Vol. 63, Issue 3, 682-689, March 2003

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, and Bernd Mayer

Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Graz, Austria (H.J.H., A.C.F.G, K.S., B.M.); Vascular Biology Center, Medical College of Georgia, Augusta, Georgia (H. L., R.C.V.); and Department of Immunology, Berlex Biosciences, Richmond, California (J.F.P.)

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 (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.