Advancements in the Research of New Modulators of Nitric Oxide Synthases Activity

Abstract

Nitric oxide (NO) has been defined as the "miracle molecule" due to its essential pleiotropic role in living systems. Besides its implications in physiologic functions, it is also involved in the development of several disease states, and understanding this ambivalence is crucial for medicinal chemists to develop therapeutic strategies that regulate NO production without compromising its beneficial functions in cell physiology. Although nitric oxide synthase (NOS), i.e., the enzyme deputed to the NO biosynthesis, is a well-recognized druggable target to regulate NO bioavailability, some issues have emerged during the past decades, limiting the progress of NOS modulators in clinical trials. In the present review, we discuss the most promising advancements in the research of small molecules that are able to regulate NOS activity with improved pharmacodynamic and pharmacokinetic profiles, providing an updated framework of this research field that could be useful for the design and development of new NOS modulators.

Keywords: activators; cancer; cardiovascular diseases; drug design; inflammation; inhibitors; natural sources; neurodegenerative diseases; nitric oxide; nitric oxide synthase.

Scheme 1

Global catalytic mechanism of NO synthesis. In the first step, L-Arg hydroxylation occurs in the presence of one molecule of oxygen and one equivalent NAPH via a sequential hydride and electron transfer through FAD, FMN up to heme and BH4. The formed N-hydroxyl-arginine requires a molecule of oxygen and one electron from 0.5 NADPH to be further oxidized to L-Citr and NO, and the final production of two H₂O molecules. Abbreviations: NADPH: nicotinamide adenine dinucleotide phosphate; FAD: flavin adenine dinucleotide; FMN: flavin mononucleotide; and BH4: tetrahydrobiopterin.

Figure 1

NOS isoforms and their regulation and effects. The nNOS is activated by its interaction with the NMDAR/PSD95 complex. The generated NO interacts with the sGC, which catalyzes the production of cGMP from the GTP, and then the cGMP mediates downstream effects. The transcription of iNOS is mediated by different proteins, such as NF-κB, STAT1, and AP1, in response to proinflammatory stimuli. NO is generated at high levels as an immune defense. In physiological conditions, eNOS is activated through its phosphorylation by different protein kinases, and the produced NO, by interacting with the sGC, maintains vascular homeostasis. Abbreviations: NMDAR: N-methyl-D-aspartate receptor; PSD95: postsynaptic density protein 95; sGC: soluble guanylate cyclase; cGMP: cyclic guanosine monophosphate; GTP: triphosphate guanosine; PTM: post-translational modifications; LPS: lipopolysaccharides; IL1β: interleukin-1β; TNFα: tumor necrosis factor α; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; STAT1: signal transducer and activator of transcription 1; AP1: activator protein 1; PI3K/AKT: phosphatidylinositol 3-kinase/protein kinase B; AMPK: 5′AMP-activated protein kinase; and PKA: protein kinase A.

Figure 2

Schematic representation of the NOS dimer. Each monomer is formed by the oxygenase domain (in red) containing the heme and the BH4 and L-Arg-binding sites and the reductase domain (in green) containing the cofactor binding sites. The oxygenase domain is bound to the reductase domain by a calcium–calmodulin (Ca²⁺-CaM) binding sequence. Zn–tetrathiolate is bound at the dimer interface between the two oxygenase domains.

Figure 3

Chemical structure of 7-nitroindazole.

Figure 4

General pharmacophoric model of 2-aminopyridine-based nNOS inhibitors and the chemical structure of selected derivatives 1–4.

Figure 5

Chemical structure of nNOS-PSD95 interaction inhibitors.

Figure 6

Chemical structures of the first generation of potent and selective iNOS inhibitors.

Figure 7

The general structure of phenyl-amidine-based compounds related to 1400W and the chemical structure of the most interesting molecule, 6. In the general structure, the phenyl ring, which was introduced to ameliorate 1400W lipophilicity, is reported in green; in red, the 1400W amidine-benzyl core is reported, and in blue, the phenyl-sulfonamide moiety responsible for the compounds’ selectivity is shown.

Figure 8

Aril-carboximidamide-based compounds for iNOS inhibitors endowed with anti-inflammatory activity.

Figure 9

Chemical structures of iNOS inhibitors containing a nitrogen heterocycle.

Figure 10

Chemical structure of iNOS dimerization inhibitors.

Figure 11

Chemical structure of chrysamide B, which is a potent natural iNOS dimerization inhibitor.

Figure 12

Chemical structure of the eNOS stimulators AVE9488 and AVE3085.

Figure 13

Chemical structure of flavonoids regulating the expression and the activity of eNOS.