A cellular model for screening neuronal nitric oxide synthase inhibitors

Fang, Jianguo; Silverman, Richard B.

doi:10.1016/j.ab.2009.04.004

Cited by 15 publications

(19 citation statements)

References 43 publications

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“…1 and 4 μM in our experiments). Also in line with our results (Figure 5C), Fang and Silverman (2009) have found that 5 μM 1400W is insufficient to attenuate the activity of recombinant nNOS in a cell-based assay.…”

Section: Discussionsupporting

confidence: 91%

On the selectivity of neuronal NOS inhibitors

Pigott

Bartus

Garthwaite

2013

British J Pharmacology

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Background and PurposeIsoform-selective inhibitors of NOS enzymes are desirable as research tools and for potential therapeutic purposes. Vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine (l-VNIO) and Nω-propyl-l-arginine (NPA) purportedly have good selectivity for neuronal over endothelial NOS under cell-free conditions, as does N-[(3-aminomethyl)benzyl]acetamidine (1400W), which is primarily an inducible NOS inhibitor. Although used in numerous investigations in vitro and in vivo, there have been surprisingly few tests of the potency and selectivity of these compounds in cells. This study addresses this deficiency and evaluates the activity of new and potentially better pyrrolidine-based compounds.Experimental ApproachThe inhibitors were evaluated by measuring their effect on NMDA-evoked cGMP accumulation in rodent hippocampal slices, a response dependent on neuronal NOS, and ACh-evoked cGMP synthesis in aortic rings of the same animals, an endothelial NOS-dependent phenomenon.Key Resultsl-VNIO, NPA and 1400W inhibited responses in both tissues but all showed less than fivefold higher potency in the hippocampus than in the aorta, implying useless selectivity for neuronal over endothelial NOS at the tissue level. In addition, the inhibitors had a 25-fold lower potency in the hippocampus than reported previously, the IC50 values being approximately 1 μM for l-VNIO and NPA, and 150 μM for 1400W. Pyrrolidine-based inhibitors were similarly weak and nonselective.Conclusion and ImplicationsThe results suggest that l-VNIO, NPA and 1400W, as well as the newer pyrrolidine-type inhibitors, cannot be used as neuronal NOS inhibitors in cells without stringent verification. The identification of inhibitors with useable selectivity in cells and tissues remains an important goal.

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Section: Discussionsupporting

confidence: 91%

On the selectivity of neuronal NOS inhibitors

Pigott

Bartus

Garthwaite

2013

British J Pharmacology

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“…[29] Although other processes may be influencing nNOS inhibition in these assays, cell permeability can be predicted from the cellular potencies of these compounds as measured by the inhibition of the production of nitric oxide in the HEK293t cells (Table 2). Comparing the IC 50 values of these compounds in the purified enzyme assay versus the cellular assay clearly indicates that membrane permeability is a major factor that limits the effective concentration of these inhibitors within cells.…”

Section: Resultsmentioning

confidence: 99%

Intramolecular hydrogen bonding: A potential strategy for more bioavailable inhibitors of neuronal nitric oxide synthase

Labby

Xue

Kraus

et al. 2012

Bioorganic & Medicinal Chemistry

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Selective neuronal nitric oxide synthase (nNOS) inhibitors have therapeutic applications in the treatment of numerous neurodegenerative diseases. Here we report the synthesis and evaluation of a series of inhibitors designed to have increased cell membrane permeability via intramolecular hydrogen bonding. Their potencies were examined in both purified enzyme and cell-based assays; a comparison of these results demonstrates that two of the new inhibitors display significantly increased membrane permeability over previous analogs. NMR spectroscopy provides evidence of intramolecular hydrogen bonding under physiological conditions in two of the inhibitors. Crystal structures of the inhibitors in the nNOS active site confirm the predicted non-intramolecular hydrogen bonded binding mode. Intramolecular hydrogen bonding may be an effective approach for increasing cell membrane permeability without affecting target protein binding.

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“…To date, the techniques of new drug high throughput screening consist of protein chip, polarized fluorescence, high content screening, model organisms, and cell models [22–27] . The cell models can simulate a variety of signalling pathways and imitate the in‐vivo effects of drugs on human or animals, and so they are successfully used in drug screening studies and are also suitable for high‐throughput screening [28] . Our previous study demonstrated that HEK293T cells expressed very lower hPPAR γ in itself, and phPPAR γ ‐IRES2‐EGFP expression vector carrying human hPPAR γ gene could be efficiently transfected and expressed in HEK293T cells [18]…”

Section: Discussionmentioning

confidence: 99%