Metabolic pathway assay
First Claim
1. A kit which is comprised of an enzyme cascade comprising a first enzyme, a second enzyme and a substrate for the first enzyme.
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Accused Products
Abstract
An in vitro screening assay which identifies enzyme inhibitors and allows for the simultaneous assay of many enzymes. Enzyme, substrate, co-factor, etc. concentrations are optimized so that inhibitors of any one of the enzymes in the pathway are equally likely to be detected. Necessarily, the flux of substrate through each enzyme should be nearly the same during the assay, i.e., each of the enzyme catalyzed steps must be equally rate-limiting. Preferably, optimal assay conditions are predicted by computer modeling. Further, the pathway conditions are optimized through variation of enzyme, starting substrate, co-substrate and co-factor concentrations. A positive response is initially detected as a change in the amount of the product generated at the end of the enzyme cascade as compared to a standard. A sample producing a positive result can be deconvoluted.
15 Citations
37 Claims
- 1. A kit which is comprised of an enzyme cascade comprising a first enzyme, a second enzyme and a substrate for the first enzyme.
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17. A method of screening for a biologically active compound which comprises:
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combining an enzyme cascade, comprising a first enzyme, a second enzyme and a substrate for the first enzyme, with a compound suspected of having biological activity; and measuring the concentration of the products of the enzymes and comparing to a standard. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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37. A high-throughput in vitro screening method for detecting a biologically active compound which is comprised of:
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(a) combining an enzyme cascade comprising a first enzyme, a second enzyme, a third enzyme, a fourth enzyme, a fifth enzyme and a sixth enzyme, and a labeled substrate for the first enzyme with a compound suspected of having biological activity;
wherein;the first enzyme is the gene product of murA, the second enzyme is the gene product of murB, the third enzyme is the gene product of murC, the fourth enzyme is the gene product of murD, the fifth enzyme is the gene product of murE, and the sixth enzyme is the product of murF; and (b) measuring the concentrations of the products of the enzymes and comparing to a standard, by absorbing the product of the sixth enzyme onto resin and detecting the amount of label and comparing the amount of label to control.
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Specification