Bacterial transglycosylases: assays for monitoring the activity using Lipid II substrates analogs and methods for discovering new antibiotics
AssigneeThe Trustees of Princeton University
InventorSuzanne Walker Kahne
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Abstract
This invention provides a direct method for monitoring bacterial transglycosylase activity using labeled substrates produced by chemo-enzymatic synthesis wherein the labels are selected to permit the detection of both polymeric and non-polymeric products simultaneously, either directly or following the separation of product from starting material. The invention promotes the discovery of new antibiotics with activity against bacterial transglycosylases by a) laying the groundwork for structural analysis of purified, active transglycosylase (which permits structure-based design); and b) providing an assay that can be used to screen for inhibitors.
Claims44
A substance comprising the chemical moiety of the formula (I):
- in which “R” is an acyl group comprising at least two carbon atoms, “R1” is a substituted or unsubstituted alkyl group comprising at least one carbon atom, “R2” is a substituted or unsubstituted alkyl or alkenyl group comprising at least five carbon atoms, “R3” is a glucosaminyl group comprising at least five carbon atoms, “A” is a substituted or unsubstituted amino acid residue or a peptide comprising at least two substituted or unsubstituted amino acid residues, said substance exhibiting a binding affinity for at least wild type transglycosylase enzyme and provided that said substance is not Lipid II, the natural substrate of wild type transglycosylase enzyme.
A method of detecting transglycosylase activity in a sample suspected of containing a protein or an active fragment thereof exhibiting transglycosylase activity comprising:
- (a) providing a sample suspected of containing a protein or an active fragment thereof exhibiting transglycosylase activity;
- (b) contacting the sample with effective amounts of a substance comprising the chemical moiety of the formula (I):
- in which “R” is an acyl group comprising at least two carbon atoms, “R1” is a substituted or unsubstituted alkyl group comprising at least one carbon atom, “R2” is a substituted or unsubstituted alkyl or alkenyl group comprising at least five carbon atoms, “R3” is a glucosaminyl group comprising at least five carbon atoms, “A” is a substituted or unsubstituted amino acid residue or a peptide comprising at least two substituted or unsubstituted amino acid residues, provided that said substance is not Lipid II, the natural substrate of wild type transglycosylase enzyme,
- under conditions effective to provide a coupling product comprising a poly(peptidosaccharide) moiety in the presence of a protein or an active fragment thereof exhibiting transglycosylase activity;
- (c) detecting the formation or presence of said coupling product, which is indicative of transglycosylase activity in said sample.
An assay for detecting transglycosylase activity in a sample suspected of containing a protein or an active fragment thereof exhibiting transglycosylase activity comprising a compound of the formula (I):
- in which “R” is an acyl group comprising at least two carbon atoms, “R1” is a substituted or unsubstituted alkyl group comprising at least one carbon atom, “R2” is a substituted or unsubstituted alkyl or alkenyl group comprising at least five carbon atoms, “R3” is a glucosaminyl group comprising at least five carbon atoms, “A” is a substituted or unsubstituted amino acid residue or a peptide comprising at least two substituted or unsubstituted amino acid residues, said substance being able to serve as a transglycosylase substrate to form a coupling product comprising a poly(peptidosaccharide) in the presence of a protein or an active fragment thereof exhibiting transglycosylase activity, provided that said substance is not Lipid II, the natural substrate of wild type transglycosylase enzyme.
A screen for compounds exhibiting potential antibacterial activity comprising (i) a protein or an active fragment thereof exhibiting transglycosylase activity, and (ii) a labeled or unlabeled substance, or a combination thereof, said substance comprising the chemical moiety of the formula (I):
- in which “R” is an acyl group comprising at least two carbon atoms, “R1” is a substituted or unsubstituted alkyl group comprising at least one carbon atom, “R2” is a substituted or unsubstituted alkyl or alkenyl group comprising at least five carbon atoms, “R3” is a glucosaminyl group comprising at least five carbon atoms, “A” is a substituted or unsubstituted amino acid residue or a peptide comprising at least two substituted or unsubstituted amino acid residues, said substance being able to serve as a transglycosylase substrate to form a coupling product comprising a poly(peptidosaccharide) in the presence of a protein or an active fragment thereof exhibiting transglycosylase activity, provided that said substance is not Lipid II, the natural substrate of wild type transglycosylase enzyme.
