title: Photocontrol of nitric oxide production in cell culture using a caged isoform selective inhibitor.
abstract: Over the past decade, multiphoton microscopy has progressed from a photonic novelty to a technique whose application is currently experiencing exponential growth in the biological sciences. A novel application of this technology with significant therapeutic potential is the control of drug activity by multiphoton photolysis of caged therapeutics. As an initial case study, the potent isoform selective inhibitor N-(3-(aminomethyl)benzyl) acetamidine (1400W) of inducible nitric oxide synthase (iNOS) has been conjugated to a caging molecule 6-bromo-7-hydroxy-4-hydroxyquinoline-2-ylmethyl acetyl ester (Bhc). Here we present the first report of a bulk therapeutic effect, inhibition of nitric oxide production, in mammalian cell culture by multiphoton photolysis of a caged drug, Bhc-1400W. Mouse macrophage RAW 264.7 cells induced with bacterial lipopolysaccharides to express iNOS were used to assess the therapeutic value of the conjugated inhibitor. Both 1400W and Bhc-1400W are stable in metabolically active cells and an optimal time interval for the photorelease of the inhibitor was determined. The ratios of the IC(50) values of Bhc-1400W over 1400W calculated in the presence of iNOS enzyme and in RAW 264.7 cell culture are 19 and 100, respectively, indicating that a broad therapeutic range exists in cell culture. Multiphoton uncaging protocols and therapeutic doses of inhibitors were not cytotoxic. Photocontrol of LPS induced nitric oxide production was achieved in mammalian cell culture using a single laser focal volume. This technology has the potential to control active drug concentrations in vivo, a lack of which is one of the main problems currently associated with systemic drug administration.
authors.affiliation: Department of Chemical Engineering, University of Waterloo, Waterloo, ON, Canada N2L 3G1.
authors.forename: Basil
authors.initials: B
authors.lastname: Perdicakis
authors.forename: Heather J
authors.initials: HJ
authors.lastname: Montgomery
authors.forename: Glenn L
authors.initials: GL
authors.lastname: Abbott
authors.forename: Dan
authors.initials: D
authors.lastname: Fishlock
authors.forename: Gilles A
authors.initials: GA
authors.lastname: Lajoie
authors.forename: J Guy
authors.initials: JG
authors.lastname: Guillemette
authors.forename: Eric
authors.initials: E
authors.lastname: Jervis
mesh_terms: D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D052247:Nitric Oxide Synthase Type II; D017785:Photons
chemical_list: D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse