NO Formation by Neuronal NO‐Synthase can be Controlled by Ultrafast Electron Injection from a Nanotrigger

Beaumont, Edward; Lambry, Jean−Christophe; Blanchard‐Desce, Mireille; Martásek, Pavel; Panda, Satya Prakash; Faassen, E.E.H. van; Brochon, Jean Claude; Deprez, Eric; Slama‐Schwok, Anny

doi:10.1002/cbic.200800721

Cited by 16 publications

(51 citation statements)

References 33 publications

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“…This choice of a shorter stilbene moiety was guided by initial modelling studies, which had suggested that a shorter compound than NT1 with the same terminal donor group could fit tighter to this site. Our design retained the nucleotidic recognition motif of NADPH that confers proper targeting to the NADPH site, based on previous studies that led to the discovery of NT1 (12)(13)(14)17). The designed inhibitor NS1 was docked in nNOS red by replacement of the embedded NADPH (4).…”

Section: Resultsmentioning

confidence: 99%

“…Additionally, significant modulations of the fluorescence emission by the polarity (λ max ) and the presence of water (quantum yield, Φ) of the environment are expected. (B) Close-up view of the terminal groups of NT1 diene chromophore and NS1stilbene chromophore in nNOS red ; FAD is omitted for clarity (12)(13)(14). moiety were similar, with a maximum similar to the related 4-N, N-dimethylamino-4′-nitrostilbene (16,18).…”

Section: Resultsmentioning

confidence: 99%

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“…Following photoactivation, the terminal amine group of excited NT1* was able to inject electrons to the FAD acceptor, thereby initiating the electron flow. This initiation and synchronization of NOS catalysis upon one-or two-photon excitation (1-PE or 2-PE, respectively) led to light-induced NO formation (12)(13)(14).Here, we designed a unique prototype of NOS inhibitor, called nanoshutter (NS1), with intrinsic fluorescence imaging properties. To this purpose, we designed a compound that meets the following criteria: (i) NS1 should demonstrate a good affinity for NOS, at least comparable to the affinity of the natural hydride donor NADPH; (ii) injection of electrons to FAD should be prohibited by bound NS1 to NOS; (iii) NS1 should have a good two-photon cross-section, compatible with multiphoton imaging; (iv) free and bound NS1 should possess different fluorescence properties.…”

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confidence: 99%

“…The nicotinamide part of NADPH can be flexible and undergoes conformational changes depending on the NADP(H) enzymes and their specific catalysis. In recent studies, we have characterized a NADPH analog called nanotrigger, NT1, targeting the NADPH site in NOS reductase domain (12)(13)(14) (Fig. 1A).…”

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Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation

Wang

Tarus

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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 signalto-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.chemical biology | inhibition of enzymatic catalysis | structure-based design and molecular modeling | two-photon fluorescence imaging | fluorescence spectroscopy I n mammals, NO is a gaseous signaling mediator that exerts a wide range of key physiological functions, including blood pressure regulation, neurotransmission, and immune responses (1). NO is formed by nitric-oxide synthases, a family of hemoproteins that catalyzes the oxidation of L-arginine leading to L-citrulline and NO (2, 3). NOS catalysis is driven by reducing equivalents supplied by NADPH. NOS enzymes are constituted by oxygenase and reductase domains linked by a calmodulin (CaM)-binding domain. The oxygenase domain binds the heme, L-arginine, and the cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (H 4 B). The reductase domain binds two flavins, FMN and FAD, and NADPH (4). CaM binding allows the electron transfer from FMN to the heme (2, 3). The biological activities of NO are closely linked to the specific NOS isoform involved in its synthesis and deregulation of its biosynthesis leads to various pathologies (5). Data support the notion that inhibition of the inducible and neuronal isoforms iNOS and nNOS have therapeutic utility in the treatment of a variety of diseases, including septic shock, neurodegeneration, inflammation, and cancer (5, 6). It was shown that the endothelial isoform eNOS participates in tumor initiation and maintenance, thus its inhibition could be beneficial in this context (7,8). Based on crystallographic studies (9, 10), much effort was dedicated to the development of specific NOS inhibitors by targeting the heme site. However, most of these inhibitors are not specific and cannot avoid NOS uncoupling and formation of deleterious radical oxygen species (ROS) at the reductase domain. Although the struct...

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confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 98%

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confidence: 99%

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confidence: 99%

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Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation

Wang

Tarus

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Two Photon‐Induced Electron Injection From a Nanotrigger in Native Endothelial NO‐Synthase

et al. 2008

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We have recently designed a nanotrigger (NT), a photoactive molecule addressing the NADPH sites of proteins. This nanotrigger has a 10(3) times larger two-photon cross-section compared to the ubiquitous NADPH cofactor. In this work, we tested whether two-photon excitation of the bound NT to NADPH sites may be used to initiate enzymatic catalysis by appropriate electron injection. To establish proof of principle, we monitored the ultrafast absorption of NT bound to the fully active endothelial NO-Synthase (eNOS) following excitation by one and two-photons at 405 and 810 nm, respectively. Electron injection from NT* to FAD in eNOS initiated the catalytic cycle in 15+/-3 ps at both exciting wavelengths. The data proved for the first time that electron transfer can be promoted by two-photon excitation. We also show that the nanotrigger decays faster in homogeneous solvents than in the NADPH site of proteins, suggesting that hindered environments modified the natural decay of NT. The nanotrigger provides a convenient way of synchronizing an ensemble of proteins in solution with a femtosecond laser pulse. The ability of NT to initiate NOS catalysis by two-photon excitation may be exploited for controlled and localized release of free NO in cells with enhanced spatial and temporal resolution.

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Selective probing of a NADPH site controlled light‐induced enzymatic catalysis

Lambry¹,

Beaumont²,

Tarus³

et al. 2010

J of Molecular Recognition

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Achieving molecular recognition of NADPH binding sites is a compelling strategy to control many redox biological processes. The NADPH sites recognize the ubiquitous NADPH cofactor via highly conserved binding interactions, despite differences in the regulation of the hydride transfer in redox active proteins. We recently developed a photoactive NADPH substitute, called nanotrigger NT synchronizing the initiation of enzymatic catalysis of the endothelial NO-synthase (eNOS) with a laser pulse. Spatial and temporal control of enzymatic activity by such a designed light-driven activator would benefit from achieving molecular selectivity, i.e. activation of a single NADPH-mediated enzyme.In this work, we probe the ability of NT to discriminate between two NADPH sites with light. The selected NADPH sites belong to dihydrofolate reductase dihydrofolate reductase enzyme (DHFR) and endothelial NO-synthase (eNOS). Ultrafast kinetics showed that NT could not activate DHFR catalysis with a laser pulse in contrast with the observed trigger of eNOS catalysis leading to NO formation. Homology modelling, molecular dynamics simulations showed that NT discriminated between the two NADPH sites by different donor to acceptor distances and by local steric effects hindering light activation of DHFR catalysis. The data suggested that the narrow NADPH site required a tight fit of the nanotrigger at a suitable distance/angle to the electron acceptor for a specific activation of the catalysis. The ability of the nanotrigger to activate eNOS combined with a low reactivity in unfavourable NADPH sites makes NT a highly promising tool for targeting eNOS in endothelial cells with a laser pulse.

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NO Formation by Neuronal NO‐Synthase can be Controlled by Ultrafast Electron Injection from a Nanotrigger

Cited by 16 publications

References 33 publications

Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation

Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation

Two Photon‐Induced Electron Injection From a Nanotrigger in Native Endothelial NO‐Synthase

Selective probing of a NADPH site controlled light‐induced enzymatic catalysis

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