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 TNFα 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.