Photo-control of nitric oxide synthase activity using a caged isoform specific inhibitor.
Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited.
Montgomery
Heather J
HJ
Department of Chemistry, University of Waterloo, Waterloo, N2L 3G1, Ontario, Canada.
Perdicakis
Basil
B
Fishlock
Dan
D
Lajoie
Gilles A
GA
Jervis
Eric
E
Guy Guillemette
J
J
D000578:Amidines / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001596:Benzylamines / Q000737:chemistry / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D003374:Coumarins / Q000737:chemistry / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D016716:PC12 Cells; D010782:Photolysis; D051381:Rats; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013997:Time Factors
D000578:Amidines; D001596:Benzylamines; D003374:Coumarins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C104820:N-(3-(aminomethyl)benzyl)acetamidine; C470914:N-(6-bromo-7-hydroxycoumarin-4-yl)methoxycarbonyl-N-(3-(aminomethyl)benzyl)acetamide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat