Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation.
We report the structure-based design and synthesis of a unique NOS inhibitor, called nanoshutter NS1, with two-photon absorption properties. NS1 targets the NADPH site of NOS by a nucleotide moiety mimicking NADPH linked to a conjugated push-pull chromophore with nonlinear absorption properties. Because NS1 could not provide reducing equivalents to the protein and competed with NADPH binding, it efficiently inhibited NOS catalysis. NS1 became fluorescent once bound to NOS with an excellent signal-to-noise ratio because of two-photon excitation avoiding interference from the flavin-autofluorescence and because free NS1 was not fluorescent in aqueous solutions. NS1 fluorescence enhancement was selective for constitutive NOS in vitro, in particular for endothelial NOS (eNOS). Molecular dynamics simulations suggested that two variable residues among NOS isoforms induced differences in binding of NS1 and in local solvation around NS1 nitro group, consistent with changes of NS1 fluorescence yield. NS1 colocalized with eNOS in living human umbilical vein endothelial cells. Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800-950-nm range, with great perspectives for eNOS imaging in living tissues.
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Centre National de la Recherche Scientifique UMR8601, Université Paris Descartes, 75270 Paris, France.
Yun
Y
Li
Huan
H
Wang
Bogdan
B
Tarus
Miguel Romero
MR
Perez
Laurence
L
Morellato
Etienne
E
Henry
Vladimir
V
Berka
Ah-Lim
AL
Tsai
Booma
B
Ramassamy
Hamid
H
Dhimane
Chantal
C
Dessy
Patrick
P
Tauc
Jean-Luc
JL
Boucher
Eric
E
Deprez
Anny
A
Slama-Schwok
D002384:Catalysis; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D056004:Molecular Dynamics Simulation; D009249:NADP / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis / Q000737:chemistry; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000378:metabolism
D005456:Fluorescent Dyes; D007527:Isoenzymes; D009569:Nitric Oxide; D009249:NADP; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III