Mcgill University

Canadian Patents & Development Ltd.

The Royal Institution for the Advancement of Learning / Mcgill University

Elitra Pharmaceuticals Inc.

Howard Bussey

Terry Roemer

Charles Boone

John Davison

Marc Lussier

Anne-Marie Sdicu

Bo Jiang

Sarkis Serge Shahinian

David Y Thomas

Aleksandra Dmochowska

Philip Meaden

Kathryn Hill

Steve S Sommer

The invention relates to the identification and disruption of essential fungal specific genes isolated in the yeast pathogennamely CaKRE5, CaALR1, and CaCDC24 and to the use thereof in antifungal diagnosis and as essential antifungal targets in a fungal species for antifungal drug discovery. More specifically, the invention relates to the CaKRE5, CaALR1, and CaCDC24 genes, to their use to screen for antifungal compounds and to the drugs identified by such.

[object Object]

The present invention provides methods and compositions that enable the experimental determination as to whether any gene in the genome of a diploid pathogenic organism is essential, and whether it is required for virulence or pathogenicity. The methods involve the construction of genetic mutants in which one allele of a specific gene is inactivated while the other allele of the gene is placed under conditional expression. The identification of essential genes and those genes critical to the development of virulent infections, provides a basis for the development of screens for new drugs against such pathogenic organisms. The present invention further provides genes that are demonstrated to be essential and are potential targets for drug screening. The nucleotide sequence of the target genes can be used for various drug discovery purposes, such as expression of the recombinant protein, hybridization assay and construction of nucleic acid arrays. The uses of proteins encoded by the essential genes, and genetically engineered cells comprising modified alleles of essential genes in various screening methods are also encompassed by the invention.

Gene disruption methodologies for drug target discovery

The present invention to an isolated DNA which codes for a gene essential for cell wall glucan synthesis of , wherein the gene is referred to as CaKRE9, wherein the sequence of the DNA is as set forth in FIG. . The present invention relates to antifungal in vitro and in vivo screening assays for identifying compounds which inhabit the synthesis, assembly and/or regulation of &bgr;1,6-glucan. There is also disclosed an in vitro method for the diagnosis of disease caused by fungal infection in a patient.

Candida albicans KRE9 and uses thereof

The present invention provides a novel dominant selectable marker system in yeast that is based on an aminoglycoside, nourseothricin (NST). This compound possesses a powerful antifungal activity against and . The invention provides a cognate drug resistance marker for use in gene transformation and disruption experimentation in and . In particular, the invention presents: 1) direct utility for gene manipulations in both clinically and experimentally relevant strains regardless of genotype and without affecting growth rate, or hyphal formation; and 2) applicability to antifungal drug discovery, including target validation and various forms of drug screening assays.

Dominant selectable marker for gene transformation and disruption in yeasts

The present invention relates to an antifungal in vitro and in vivo screening assays for identifying compounds which inhibit mannosyltransferases involved in protein O- and N-glycosylation. The antifungal screening assay for identifying a compound which inhibit mannosyltransferases involved in protein O- and N-glycosylation, comprises the steps of: a) subjecting proteins to a specific mannosyltransferase protein encoded by a Saccharomyces cerevisiae mannosyltransferase encoding gene, wherein said gene is selected from the group consisting of KRE2/MNT1, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6 and KTR7; b) subjecting step a) to a screened compound and determining the absence or presence of protein O- and N-glycosylation, wherein the absence of protein O- and N-glycosylation is indicative of an antifungal compound. There is also disclosed an in vitro method for the diagnosis of diseases caused by fungal infection in a patient.

Screening using S. cerevisiae mannosyltransferase encoding genes

The present invention relates to DNA sequences coding for genes which participate in a yeast cell wall .beta.-glucan assembly pathway. These genes are essential for normal cell growth and are tools whicn enable the `in vitro` or `in vivo` screening for differential inhibition of fungi pathogenic to plants and animals, including man by specific antifungal agents. Further, a method which allows one to produce structurally modified glucans by using the DNA sequences of the present invention is also provided. Such modified glucans can be used to facilitate protein extraction from a microorganism; as food additives or as immunomodulators. Also, a method for selecting yeast mutants resistant to killer toxins from other yeasts or fungi which comprises exposing sensitive yeast cells to such killer toxins on agar plates, and selecting for yeast cells that survived such exposure by their ability to grow and form colonies.

Genes which participate in .beta.-glucan assembly and use thereof

This invention is concerned with the specific processing of secreted proteins in genetically modified yeast cells. The yeast KEX1 gene was cloned and the KEX1 product was shown to be a serine protease, evidently a carboxypeptidase B-like protease. A probable site of processing of polypeptides by the KEX1 gene product is at the C-terminus of the .alpha. subunit of the killer toxin, where the mature toxin subunit is followed in the precursor by a pair of basic amino acid residues. Processing likely involves an endoprotease cut following these basic residues, and their subsequent C-terminal trimming by a carboxypeptidase. Consistent with the KEX1 product being this carboxypeptidase is the finding that it is also involved in .alpha.-factor pheromone production. In wildtype yeast, KEX1 is not essential for .alpha.-factor production, as the final hormone repeat in the prepro .alpha.-hormone precursor does not need C-terminal processing to form one copy of the active hormone. However, in a mutant strain where .alpha.-factor production requires carboxypeptidase B-like processing, pheromone production is KEX1 dependent. Besides the processing of yeasts' naturally secreted proteins, of which .alpha.-factor pheromone and K1 killer toxin are the best characterized examples, the products of KEX1 and KEX2 are required for the processing of some proteins and peptides of commercial importance for example hormones and neuropeptides. High level production of certain commercially important proteins and peptides appears to require the overproduction of the appropriate processing proteases. The cloning of KEX1 on a multicopy plasmid can provide for this overproduction.

Growing community of inventors

Westmount, Canada

Howard Bussey