Method and reagent for measuring nitroreductase enzyme activity

1. A method for detecting nitroreductase enzyme activity in a composition comprising:

i) mixing said composition under conditions to promote nitroreductase activity with a dye molecule; and

ii) measuring a change in an optical property of said dye molecule wherein said change is a measure of the amount of nitroreductase activity;

wherein said dye molecule is a squaraine dye comprising at least one NO 2 group.

2. The method of claim 1 , wherein said composition comprises at least one cell or a cell extract.

3. The method of claim 1 , wherein said method is conducted in the presence of a test agent whose effect on nitroreductase enzyme activity is to be determined.

4. A method which comprises:

i) contacting a host cell with a dye molecule wherein said host cell has been transfected with a nucleic acid molecule comprising expression control sequences operably linked to a sequence encoding a nitroreductase; and

ii) measuring a change in an optical property of said dye molecule wherein said change is a measure of the amount of nitroreductase activity;

wherein said dye is a squaraine dye comprising at least one NO 2 group.

5. The method of claim 1 or 4 , wherein said change in an optical property is an increase in the fluorescence intensity of the dye molecule, whereby said increase is a measure of the amount of nitroreductase activity.

6. The method of claim 1 or 4 , wherein said squaraine dye is of formula (I):

wherein:

R 3 is attached to the Z 1 ring structure and R 4 is attached to the Z 2 ring structure;

Z 1 and Z 2 independently represent a phenyl or a naphthyl ring system;

X and Y are the same or different and are selected from oxygen, sulphur, —CH═CH— and the group:

groups R 1 and R 2 are independently selected from C 1 -C 4 alkyl, —(CH 2 ) n —P , —{(CH 2 ) 2 —O} p —R 6 and the group W; where P is selected from COOR 7 , SO 3 − and OH, W is mono- or di-substituted nitrobenzyl, R 6 is methyl or ethyl, R 7 is selected from H, C 1 -C 4 alkyl and CH 2 OC(O)R 8 , where R 8 is methyl, or t-butyl, n is an integer from 1 to 10, and p is an integer from 1to 3;

groups R 3 and R 4 are independently selected from hydrogen, NO 2 , halogen, SO 3 − , C 1 -C 4 alkoxy and —(CH 2 ) m —COOR 7 ; where R 7 is hereinbefore defined and m is 0 or an integer from 1 to 5;

R 5 is C 1 -C 6 alkyl optionally substituted with COOR 7 , SO 3 − , or OH; where R 7 is hereinbefore defined; and

at least one of groups R 1 , R 2 , R 3 and R 4 comprises at least one NO 2 group.

7. The method of claim 6 , wherein said dye is, or is rendered, cell permeable.

8. The method of claim 1 or 4 , wherein said squaraine dye

is a compound selected from dyes of formula:

wherein:

X and Y are the same or different and are selected from oxygen, sulphur, —CH═CH— and the group:

wherein R 5 is hereinbefore defined;

at least one of groups R 1 and R 2 is the group W; where W is hereinbefore defined;

any remaining group R 1 or R 2 is selected from C 1 -C 4 alkyl, —(CH 2 ) n —P and —{(CH 2 ) 2 —O} p —R 6 ; where P is selected from COOR 7 , SO 3 − and OH, R 6 is methyl or ethyl, R 7 is selected from H, C 1 -C 4 alkyl and CH 2 OC(O)R 8 , where R 8 is methyl, or t-butyl, n is an integer from 1 to 10 and p is an integer from 1 to 3; and

groups R 3 and R 3 are independently selected from hydrogen, halogen, SO 3 − , C 1 -C 4 alkoxy and —(CH 2 ) m —COOR 7 ; where R 7 is hereinbefore defined and m is 0 or an integer from 1 to 5.

9. The method of claim 8 , wherein one of groups R 1 and R 2 is selected from group W where W is selected from:

remaining R 1 or R 2 is selected from methyl and ethyl, or is the group —(CH 2 ) n —COOR 7 where R 7 is selected from H, C 1 -C 4 alkyl and CH 2 OC(O)R 8 , where R 8 is methyl, or t-butyl, n is an integer from 1 to 10, preferably 5 or 6.

10. The method of claim 9 , wherein W is the group:

and remaining R 1 or R 2 is hereinbefore defined.

11. The method of claim 1 or 4 , wherein said squaraine dye is a compound of formula:

wherein:

X and Y are the same or different and are selected from oxygen, sulphur, —CH═CH— and the group:

wherein R 5 is hereinbefore defined;

groups R 1 or R 2 are independently selected from C 1 -C 4 alkyl, —(CH 2 ) n —P and —{(CH 2 ) 2 —O} p —R 6 ; where P is selected from COOR 7 , SO 3 − and OH, R 6 is methyl or ethyl, R 7 is selected from H, C 1 -C 4 alkyl and CH 2 OC(O)R 8 , where R 8 is methyl, or t-butyl, n is an integer from 1 to 10 and p is an integer from 1 to 3;

at least one of groups R 3 and R 4 is NO 2 ; and

any remaining group R 3 or R 4 is selected from hydrogen, SO 3 − , C 1 -C 4 alkoxy and —(CH 2 ) m —COOR 7 ; where R 7 is selected from H, C 1 -C 4 alkyl and CH 2 OC(O)R 8 , where R 8 is methyl, or t-butyl, and m is 0 or an integer from 1 to 5.

12. A method for screening for a test agent whose effect upon nitroreductase gene expression is to be determined, said method comprising:

a) performing the method of claim 4 in the absence and in the presence of said test agent; and

b) determining the amount of nitroreductase gene expression in the absence and in the presence of said agent;

wherein a difference between the amount of nitroreductase gene expression in the absence and in the presence of said agent is indicative of the effect of said agent on nitroreductase gene expression.

13. A method of screening for a test agent whose effect upon nitroreductase gene expression is to be determined, said method comprising:

a) performing the method of claim 4 in the presence of said agent; and

b) comparing the amount of nitroreductase gene expression with a control value for the amount of nitroreductase gene expression in the absence of the test agent.

14. The method of claim 13 , wherein said control value is stored electronically in a database or other electronic format.