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Originally published In Press as doi:10.1074/jbc.M105691200 on October 31, 2001

J. Biol. Chem., Vol. 277, Issue 1, 295-302, January 4, 2002
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Mechanistic Studies with Potent and Selective Inducible Nitric-oxide Synthase Dimerization Inhibitors*

Eric Blaskoa, Charles B. Glaserb, James J. Devlinb, Wei Xiab, Richard I. Feldmanc, Mark A. Polokoffd, Gary B. Phillipsd, Marc Whitlowe, Douglas S. Auldf, Kirk McMillanfg, Sanjay Ghoshhi, Dennis J. Stuehrhi, and John F. Parkinsonjk

From the a Cardiovascular Research, b Protein Expression and Cell Sciences, c Cancer Research, d Discovery Research, e Biophysics, and j Immunology Departments, Berlex Biosciences, Richmond, California 94804-0099, the h Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, and f Pharmacopeia Inc., Princeton, New Jersey 08512

A series of potent and selective inducible nitric-oxide synthase (iNOS) inhibitors was shown to prevent iNOS dimerization in cells and inhibit iNOS in vivo. These inhibitors are now shown to block dimerization of purified human iNOS monomers. A 3H-labeled inhibitor bound to full-length human iNOS monomer with apparent Kd ~1.8 nM and had a slow off rate, 1.2 × 10-4 s-1. Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent Kd values from 330 pM to 27 nM) correlated with potency in a cell-based iNOS assay (IC50 values from 290 pM to 270 nM). Inhibitor potency in cells was not prevented by medium supplementation with L-arginine or sepiapterin, but inhibition decreased with time of addition after cytokine stimulation. The results are consistent with a mechanism whereby inhibitors bind to a heme-containing iNOS monomer species to form an inactive iNOS monomer-heme-inhibitor complex in a pterin- and L-arginine-independent manner. The selectivity for inhibiting dimerization of iNOS versus endothelial and neuronal NOS suggests that the energetics and kinetics of monomer-dimer equilibria are substantially different for the mammalian NOS isoforms. These inhibitors provide new research tools to explore these processes.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

g Current address: Exelixis Inc., 170 Harbor Way, P.O. Box 511, South San Francisco, CA 94083-0511.

i Supported by National Institutes of Health Grant CA53914.

k To whom correspondence should be addressed: Immunology Dept., Berlex Biosciences, 15049 San Pablo Ave, Richmond, CA 94804-0099. Tel.: 510-669-4145; Fax: 510-669-4244; E-mail: john_parkinson@berlex.com.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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