Acetamidine-Based iNOS Inhibitors as Molecular Tools to Counteract Inflammation in BV2 Microglial Cells

Abstract

Neurodegenerative diseases are associated with increased levels of nitric oxide (NO) mainly produced by microglial cells through inducible nitric oxide synthase (iNOS) whose expression is induced by inflammatory stimuli. NO can both exert cytotoxic functions and induce a metabolic switch by inhibiting oxidative phosphorylation and upregulating glycolytic flux. Here, we investigated whether two newly synthesized acetamidine based iNOS inhibitors, namely CM292 and CM544, could inhibit lipopolysaccharide (LPS)-induced BV2 microglial cell activation, focusing on both inflammatory and metabolic profiles. We found that CM292 and CM544, without affecting iNOS protein expression, reduced NO production and reverted LPS-induced inflammatory and cytotoxic response. Furthermore, in the presence of the inflammatory stimulus, both the inhibitors increased the expression of glycolytic enzymes. In particular, CM292 significantly reduced nuclear accumulation of pyruvate kinase M2, increased mitochondrial membrane potential and oxygen consumption rate, and augmented the expression of pyruvate dehydrogenase, pointing to a metabolic switch toward oxidative phosphorylation. These data confirm the role played by NO in the connection between cell bioenergetics profile and inflammation, and suggest the potential usefulness of iNOS inhibitors in redirecting microglia from detrimental to pro-regenerative phenotype.

UPLC-ESI-Q-TOF-MS-Based Metabolite Profiling, Antioxidant and Anti-Inflammatory Properties of Different Organ Extracts of Abeliophyllum distichum

“…Consistently with these findings, Western blot analyses elucidated an increase in iNOS protein expression in response to LPS, which was almost completely inhibited by AL ( Figure 3 B), suggesting that the inhibition of LPS-induced NO production by AL treatment was due to the inhibition of iNOS expression. iNOS-generated NO reportedly interacts with mitochondrial cytochrome c oxidase and competes with oxygen, resulting in a decrease in adenosine triphosphate production [ 31 , 32 ]. Therefore, the inhibitory effect of AL on iNOS-derived NO production in LPS-stimulated cells suggested that AL could be used as a plant-derived drug for the removal of cytotoxic NO.…”

The Inhibition of the Inducible Nitric Oxide Synthase Enhances the DPSC Mineralization under LPS-Induced Inflammation

“…In this research context we have previously disclosed the acetamidines CM544 and FAB1020 , which are two compounds structurally related to the iNOS inhibitor 1400W ( Figure 1 ) [ 15 , 16 ]. These two acetamidines inhibit the iNOS with high potency and selectivity of action with respect to the constitutive NOSs, and, remarkably, they showed an ameliorated biological profile as anticancer, neuroprotective and immunomodulatory agents, in comparison with 1400W [ 17 , 18 , 19 ]. The lipophilicity of CM544 and FAB1020 also appears somewhat increased with respect to the very polar and poorly cell-permeable 1400W ( Table 1 ).…”

Selective Inhibitors of the Inducible Nitric Oxide Synthase as Modulators of Cell Responses in LPS-Stimulated Human Monocytes

“…This last interaction is not observed when CM544 is docked into the eNOS binding site, and this could explain the isoform selectivity. Very interestingly, CM544 and FAB1020 demonstrated promising activities as antiglioma agents, compromising cell cycle progression in C6 rat glioma cells without affecting astrocytes, and exhibiting an improved biological profile with respect to 1400 W. CM544 was also able to counteract inflammation in BV2 microglia cells [ 20 ].…”