Methods for the identification of antimicrobial compounds
AssigneeMycologics, Inc.
InventorsClaude P. Selitrennikoff, Mitsunori Nakata
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Abstract
The present invention relates to methods to assay 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase activity. The present invention also relates to methods for drug screening to identify compounds having antimicrobial activity, wherein the compounds have the ability to inhibit the enzymatic activity of a microbial ketol-isomerase. In other embodiments, methods are provided for the identification of compounds that selectively inhibit microbial ketol-isomerase activity compared to the ketol-isomerase activity of the subject being treated for an infection.
Claims29
A method for the detection of glutamate in an experimental sample, comprising the steps of:
- a) providing:
- i) an experimental sample,
- ii) glutamate dehydrogenase,
- iii) nicotinamide adenine dinucleotide,
- iv) nitro blue tetrazolium chloride, and
- v) phenazine methosulfate;
- and
- b) combining said experimental sample, said glutamate dehydrogenase activity, said nicotinamide adenine dinucleotide, said nitro blue tetrazolium chloride, and said phenazine methosulfate under conditions in which said nitro blue tetrazolium chloride is reduced to yield a chromogenic product, and wherein production of said chromogenic product is indicative of glutamate in said experimental sample.
A method for the detection of ketol-isomerase activity in a sample, comprising the sequential steps of:
- a) providing:
- i) a sample,
- ii) fructose-6-phosphate,
- iii) glutamine,
- iv) glutamate dehydrogenase,
- v) nicotinamide adenine dinucleotide,
- vi) nitro blue tetrazolium chloride, and
- vii) phenazine methosulfate;
- b) combining said sample, said fructose-6-phosphate, and said glutamine under conditions to yield a reaction product comprising glucosamine-6-phosphate and glutamate;
- c) inactivating said ketol-isomerase activity;
- and
- d) combining said reaction product with said glutamate dehydrogenase activity, said nicotinamide adenine dinucleotide, said nitro blue tetrazolium chloride, and said phenazine methosulfate under conditions wherein said nitro blue tetrazolium chloride is reduced to yield a chromogenic product, wherein production of said chromogenic product is indicative of ketol-isomerase activity in said sample.
A method for the identification of a compound having the ability to inhibit a microbial ketol-isomerase activity, comprising the sequential steps of:
- a) providing:
- i) microbial ketol-isomerase,
- ii) fructose-6-phosphate,
- iii) glutamine,
- iv) glutamate dehydrogenase,
- v) nicotinamide adenine dinucleotide,
- vi) nitro blue tetrazolium chloride,
- vii) phenazine methosulfate, and
- viii) a candidate compound;
- and
- b) preparing a first and a second reaction mixture, wherein said first reaction mixture comprises said microbial ketol-isomerase, said fructose-6-phosphate and said glutamine, and wherein said second reaction mixture comprises said microbial ketol-isomerase, said fructose-6-phosphate, said glutamine and said candidate compound;
- c) exposing said first and second reaction mixtures to conditions wherein said microbial ketol-isomerase is capable of producing glucosamine-6-phosphate and glutamate;
- d) inactivating said microbial ketol-isomerase;
- e) combining said first and second reaction mixtures with said glutamate dehydrogenase, said nicotinamide adenine dinucleotide, said nitro blue tetrazolium chloride, and said phenazine methosulfate under conditions wherein said nitro blue tetrazolium chloride is capable of yielding a chromogenic product, wherein the quantity of chromogenic product produced is proportional to microbial ketol-isomerase activity;
- f) comparing ketol-isomerase activities in said first and second reaction mixtures, and
- g) identifying a candidate compound having the ability to inhibit said microbial ketol-isomerase activity, wherein the microbial ketol-isomerase activity of said first reaction mixture is greater than the ketol-isomerase activity of said second reaction mixture.
Dependencies are inferred from the claim text.