Photoactivatable antimicrobial agents and therapeutic and diagnostic methods of using same
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.
1. A method of measuring susceptibility of bacteria in a sample to a beta-lactam antibiotic, wherein the method comprises:
(a) contacting the sample with:
(i) a composition comprising a compound according to formula I: X-L-X′,
wherein L is a cephalosporin linker or fragment thereof, and both X and X′ are bodipy dyes, wherein the bodipy dyes are quenched by a static quenching mechanism when L is uncleaved, to form a standard; and
(ii) a composition comprising the beta-lactam antibiotic and the composition of (i), to form a test sample;
(b) light-activating the standard and the test sample to produce a fluorescence signal;
(c) quantifying the fluorescence signal with a detector to obtain a result;
(d) comparing the test sample result to the standard result; and
(e) if the amount of the measured fluorescence in the test sample is less than the amount of measured fluorescence in the standard, then the bacteria is determined to be susceptible to the β-lactam antibiotic.
2. The method of claim 1 , wherein the sample comprises whole blood, plasma, serum, nasal secretions, sputum, saliva, urine, sweat, transdermal exudates or cerebrospinal fluid.
3. The method of claim 1 , wherein the sample is a biological sample isolated from an infection in a subject.
4. The method of claim 3 , wherein the infection is caused by an antibiotic-resistant pathogen.
5. The method of claim 1 , wherein the bodipy dye is selected from the group consisting of: 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester; 6-dibromo-4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid; 4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid succinimidyl ester; 4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid sulfosuccinimidyl ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-s-indacene-3-propionyl) amino) hexanoic acid; 6-((4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino) hexanoic acid or succinimidyl ester; N-(4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) cysteic acid, succinimidyl ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino) hexanoic acid or succinimidyl ester; 4,4-difluoro-5-(4-phenyl-1, 3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl) styryloxy) acetyl) aminohexanoic acid or succinimidyl ester; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid; 4,4-difluoro-1,3,5, 7-tetramethyl-4-bora-3a,4a diaza-s-indacene-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza s-indacene-3-yl) phenoxy) acetyl) amino) hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy) acetyl) aminohexanoic acid or succinimidyl ester.
6. The method of claim 1 , wherein the bodipy dye is selected from the group consisting of: 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester.