Methods for the identification of fungal glucose utilization inhibitors and antifungal agents
AssigneeMycologics, Inc.
InventorsClaude P. Selitrennikoff, Mitsunori Nakata
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Abstract
The present invention provides methods for simultaneously assessing microbial phosphoglucose isomerase, ketol-isomerase and glucosamine-6-phosphate acetyltransferase activities, by measuring the production of Coenzyme A (CoA). The present invention finds use in the isolation of new classes of antifungal drugs, wherein the compounds have the ability to inhibit fungal glucose utilization.
Claims21
A method for the detection of phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities in a sample, comprising the steps of:
- a) providing:
- i) a sample suspected to contain phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities,
- ii) glucose-6-phosphate,
- iii) glutamine,
- iv) acetyl coenzyme A, and
- v) 5,5′-dithiobis(2-nitrobenzoic acid);
- b) combining said sample, said glucose-6-phosphate, said glutamine, and said acetyl coenzyme A under conditions to yield reaction products comprising coenzyme A and N-acetylglucosamine-6-phosphate;
- c) inactivating said phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities;
- and
- d) combining said reaction product comprising coenzyme A and 5,5′-dithiobis(2-nitrobenzoic acid) under conditions to yield a chromogenic reaction product comprising 2-nitro-thiobezoate anion, wherein said chromogenic reaction product is indicative of phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities
A method for the detection of a compound having the ability to inhibit phosphoglucose isomerase, ketol-isomerase and/or acetyltransferase activities in a sample, comprising the steps of:
- a) providing:
- i) a sample suspected to contain phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities,
- ii) glucose-6-phosphate,
- iii) glutamine,
- iv) acetyl coenzyme A,
- v) 5,5′-dithiobis(2-nitrobenzoic acid), and
- vi) a candidate compound;
- b) preparing a first and second reaction mixture, wherein said first reaction mixture comprises said sample, said glucose-6-phosphate, said glutamine, and said acetyl coenzyme A, and wherein said second reaction mixture comprises said sample, said glucose-6-phosphate, said glutamine, said acetyl coenzyme A and said candidate compound;
- c) incubating said first and second reaction mixtures under conditions to yield reaction products comprising coenzyme A and N-acetylglucosamine-6-phosphate;
- d) inactivating said phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities;
- e) combining said first and second reaction mixtures with said 5,5′-dithiobis(2-nitrobenzoic acid) under conditions to yield a chromogenic reaction product comprising 2-nitro-thiobezoate anion;
- and
- f) comparing the quantity of said chromogenic reaction product in said first and second reaction mixtures.
Dependencies are inferred from the claim text.