Pharmacological interference with dimerization of human neuronal nitric-oxide synthase expressed in adenovirus-infected DLD-1 cells

Hans jörg Habisch, Antonius C.F. Gorren, Haiying Liang, Richard C Venema, John F. Parkinson, Kurt Schmidt, Bernd Mayer

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Abstract

A recombinant adenovirus containing the cDNA of human neuronal nitric-oxide synthase (nNOS) was constructed to characterize the interaction of nNOS with N-[(1,3-benzodioxol-5-yl)methyl]-1-[2-(1H-imidazole-1-yl)pyrimidin-4-yl]- 4-(methoxycarbonyl)-pipera-zine-2-acetamide (BBS-1), a potent inhibitor of inducible NOS dimerization [Proc Natl Acad Sci USA 97:1506-1511, 2000]. BBS-1 inhibited de novo expression of nNOS activity in virus-infected cells at a half-maximal concentration (IC50) of 40 ± 10 nM in a reversible manner. Low-temperature gel electrophoresis showed that BBS-1 attenuated the formation of SDS-resistant nNOS dimers with an IC50 of 22 ± 5.2 nM. Enzyme inhibition progressively decreased with increasing time of addition after infection. BBS-1 did not significantly inhibit dimeric nNOS activity (IC50 > 1 mM). Long-term incubation with BBS-1 of human embryonic kidney cells stably transfected with nNOS or endothelial NOS revealed a slow time- and concentration-dependent decrease of NOS activity with half-lives of 30 and 43 h and IC50 values of 210 ± 30 nM and 12 ± 0.5 μM, respectively. These results establish that BBS-1 interferes with the assembly of active nNOS dimers during protein expression. Slow inactivation of constitutively expressed NOS in intact cells may reflect protein degradation and interference of BBS-1 with the de novo synthesis of functionally active NOS dimers. As time-dependent inhibitors of NOS dimerization, BBS-1 and related compounds provide a promising strategy to develop a new class of selective and clinically useful NOS inhibitors.

Original languageEnglish (US)
Pages (from-to)682-689
Number of pages8
JournalMolecular Pharmacology
Volume63
Issue number3
DOIs
StatePublished - Mar 1 2003

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Nitric Oxide Synthase Type I
Dimerization
Adenoviridae
Pharmacology
Inhibitory Concentration 50
Proteolysis
Electrophoresis
Complementary DNA
Gels
Viruses
Kidney
Temperature
Enzymes
Infection

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

Habisch, H. J., Gorren, A. C. F., Liang, H., Venema, R. C., Parkinson, J. F., Schmidt, K., & Mayer, B. (2003). Pharmacological interference with dimerization of human neuronal nitric-oxide synthase expressed in adenovirus-infected DLD-1 cells. Molecular Pharmacology, 63(3), 682-689. https://doi.org/10.1124/mol.63.3.682

Pharmacological interference with dimerization of human neuronal nitric-oxide synthase expressed in adenovirus-infected DLD-1 cells. / Habisch, Hans jörg; Gorren, Antonius C.F.; Liang, Haiying; Venema, Richard C; Parkinson, John F.; Schmidt, Kurt; Mayer, Bernd.

In: Molecular Pharmacology, Vol. 63, No. 3, 01.03.2003, p. 682-689.

Research output: Contribution to journalArticle

Habisch, HJ, Gorren, ACF, Liang, H, Venema, RC, Parkinson, JF, Schmidt, K & Mayer, B 2003, 'Pharmacological interference with dimerization of human neuronal nitric-oxide synthase expressed in adenovirus-infected DLD-1 cells', Molecular Pharmacology, vol. 63, no. 3, pp. 682-689. https://doi.org/10.1124/mol.63.3.682
Habisch, Hans jörg ; Gorren, Antonius C.F. ; Liang, Haiying ; Venema, Richard C ; Parkinson, John F. ; Schmidt, Kurt ; Mayer, Bernd. / Pharmacological interference with dimerization of human neuronal nitric-oxide synthase expressed in adenovirus-infected DLD-1 cells. In: Molecular Pharmacology. 2003 ; Vol. 63, No. 3. pp. 682-689.
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