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

Lambry, Jean−Christophe; Beaumont, Edward; Tarus, Bogdan; Blanchard‐Desce, Mireille; Slama‐Schwok, Anny

doi:10.1002/jmr.1009

Cited by 7 publications

(11 citation statements)

References 42 publications

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“…NS1 competed with NADPH on nNOS reduction in a dose-dependent manner, presenting a typical saturation curve, and led to an apparent inhibition constant K i ¼ 15 AE 4 μM. This value compares well with those obtained previously with NT1, K i ¼ 7 AE 3 μM (13,17), showing that NS1 and NT1 display similar affinities for the NADPH site of constitutive NOS, as suggested by our molecular modelling study.…”

Section: Resultssupporting

confidence: 88%

“…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%

“…We attempted to explain the modulation in fluorescence yields of NS1 bound to the three NOS isoforms by additional structural insight; homology modelling was used to generate eNOS and iNOS reductase domains (17). NS1 bound to nNOS or eNOS by three arginine residues interacting with its phosphate group (SI Appendix, Fig.…”

Section: Structural Insight Of Ns1 Binding To Nos Isoforms Based On Mmentioning

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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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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Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Structural Insight Of Ns1 Binding To Nos Isoforms Based On Mmentioning

confidence: 99%

See 1 more Smart Citation

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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“…A rational way to regulate redox stress would be to make use of compounds modulating NADPH levels, which requires selectivity toward specific NADPH-dependent enzymes without strong interference with normal cellular processes. Recently, we designed a novel photoactive probe, called nanoshutter (NS1) that efficiently bound to constitutive NOS (eNOS and nNOS) [19] in a similar manner than our previously reported dienic nanotrigger [20–23]. NS1 reversibly inhibited NO formed by recombinant eNOS in endothelial cells by competing with NADPH binding and presented anti-angiogenic effects [19].…”

Section: Introductionmentioning

confidence: 99%

Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue

et al. 2016

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Melanoma is one of the most lethal cancers when it reaches a metastatic stage. Despite the spectacular achievements of targeted therapies (BRAF inhibitors) or immuno-therapies (anti-CTLA4 or anti-PD1), most patients with melanoma will need additional treatments. Here we used a photoactive NADPH analogue called NS1 to induce cell death by inhibition of NADPH oxidases NOX in melanoma cells, including melanoma cells isolated from patients. In contrast, healthy melanocytes growth was unaffected by NS1 treatment.NS1 established an early Endoplasmic Reticulum stress by the early release of calcium mediated by (a) calcium-dependent redox-sensitive ion channel(s). These events initiated autophagy and apoptosis in all tested melanoma cells independently of their mutational status. The autophagy promoted by NS1 was incomplete. The autophagic flux was blocked at late stage events, consistent with the accumulation of p62, and a close localization of LC3 with NS1 associated with NS1 inhibition of NOX1 in autophagosomes. This hypothesis of a specific incomplete autophagy and apoptosis driven by NS1 was comforted by the use of siRNAs and pharmacological inhibitors blocking different processes. This study highlights the potential therapeutic interest of NS1 inducing cell death by triggering a selective ER stress and incomplete autophagy in melanoma cells harbouring wt and BRAF mutation.

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“…The resulting structure was refined by allowing the system to relax over molecular dynamics (MD) simulation. The model was further tested by Verify 3D and Ramachandran plots [23].…”

Section: Homology Modelingmentioning

confidence: 99%

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

et al. 2020

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A specific light trigger for activating endothelial Nitric Oxide-Synthase (eNOS) in real time would be of unique value to decipher cellular events associated with eNOS activation or to generate on demand cytotoxic levels of NO at specific sites for cancer research. We previously developed novel tools called nanotriggers (NT), which recognized constitutive NO-synthase, eNOS or neuronal NOS (nNOS), mainly via their 2’ phosphate group which is also present in NADPH in its binding site. Laser excitation of NT1 bound to eNOS triggered recombinant NOS activity and released NO. We recently generated new NTs carrying a 2’ or 3’ carboxylate group or two 2’ and 3’ carboxylate moieties replacing the 2’ phosphate group of NADPH. Among these new NT, only the 3’ carboxylate derivative released NO from endothelial cells upon laser activation. Here, Molecular Dynamics (MD) simulations showed that the 3’ carboxylate NT formed a folded structure with a hydrophobic hub, inducing a good stacking on FAD that likely drove efficient activation of nNOS. This NT also carried an additional small charged group which increased binding to e/nNOS; fluorescence measurements determined a 20-fold improved affinity upon binding to nNOS as compared to NT1 affinity. To gain in specificity for eNOS, we augmented a previous NT with a “hook” targeting variable residues in the NADPH site of eNOS. We discuss the potential of exploiting the chemical diversity within the NADPH site of eNOS for reversal of endothelial dysfunction in cells and for controlled generation of cytotoxic NO-derived species in cancer tissues.

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

Cited by 7 publications

References 42 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

Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue

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

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