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

Abstract

This invention provides methods for the discovery of molecules that target an essential step of bacterial DNA replication—the sealing of DNA strands by NAD + -dependent DNA ligase. Specific structural components of NAD + -dependent DNA ligase that are important for the reaction of DNA ligase with NAD + and that comprise a putative binding site for the NMN component of NAD + were identified. The invention also includes recombinant DNA ligase enzymes that are defective in their reaction with NAD + , but active in the ligation of pre-adenylated DNA nicks. An underlying principle of this invention is the use of NAD + -reactive and NAD + -defective ligases to identify molecules that specifically bind to the NAD + -binding site of DNA ligase and thereby interfere with the reaction of DNA ligase with NAD + .

1 . An isolated DNA molecule encoding a mutated 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 enzyme is mutated by deleting domain Ia of said DNA ligase or by substituting one or more conserved residues in said domain Ia.  
     
     
         2 . The isolated DNA molecule of  claim 1 , wherein said DNA encodes an  Amsacta moorei  entomopoxvirus NAD + -dependent DNA ligase comprising substitution mutation at one or more of said conserved residues selected from the group consisting of Tyrosine-39, Tyrosine-40, Aspartate-48, Aspartate-52 and Tyrosine-51.  
     
     
         3 . The isolated DNA molecule of  claim 1 , wherein said DNA encodes an  Escherichia coli  NAD + -dependent DNA ligase comprising substitution mutation at one or more of said conserved residues selected from the group consisting of Tyrosine-22, Histidine-23, Aspartate-32, Tyrosine-35 and Aspartate-36.  
     
     
         4 . A recombinant expression vector comprising the isolated DNA of  claim 1  and regulatory elements necessary for expressing said DNA in a host cell.  
     
     
         5 . A recombinant expression vector comprising the isolated DNA of  claim 2  and regulatory elements necessary for expressing said DNA in a host cell.  
     
     
         6 . A recombinant expression vector comprising the isolated DNA of  claim 2  and regulatory elements necessary for expressing said DNA in a host cell.  
     
     
         7 . A host cell comprising the expression vector of  claim 4 .  
     
     
         8 . A host cell comprising the expression vector of  claim 5 .  
     
     
         9 . A host cell comprising the expression vector of  claim 6 .  
     
     
         10 . 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.  
     
     
         11 . The isolated DNA ligase of  claim 10 , wherein said DNA ligase is an  Amsacta moorei  entomopoxvirus NAD + -dependent DNA ligase comprising substitution mutation at one or more of said conserved residues selected from the group consisting of Tyrosine-39, Tyrosine-40, Aspartate-48, Tyrosine-51 and Aspartate-52.  
     
     
         12 . The isolated DNA ligase of  claim 11 , wherein said DNA ligase is an  Escherichia coli  NAD + -dependent DNA ligase comprising substitution mutation at one or more of said conserved residues selected from the group consisting of Tyrosine-22, Histidine-23, Aspartate-32, Tyrosine-35 and Aspartate-36.  
     
     
         13 . 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.    
     
     
         14 . The method of  claim 13 , wherein said mutated DNA ligase is an  Escherichia coli  NAD + -dependent DNA ligase compromising a substitution mutation in said domain Ia at one or more conserved residues selected from the group consisting of Tyrosine-22, Histidine-23, Aspartate-32, Tyrosine-35, and Aspartate-36.  
     
     
         15 . 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.    
     
     
         16 . 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.    
     
     
         17 . 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.