Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-05-08
2004-10-26
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S254110, C435S029000
Reexamination Certificate
active
06808892
ABSTRACT:
The present invention relates to a cell-based screen for inhibitors of fungal inositolphosphoryl-ceramide (IPC) synthase, an important antifungal target.
Inhibitors of fungal IPC synthase are potent and selective antifungal agents for example Aureobasidin, Khafrefungin and Rustmicin) as identified by several research groups and pharmaceutical companies.
However, all such compounds are natural products that are difficult to produce, handle and administer to a patient (for example, they may have unsuitable pharmacokinetics). Therefore it is highly desirable to obtain other novel chemical compounds selectively inhibiting the same target (a fungal IPC synthase) but without the intrinsic disadvantages displayed by the currently known inhibitors. Screening for such novel chemicals as well as optimisation of already available “leads” (ie. optimisation of a known inhibitor in a structure-based design or lead optimisation) will require an assay for IPC synthase activity that can be performed at a sufficiently high throughput.
All currently available biochemical assays for IPC synthase are involved and very labour-intensive.
Nagiec et al (Journal of Biological Chemistry, Vol 272 No 15, pp 9809-9817 (1997))) describe the complementation of an IPC synthase gene defect in a mutant strain of
S. Cerevisiae
by the AUR1 gene. The mutant strain has a deletion of the LCB1 gene and a point mutation that creates the suppressor gene SLC1-1. The lcb1 mutation prevents sphingolipid synthesis and the SLC-1-1 gene enables the cells to make phospholipids and remain viable. (Use of capital letters implies a functional gene or a gain of function mutation such as SLC1-1 whereas small letters indicate a non functional allele such as lcb1). Using this the authors were able to isolate a mutant strain defective in IPC synthase and to isolate a gene AUR1 which complemented the IPC synthase defect and restored IPC synthase activity. The authors conclude that IPC synthase is the target for antifungal agents such as aureobasidin. They postulate that it should be possible to develop high throughput screens to identify new inhibitors of IPC synthase to combat fungal diseases.
However we have found that whilst a similar strain of
S. cerevisiae
(lcb1/SLC1-1) is viable, the strain grows very poorly and is extremely sensitive to any environmental influences such as for example freezing. This strain is simply not robust enough for screening purposes.
We now provide a robust cell-based assay for identifying selective IPC synthase inhibitors. This assay is based on our development of an
S. cerevisiae
strain wherein the production of compensatory phospholipids is enhanced.
Therefore in a first aspect of the present invention we provide a screening assay for identifying a selective IPC synthase inhibitor which assay comprises contacting a test compound with engineered cells whose capability to synthesize sphingolipids depends on the addition of exogenous phytosphingosine and which are capable of sustained growth via compensatory phospholipids, adding phytosphingosine, and determining IPC synthase inhibition by the test compound by reference to any cell growth inhibition.
Any convenient host cell strain may be used provided that it can function as a host for a fungal IPC synthase gene. Convenient hosts include fungi that are manipulatable C) genetically such as
S. cerevisiae
but also others such as
Candida albicans, Candida glabrata, Aspergillus
sp. or
Schizosaccharomyces pombe
. Convenient sources for the AUR1 gene are pathogenic (also phytopathogenic) fungi as outlined above and others such as
Ashbya
sp.,
Fusarium
sp.,
Trichoderma
sp.,
Cryptococci, Blastomyces
, and
Histoplasma.
Whilst we do not wish to be bound by theoretical considerations the compensatory phospholipids are believed to be novel glycerophospholipids that may compensate for one or more functions of sphingolipids essential for vegetative growth (Lester et al, J. Biol. Chem., 1993, 268, 845-856).
In a further aspect of the invention we provide engineered cells whose capability to synthesize sphingolipids depends on the addition of exogenous phytosphingosine and which are capable of sustained growth via compensatory phospholipids
By “sustained growth” we mean no significant decrease of viable cell counts during a growth period (ie. cell-death is negligible compared to cell growth). The strain also has to be capable of one or more of the following: being stored for prolonged periods, for example up to three or six months or longer; storage in liquid medium; or capable of being frozen and revived. The engineered cells of the invention are capable and robust enough for routine use in high throughput assay procedures. In general they will have generation times compatible with growth assays (ie. not more than 4 hours per doubling) and final optical densities reached of more than 4 OD (at 600 nm and 1 cm path length). These parameters allow complete assessment of a host strain's growth within less than 30 hours.
A convenient host strain for use in the assay methods of the invention is an lcb1/SLC1-1 strain. More conveniently it will include a selection marker, for example the lcb1 gene may be directly replaced by an amino acid biosynthetic gene (such as LEU2, TRP1 or HIS3) or antibiotic resistance such as Geneticin (G418).
Adapting host cells for sustained growth is for example achieved by enhancing expression of the compensatory mutant SLC1-1 allele. We have surprisingly found that can be achieved by cloning the SLC1-1 gene onto a multi-copy plasmid (pYES2-LEU2d-GPD3-SLC1-1=pNS149) under control of the glyceraldehyde 3-phosphate dehydrogenase promoter. Use of a multi-copy pGPD-SLC1-1 promoter/gene construct yielded a strain with much improved growth characteristics, improved growth rate, final optical density and resistance to freezing. In summary it provided for the first time a host strain which is robust enough for screening purposes.
The GPD3 is an example of a very strong constitutive promoter in
S. cerevisiae
. Other glycolytic enzymes such as Phosphoglycerate Kinase (PGK), Enolase 1 (ENO), Pyruvate Kinase (PYK) and Fructose-Bisphosphate Aldolase II FBA are convenient sources of other such promoters.
Therefore in a further aspect of the invention we provide an engineered host strain
S. cerevisiae
(lcb1/pGPD-SLC1-1).
The invention will now be illustrated but not limited by reference to the following Examples and Figures:
REFERENCES:
patent: 5667986 (1997-09-01), Goodey et al.
patent: 63129/94 (1994-12-01), None
patent: 0424117 (1991-04-01), None
patent: 0644262 (1995-03-01), None
patent: WO-96/38573 (1996-12-01), None
Nagiec et al. Sphingolipid synthesis as a target for antifungal drugs. Complementation of the inositol phosphorylceramide synthase defect in a mutant strain of Saccharomyces cerevisiae by the AUR1 gene. J Biol Chem. Apr. 11, 1997;271(15):9809-17.
Chavada Jini S
Schnell Norbert F.
AstraZeneca AB
Guzo David
Ropes & Gray LLP
Sullivan Daniel M.
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