Targeting enzymes of the trna splicing pathway for identification of anti-fungal and/or anti-proliferative molecules

A method of identifying an anti-fungal compound that inhibits or reduces the activity of a fungal tRNA splicing ligase, said method comprising:

• (a) contacting a population of 5′ tRNA half molecules and a population of 3′ tRNA half molecules with a fungal cell-free extract or a purified fungal tRNA splicing ligase and a compound or a member of a library of compounds, wherein the termini of one of the populations of tRNA molecules is labeled with a fluorophore and the other is labeled with a quencher;
• and
• (b) measuring the fluorescence of the half molecules, wherein an anti-fungal compound that inhibits or reduces the activity of a fungal tRNA splicing ligase is identified if an increase in the fluorescent signal is detectable relative to the signal in the absence of the compound or the presence of a negative control.

A method of identifying an anti-fungal compound that inhibits or reduces the activity of a fungal tRNA splicing ligase, said method comprising:

• (a) contacting a population of 5′ tRNA half molecules and a population of 3′ tRNA half molecules with a fungal cell-free extract or a purified fungal tRNA splicing ligase and a compound or a member of a library of compounds, wherein the termini of one of the populations of tRNA molecules is labeled with a fluorescent acceptor moiety and the other is labeled with a fluorescent donor moiety;
• and
• (b) measuring the fluorescence of the half molecules, wherein an anti-fungal compound that inhibits or reduces the activity of a fungal tRNA splicing ligase is identified if a decrease the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to the fluorescence emission in the absence of the compound or the presence of a negative control is detected.

A method of identifying an anti-proliferative compound that inhibits or reduces the activity of an animalia tRNA splicing ligase, said method comprising:

• (a) contacting a population of 5′ tRNA half molecules and a population of 3′ tRNA half molecules with an animalia cell-free extract or a purified animalia tRNA splicing ligase and a compound or a member of a library of compounds;
• and
• (b) measuring the ligation of the half molecules, wherein an anti-proliferative compound that inhibits or reduces the activity of an animalia tRNA splicing ligase is identified if an increase in ligated half molecules is detected relative to the signal in the absence of the compound or the presence of a negative control.

A method of identifying an anti-proliferative compound that inhibits or reduces the activity of an animalia tRNA splicing ligase, said method comprising:

• (a) contacting a population of 5′ tRNA half molecules and a population of 3′ tRNA half molecules with an animalia cell-free extract or a purified animalia tRNA splicing ligase and a compound or a member of a library of compounds, wherein the termini of one of the populations of tRNA molecules is labeled with a fluorophore and the other is labeled with a quencher;
• and
• (b) measuring the fluorescence of the half molecules, wherein an anti-proliferative compound that inhibits or reduces the activity of an animalia tRNA splicing ligase is identified if an increase in the fluorescent signal is detectable relative to the signal in the absence of the compound or the presence of a negative control.

A method of identifying an anti-proliferative compound that inhibits or reduces the activity of an animalia tRNA splicing ligase, said method comprising:

• (a) contacting a population of 5′ tRNA half molecules and a population of 3′ tRNA half molecules with an animalia cell-free extract or a purified animalia tRNA splicing ligase and a compound or a member of a library of compounds, wherein the termini of one of the populations of tRNA molecules is labeled with a fluorescent acceptor moiety and the other is labeled with a fluorescent donor moiety;
• and
• (b) measuring the fluorescence of the half molecules, wherein an anti-proliferative compound that inhibits or reduces the activity of an animalia tRNA splicing ligase is identified if a decrease the fluorescence emission of the fluorescent acceptor moiety at the wavelength of the fluorescent donor moiety relative to the fluorescence emission in the absence of the compound or the presence of a negative control is detected.