US 5,591,576 A · Sterol .DELTA.14 reductase screen

Abstract

A binary assay identifies agents that inhibit sterol .DELTA.14 reductase involved in ergosterol biosynthesis. In the primary screen, sterol .DELTA.14 reductase inhibition by a test sample is assayed by adding the test sample to a culture of Neurospora crassa having an erg-3 mutation and also to a culture of a strain having an erg-1 mutation, comparing the extent of growth inhibition after incubation in the two cultures, and identifying as positives those samples that show growth inhibition in the erg-3 culture exceeding that in the erg-1 culture. In the secondary screen, samples that test positive in the primary screen are reassayed by adding the test sample to a culture of a Saccharomyces cerevisiae strain into which has been introduced multiple copies of a gene encoding sterol .DELTA.14 reductase and also to a strain of S. cerevisiae that does not have the introduced gene; positive samples are identified after incubation by observation that growth inhibition in the culture having no introduced reductase gene exceeds growth inhibition in the culture having the introduced reductase gene. In preferred embodiments, a known inhibitor of sterol .DELTA.14 reductase is employed in solidified media in both the primary and the secondary screens, resulting in an assay that is highly sensitive and specific for the detection of sterol .DELTA.14 reductase inhibitors.

Claims21

Dependent claims

A method for screening for the presence or absence of sterol .DELTA.14 reductase inhibition by a test sample, said method comprising:

(a) adding the test sample to a culture of a Neurospora crassa strain having an erg-3 mutation;
(b) adding the test sample to a second culture of a Neurospora crassa strain having an erg-1 mutation;
(c) incubating the cultures for such time under such conditions sufficient to observe fungal cell growth in corresponding cultures containing no test sample;
(d) comparing the extent of growth inhibition in the culture containing the erg-3 mutation with the extent of growth inhibition in the culture containing the erg-1 mutant;
and
(e) determining the presence of sterol biosynthesis inhibition by observation that growth inhibition in the erg-3 culture exceeds growth inhibition in the erg-1 culture.

3 more elements

A method for screening for the presence or absence of sterol .DELTA.14 reductase inhibition by a test sample, which comprises:

(a) first screening the test sample in a primary screen comprising the steps of:
(i) adding the test sample to a culture of a Neurospora crassa strain having an erg-3 mutation;
(ii) adding the test sample to a second culture of a Neurospora crassa strain having an erg-1 mutation;
(iii) incubating the test samples in the cultures for such time under such conditions sufficient to observe fungal cell growth in corresponding cultures containing no test sample;
(iv) comparing the extent of growth inhibition in the culture containing the erg-3 mutation with the extent of growth inhibition in the culture containing the erg-1 mutant;
and
(v) identifying a positive test sample by observing that growth inhibition in the erg-3 culture containing the test sample exceeds growth inhibition in the erg-1 culture containing the test sample;
followed by
(b) screening said positive test sample in a secondary screen comprising the steps of:
(vi) adding a positive test sample from the primary screen to a culture of a Saccharomyces cerevisiae strain into which has been introduced at high copy a gene encoding sterol .DELTA.14 reductase;
(vii) adding the positive test sample to a culture of a corresponding Saccharomyces cerevisiae strain which does not have an introduced sterol .DELTA.14 reductase gene;
(viii) incubating the samples in the cultures for such time under such conditions sufficient to observe yeast cell growth in corresponding cultures containing no test sample;
(ix) comparing the extent of growth inhibition in the culture having the reductase gene with the extent of growth inhibition in the corresponding culture having no introduced gene;
and
(x) determining the presence of sterol .DELTA.14 reductase inhibition by observation that inhibition in the culture having no introduced reductase gene exceeds growth inhibition in the corresponding culture having the introduced reductase gene.

12 more elements

Dependencies are inferred from the claim text.

Sterol .DELTA.14 reductase screen

Life of this patent

Sterol .DELTA.14 reductase screen

Life of this patent

Abstract

Claims21