapeutic agents, such as effective vaccines and other immunotherapeutic agents, for the prevention and/or treatment of disease caused by such microorganisms.
SUMMARY OF THE INVENTION
The present invention fulfills this need by providing methods and compositions for identifying nucleic acids and polypeptides derived from fungi that are useful as antifungal targets. The methods are carried out by:
(i) assessing whether a fungal-derived nucleic acid, or a polypeptide encoded therein, is important for viability of the fungal species from which it is derived; and
(ii) determining if the nucleic acid or polypeptide shares substantial homology with any nucleic acids or polypeptides derived from bacterial species, other fungal species, and higher eukaryotic species.
According to the invention, an antifungal target comprises a nucleic acid or polypeptide which
(a) is important for viability of the fungus from which it is derived;
(b) does not share substantial sequence homology with any nucleic acid or polypeptide derived from bacterial or higher eukaryotic species; and
(c) is present in at least one pathogenic fungal species.
The invention also provides libraries of antifungal targets derived from different fungal species, including without limitation
Saccharomyces cerevisiae, Candida albicans, Aspergillus fumigatus, Schizosaccharomyces pombe, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Paracoccidioides brasiliensis
, and
Pneumocystis carinii
. The libraries may comprise a plurality of isolated nucleic acids, a plurality of isolated polypeptides, or a plurality of bacterial cells, each of which contains a vector comprising a particular nucleic acid and which may also contain the fungal polypeptide encoded therein.
Another aspect of the invention encompasses the use of such fungal specific targets, or fungal specific antibodies, in diagnostic applications.
Still another aspect of the invention encompasses methods for identifying antifungal agents that interact with and/or alter the function either directly or indirectly of the antifungal targets described above. Such methods include without limitation overexpression assays and ligand-binding assays, and are used in low-throughput or high-throughput modes to screen compounds and extracts for antifungal activity, whether fungicidal or fungistatic. In one series of embodiments, the methods comprise: (i) contacting a test compound with an fungal target protein; and (ii) selecting as candidate antifungal agents those test compounds that bind to the protein.
In yet another aspect, the invention encompasses antifungal agents that bind to, or otherwise influence the activity or function of, any of the antifungal target genes or polypeptides of the invention. Antifungal agents may include nucleic acids, particularly antisense oligonucleotides; peptides; oligosaccharides; lipids; derivatives of any of the foregoing, or other molecules.
DETAILED DESCRIPTION OF THE INVENTION
All patent applications, patents, and literature references cited in this specification are hereby incorporated herein by reference in their entirety. In the case of conflict, the present description, including definitions, is intended to control.
Definitions:
1. “Nucleic acid” or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.
2. An “open reading frame” (ORF) as used herein is a region of a polynucleotide sequence having a start and stop codon and which may encode a polypeptide. This region may represent a portion of a coding sequence or may comprise a total coding sequence for the polypeptide.
3. A “coding sequence” or a “protein-coding sequence” is a polynucleotide sequence capable of being transcribed into mRNA and/or capable of being translated into a polypeptide. The boundaries of the coding sequence are typically determined by a translation start codon at the 5′-terminus and a translation stop codon at the 3′-terminus.
4. A “complement” of a nucleic acid sequence as used herein refers to the “antisense” sequence that participates in Watson-Crick base-pairing with the original sequence.
5. An “isolated” nucleic acid or polypeptide as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring). An isolated nucleic acid or polypeptide contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated.
6. A nucleic acid or polypeptide sequence that is “derived from” a designated sequence refers to a sequence that corresponds to a region of the designated sequence. For nucleic acid sequences, this encompasses sequences that are homologous or complementary to the sequence, as well as “sequence-conservative variants” and “function-conservative variants.” For polypeptide sequences, this encompasses “function-conservative variants.” Sequence-conservative variants are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. Function-conservative variants are those in which a given amino acid residue in a polypeptide has been changed without altering the overall conformation and function of the native polypeptide, including, but not limited to, replacement of an amino acid with one having similar physico-chemical properties (such as, for example, acidic, basic, hydrophobic, and the like). “Function-conservative” variants also include any polypeptides that have the ability to elicit antibodies specific to a designated polypeptide.
7. A “
S. cerevisiae
-derived” nucleic acid or polypeptide sequence may or may not be present in other yeast or fungal species, and may or may not be present in all
S. cerevisiae
Hare Roberta S.
Miller George H.
Shaw Karen J.
Shimer, Jr. George H.
Brusca John S.
Moran Majorie A.
Morgan & Finnegan , LLP
Schering-Plough Corporation
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