Crystal structure of bifunctional transglycosylase pbp1b from e. coli and inhibitors thereof

Abstract

The crystal structure at 2.16 Å resolution of the full-length bacterial bifunctional transglycosylase penicillin-binding protein 1b (PBP1b) from Escherichia coli , in complex with its inhibitor moenomycin, is provided. The atomic coordinates of the complex as well as the moenomycin binding site are provided. Three dimensional structures of amino acid residues involved in moenomycin binding and transglycosylation activity are identified. Binding site for peptidoglycan synthesis inhibitors comprising inhibitor-binding site comprises amino acid residues from at least one of transglycosylase (TG), UvrB domain 2 homolog (UB2H) and transmembrane (TM) domains of PBP1b are identified at an atomic level of resolution. Methods for rational drug design based on the atomic coordinates are provided. Methods for screening for antibiotics based on anisotropic binding assay and transglycosylase inhibitor assays are provided. Novel antibiotics based on the screening assays of the invention are disclosed.

1 . A method for identifying a potential transglycosylase (peptidoglycan glycosyltransferase) inhibitor compound, the method comprising the steps of:
 a) using a three-dimensional structure of  E. coli  Penicillin binding protein 1b (PBP1b) as defined by atomic coordinates according to  FIGS. 8-1  through  8 - 117 ;   b) employing said three-dimensional structure to design or select said potential inhibitor such that said potential inhibitor is capable of binding to at least one amino acid in an active site of PBP1b transglycosylase;   c) synthesizing the potential inhibitor;   d) in an assay, contacting the potential inhibitor with the PBP1b transglycosylase in the presence of lipid II, or derivative thereof; and   e) determining the PBP1b transglycosylase inhibitory activity of the potential inhibitor.   
     
     
         2 . The method of  claim 1 , wherein the potential transglycosylase inhibitor is designed or selected using computer modeling. 
     
     
         3 . The method of  claim 1 , wherein the potential transglycosylase inhibitor is designed de novo. 
     
     
         4 . The method of  claim 1 , wherein the potential transglycosylase inhibitor is designed based on a known inhibitor. 
     
     
         5 . The method of  claim 4 , wherein the known inhibitor is moenomycin A. 
     
     
         6 . The method of  claim 1 , wherein the transglycosylase active site comprises one or more of the amino acid residues E114, E171, E233, E290, S398, and S510. 
     
     
         7 . The method of  claim 1 , wherein the inhibitor-binding site comprises one or more of residues Thr269, Val273, Phe277, Tyr315, Gln318, Lys355, Gly356, and Ser 358 residues of PBP1b. 
     
     
         8 . The method of  claim 1 , wherein the inhibitor-binding comprises a hydrogen-bonding interaction with one or more of Glu233, Gln271, Asn275, Lys355, Arg286, Glu290 and Ser358 residues of  E. Coli  PBP1b. 
     
     
         9 . The method of  claim 7 , wherein the inhibitor-binding site comprises amino acid residues from at least one of transglycosylase (TG), UvrB domain 2 homolog (UB2H) and transmembrane (TM) domains of PBP1b. 
     
     
         10 . The method of  claim 1 , wherein the inhibitor prevents peptidoglycan elongation by structurally mimicking lipid IV at the binding site of transglycosylase. 
     
     
         11 . A method of using a co-crystal of an  E. coli  PBP1b transglycosylase enzyme with moenomycin A for screening for a novel drug capable of inhibiting a transglycosylase (peptidoglycan glycosyltransferase), the method comprising
 wherein said crystal effectively diffracts X-rays for the determination of the atomic coordinates of said PBP1b-moenomycin complex to a resolution of greater than 2.16 Å, and according to  FIGS. 8-1  through  8 - 117 , and wherein said method comprises:   a) selecting a potential ligand by performing rational drug design with the three-dimensional structure of the moenomycin binding site determined for the crystal;   b) in an assay, contacting the potential ligand with the ligand binding domain of the enzyme; and   c) detecting the binding potential of the potential ligand for the ligand binding domain, wherein the potential ligand is selected as a novel drug based on the potential ligand having a greater affinity for the ligand binding domain than that of a known drug.   
     
     
         12 . The method of  claim 11 , wherein the potential transglycosylase inhibitor is designed or selected using computer modeling. 
     
     
         13 . The method of  claim 11 , wherein the potential transglycosylase inhibitor is designed de novo. 
     
     
         14 . The method of  claim 11 , wherein the potential transglycosylase inhibitor is designed based on a known inhibitor. 
     
     
         15 . The method of  claim 14 , wherein the known inhibitor is moenomycin A. 
     
