Photoactivatable Antimicrobial Agents And Therapeutic And Diagnostic Methods Of Using Same
AssigneeThe General Hospital Corporation
InventorsTayyaba Hasan, Ulysses W. Sallum, Sarika Verma, Gerard Nau
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Abstract
The present invention provides photosensitizer compounds for use in detecting beta-lactamase activity. Methods and kits that utilize the photosensitizer compounds of the invention for the detection of, quantitation of, and classification or typing of microbial beta-lactamases.
Claims90
A photosensitizer composition, wherein the composition comprises at least one benzophenothiazinium chloride (EtNBS) photosensitizer conjugated to a cephalosporin linker or fragment thereof.
A photosensitizer composition according to formula I:
- X-L-X′,
- wherein L is a cephalosporin linker or fragment thereof, X is benzophenothiazinium chloride (EtNBS) and X′ is benzophenothiazinium chloride (EtNBS), wherein the photosensitizers are in a quenched state when L is uncleaved.
A method for detecting a beta-lactamase activity in a sample, comprising the steps of:
- contacting the sample with a photosensitizer composition comprising two or more photosensitizers that are conjugated to a linker, wherein the linker comprises a cleavage site for a beta-lactamase and wherein the photosensitizers are quenched when the cleavage site is intact but unquenched when the cleavage site is hydrolyzed;
- detecting cleavage of said linker, wherein cleavage of said linker is indicative of a beta-lactamase activity in the sample.
A method for determining the substrate specificity of a beta-lactamase enzyme in a sample, comprising:
- contacting the sample with a photosensitizer composition comprising two photosensitizers that are conjugated to a linker, wherein the linker comprises a cleavage site for a beta-lactamase enzyme and wherein the photosensitizers are quenched when the cleavage site is intact but unquenched when the cleavage site is cleaved;
- determining whether the linker is cleaved;
- wherein cleavage of the linker indicates the linker is a substrate of the beta-lactamase enzyme.
A method of typing a beta-lactamase enzyme in a sample, comprising:
- performing a competitive reaction comprising the steps of (a) contacting the sample with a photosensitizer composition comprising two photosensitizers that are conjugated to a linker, wherein the linker comprise a cleavage site for a beta-lactamase and wherein the photosensitizers are in a quenched state;
- (b) cleaving the linker to dequench the photosensitizers;
- (c) light-activating the composition to produce a fluorescence signal;
- and (d) quantifying the fluorescence signal with a detector to obtain a result, said competitive reaction being performed in the presence of a competing beta-lactam antibiotic;
- comparing the result of the competitive reaction to a standard to type the beta-lactamase.
Dependencies are inferred from the claim text.