A substrate analog of Lipid II (i) having a structure that is accepted by at least wild type transglycosylase enzyme such that a coupling product is produced by the transglycosylase activity of the enzyme in the presence of said substrate analog, and (ii) having structural features that facilitate the separation of said substrate analog from said coupling product.
A substance comprising the chemical moiety of the formula (I):
- in which “R” is an acyl group comprising at least two carbon atoms, “R1” is a substituted or unsubstituted alkyl group comprising at least one carbon atom, “R2” may be selected from H, an aliphatic group comprising 1 to about 50 carbon atoms, an aromatic or heteroaromatic group comprising 3 to about 55 carbon atoms and pyrophosphate protecting groups, “R3” is a glucosaminyl group comprising at least five carbon atoms, “A” is a substituted or unsubstituted amino acid residue or a peptide comprising at least two substituted or unsubstituted amino acid residues, and pharmaceutically acceptable salts thereof, said substance exhibiting a binding affinity for at least wild type transglycosylase enzyme and provided that said substance is not Lipid II, the natural substrate of wild type transglycosylase enzyme.
A method of detecting transglycosylase activity in a sample suspected of containing a protein or an active fragment thereof exhibiting transglycosylase activity comprising:
- (a) providing a sample suspected of containing a protein or an active fragment thereof exhibiting transglycosylase activity;
- (b) contacting the sample with effective amounts of at least one peptidodisaccharide moiety, each exhibiting (i) a binding affinity for at least wild type transglycosylase enzyme, (ii) substantial solubility in water, (iii) a substituted pyrophosphate leaving group, and (iv) a capacity to serve as a substrate for said at least wild type transglycosylase enzyme, provided that each is not Lipid II, the natural substrate of wild type transglycosylase enzyme,
- under conditions effective to provide a coupling product comprising dimers or polymers of said moieties in which the substituted pyrophosphate leaving group of one moiety has been displaced by a C-4 hydroxyl group of another moiety in the presence of a protein or an active fragment thereof exhibiting transglycosylase activity;
- (c) detecting the formation or presence of said coupling product, which is indicative of transglycosylase activity in said sample.
A method of detecting transglycosylase activity in a sample suspected of containing a protein or an active fragment thereof exhibiting transglycosylase activity comprising:
- (a) contacting a sample suspected of containing a protein or an active fragment thereof exhibiting transglycosylase activity with a labeled first substrate comprising a substance having (i) a binding affinity for at least wild type transglycosylase enzyme, (ii) a detectable label, (iii) a substituted pyrophosphate leaving group, and (iv) a capacity to serve as a substrate for said at least wild type transglycosylase enzyme and a labeled second substrate comprising a substance having a N-acetylglucosamyl residue with a free, protected, or releasable C-4 hydroxyl group,
- under conditions effective to provide a coupling product in which the substituted pyrophosphate leaving group of the first substrate has been displaced by the C-4 hydroxyl group of the N-acetylglucosamyl residue of the second substrate in the presence of a protein or an active fragment thereof exhibiting transglycosylase activity;
- and
- (b) detecting the formation or presence of said coupling product, which is indicative of transglycosylase activity in said sample.
A substrate for an enzyme that exhibits transglycosylase activity comprising a structure that is accepted by an enzyme that exhibits transglycosylase activity such that a coupling product comprising the substrate coupled to an acceptor moiety is produced, which substrate further comprises one or more features that facilitate the detection of the coupling product.
A method of discovering an inhibitor of transglycosylase activity, which comprises contacting a substance suspected of having transglycosylase inhibitory activity with an enzyme exhibiting transglycosylase activity in the presence of a substrate therefor, which substrate comprises at least one feature that facilitates the detection or measurement of a coupling product formed from the coupling of the substrate and an acceptor moiety, measuring the amount of coupling product formed, and comparing this amount with that measured in the absence of said substance.
Dependencies are inferred from the claim text.