American Cyanamid Company

Basf Aktiengesellschaft

E. R. Squibb Sons, Inc.

Wyeth

Donald R Kirsch

Bradley Alton Ozenberger

Mark Henry Pausch

John Richard Hadcock

Deborah Tardy Chaleff

Eileen Marie Kajkowski

Margaret H Lai

Joseph O'Sullivan

Laura Price

Martin Bard

Laura A Price

Adrienne A Tymiak

Margaret Hsien-Fen Kuh Lai

Waldemar F Pfrengle

Guido Albert

Edward Meyers

John E McCullough

Myra B Kurtz

Sanford Jay Silverman

Douglas W Phillipson

J Scott Wells

Julia N Heinrich

Carol A Aklonis

Mark R Schmitt

Jane E Harris

Fernando Dela Cruz

Carl F Beyer

Helen A Ax

Klaus-Jüergen Pees

Margaret Hsien-fen Kuh Lai

Paul A Carter

Joseph E Biskupiak

The invention provides a method of treating or inhibiting the growth of cancerous tumor cells and associated diseases in a mammal in need thereof which comprises administering to said mammal an effective amount of a substituted triazolopyrimidine derivative or a pharmaceutically acceptable salt thereof and further provides a method of treating or inhibiting the growth of cancerous tumor cells and associated diseases in a mammal in need thereof by interacting with tubulin and microtubules and promoting microtubule polymerization which comprises administering to said mammal an effective amount of a substituted triazolopyrimidine derivative or a pharmaceutically acceptable salt thereof.

Substituted-triazolopyrimidines as anticancer agents

Expression vectors and yeast cells that contain a heterologous G protein-coupled receptor gene and a gene mutation that causes increased sensitivity to receptor activation or a gene mutation that permits transcriptional activation of pheromone-responsive genes without cell cycle arrest. Methods of making the yeast cells.

Heterologous G protein coupled receptors expressed in yeast, their fusion with G proteins and use thereof in bioassay

A first aspect of the present invention is directed to a transformed yeast cell containing a first heterologous DNA sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second heterologous DNA sequence which codes for a G protein a subunit or portions thereof fused to DNA sequences from the yeast G protein in a subunit. A second aspect of the present invention is a transformed yeast cell containing a heterologous DNA sequence which codes for a G protein coupled receptor. A third aspect of the present invention is a method of assaying compounds to determine effects on cell growth.

A method for the identification of agents which inhibit chitin synthesis, thus exhibiting potential fungicidal and insecticidal activity, involves the incubation of test samples in neutral cultures containing calcofluor white, a fluorochrome that causes lethal chitin hyper-polymerization in the yeast. Cultures containing samples that inhibit chitin synthesis exhibit enhanced growth because the growing fungus is rescued from the adverse effects of calcofluor white. In the practice of the invention, the test sample is added to a culture or culture area containing calcofluor white and the culture is incubated with the test sample for such time under such conditions sufficient to observe yeast cell growth inhibition in a corresponding culture or culture area containing calcofluor but no test sample. The extent of growth in the culture or culture area containing test sample is then compared with the extent of growth in the culture or culture area containing no test sample, and the presence of chitin synthesis inhibition is determined by observation of whether culture growth in the presence of test sample exceeds growth in its absence. Preferred embodiments employ test samples on disks or in wells in solidified cultures and a known chitin synthesis inhibitor as a positive control which is compared to the test sample.

Calcafluor screen for chitin biosynthesis inhibitors

The present invention is directed to expression vectors and yeast cells transformed therewith containing a first heterologous nucleotide sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second nucleotide sequence which codes for all or a portion of a G protein &agr;&bgr;&ggr; complex. Said heterologous protein is physically expressed in a host cell membrane in proper orientation for both stereoselective binding of ligands, as well as functional interaction with G proteins on a cytoplasmic side of the cell membrane. In some embodiments, a nucleotide sequence encoding a heterologous or chimeric G&agr; protein is expressed in conjunction with nucleotide sequences from the yeast G protein &bgr;&ggr; subunits. A second aspect of the present invention provides expression vectors and yeast cells transformed therewith encoding chimeric yeast/heterologous G protein coupled receptors. A third aspect of the present invention is directed to methods of assaying compounds using such expression constructs and yeast cell expression systems to determine the effects of ligand binding to the heterologous receptors expressed in the systems.

Method for detecting ligand binding to G protein coupled receptors

A method for screening for potential fungicides identifies biochemical or chemical agents that inhibit the formation of the fungal sterol ergosterol by inhibiting cytochrome P450 reductase or reductase related enzymes or proteins involved in its biosynthesis. Test samples are incubated in two yeast strain cultures, and the extent of growth inhibition is compared between them. One culture is supersensitive to cytochrome P450 reductase inhibitors; the yeast strain typically contains a disrupted or deleted cytochrome P450 reductase gene. The other culture is less sensitive to the compounds, typically exhibiting enhanced permeability such as that observed in erg6 mutants. In preferred embodiments, a known cytochrome P450 reductase related electron-transfer protein inhibitor such as keto-conazole, miconazole, dinaconazole or econazole is employed as a positive control. Preferred embodiments employ solidified media, and test samples are applied to disks or wells. Actives are identified by the production of a zone differential of approximately 8 mm or greater on the yeast culture lawns.

