Molecular mechanisms of 10-Butyl Ether Minocycline (BEM), a novel non-antibiotic tetracycline, as a potential treatment for inflammatory and neuroimmune-related disorders

Abstract The pleiotropy of minocycline (MINO), including anti-inflammatory, antioxidant, anti-migratory, anti-MMP, and neuroprotective effects, has been extensively reported. A novel non-antibiotic minocycline derivative, 10-butyl ether minocycline (BEM), was synthesized to retain the pleiotropy of minocycline 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 (AUD). 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 AUD. Here, we report that BEM showed a nearly complete loss of antimicrobial activity against E. coli, S. typhi, and C. albicans. BEM showed a dose-dependent reduction in cell viability as measured by the MTT assay, similarly to MINO. BEM also suppressed LPS-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) while MMP-8 inhibition was moderate (IC50: BEM = 69.4 µM; MINO = 45.4 µM). BEM was found to be effective in inhibiting VEGF-induced endothelial cell migration and L-glutamine-induced ROS levels. Limited inhibition of 15-LOX 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. Impact Our current approach to correlate non-antibacterial actions of BEM to MINOs established mechanistic effects will enable the informed use of BEM for several medical indications including for inflammation and neuroimmune conditions. The focus on BEM’s multimodal actions and long-term safety during drug discovery represents a paradigm shift toward complex therapeutic drug development and repositioning, improving upon traditional singular high-affinity target-based approaches. Such new drug discovery attempts could potentially enhance treatment relevance in complex disorders with multiple targets and theoretically guide the creation of second-generation analogs. Significance statement We report mechanisms of action for BEM, 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 BEM had a nearly complete loss of antimicrobial action, yet retained the pleiotropy of MINO, likely making it a better multimodal therapeutic for long-term treatment of complex diseases with neuroimmune-related components. Competing Interest Statement The authors have declared no competing interest. Footnotes Sources of support: NIAAA grant U01 AA028957 Recommended section assignment: Drug discovery and translational medicine This is a revised version of our manuscript based on feedback from reviewers at the Journal of Pharmacology and Experimental Therapeutics.