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)-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.