Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry

Abstract

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.

Figure 1

(A) The synthetic steps used to prepare the encoded combinatorial chemical library on a polymeric support. (B) The distribution frequency of synthons in active compounds from the combinatorial library. (C) Compound 1 (N-[(1,3-benzodioxol-5-yl)methyl]-1-[6-chloro-2-(1H-imidazol-1-yl)pyrimidin-4-yl]piperazine-2-acetamide) is one of the compounds that was found by screening the library. Compound 2 is (N-[(1,3-benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)pyrimidin-4-yl]-4-(methoxycarbonyl)-piperazine-2-acetamide. Compound 3, tritiated on the propionamide moiety, is the analog used in the competitive binding assay (N-[(1,3-benzodioxol-5-yl)methyl]-4-(ethylcarbonyl)-1-[2-(1H-imidazol-1-yl)pyrimidin-4-yl]piperazine-2-acetamide).

Figure 2

Inhibition of dimer formation in RAW264.7 cells. The effects of inhibitor treatment on the iNOS monomer/dimer ratio in cells was determined by size-exclusion chromatography of lysates from cells grown in the presence (○) or absence (●) of compound 2. (A) Cytochrome c reductase activity (a property of both iNOS monomers and dimers). (B) iNOS activity, indicating the presence of active dimer. This figure shows one of two experiments, which gave similar results.

Figure 3

Binding of 2 to purified iNOS monomers. The amount of bound radioligand 3 (cpm) is plotted versus the concentration of 2 (K_i = 2.2 nM). Error bars indicate the SD for each value.

Figure 4

Inhibition of LPS-induced elevations of plasma NO_x levels in rats. Plasma levels of NO_x were 24.3 ± 1.5 μM in naive rats. Error bars show the SEM; this experiment has been repeated in rats and mice with similar results.

Figure 5

The 2.25-Å resolution crystal structure of the 2–murine iNOS Δ114 complex and comparison to the murine iNOS dimer structure. Two views of the refined model of the 2–murine iNOS Δ114 complex are shown. Compound 2 (green) and heme (dark red) both are shown with blue nitrogens and red oxygens. The sulfur (yellow) of Cys-194 (magenta) coordinates the heme iron (yellow sphere). The water molecules described in the text are shown as red spheres, and in Tyr-367, Tyr-485, and Gln-257 side chains are shown in orange with red oxygens and blue nitrogens. Both iron coordination and hydrogen bonds are shown as dashed lines; other iNOS Δ114 atoms are shown in gray. Numerous residues are not shown for clarity, including residues 486–496 and the side chains of Val-346, Asn-348, and Met-349 in A and Gln-257 and surrounding residues (241–345) in B.

Figure 6

Comparison of the 2–iNOS Δ114 complex with the murine iNOS oxygenase domain dimer. Compound 2 occupies the arginine-binding site. The monomers of the iNOS dimer structure (11) are shown in dark and light blue. The 2–iNOS Δ 114 structure (tan) has been aligned with a monomer (light blue) of the dimer structure; the hemes are shown in dark red. The iNOS Δ 114 heme and the dimer heme align very closely (for clarity only the iNOS Δ 114 heme is shown). Compound 2 (dark gray) displaces helix 7a (red) side chains from the arginine-binding site in the 2–iNOS Δ 114 structure, e.g., Glu-371 (red). The region from the beginning of helix 7a to the middle half of helix 8 is disordered in 2–iNOS Δ 114 structure.