Methods for identifying targets for antimicrobial and antiproliferative compounds and compositions therefrom

Abstract

The invention is directed to methods for identifying targets for antimicrobial and antiproliferative compounds as well as methods for identifying novel compounds for treating cancer and microbial infections.

1 - 48 . (canceled) 
     
     
         49 . A method of identifying a compound having antimicrobial activity against budding yeast cells, comprising:
 (a) growing the budding yeast cells in vitro under infection mimetic conditions to produce a metabolically synchronous population of budding yeast cells;   (b) adding a test compound to the metabolically synchronous population of budding yeast cells;   (c) identifying reduced growth or killing of the budding yeast cells in the presence of the test compound; and   (d) identifying the compound having antimicrobial activity against budding yeast cells under infection mimetic conditions.   
     
     
         50 . The method of  claim 49 , wherein the infection mimetic conditions mimic blood of the infected animal and comprise growing the budding yeast cell in a growth media comprising 150 mM NaCl, 5.5 mM glucose, free amino acids, macrophages, neutrophils, and human serum factors at a temperature of greater than 37° C. and a pH of <7.35 with normoxic oxygen levels. 
     
     
         51 . The method of  claim 49 , wherein the infection mimetic conditions mimic a wound infection in the animal and comprise growing the budding yeast cell in a media comprising less than 150 mM NaCl, 5.5 mM glucose, free amino acids, collagen, elastin, fibronectin macrophages, neutrophils, interleukin, and tumor necrosis factor at a temperature of greater than 37° C. and a pH ranging from 7.15 to 8.9 with hypoxic oxygen levels. 
     
     
         52 . The method of  claim 49 , wherein the infection mimetic conditions mimic cerebral spinal fluid of the infected animal and comprise growing the budding yeast cell in a media comprising phosphate buffered saline (PBS), 5 g/L of urea and 0.1% dextrose or in a media comprising 150 mM NaCl, 3 mM glucose, and 4 mM urea at a temperature of greater than 37° C. and a pH of about 7.35 with normoxic oxygen levels. 
     
     
         53 . The method of  claim 49 , wherein the budding yeast cell is a  Cryptococcus  spp.,  Candida  spp., or  Mucor  spp. 
     
     
         54 . A method of identifying a modification in a gene expression pattern in a budding yeast cell comprising:
 (a) growing the budding yeast cell in vitro under infection mimetic conditions to produce a metabolically synchronous population of budding yeast cells;   (b) sampling continuous cultures of such metabolically synchronous population of cells over time; and   (c) identifying one or more genes that are differentially expressed when the budding yeast cells are grown in vitro under infection mimetic conditions as compared to when grown under standard laboratory conditions, thereby identifying a modification in a gene expression pattern during growth of budding yeast cells under infection mimetic conditions.   
     
     
         55 . The method of  claim 54  wherein the infection mimetic conditions mimic blood of the infected animal and comprise growing the budding yeast cell in a growth media comprising 150 mM NaCl, 5.5 mM glucose, free amino acids, macrophages, neutrophils, and human serum factors at a temperature of greater than 37° C. and a pH of <7.35 with normoxic oxygen levels. 
     
     
         56 . The method of  claim 54 , wherein the infection mimetic conditions mimic a wound infection in the animal and comprise growing the budding yeast cell in a media comprising less than 150 mM NaCl, 5.5 mM glucose, free amino acids, collagen, elastin, fibronectin macrophages, neutrophils, interleukin, and tumor necrosis factor at a temperature of greater than 37° C. and a pH ranging from 7.15 to 8.9 with hypoxic oxygen levels. 
     
     
         57 . The method of  claim 54 , wherein the infection mimetic conditions mimic cerebral spinal fluid of the infected animal and comprise growing the budding yeast cell in a media comprising phosphate buffered saline (PBS), 5 g/L of urea and 0.1% dextrose or in a media comprising 150 mM NaCl, 3 mM glucose, and 4 mM urea at a temperature of greater than 37° C. and a pH of about 7.35 with normoxic oxygen levels. 
     
     
         58 . The method of  claim 54 , wherein the budding yeast cell is a  Cryptococcus  spp.,  Candida  spp., or  Mucor  spp. 
     
     
         59 . A method of identifying differentially expressed genes in budding yeast cells in response to a test compound, comprising:
 (a) growing the budding yeast cell in vitro under infection mimetic conditions to produce a metabolically synchronous population of budding yeast cells;   (b) adding a test compound to the metabolically synchronous population of budding yeast cells;   (c) sampling continuous cultures of the metabolically synchronous population of budding yeast cells of (b) over time; and   (d) identifying one or more genes that are differentially expressed when the budding yeast cells are grown under infection mimetic conditions and in the presence of the test compound as compared to when grown under standard laboratory conditions and in the presence of a test compound.   
     
     
         60 . The method of  claim 59  wherein the infection mimetic conditions mimic blood of the infected animal and comprise growing the budding yeast cell in a growth media comprising 150 mM NaCl, 5.5 mM glucose, free amino acids, macrophages, neutrophils, and human serum factors at a temperature of greater than 37° C. and a pH of <7.35 with normoxic oxygen levels. 
     
     
         61 . The method of  claim 59 , wherein the infection mimetic conditions mimic a wound infection in the animal and comprise growing the budding yeast cell in a media comprising less than 150 mM NaCl, 5.5 mM glucose, free amino acids, collagen, elastin, fibronectin macrophages, neutrophils, interleukin, and tumor necrosis factor at a temperature of greater than 37° C. and a pH ranging from 7.15 to 8.9 with hypoxic oxygen levels. 
     
     
         62 . The method of  claim 59 , wherein the infection mimetic conditions mimic cerebral spinal fluid of the infected animal and comprise growing the budding yeast cell in a media comprising phosphate buffered saline (PBS), 5 g/L of urea and 0.1% dextrose or in a media comprising 150 mM NaCl, 3 mM glucose, and 4 mM urea at a temperature of greater than 37° C. and a pH of about 7.35 with normoxic oxygen levels. 
     
     
         63 . The method of  claim 59 , wherein the budding yeast cell is a  Cryptococcus  spp.,  Candida  spp., or  Mucor  spp.