Pharmacological interference with dimerization of human neuronal nitric-oxide synthase expressed in adenovirus-infected DLD-1 cells.
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)-piperazine-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 (IC(50)) 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 IC(50) 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 (IC(50) > 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 IC(50) values of 210 +/- 30 nM and 12 +/- 0.5 microM, 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.
Habisch
Hans-Jörg
HJ
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Graz, Austria.
Gorren
Antonius C F
AC
Liang
Haiying
H
Venema
Richard C
RC
Parkinson
John F
JF
Schmidt
Kurt
K
Mayer
Bernd
B
D000256:Adenoviridae / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured / Q000821:virology; D019281:Dimerization; D006801:Humans; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I
C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I