     
         16 . The method of  claim 11 , wherein the affinity of the inhibitor for PBP1b is determined by a fluorescence anisotropy assay. 
     
     
         17 . The method of  claim 11 , wherein the inhibitor-binding site comprises one or more of moenomycin-binding residues Thr269, Val273, Phe277, Tyr315, Gln318, Lys355, Gly356, and Ser 358 residues of PBP1b. 
     
     
         18 . The method of  claim 17 , wherein the inhibitor binding further comprises a hydrogen-bonding interaction with one or more of Glu233, Gln271, Asn275, Lys355, Arg286, Glu290 and Ser358 residues of  E. Coli  PBP1b. 
     
     
         19 . The method of  claim 17 , wherein the inhibitor-binding site comprises amino acid residues from at least one of transglycosylase (TG), UvrB domain 2 homolog (UB2H) and transmembrane (TM) domains of PBP1b. 
     
     
         20 . The method of  claim 11 , herein the inhibitor inhibits peptidoglycan elongation by structurally mimicking lipid IV at the binding site of transglycosylase. 
     
     
         21 . A method of evaluating the binding properties of a potential PBP1b transglycosylase inhibitor compound comprising the steps of:
 (a) co-crystallizing said compound with PBP1b;   (b) determining the three-dimensional structure of said PBP1b-potential inhibitor complex co-crystal by molecular replacement using the three-dimensional structure of PBP1b as defined by atomic coordinates according to  FIGS. 8-1  through  8 - 117 ; and   (c) analyzing said three-dimensional structure of said PBP1b bound to said potential inhibitor compound to evaluate the binding characteristics of said potential inhibitor compound.   
     
     
         22 . A method for identifying a potential inhibitor compound for  E. coli  Penicillin binding protein 1b (PBP1b) transglycosylase, the method comprising the steps of:
 (a) providing a candidate agent;   (b) in an anisotropy measurement assay, determining an effectiveness of the candidate agent to bind PBP1b; and   (c) in a transglycosylation assay, contacting the candidate agent with the PBP1b transglycosylase in the presence of lipid II, or derivative thereof, and determining a PBP1b transglycosylase inhibitory activity of the candidate agent.   
     
     
         23 . The method of  claim 22 , further comprising:
 (d) co-crystallizing said candidate agent with PBP1b;   (e) determining the three-dimensional structure of the co-crystal of said PBP 1b-candidate agent complex by comparing with the three-dimensional structure of PBP1b-moenomycin as defined by atomic coordinates according to  FIGS. 8-1  through  8 - 117 ; and   (f) analyzing said three-dimensional structure of said PBP1b bound to said candidate agent to evaluate the binding characteristics of said potential inhibitor compound.   
     
     
         24 . The method of  claim 22 , wherein the candidate agent is a compound of the formula: 
       
         
           
           
               
               
           
         
       
     
     
         25 . The method of  claim 24 , wherein the candidate agent is selected from the group consisting of:
 (a) a compound of the formula (WCKTS-A1N1):   
       
         
           
           
               
               
           
         
         and (b) a compound of the formula (WCKTS-A1N3): 
       
       
         
           
           
               
               
           
         
       
     
     
         26 . The method of  claim 22 , wherein the candidate agent has the formula: 
       
         
           
           
               
               
           
         
         wherein R 1 ═Br, Cl, I, H or OH; 
         R 2 ═H, OH or Cl; 
         R 3 ═Br, Cl, I, H, or 
       
       
         
           
           
               
               
           
         
         R 4 ═H, OH, Cl, 
       
       
         
           
           
               
               
           
         
         R 5 ═H, Cl, 
       
       
         
           
           
               
               
           
         
         R 6 ═H, CH 3 , OH, OCH 3 , C 1 , NO 2 , or 
       
       
         
           
           
               
               
           
         
         R 7 ═H, Cl, 
       
       
         
           
           
               
               
           
         
       
     
     
         27 . The method of  claim 26 , further comprising:
 (d) selecting a candidate agent that binds PBP1b, and exhibits transglycosylase activity;   (e) co-crystallizing said candidate agent with PBP1b;   (f) determining the three-dimensional structure of said PBP1b-candidate agent complex co-crystal by comparing the three-dimensional structure of PBP1b-moenomycin as defined by atomic coordinates according to  FIGS. 8-1  through  8 - 117 ; and   (g) analyzing said three-dimensional structure of said PBP1b bound to said candidate agent to evaluate the binding characteristics of said potential inhibitor compound.   
     
     
         28 . The method of  claim 27 , wherein the binding comprises one or more interactions with amino acid residues from transglycosylase (TG), UvrB domain 2 homolog (UB2H) or transmembrane (TM) domains of PBP1b. 
     