Cytochrome P-450 reductase screen for ergosterol biosynthesis inhibitors

This invention discloses methods for identifying compounds, variant nuclear proteins and other auxiliary proteins that interfere with the Drosophila ultraspiracle protein ('Usp') and homologs thereof. The methods disclosed involve transformed yeast cells which contain a Usp binding partner, Usp or a homolog thereof which can bind with the Usp binding partner, and a reporter gene which requires a functional Usp--Usp binder partner complex for expression. The transformed yeast cells are incubated in the presence of a test compound to form a test culture and in the absence of a test compound to form a control culture. The expression of the reporter gene is monitored in one example by exposing the test and control cultures to canavanine under conditions in which control cultures exhibit reduced growth and detecting test cultures in which growth is increased relative to growth of control cultures.

Screen for ultraspiracle inhibitors

The present invention is directed to a expression vectors and yeast cells transformed therewith containing a first heterologous nucleotide sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second nucleotide sequence which codes for all or a portion of a G protein .alpha..beta..gamma. complex. Said heterologous protein is physically expressed in a host cell membrane in proper orientation for both stereoselective binding of ligands, as well as functional interaction with G proteins on the cytoplasmic side of the cell membrane. In some embodiments, a nucleotide sequence encoding a heterologous or chimeric G.alpha. protein is expressed in conjunction with nucleotide sequences from the yeast G protein .beta..gamma. subunits. A second aspect of the present invention provides expression vectors and transformed yeast cells encoding chimeric yeast/heterologous G protein coupled receptors. A third aspect of the present invention is directed to methods of assaying compounds using these expression constructs and yeast cell expression systems to determine the effects of ligand binding to the heterologous receptors expressed in the systems.

Recombinant expression vectors for expression of heterologous proteins

A first aspect of the present invention is directed to a transformed yeast cell containing a first heterologous DNA sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second heterologous DNA sequence which codes for a G protein .alpha. subunit or portions thereof fused to DNA sequences from the yeast G protein a subunit. A second aspect of the present invention is a transformed yeast cell containing a heterologous DNA sequence which codes for a G protein coupled receptor. A third aspect of the present invention is a method of assaying compounds to determine effects on cell growth.

Transformed yeast cells expressing heterologous G-protein coupled

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.

Sterol .DELTA.14 reductase screen

A method for screening for sterol biosynthesis inhibitors of potential use as fungicides or antihypercholesterolemic agents identifies agents by the induction of lanosterol 14-.alpha.-demethylase, an enzyme in the biosynthetic pathway of ergosterol and cholesterol, in cultures containing the agents. In one screening test, test samples are incubated in a culture of a Saccharomyces cerevisiae strain sensitive to ergosterol biosynthesis and containing a gene fusion of a lanosterol 14-.alpha.-demethylase clone with a gene for bacterial .beta.-galactosidase. After incubation of the culture, an increase in lancsterol 14-.alpha.-demethylase activity is determined indirectly by measuring .beta.-galactosidase activity. The culture media contains a chromogenic substrate of .beta.-galactosidase such as orthonitrophenyl-.beta.-D-galactoside or 5-bromo-4-chloro-3-indoyl-.beta.-D-galactoside, so that active samples are identified by the production of colored product. For comparison purposes, screening tests may employ a lanosterol 14-.alpha.-demethylase inhibitor such as dinaconazole as a positive control.

Enzyme induction screen for ergosterol biosynthesis inhibitors

A gene encoding Saccharomyces cerevisiae sterol .DELTA.14 reductase of the ergosterol biosynthetic pathway is isolated and cloned by selecting strains carrying sequences on a 2.mu. based vector for resistance to a morpholine fungicide such as fenpropimorph. Four distinct plasmid inserts which produce morpholine resistance are obtained, and one of these is characterized and sequenced. The purified and isolated DNA sequence encoding sterol .DELTA.14 reductase encodes a polypeptide exhibiting homology to the S. cerevisiae sterol C-24(28) reductase enzyme in the ergosterol biosynthetic pathway.

DNA sequence encoding sterol .DELTA.14 reductase

A method for the identification of agents which inhibit spindle pole body formation or function, thus exhibiting selective fungicidal activity, involves the incubation of test samples in cultures of a Saccharomyces cerevisiae strain that produces excess numbers of spindle pole bodies. Cultures containing samples that inhibit spindle pole bodies exhibit enhanced growth because the growing yeast is rescued from the adverse effects of excess spindle bodies. In the preferred practice of the invention, the test sample is added to a S. cerevisiae culture or culture area containing a strain that has a conditional mutation producing excess spindle pole bodies, such as diploid esp1-1 strains. The culture or culture area is preincubated under permissive conditions wherein the strain can grow to some extent, and then conditions are shifted to restrictive conditions so that the mutant strain either cannot grow or grows poorly. The extent of growth in the culture or culture area containing test sample is then compared with the extent of growth in the absence of test sample, and the presence of spindle pole body inhibition is determined by observation of whether culture growth in the presence of test sample exceeds growth in its absence. Preferred embodiments employ test samples on disks or in wells in solidified cultures, facilitating easy visual inspection of growth. A presumptive spindle pole body inhibitor, identified in random screening, is used as a positive control which can be compared to the test sample.

Spindle pole body screen for fungicides

A novel antibiotic substance, Scopularin, is prepared by cultivation of a strain of the microorganism, Scopulariopsis, A.T.C.C. No. 20,914. This compound and its alcohol derivative have also been found to have cytochrome P450 inhibition activity.

Growing community of inventors

Princeton, NJ, United States of America

Donald R Kirsch