US2015232417A1PendingUtilityA1
Crystal structure of bifunctional transglycosylase pbp1b from e. coli and inhibitors thereof
Est. expiryJul 21, 2028(~2 yrs left)· nominal 20-yr term from priority
C12Q 1/48G01N 2333/91102C07C 235/84C07C 235/64G01N 2500/20C12Q 1/18G01N 2500/04G01N 2500/00C07K 2299/00G01N 33/573
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Claims
Abstract
Crystal structure at 2.16 Å resolution of full-length Escherichia coli penicillin-binding protein 1b (PBP1b) in complex with its inhibitor moenomycin, is provided. 3D structures of amino acid residues involved in moenomycin binding and transglycosylation activity are identified. Binding sites for peptidoglycan synthesis inhibitors comprising amino acid residues from transglycosylase (TG), UvrB domain 2 homolog (UB2H) and transmembrane (TM) domains of PBP1b are identified at atomic level resolution. Rational drug design, based on the atomic coordinates, are disclosed. Methods for screening for antibiotics using anisotropic binding and transglycosylase inhibitor assays and novel antibiotics based on the screening assays are provided.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A method for identifying a transglycosylase (peptidoglycan glycosyltransferase) inhibitor compound, the method comprising the steps of:
a) employing a three-dimensional structure of E. coli Penicillin binding protein 1b (PBP1b) as defined by atomic coordinates according to FIGS. 8-1 through 8 - 82 , to design or select said potential inhibitor such that said potential inhibitor is capable of binding to at least one amino acid located in an active site of PBP1b transglycosylase; b) determining an ability of the potential inhibitor to bind PBP1b; and c) determining a 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 comprises a structure 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 . The method of claim 1 , wherein the transglycosylase is E. coli Penicillin binding protein 1b (PBP1b) transglycosylase.
12 . The method of claim 1 , wherein the PBP1b transglycosylase inhibitory activity of the potential inhibitor is determined by a transglycosylation assay comprising:
(a) contacting the candidate agent with the PBP1b transglycosylase in the presence of lipid II, or derivative thereof; and (b) determining a PBP1b transglycosylase inhibitory activity of the candidate agent.
13 . The method of claim 1 , wherein the affinity of the inhibitor for PBP1b is determined by an anisotropy measurement assay.
14 . The method of claim 13 , wherein the anisotropy measurement assay is a fluorescence anisotropy assay.
15 . The method of claim 1 , further comprising: evaluating the binding properties of a potential PBP1b transglycosylase inhibitor compound by a method 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 - 82 ; 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.
16 . The method of claim 1 , wherein the potential inhibitor compound is a compound of 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 , Cl, NO 2 , or
R 7 ═H, Cl,
17 . The method of claim 16 , wherein the potential inhibitor compound is selected from the group consisting of:
(a) a compound of the formula (WCKTS-A1N1):
and (b) a compound of the formula (WCKTS-A1N3):
18 . The method of claim 1 , wherein the potential inhibitor compound is an anti-bacterial compound.
19 . The anti-bacterial compound of claim 18 , 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), Escherichia coli (ATCC 25922, EC), Streptococcus pneumonia (ATCC 33400), Bacillus subtilis (ATCC 6051), Enterococcus faecalis (ATCC 51299), Acinetobacter baumannii (ATCC 17978), Pseudomonas aeruginosa (ATCC 9027), Stenotrophomonas maltophilia (ATCC 13637), and Aquifex aeolicus (Str. VF5).
20 . 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 , Cl, NO 2 , or
R 7 ═H, Cl,
and
wherein the compound (a) binds PBP1b such that in a co-crystal of the compound with E. coli PBP1b, the compound contacts the moenomycin-binding site of PBP1b as defined by atomic coordinates according to FIGS. 8-1 through 8 - 82 , and (b) exhibits transglycosylase activity.
21 . The anti-bacterial compound of claim 20 , wherein the binding of the compound to E. coli PBP1b comprises binding to at least one portion of the transmembrane (TM) domain of PBP1b.
22 . The compound of claim 20 , 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.
23 . The compound of claim 22 , wherein the UB2H binding further inhibits cell wall synthesis.
24 . The compound of claim 22 , wherein the UB2H binding further inhibits DNA repair.
25 . The compound of claim 20 , wherein the compound prevents peptidoglycan elongation by structurally mimicking lipid IV at the binding site of transglycosylase.
26 . The compound of claim 20 , wherein the compound inhibits a peptidoglycan glycosyltransferase.
27 . The compound of claim 26 , wherein the peptidoglycan glycosyltransferase is PBP1b, SaPBP2 or AaPGT.
28 . The compound of claim 20 , wherein the potential inhibitor compound is selected from the group consisting of:
(a) a compound of the formula (WCKTS-A1N1):
and (b) a compound of the formula (WCKTS-A1N3):
29 . The compound of claim 20 , wherein the compound comprises a pharmaceutical composition.
30 . The compound of claim 20 , wherein the compound is identified by a method according to claim 1 .Join the waitlist — get patent alerts
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