Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-10-08
2003-03-25
Ketter, James (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S006120, C435S007200, C435S320100
Reexamination Certificate
active
06537767
ABSTRACT:
The invention relates to a process for screening antimycotically active substances, in which process essential genes from mycetes, in particular
Saccharomyces cerevisiae
, as well as mycete genes which are homologous with regard to function and/or sequence, are employed as targets.
The spectrum of known fungal infections extends all the way from fungal infestations of the skin surface or the nails to potentially life-threatening mycotic infections of the internal organs. Infections of this nature, and the sequelae which accompany them, are termed mycoses.
Antimycotically (fungistatically or fungicidally) active substances are employed for treating mycoses. However, only relatively few pharmacologically active substances, such as amphotericin B, nystatin, pimaricin, griseofulvin, clotrimazole, 5-fluorocytosine and batrafen, have so far become available. It is exceptionally difficult to treat fungal infections medicinally, particularly because both the mycetes and the host cells are eukaryotic cells. For this reason, taking drugs which comprise the known antimycotic active substances is often associated with undesirable side-effects; for example, amphotericin B has a nephrotoxic effect. There is therefore a great need for pharmacologically active substances which can be used for producing medicaments which can be employed for treating mycoses, both prophylactically, when the immune system is impaired, and in the case of an infection which is already present. At the same time, the substances should display a specific action profile such that the growth and replication of the mycetes can be prevented selectively without concomitantly damaging the host organism.
There has to date been a lack of compatibile, informative test processes for identifying antimycotically active substances.
WO 95/11969 describes a process for screening antimycotic substances, in which process the effect of the substance to be tested is measured by its effect on the translation of a protein.
An object of the present invention is to develop a process for identifying antimycotically active substances, which process can be employed as universally as possible and enables a large number of potential active compounds to be tested in as efficient a manner as possible. An important feature of the process is that essential mycete genes are used as targets for the screening. This process differs from known processes in particular due to the fact that there is no requirement for any detailed knowledge of the biochemical function of the protein which is encoded by the essential gene.
The invention relates to a process for finding antimycotically active substances, which process employs essential mycete genes and/or the products of these essential genes as targets. In particular, antimycotically active substances are found as a result of the fact that they totally or partially inhibit the functional expression of the essential mycete genes (transcription and translation) or the functional activity of the encoded proteins.
The invention relates to a process for finding antimycotically active substances, in which process
a) a nucleic acid which controls the expression of an essential
Saccharomyces cerevisiae
protein and/or which encodes an essential
Saccharomyces cerevisiae
protein, or a part thereof, or the encoded essential protein itself, or
b) another nucleic acid which controls the expression of a protein which is derived from another mycete species and which is functionally similar to the protein mentioned under a) and/or encodes a protein which is derived from another mycete species and which is functionally similar to the protein mentioned under a), or the encoded functionally similar protein itself, is used as the target, with either
a) the effect of a substance to be investigated on the expression of the essential
Saccharomyces cerevisiae
protein or the functional activity of the encoded essential protein itself, or
b) the effect of a substance to be investigated on the expression of the functionally similar protein which is derived from another mycete species, or the functional activity of the encoded functionally similar protein itself, then being determined.
In one embodiment of the process, the nucleic acid is an essential gene or a part thereof, for example the promotor of the essential gene or an enhancer of the essential gene.
The invention involves identifying essential genes in mycetes, which genes can then be employed in the screening process.
The invention involves first of all identifying essential genes in
Saccharomyces cerevisiae
. The invention also involves using essential genes which have been identified in
Saccharomyces cerevisaie
(
S. cerevisiae
) to identify functionally similar genes in other mycetes. Where appropriate, these functionally similar genes can be essential genes in other mycetes.
In order to identify essential genes in
S. cerevisiae
, individual
S. cerevisiae
genes are removed from the
S. cerevisiae
genome by means of homologous recombination. The deletion is then lethal for the
S. cerevisiae
cells if the DNA segment which has been removed is an essential gene.
In order to produce appropriate deletions in the
S. cerevisiae
genome and to be able to select those
S. cerevisiae
cells which carry the deletion, use is made of a method in which the
S. cerevisiae
gene to be investigated is replaced by a marker gene. This marker gene (gene for a selection marker) can be used to select the cells in which a homologous recombination has taken place since, in these cells, the gene to be investigated has been replaced by the gene for the selection marker. Examples of selection markers which can be used are dominant selection markers or auxotrophic markers.
The auxotrophic markers used are genes which encode key enzymes of the amino acid or nucleobase synthetic pathways. For example,
S. cerevisiae
genes which encode enzymes from the amino acid metabolism of leucine (e.g. LEU2 gene), histidine (e.g. HIS3 gene) or tryptophan (e.g. TRP1 gene) or from the metabolism of the nucleobase uracil (e.g. URA3 gene) can be used as marker.
The process involves being able to use auxotrophic
S. cerevisiae
cells or strains, i.e. cells or strains in which the gene encoding the marker which is used in each case possesses one or more mutations thereby ensuring that no functionally active enzyme is expressed. These auxotrophic cells or strains are only able to grow in nutrient media which contain the corresponding amino acids or nucleobases. Examples of strains which can be used are all the
S. cerevisiae
laboratory strains which possess auxotrophic and/or nucleobase markers. If diploid
S. cerevisiae
cells or strains are used, the corresponding marker genes then have to be present in homozygously mutated form. Use is made, in particular, of the train CEN.PK2 (Scientific Research & Development GmbH, Oberursel, Germany) or isogenic derivatives of the strain.
The process also involves using
S. cerevisiae
cells or strains which do not possess any suitable auxotrophic markers, for example prototrophic
S. cerevisiae
cells or strains. Dominant selection markers, for example resistance genes such as the kanamycin resistance gene, can then be used as markers.
In order to achieve homologous recombination in which, in
S. cerevisiae
genes, the DNA sequence of the
S. cerevisiae
gene to be investigated is replaced totally or partly by the sequence of the marker gene, use is made of DNA fragments in which the marker gene is flanked, at its 5′ and 3′ ends, by sequences which are homologous with sequence segments at the 5′ and 3′ ends of the
S. cerevisiae
gene to be investigated.
A variety of methods, which are more or less equally well suited for deleting specific
S. cerevisiae
genes, are available for preparing appropriate DNA fragments. A linear DNA fragment is employed for the transformation into a suitable
S. cerevisiae
cell or strain. The homologous recombination integrates this fragment into the
S. cerevisiae
genome.
Three different methods can be used
Entian Karl-Dieter
Feldmann Horst
Hegemann Johannes
Hinnen Albert
Kötter Peter
Aventis Pharma Deutschland GmbH
Heller Ehrman White & McAuliffe LLP
Ketter James
Schnizer Richard
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