FGA139, a Novel Cysteine Protease Inhibitor, Exhibits Anti-Inflammatory and Neuroprotective Activity and Reveals Microglial Modulation via Multi-Omics Profiling

Neuroinflammation is a key driver in the progression of numerous brain disorders, with cysteine proteases such as calpains, caspases, and cathepsins playing central roles in inflammatory signaling. This study investigates FGA139, a novel irreversible inhibitor targeting cysteine proteases. We evaluated the anti-inflammatory properties of FGA139 in lipopolysaccharide (LPS)-activated macrophages (RAW264.7) and microglia (HMC3). In addition, its neuroprotective effects were assessed in differentiated SH-SY5Y neuron-like cells exposed to conditioned media (CM) derived from the activated immune cells. FGA139 exhibited a favorable safety profile and robust anti-inflammatory activity, significantly reducing nitric oxide (NO) production in macrophages and TNF levels in microglia. Conditioned media from both LPS-stimulated immune cells lines (CM+) reduced neurite length in neuronal cells. However, CM from HMC3 cells impaired neuronal viability, whereas CM from RAW264.7 cells elevated reactive oxygen species (ROS) and NO levels--indicating distinct neurotoxic signatures. Preincubation of neuron-like cells with FGA139 effectively mitigated most of these adverse effects. Metabolomic analysis of the activated microglia supernatant revealed that FGA139 increased extracellular levels of neuroprotective metabolites, including purines, linoleic acid, and phenyllactic acid. Proteomic data confirmed that FGA139 attenuated M1-like microglial polarization, likely through modulation of pathways associated with zinc transport and vesicle trafficking. In conclusion, FGA139 demonstrates potent neuroprotective effects and modulates microglial activation. These findings uncover novel mechanisms underlying the beneficial effects of cysteine protease inhibition and support the therapeutic potential of FGA139 in treating neuroinflammatory conditions, positioning it as a promising modulator of microglial function. HighlightsO_LIFGA139, an irreversible cysteine protease inhibitor, exhibits anti-inflammatory effects, significantly reducing LPS-induced pro-inflammatory markers (NO in macrophages, TNF in microglia). C_LIO_LIFGA139 prevented the damaged neurons exposed to conditioned media (CM) from LPS-stimulated immune cells (reduced viability, reduced neurite length and increased ROS/NO). C_LIO_LIFGA139 boosted secretion of neuroprotective metabolites (e.g., purines, linoleic acid) in LPS-stimulated microglia C_LIO_LIProteome analysis in FGA139 pretreated microglia showed a prevention of LPS-induced M1-like polarization, revealing novel mechanistic pathways. C_LIO_LIFGA139s ability to modulate microglial activation and protect neurons positions it as a promising candidate for neuroinflammatory diseases. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/652036v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1968c35org.highwire.dtl.DTLVardef@ceef77org.highwire.dtl.DTLVardef@1cb849corg.highwire.dtl.DTLVardef@6f4190_HPS_FORMAT_FIGEXP M_FIG C_FIG