Materials and methods for identifying gyrase inhibitors

Patent metadata

Field	Value
Publication number	US-11613780-B2
Application number	US-201816009735-A
Country	US
Kind code	B2
Filing date	Jun 15, 2018
Priority date	Sep 1, 2016
Publication date	Mar 28, 2023
Grant date	Mar 28, 2023

Abstract

The disclosure describes the effects of transcription mediated from a promoter on the transcription mediated by divergently coupled supercoiling-sensitive promoter. Transcription initiated from a promoter inhibits transcription mediated by a specific supercoiling-sensitive promoter that is divergently coupled to the promoter. A gyrase inhibitor relieves this inhibition and substantially increases the transcription mediated by the specific supercoiling-sensitive promoter that is divergently coupled to another promoter. Accordingly, the invention pertains to a method for identifying a compound as a gyrase inhibitor or not a gyrase inhibitor based on differential expression of genes under the control of divergently coupled promoters in the presence of the compound. Another embodiment of the invention provides an assay for identifying one or more compounds from a library of compounds as a gyrase inhibitor. Polynucleotides and cells containing such polynucleotides that are suitable for carrying out the methods described herein are also provided.

First claim

We claim:

1. A method for identifying a compound as a gyrase inhibitor or not a gyrase inhibitor, the method comprising the steps of:
a) providing a cell or a culture of a cell, wherein the cell comprises a linear polynucleotide comprising:
i) a first inducible promoter under the control of an inducer and operably linked to a first gene and at least one terminator, and
ii) a second promoter operably linked to a second gene;
wherein the first inducible promoter is P T7lac , P trc , P lac , P T7Al/O4 , P lacUV5 , P rhaBAD , P ara , P tetA , P recA , P phoA , P trp , P nar , P PL , P cspA , P proU , or P cst-4 and the second promoter is P gyrA or P gyrB ; wherein the second promoter is divergently coupled to the first inducible promoter such that the first inducible promoter is located between the second promoter and the first gene and the second promoter is located between the first inducible promoter and the second gene, wherein transcription of the first gene under the control of the first inducible promoter causes negative supercoiling of the second promoter, and said negative supercoiling of the second promoter inhibits transcription of the second gene from the second promoter;
b) optionally, culturing the cell or the culture of the cell;
c) incubating a first portion of the culture, provided in step a) or cultured in step b), in the presence of the compound and incubating a second portion of the culture, provided in step a) or cultured in step b), in the absence of the compound;
d) measuring the expression of the first gene and/or the second gene in the first portion and the second portion after the incubation of step c); and
e) identifying the compound as:
i) the gyrase inhibitor:

A) if the expression of the second gene is higher in the first portion compared to the expression of the second gene in the second portion, or
B) if the expression of the first gene is higher in the second portion compared to the expression of the first gene in the first portion; or
ii) not the gyrase inhibitor:

A) if the expression of the second gene is not higher in the first portion compared to the expression of the second gene in the second portion, or
B) if the expression of the first gene is not higher in the second portion compared to the expression of the first gene in the first portion.

2. The method of claim 1 , wherein the cell is a bacterial cell.

3. The method of claim 2 , wherein the bacterial cell is Escherichia coli.

4. The method of claim 1 , wherein the first inducible promoter is P T7lac , P lac , or P lacUV5 .

5. The method of claim 1 , wherein the second promoter is or P gyrB .

6. The method of claim 1 , wherein at least the first inducible promoter is heterologous to the first gene or the second promoter is heterologous to the second gene.

7. The method of claim 1 , wherein the linear polynucleotide consists of:
i) the first inducible promoter under the control of an inducer and operably linked to the first gene and at least one terminator, and
ii) the second promoter operably linked to the second gene,
wherein the first inducible promoter is P trc , P lac , P lacUV5 , P rhaBAD , P ara , P tetA , P recA , P phoA , P trp , P nar , P PL , P cspA , P proU , or P cst-4 and the second promoter is P gyrA or P gyrB ;
wherein the second promoter is divergently coupled to the first promoter such that the first inducible promoter is located between the second promoter and the first gene, the second promoter is located between the first inducible promoter and the second gene, and at least the first promoter is heterologous to the first gene or the second promoter is heterologous to the second gene; and wherein transcription of the first gene under the control of the first promoter causes negative supercoiling of the second promoter, and said negative supercoiling of the second promoter inhibits transcription of the second gene from the second promoter.

