Targeting the NADPH Binding Site of Nitric Oxide Synthase by a Ligand with Two-Photon Absorption Properties

Abstract

Synthesis of nitric oxide is performed by NO-synthase. The catalysis is initiated by the combined transfer of electrons/proton from NADPH to the flavin FAD. We have previously characterized one photoactive compound (named Nanotrigger)1,2 allowing to trigger and synchronize NOS activity upon light illumination. Here, we described a new compound (C1) with two-photon absorption properties suitable to assess the binding to the NOS protein. This family of compounds combines a docking moiety (NADPH analog) and a chromophore moiety responsive for light illumination. C1 was characterized in DMSO by an absorption maximum at 460nm and was found to be fluorescent with an emission peak centered at 740nm upon one-photon excitation. However, the fluorescence emission was strongly sensitive to solvent polarity as evidenced by the significant decrease in the emission intensity in polar solvents. The same behaviour was observed under two-photon excitation (Exc, 940nm). One- and two-photon fluorescence approaches were used to assess the binding of C1 to the neuronal NOS. Under one-photon excitation, the emission properties of the complex were rather difficult to interpret due to the overlapping between the intrinsic fluorescence of nNOS and C1 fluorescence. As the two-photon fluorescence of nNOS protein is very weak, the complex formation was measured by monitoring the two-photon fluorescence recovery of C1 upon binding to nNOS. The calculated Kd value was found to be consistent with the value characterizing NADPH binding. However, competition experiments suggest that the competition between C1 and NADPH is more complex than expected from a one-binding site model. Our results suggest that C1 represents a promising compound for cellular applications in two-photon fluorescence imaging.1 Beaumont et al., J. Am. Chem. Soc. 2007, 129, 2178-2186.2 Beaumont et al., ChemBioChem 2009, 10, 690-701.