A new hydrocyanine probe for imaging reactive oxygen species in the mitochondria of live cells.

A new hydrocyanine probe containing a ROS-responsive element and a mitochondrial-targeting moiety, as well as a thiol-reactive chloromethyl group, has been designed for imaging reactive oxygen species (ROS) in the mitochondria of live cells. This hydrocyanine probe is cell-permeable, selectively targets mitochondria and responds to superoxide and hydroxyl radical, and can be used for detection of ROS production in the mitochondria of live cells caused by an oxidative stress.

Influence of sulfur oxidation state and steric bulk upon trifluoromethyl ketone (TFK) binding kinetics to carboxylesterases and fatty acid amide hydrolase (FAAH).

Carboxylesterases metabolize numerous exogenous and endogenous ester-containing compounds including the chemotherapeutic agent CPT-11, anti-influenza viral agent oseltamivir, and many agrochemicals. Trifluoromethyl ketone (TFK)-containing compounds with a sulfur atom beta to the ketone moiety are some of the most potent carboxylesterase and amidase inhibitors identified to date. This study examined the effects of alkyl chain length (i.e., steric effects) and sulfur oxidation state upon TFK inhibitor potency (IC50) and binding kinetics (k(i)). The selective carboxylesterase inhibitor benzil was used as a non-TFK containing control. These effects were examined using two commercial esterases (porcine and rabbit liver esterase) and two human recombinant esterases (hCE-1 and hCE-2) as well as human recombinant fatty acid amide hydrolase (FAAH). In addition, the inhibition mechanism was examined using a combination of 1H NMR, X-ray crystallography, and ab initio calculations. Overall, the data show that while sulfur oxidation state profoundly affects both inhibitor potency and binding kinetics, the steric effects dominate and override the contributions of sulfur oxidation. In addition, the data suggest that inclusion of a sulfur atom beta to the ketone contributes an increase (approximately 5-fold) in inhibitor potency due to effects upon ketone hydration and/or intramolecular hydrogen bond formation. These results provide further information on the nature of the TFK binding interaction and will be useful in increasing our understanding of this basic biochemical process.