Molecular mechanisms of 10-butyl ether minocycline, a novel nonantibiotic tetracycline, as a potential treatment for inflammatory and neuroimmune-related disorders.

The pleiotropy of minocycline (MINO), including anti-inflammatory, antioxidant, antimigratory, anti-matrix metalloproteinase (MMP), and neuroprotective effects, has been extensively reported. A novel nonantibiotic MINO derivative, 10-butyl ether minocycline (BEM), was synthesized to retain the pleiotropy of MINO while minimizing side effects such as antibiotic resistance and gut dysbiosis. Previously, we showed that BEM reduced alcohol consumption in dependent murine and porcine models of alcohol use disorder. In this study, we investigated the molecular mechanisms of BEM to determine its potential as a therapeutic agent for neuroimmune and inflammatory conditions such as alcohol use disorder. Here, we report that BEM showed a nearly complete loss of antimicrobial activity against Escherichia coli, Salmonella typhi, and Candida albicans. BEM showed a dose-dependent reduction in cell viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, similarly to MINO. BEM also suppressed lipopolysaccharide-induced microglial activation as shown by reduced Iba1 expression in immunohistochemistry and western blot analyses. Inhibition of MMP-9 by BEM (IC50 = 42.2 μM) was improved compared to MINO (IC50 = 60.3 μM), whereas MMP-8 inhibition was moderate (IC50: BEM = 69.4 μM; MINO = 45.4 μM). BEM was found to be effective in inhibiting vascular endothelial growth factor-induced endothelial cell migration and L-glutamine-induced reactive oxygen species levels. Limited inhibition of 15-lipoxygenase activity was observed (IC50: BEM = 92.6 μM; MINO = 65.6 μM). BEM was not toxic to mitochondria, even at high concentrations (200 μM). By eliminating antimicrobial properties while preserving therapeutic pleiotropy, BEM presents an advancement in the development of a promising candidate with multimodal mechanisms to treat neuroimmune-inflammatory pathologies. SIGNIFICANCE STATEMENT: We report mechanisms of action for butyl ether minocycline, a minocycline analog under evaluation for the treatment of alcohol use disorder, which may also show efficacy for other complex disease processes that involve inflammatory or neuroimmune components. We show that butyl ether minocycline had a nearly complete loss of antimicrobial action, yet retained the pleiotropy of minocycline, likely making it a better multimodal therapeutic for long-term treatment of complex diseases with neuroimmune-related components.