Development of a platform for the discovery of new Alzheimer’s disease drugs targeting stress‐induced neuroinflammation

Background Sterile inflammation plays a detrimental role in neurodegenerative disorders, having an extraordinary impact on Alzheimer’s disease (AD). Stress signals, such as the alarmin high mobility‐group box‐1 (HMGB‐1), trigger activation of inflammasome complexes —as NLRP3 or NLRC4— and the subsequent oligomerization of the associated speck‐like protein containing‐CARD (ASC), which is critical for stress‐induced depressive behavior and promotes AD phenotypes. Indeed, ASC specks promote Aβ aggregation, presenting both intracellular and extracellular prionoid activities. Method We developed a platform for the discovery of new anti‐inflammasome drugs. We modeled the target and performed a chemical library. We validated in‐vitro , using murine primary microglial cultures, the ability of the hits from the screening to inhibit NLRP3 and NLRC4‐mediated IL‐1β production. We further characterized the most potent inhibitors, studying their ability to inhibit processes downstream of inflammasome activation. Thus, we evaluated whether our compounds inhibit ASC oligomerization using fluorescently tagged‐ASC murine macrophages, and we determined their anti‐pyroptotic effect. Finally, we analyzed the ability of a lead compound to reduce neuroinflammation in the brain of LPS injected mice. Result We built a 3D homology model for NLRP3 that provides an accurate description of the active site. Through computational screening, we identified 100 hits. The 20% of the hits inhibited NLRP3 in‐vitro , and six among them reduced the activity of the inflammasome below 80%. The three most potent compounds presented IC50 values, for NLRP3‐mediated IL‐1β secretion, in the low micromolar range (9.24, 8.26, and 3.5 μM). Among them, we selected the third one, C77, to characterize it further. C77 inhibited NLRP3 at the protein level, being its IC50 for the ATPase activity 40 nM. Interestingly, C77 inhibited NLRC4 with similar potency, as well as processes downstream of both inflammasomes. Noticeable, C77 inhibited ASC specks formation in response to the stress alarmin HMGB1, at concentrations as low as 10 nM. Finally, C77 also reduced IL‐1β in the brain of mice LPS‐injected. Conclusion We developed a platform to identify and characterize new AD drugs, targeting neuroinflammation, and using this methodology to identify a lead compound. Future experiments will demonstrate the beneficial effect of our lead compound in AD murine models.