Adjuvant activity of a small molecule TLR4 agonist discovered via structure-based virtual screening

Monophosphoryl lipid A (MPLA), a TLR4 agonist, is a clinically approved vaccine adjuvant, but its complex structure and occasional toxicity limit broader use. Synthetic small-molecule TLR4 agonists offer advantages such as ease of synthesis, lower cost, and reduced toxicity. In this study, we conducted structure-based virtual screening of the ZINC database to identify novel TLR4-targeting small molecules across human, murine, and bovine species. Three lead compounds—NSF-418, NSF-501, and NSF-951—were selected based on favorable binding interactions and subjected to in vitro and in vivo evaluation. NSF-951 emerged as a potent TLR4 agonist, inducing strong proinflammatory cytokine responses (IL-6, TNF-α), upregulating CD80 and CD86 expression, and promoting macrophage maturation. Conversely, NSF-418 and NSF-501 acted as antagonists by suppressing MPLA-induced responses. In murine immunization studies, NSF-951, alone or with Alum (AF007), significantly enhanced OVA-specific antibody and T-cell responses without observable toxicity. These findings suggest that NSF-951 is a promising, cost-effective TLR4 agonist with strong immunostimulatory and adjuvant potential. Further studies are warranted to assess its performance with other antigens and adjuvant combinations, supporting its development as a next-generation adjuvant for veterinary and human vaccines. Structure-based screening identified three TLR4-targeting small molecules. NSF-951 showed strong agonist and adjuvant activity, while NSF418 and NSF-501 acted as antagonists. NSF-951 is a promising, cost-effective alternative to MPLA for vaccine development