Antibiotic compositions and related screening methods

Abstract

Moenomycin inhibits bacterial growth by clocking the transglycosylase activity of class A penicillin-binding proteins (PBPs), which are key enzymes in bacterial cell wall synthesis. The binding affinities of moenomycin A with various truncated PBPs were compared showing that the transmembrane domain is important for moenomycin binding. Full-length class-A PBPs from 16 bacterial species were produced, and their binding activities showed a correlation with the antimicrobial activity of moenomycin against Enterococcus faecalis and Staphylococcus aureus . Moreover, a fluorescence anisotropy-based high-throughput assay was developed and used successfully for identification of transglycosylase inhibitors.

We claim: 
     
         1 . A composition comprising:
 an antibiotic agent for inhibiting transglycosylase activity of bacterial class A penicillin-binding proteins (PBPs); and   pharmaceutically acceptable carrier,   wherein the PBP comprises at least a transmembrane (TM) domain and a transglycosylase (TG) domain, and   wherein the agent exhibits a higher first binding affinity to a transmembrane (TM) domain-containing class A penicillin-binding protein (PBP), or fragment thereof, as compared to a second binding affinity to the PBP fragment that lacks the TM domain.   
     
     
         2 . The composition of  claim 1 , wherein the binding affinity between the antibiotic agent and the class A penicillin-binding protein is determined by anisotropy measurement assay. 
     
     
         3 . The composition of  claim 2 , wherein the anisotropy measurement assay PBP binding is at least one of a competition displacement assay or a direct binding assay. 
     
     
         4 . The composition of  claim 3 , wherein the competition displacement assay competes a labeled moenomycin with the antibiotic agent. 
     
     
         5 . The composition of  claim 2 , wherein the anisotropy measurement of PBP binding, comprises a fluorescence anisotropy (FA) assay. 
     
     
         6 . The composition of  claim 1 , wherein the class A penicillin-binding protein (PBP) comprises three domains which, from N terminus to C terminus, are transmembrane, transglycosylase, and transpeptidase. 
     
     
         7 . The composition of  claim 1 , wherein the antibiotic agent is selected from (Z)-5-(4-bromophenyl)-3-((5-nitrofuran-2-yl)methylene)furan-2(3H)-one, (Z)-1,3-diphenyl-4-(2-(thiazol-2-yl)hydrazono-1H-pyrazol-5(4H)-one, and (E)-4-(2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-enyl)benzoic acid. 
     
     
         8 . The composition of  claim 1 , wherein the antibiotic agent inhibits bacterium growth, wherein the bacterium is  Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus  (MRSA Mu50),  Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica  or  Neisseria gonorrhoeae.    
     
     
         9 . The composition of  claim 8 , wherein the bacterium is  E. faecalis  or  S. aureus.    
     
     
         10 . The composition of  claim 1 , wherein the antibacterial agent is a small molecule. 
     
     
         11 . The composition of  claim 1 , wherein the antibacterial agent has a formula selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         12 . An antibiotic agent for inhibiting transglycosylase activity of bacterial class A penicillin-binding proteins (PBPs), wherein the PBP comprises at least a transmembrane (TM) domain and a transglycosylase (TG) domain, wherein the agent exhibits a higher first binding affinity to a transmembrane (TM) domain-containing class A penicillin-binding protein (PBP), or fragment thereof, as compared to a second binding affinity to the PBP fragment that lacks the TM domain and
 wherein the antibiotic agent has a formula selected from the group consisting of:   
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         13 . A method for inhibiting bacterial growth in a subject, the method comprising treating a subject having a bacterial infection or susceptible to a bacterial infection with a pharmaceutical composition comprising:
 an antibiotic agent for inhibiting transglycosylase activity of bacterial class A penicillin-binding proteins (PBPs); and   pharmaceutically acceptable carrier,   wherein the PBP comprises at least a transmembrane (TM) domain and a transglycosylase (TG) domain, and   wherein the agent exhibits a higher first binding affinity to a transmembrane (TM) domain-containing class A penicillin-binding protein (PBP), or fragment thereof, as compared to a second binding affinity to the PBP fragment that lacks the TM domain.   
     
     
         14 . The method of  claim 13 , wherein the class A penicillin-binding protein (PBP) comprises three domains which, from N terminus to C terminus, are transmembrane, transglycosylase, and transpeptidase. 
     
     
         15 . The method of  claim 13 , wherein the antibiotic agent is selected from (Z)-5-(4-bromophenyl)-3-((5-nitrofuran-2-yl)methylene)furan-2(3H)-one, (Z)-1,3-diphenyl-4-(2-(thiazol-2-yl)hydrazono-1H-pyrazol-5(4H)-one, and (E)-4-(2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-enyl)benzoic acid. 
     
     
         16 . The method of  claim 13 , wherein the antibiotic agent inhibits bacterium growth, wherein the bacterium is  Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus  (MRSA Mu50),  Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica  or  Neisseria gonorrhoeae.    
     
     
         17 . The method of  claim 13 , wherein the antibiotic agent inhibits bacterial growth, wherein the bacteria are gram-positive bacteria. 
     
     
         18 . The method of  claim 16 , wherein the bacterium is  E. faecalis  or  S. aureus.    
     
     
         19 . The method of  claim 13 , wherein the antibiotic agent has a formula selected from the group consisting of: