New Glucosamine-Based TLR4 Agonists: Design, Synthesis, Mechanism of Action, and In Vivo Activity as Vaccine Adjuvants

Abstract

We disclose here a panel of small-molecule TLR4 agonists (the <b>FP20</b> series) whose structure is derived from previously developed TLR4 ligands (<b>FP18</b> series). The new molecules have increased chemical stability and a shorter, more efficient, and scalable synthesis. The <b>FP20</b> series showed selective activity as TLR4 agonists with a potency similar to <b>FP18</b>. Interestingly, despite the chemical similarity with the <b>FP18</b> series, <b>FP20</b> showed a different mechanism of action and immunofluorescence microscopy showed no NF-κB nor p-IRF-3 nuclear translocation but rather MAPK and NLRP3-dependent inflammasome activation. The computational studies related a 3D shape of <b>FP20</b> series with agonist binding properties inside the MD-2 pocket. <b>FP20</b> displayed a CMC value lower than 5 μM in water, and small unilamellar vesicle (SUV) formation was observed in the biological activity concentration range. <b>FP20</b> showed no toxicity in mouse vaccination experiments with OVA antigen and induced IgG production, thus indicating a promising adjuvant activity.