Inducible nitric oxide synthase: Regulation, structure, and inhibition

Abstract

A considerable number of human diseases have an inflammatory component, and a key mediator of immune activation and inflammation is inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO) from l-arginine. Overexpressed or dysregulated iNOS has been implicated in numerous pathologies including sepsis, cancer, neurodegeneration, and various types of pain. Extensive knowledge has been accumulated about the roles iNOS plays in different tissues and organs. Additionally, X-ray crystal and cryogenic electron microscopy structures have shed new insights on the structure and regulation of this enzyme. Many potent iNOS inhibitors with high selectivity over related NOS isoforms, neuronal NOS, and endothelial NOS, have been discovered, and these drugs have shown promise in animal models of endotoxemia, inflammatory and neuropathic pain, arthritis, and other disorders. A major issue in iNOS inhibitor development is that promising results in animal studies have not translated to humans; there are no iNOS inhibitors approved for human use. In addition to assay limitations, both the dual modalities of iNOS and NO in disease states (ie, protective vs harmful effects) and the different roles and localizations of NOS isoforms create challenges for therapeutic intervention. This review summarizes the structure, function, and regulation of iNOS, with focus on the development of iNOS inhibitors (historical and recent). A better understanding of iNOS' complex functions is necessary before specific drug candidates can be identified for classical indications such as sepsis, heart failure, and pain; however, newer promising indications for iNOS inhibition, such as depression, neurodegenerative disorders, and epilepsy, have been discovered.

Keywords: animal models; cancer; enzyme inhibition; immune regulation; immune system activation; inducible nitric oxide synthase; inflammation; neurodegeneration; nitrergic signaling; nitric oxide; pain; reactive oxygen species; sepsis.

FIGURE 1

Schematic representation of electron transfers from NADPH to heme through FAD and FMN (via tetrahydrobiopterin). The double-headed arrow indicates a conformational change to bring FMN into proximity with heme. The exact catalytic mechanism is not shown. Figure adapted with permission

FIGURE 2

Assembled dimeric iNOS X-ray crystal structures organized based on cryo-EM data, from PDB IDs 3HR4 and 4CX7.^, The reductase domain shown is a 212-residue fragment of the full-length domain. Domains are annotated on the right side, cofactors on the left. The dimer interface is located between the orange- and cyan-colored domains.

FIGURE 3

Regulation of iNOS activity by the availability of its substrate L-Arginine. CAT: cation amino acid transporters. ASS: Arginosuccinate synthase.

FIGURE 4

Different signaling pathways triggered by different iNOS inducers that regulate iNOS expression (left: activation of NF-κB pathway; right: activation of JAK/STAT-1α pathway).

FIGURE 5

Arginine and amino acid amidine derivatives.

FIGURE 6

Aliphatic and aromatic amidines. When stereochemistry is unknown, a solid line is used (11).

FIGURE 7

Cyclic amidine derivatives. When stereochemistry is unknown, a solid line is used (19).

FIGURE 8.

X-ray crystal structure of compound 21 (purple) bound in the murine iNOS active site (PDB ID: 3EBF. Heme is depicted in magenta, H₄B in cyan, and amino acid residues of the enzyme in green. Distances are measured in Ångstroms. Figure prepared using PyMol (www.pymol.org).

FIGURE 9

Pyridine and aminopyridine derivatives.

FIGURE 10

Various iNOS dimerization inhibitors. When stereochemistry is unknown, a solid line is used (27).

FIGURE 11.

X-ray crystal structure of 27 (purple) bound to iNOS monomer (with N-terminal 114 residues truncated). (PDB ID: 1DD7). Note the coordination of the heme (magenta) iron by the inhibitors’s imidazole. The helix containing the catalytic Glu371 is disordered. Figure prepared using PyMol (www.pymol.org).

FIGURE 12

Neuroprotective iNOS inhibitors.