8. The method of claim 1 , the inducer being a condition.

9. A screening assay to identify one or more compounds from a plurality of compounds, wherein the screening assay comprises the steps of:
a) providing a cell or a culture of a cell wherein the cell comprises a linear polynucleotide comprising:
i) a first inducible promoter under the control of an inducer and operably linked to a first gene and at least one terminator, and
ii) a second promoter operably linked to a second gene;
wherein the first inducible promoter is P T7lac , P trc , P lac , P T7Al/O4 , P lacUV5 , P rhaBAD , P ara , P tetA , P recA , P phoA , P trp , P nar , P PL , P cspA , P proU , or P cst-4 and the second promoter is P gyrA or P gyrB ; wherein the second promoter is divergently coupled to the first inducible promoter such that the first inducible promoter is located between the second promoter and the first gene and the second promoter is located between the first inducible promoter and the second gene, wherein transcription of the first gene under the control of the first inducible promoter causes negative supercoiling of the second promoter, and said negative supercoiling of the second promoter inhibits transcription of the second gene from the second promoter;
b) optionally, culturing the cell or the culture of the cell;
c) incubating a plurality of test portions of the culture, provided in step a) or cultured in step b), each test portion in the plurality of test portions in the presence of one compound from the plurality of compounds and incubating a control portion of the culture, provided in step a) or cultured in step b), in the absence of any compound from the plurality of compounds;
d) measuring the expression of the first gene and/or the second gene in each of the plurality of test portions and the control portion after the incubation of step c); and
e) identifying each compound in the plurality of compounds as:
i) a gyrase inhibitor:

A) if the expression of the second gene is higher in the test portion compared to the expression of the second gene in the control portion, or
B) if the expression of the first gene is higher in the control portion compared to the expression of the first gene in the test portion; or
ii) not a gyrase inhibitor:

A) if the expression of the second gene is not higher in the test portion compared to the expression of the second gene in the control portion, or
B) if the expression of the first gene is not higher in the control portion compared to the expression of the first gene in the test portion.

10. The method of claim 9 , wherein at least the first inducible promoter is heterologous to the first gene or the second promoter is heterologous to the second gene.

11. The method of claim 9 , wherein the cell is a bacterial cell.

12. The method of claim 11 , wherein the bacterial cell is Escherichia coli.

13. The method of claim 9 , wherein the first inducible promoter is P T7lac , P lac , or P lacUV5 .

14. The method of claim 9 , wherein the second promoter is P gyrB .

15. A method comprising the steps of:
a) providing a cell or a culture of a cell, wherein the cell comprises a linear polynucleotide comprising:
i) a first inducible promoter under the control of an inducer and operably linked to a first gene and at least one terminator, and
ii) a second promoter operably linked to a second gene;

wherein the first inducible promoter is P T7lac , P trc , P lac , P T7Al/O4 , P lacUV5 , P rhaBAD , P ara , P tetA , P recA , P phoA , P trp , P nar , P PL , P cspA , P roU , or P est-4 and the second promoter is P gyrA or P gyrB ; wherein the second promoter is divergently coupled to the first inducible promoter s

Assignees

• Leng Fenfei
• Zhi Xiaoduo
• Dages Samantha
• Dages Kelley
• The Florida International Univ Board Of Trustees

Inventors

• Leng Fenfei
• Zhi Xiaoduo
• Dages Samantha
• Dages Kelley

Classifications

• C12N9/90 Primary

  Isomerases (5.) · CPC title

• G01N33/5023

  on expression patterns · CPC title

• C12Y599/01003

  DNA topoisomerase (ATP-hydrolysing) (5.99.1.3) · CPC title

• C12N15/1086

  Preparation or screening of expression libraries, e.g. reporter assays · CPC title

• C12Q1/025

  for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18) · CPC title