Screening for enzyme inhibitors

A method for screening a candidate compound for effective inhibition of a target enzyme, employing as reagents:

• (1) an ED-conjugate comprising a substantially inactive first portion of β-galactosidase conjugated to an inhibitor of the target enzyme;
• (2) an enzyme acceptor (EA) comprising a substantially inactive second portion of β-galactosidase, whereby the ED-conjugate and the EA complex to form an active â-galactosidase;
• and
• (3) a substrate for the β-galactosidase, wherein the substrate reacts to form a product, and at least one of the substrate and product provides a detectable signal, the method comprising steps:
• a) combining the candidate compound, ED-conjugate, EA, target enzyme, and β-galactosidase substrate under binding conditions, wherein the candidate compound and the ED-conjugate compete for the target enzyme;
• and
• b) determining the signal from at least one of the substrate and product, whereby a difference in the signal observed in the absence of the candidate compound indicates the candidate compound inhibits the target enzyme.

A method for screening a candidate compound for effective inhibition of at least one target kinase, employing as reagents:

• (1) an ED-conjugate comprising the alpha-portion of β-galactosidase conjugated to an inhibitor of the at least one kinase;
• (2) an enzyme acceptor (EA) comprising at least a major portion of the remaining portion of â-galactosidase, whereby when the ED-conjugate and the EA complex an active β-galactosidase is formed;
• and
• (3) a substrate for the β-galactosidase, wherein the substrate reacts to form a product, and at least one of the substrate and the product provides a detectable signal, the method comprising steps:
• a) combining the candidate compound, ED-conjugate, EA, at least one target kinase and β-galactosidase substrate under binding conditions, wherein the candidate compound and the ED-conjugate compete for the at least one target kinase;
• and
• b) determining the signal from at least one of substrate and product, whereby a difference in signal observed in the absence of the candidate compound indicates the candidate compound inhibits the at least one kinase.

A method for screening a candidate compound for effective inhibition of at least one target kinase, employing as reagents:

• (1) an ED-staurosporine conjugate comprising the alpha-portion of β-galactosidase;
• (2) an enzyme acceptor (EA) comprising at least a major portion of the remaining portion of β-galactosidase, whereby when the ED-conjugate and the EA complex an active β-galactosidase is formed;
• and
• (3) a substrate for the β-galactosidase, wherein the substrate reacts to form a product, and at least one of the substrate and the product provides a detectable signal, the method comprising steps:
• a) incubating the candidate compound, ED-staurosporine conjugate, EA, and at least one target kinase under binding conditions, wherein the candidate compound and the ED-staurosporine conjugate compete for at least one target kinase;
• b) adding substrate for the β-galactosidase;
• and
• c) determining the signal from at least one of the substrate and product, whereby a difference in the signal observed in the absence of the candidate compound indicates the candidate compound inhibits the at least one kinase.

A method for high throughput screening of candidate compounds for effective inhibition of at least one target kinase, employing as reagents:

• (1) an ED-conjugate comprising the alpha-portion of β-galactosidase conjugated to an inhibitor of the at least one kinase;
• (2) an enzyme acceptor (EA) comprising at least a major portion of the remaining portion of β-galactosidase, whereby when the ED-conjugate and the EA complex an active β-galactosidase is formed;
• and
• (3) a substrate for the β-galactosidase, wherein the substrate reacts to form a product, and at least one of the substrate and the product provides a detectable signal, the method comprising steps:
• a) combining in separate wells of a microtiter well plate different ones of the candidate compounds, ED-conjugate, EA, at least one target kinase, and β-galactosidase substrate under binding conditions, wherein the candidate compound and the ED-conjugate compete for at least one target kinase;
• and
• b) determining the signal from at least one of the substrate and product in each of the wells, whereby a difference in signal observed in the absence of the candidate compound indicates the candidate compound inhibits the at least one kinase.

A compound of the formula:

• (deNHCH3)STA-(NH)-R-L
• wherein (deNHCH3)STA is staurosporine without the methylamino group on the tetrahydropyran ring;
• wherein NH is amino;
• and
• wherein L is a particle or a ligand of at least about 100 Dal and not more than about 5 kDal.

A compound of the formula

• 4
• wherein A is
• 5
• wherein R is a linking group having a chain of from 1 to 20 atoms in the chain, wherein said chain is composed of C, N, O, S, P and is aliphatic, alicyclic, aromatic or heterocyclic, or a combination thereof;
• and
• wherein any one hydrogen on the ring or a substituent on the ring is replaced with a bond or linking group to an enzyme donor from β-galactosidase.