Pharmacological targeting of bacterial DNA ligase for treatment and prevention of bacterial infections

An isolated NAD+-dependent DNA ligase enzyme that is defective in its reaction with NAD+ but is active in the ligation of pre-adenylated DNA nicks, wherein said DNA ligase is mutated by deleting domain Ia of said DNA ligase or by substituting one or more conserved residues in said domain Ia.

A method of screening for a compound that binds to the NAD+ substrate recognition site of NAD+-dependent DNA ligase, comprising the steps of:

• contacting a NAD+-dependent DNA ligase or a fragment thereof comprising domain Ia with a test compound;
• contacting a mutated NAD+-dependent DNA ligase or a fragment thereof with said test compound, wherein said mutated DNA ligase or a fragment thereof comprises a substitution mutation at one or more conserved residues in said domain Ia;
• comparing the binding of said test compound to said NAD+-dependent DNA ligase or a fragment thereof and said mutated NAD+-dependent DNA ligase or a fragment thereof, wherein binding of said test compound to said NAD+-dependent DNA ligase or a fragment thereof but not to said mutated NAD+-dependent DNA ligase or a fragment thereof indicates that said test compound binds to the NAD+ substrate recognition site of NAD+-dependent DNA ligase.

A method of screening for a compound that binds to the NAD+ substrate recognition site of NAD+-dependent DNA ligase, comprising the steps of:

• contacting a NAD+-dependent DNA ligase or a fragment thereof comprising domain Ia with a test compound;
• contacting a mutated NAD+-dependent DNA ligase or a fragment thereof with said test compound, wherein said mutated DNA ligase or a fragment thereof comprises a substitution mutation at one or more conserved residues in said domain Ia, wherein said substitution mutation is selected from the group consisting of Tyrosine-22, Histidine-23, Aspartate-32, Tyrosine-35, and Aspartate-36;
• comparing the binding of said test compound to said NAD+-dependent DNA ligase or a fragment thereof and said mutated NAD+-dependent DNA ligase or a fragment thereof, wherein binding of said test compound to said NAD+-dependent DNA ligase or a fragment thereof but not to said mutated NAD+-dependent DNA ligase or a fragment thereof indicates that said test compound binds to the NAD+ substrate recognition site of NAD+-dependent DNA ligase.

A method of screening for a compound that inhibits the nucleotidyl transferase activity of NAD+-dependent DNA ligase, comprising the steps of:

• contacting a NAD+-dependent DNA ligase with a test compound in the presence of NAD+ and a divalent cation;
• and
• detecting the reaction of said DNA ligase with NAD+ to form covalent ligase-AMP intermediate in the presence or absence of said test compound, wherein the lack of formation of ligase-AMP intermediate or a reduction in the formation of ligase-AMP intermediate in the presence of said compound indicates said compound inhibits nucleotidyl transferase activity of NAD+-dependent DNA ligase.

A method of screening for a compound that inhibits the DNA ligase activity of NAD+-dependent DNA ligase, comprising the steps of:

• contacting a NAD+-dependent DNA ligase with a test compound in the presence of NAD+, a divalent cation, and a nicked duplex DNA substrate containing 3′-OH and 5′-PO4 termini at the nick;
• and
• detecting the sealing of said DNA nick to form a phosphodiester in the presence or absence of said test compound, wherein the lack of sealing of said DNA nick or a reduction in the sealing of said DNA nick in the presence of said compound indicates said compound inhibits DNA ligase activity of NAD+-dependent DNA ligase.