     
         29 . An anti-bacterial compound comprising the formula: 
       
         
           
           
               
               
           
         
       
       wherein the compound (a) binds PBP1b, and (b) exhibits transglycosylase activity. 
     
     
         30 . The anti-bacterial compound of  claim 29 , wherein the compound is effective in inhibiting the growth of at least one of  Staphylococcus aureus  (ATCC29213, SA), methicillin-resistant  Staphylococcus aureus  (ATCC33592, MRSA),  Mycobacterium smegmatis  (ATCC11565, MS), and  Escherichia coli  (ATCC 25922, EC),  Streptococcus pneumonia, Bacillus subtilis, Enterococcus faecalis, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia , and  Aquifex aeolicus.    
     
     
         31 . The anti-bacterial compound of  claim 29 , wherein, in a co-crystal of the compound with PBP1b, the compound contacts the moenomycin-binding site of PBP1b as defined by atomic coordinates according to  FIGS. 8-1  through  8 - 117 . 
     
     
         32 . The anti-bacterial compound of  claim 29 , wherein the compound is selected from the group consisting of:
 (a) WCKTS-A1N1 having the formula:   
       
         
           
           
               
               
           
         
         and 
         (b) WCKTS-A1N1 having the formula: 
       
       
         
           
           
               
               
           
         
       
     
     
         33 . The anti-bacterial compound of  claim 32 , wherein, in a co-crystal of the compound with PBP1b, the compound contacts the moenomycin-binding site of PBP1b as defined by atomic coordinates according to  FIGS. 8-1  through  8 - 117 . 
     
     
         34 . The compound of  claim 29  wherein the PBP1b binding is determined by an anisotropic assay. 
     
     
         35 . The compound of  claim 34 , wherein the anisotropic assay is a fluorescent anisotropic assay. 
     
     
         36 . The compound of  claim 29  wherein the transglycosylase activity is determined by a polymerization assay using lipid II, or a derivative thereof. 
     
     
         37 . An anti-bacterial compound having the formula: 
       
         
           
           
               
               
           
         
         wherein R 1 ═Br, Cl, I, H or OH; 
         R 2 ═H, OH or Cl; 
         R 3 ═Br, Cl, I, H, or 
       
       
         
           
           
               
               
           
         
         R 4 ═H, OH, Cl, 
       
       
         
           
           
               
               
           
         
         R 5 ═H, Cl, 
       
       
         
           
           
               
               
           
         
         R 6 ═H, CH 3 , OH, OCH 3 , C 1 , NO 2 , or 
       
       
         
           
           
               
               
           
         
         R 7 ═H, Cl, and 
       
       
         
           
           
               
               
           
         
         wherein the compound (a) binds PBP1b, and (b) exhibits transglycosylase activity. 
       
     
     
         38 . The anti-bacterial compound of  claim 37 , wherein, in a co-crystal of the compound with PBP1b, the compound contacts the moenomycin-binding site of PBP1b as defined by atomic coordinates according to  FIGS. 8-1  through  8 - 117 . 
     
     
         39 . The compound of  claim 37  wherein the PBP1b binding is determined by an anisotropic assay. 
     
     
         40 . The compound of  claim 39 , wherein the anisotropic assay is a fluorescent anisotropic assay. 
     
     
         41 . The compound of  claim 37  wherein the transglycosylase activity is determined by a polymerization assay using lipid II, or a derivative thereof. 
     
     
         42 . The compound of  claim 37 , wherein the binding of the compound to  E. coli  PBP1b comprises binding to at least one portion of the transmembrane (TM) domain of PBP1b. 
     
     
         43 . The compound of  claim 37 , wherein the binding of the compound to  E. coli  PBP1b comprises binding to at least one portion of the UvrB domain 2 homolog (UB2H) domain of PBP1b. 
     
     
         44 . The compound of  claim 43 , wherein the UB2H binding further inhibits cell wall synthesis. 
     
     
         45 . The compound of  claim 43 , wherein the UB2H binding further inhibits DNA repair. 
     
     
         46 . The compound of  claim 37 , wherein the compound prevents peptidoglycan elongation by structurally mimicking lipid IV at the binding site of transglycosylase. 
     
     
         47 . The compound of  claim 37 , wherein the compound inhibits a peptidoglycan glycosyltransferase. 
     
     
         48 . The compound of  claim 47 , wherein the peptidoglycan glycosyltransferase is PBP1b, SaPBP2 or AaPGT. 
     
     
         49 . The compound of  claim 37 , wherein the compound comprises a pharmaceutical composition.