Light-induced formation of NO in endothelial cells by photoactivatable NADPH analogues targeting nitric-oxide synthase

Design of Light-Sensitive Triggers for Endothelial NO-Synthase Activation

“…In contrast, NT3_7, which bears a single carboxylate at the 2' position, had a smaller interaction energy with eNOS than had NT3_5. NT3_7 chromophore did not stack well on the FAD isoalloxazine ring, suggesting a reduced electron transfer ability in the excited state, in agreement with its parent NT2_7 being unable to generate NO [18,19]. This is in agreement with previous studies on the effect of stacking interactions between flavin derivatives and aromatic hydride-donor model compounds [32].…”

“…Once bound, the fluorescence intensities of NT1 were enhanced, the absorption maxima were red-shifted while the emission maxima blue-shifted. A similar spectroscopic behavior was observed for the new NT derivatives bearing a carboxylate group instead of the phosphate group present in NT1 when bound to recombinant eNOS and in eNOS-containing cells [19]. Interestingly, enhanced two-photon emissions of several NT's were observed in human umbilical vein endothelial (A) (B) In the prototype NT1 [16], the nucleotidic part boxed in red was kept identical to that of NADPH, as this moiety is responsible for interactions with conserved arginines found in the docking pocket in many NADPH-proteins [20,21].…”

“…NT1 [15] and NT2_5 [19] incorporated into HUVEC cells produced NO upon light activation. Based on our previous results with NT1 with its 2' phosphate group, we anticipated better binding to NOS and more NO formed in HUVECs by NT2_7, with its 2' carboxylate, over NT2_5, which has a 3' carboxylate.…”

“…14.7 ± 1.1 a NA − 21.5 ± 0,7 a ND NA [19] [19] [19] NT 3_3 Table 1 also demonstrates the ability of the compounds to reduce nNOS flavins upon light excitation, a process initiated by injection of electrons from the NT* in its excited state to FAD (Scheme 1). NT1, NT2_5 and NT3_5 reduced FAD upon light excitation, with some ground state reduction only for NT2_5 (Table 1, Figure A2).…”

“…The R1 "hook" substituent of NT1 was an ethyl group. To further characterize how the charge carried by the ribose influenced binding to endothelial Nitric Oxide-Synthase (eNOS), the nucleotidic ribose was substituted by one or two carboxylate groups at either the 2' or 3' position or both, yielding NT2_7, NT2_5, and NT2_3, respectively [18,19]. In order to increase the affinity of the nanotrigger for e/nNOS, a charged R1 "hook1" was added, which targets additional charged residues of eNOS located within 7…”