KLYP956 Is a Non-Imidazole-Based Orally Active Inhibitor of Nitric-Oxide Synthase Dimerization

Abstract

Nitric-oxide synthases (NOS) generate nitric oxide (NO) through the oxidation of L-arginine. Inappropriate or excessive production of NO by NOS is associated with the pathophysiology of various disease states. Efforts to treat these disorders by developing arginine mimetic, substrate-competitive NOS inhibitors as drugs have met with little success. Small-moleculemediated inhibition of NOS dimerization represents an intriguing alternative to substrate-competitive inhibition. An ultra-high-throughput cell-based screen of 880,000 small molecules identified a novel quinolinone with inducible NOS (iNOS) inhibitory activity. Exploratory chemistry based on this initial screening hit resulted in the synthesis of KLYP956, which inhibits iNOS at low nanomolar concentrations. The iNOS inhibitory potency of KLYP956 is insensitive to changes in concentrations of the substrate arginine, or the cofactor tetrahydrobiopterin. Mechanistic analysis suggests that KLYP956 binds the oxygenase domain in the vicinity of the active site heme and inhibits iNOS and neuronal NOS (nNOS) by preventing the formation of enzymatically active dimers. Oral administration of KLYP956 [N-(3-chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-4-methylthiazole-5-carboxamide] inhibits iNOS activity in a murine model of endotoxemia and blocks pain behaviors in a formalin model of nociception. KLYP956 thus represents the first nonimidazole-based inhibitor of iNOS and nNOS dimerization and provides a novel pharmaceutical alternative to previously described substrate competitive inhibitors.The overproduction of nitric oxide (NO) has been implicated in the pathophysiology of a broad range of human diseases including pain, inflammation, migraine and neurodegenerative disorders. Three nitric-oxide synthase (NOS) isoforms have been described, including endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). Data support the notion that the inhibition of iNOS and nNOS may have therapeutic utility in the treatment of a variety of disease states, including inflammation and pain. However, inhibition of eNOS is considered detrimental because it results in elevated systemic blood pressure. Among the three isoforms, iNOS generates stoichiometrically higher amounts of NO and is expressed at sites of inflammation. Irrespective of the source, excessive NO can generate peroxynitrite and other reactive species that can trigger protein nitrosylation, leading to tissue damage (Vallance and Leiper, 2002).NOS isoforms catalyze the NADPH-and O 2 -dependent oxidation of L-arginine to NO and citrulline, with N-hydroxy-L-arginine formed as an intermediate. NOS isoforms are flavoheme enzymes that are only active as homodimers. Each monomer has a carboxyl-terminal diflavin-reductase domain Article, publication date, and citation information can be found at

Pharmacological Characterization of KLYP961, a Dual Inhibitor of Inducible and Neuronal Nitric-Oxide Synthases

“…For example, in primary human bronchial epithelial cells, the cellular half-life for iNOS dimers was reported to be ϳ1.6 h, which contrasts with reported values of 28 h for eNOS and 20 h for nNOS (Kolodziejski et al, 2004). The feasibility of inhibition or destabilization of the iNOS dimerization process, a protein-protein interaction process, by small-molecule ligands has been successfully demonstrated (Davey et al, 2007;Symons et al, 2009). Rapid cellular turnover of the iNOS dimer suggests the possibility of achieving superior isoform selectivity by targeting the dimerization process.…”

Inducible nitric oxide synthase: Regulation, structure, and inhibition

“…Gel electrophoresis and immunoblotting of enzyme isolated from treated cells revealed dose-dependent loss of dimeric iNOS. 181 Biochemical characterization revealed that inhibition was insensitive to addition of L-Arg and H 4 B, but binding of 30 did not result in spectral shifts consistent with imidazole-like heme coordination. Although 30 and 31 have good oral bioavailability, they have high clearance and a short half-life in vivo.…”

“…Optimization was performed to improve murine iNOS activity (for animal use), and new leads 30 (KLYP596) and 31 were found to have improved potency and selectivity, with 30 having an EC 50 of 11 nM and the higher selectivity of the two (i/e of 2300 and i/n of 210) in cell‐based assays. Gel electrophoresis and immunoblotting of enzyme isolated from treated cells revealed dose‐dependent loss of dimeric iNOS . Biochemical characterization revealed that inhibition was insensitive to addition of l ‐Arg and H 4 B, but binding of 30 did not result in spectral shifts consistent with imidazole‐like heme coordination.…”

Induction of PD-L1 by Nitric Oxide <i>via</i> JNK Activation in A172 Glioblastoma Cells

“…Moreover, NOS is inhibited by many molecules including NOS dimerization inhibitors, such as BBS-2 and KLYP956, substrate competitive analogues, such as L-NAME and L-NIL, and non-amino acid derivatives, such as MEG and1400W. In addition, cell-free hemoglobin and carboxy-PTIO scavenges NO [15][16][17] . These molecules can inhibit the production or effects of NO.…”