Background: Tumor necrosis factor-alpha (TNF-α) plays a pivotal role in the pathogenesis of various autoimmune diseases (AIDs), including Rheumatoid Arthritis (RA), Inflammatory Bowel Disease (IBD), and Psoriasis. While anti-TNF-α biologics have proven effective in alleviating clinical symptoms and slowing disease progression, significant limitations remain, including suboptimal response rates, reduced long-term efficacy, limited patient accessibility, and the risk of adverse effects such as serious infections and malignancies. Small-molecule TNF-α inhibitors emerge as a promising alternative, offering distinct mechanisms of action compared to biologics and the advantage of oral administration. In this study, we report the discovery and characterization of novel small-molecule TNF-α inhibitors that specifically disrupt the TNF-α/TNFR1 interaction, demonstrating effectiveness in both in vitro and in vivo models.

Objectives: The primary objective of this study is to discover and characterize a novel class of small-molecule TNF-α inhibitors that effectively block TNF-α signaling pathways. By addressing the limitations associated with current anti-TNF-α biologics, these inhibitors aim to provide a viable therapeutic option for patients with RA etc.

Methods: A novel class of small-molecular TNF-α inhibitors were identified, characterized and optimized via in silico tools and wet-lab assays. The binding affinity of the molecules to TNF-α was evaluated using surface plasmon resonance (SPR). The inhibitory activity of these molecules on the TNF-α/TNFR1 interaction was assessed through both protein-based SPR assays and cell-based functional assays, including the HEK293-NFκB reporter gene assay and the L929 cytotoxicity assay. Additionally, the impact of the inhibitors on pro-inflammatory cytokine secretion (IL-1β and IL-8) was measured in lipopolysaccharide (LPS)- and TNF-α-stimulated human primary monocytes. The effect on IL-12 secretion was assessed during human M1 macrophage differentiation, and the expression of E-selectin in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) was evaluated. The pharmacokinetic (PK) properties were assessed in mice, in vivo efficacy was evaluated in several autoimmune disease animal models. All in vivo studies reported here were conducted in accordance to the standard operating procedures of Shanghai Medysin Biopharma Co., Ltd. Study protocols were approved by the Institutional Animal Care and Use Committee of Shanghai Medysin Biopharma Co., Ltd.

Results: These novel inhibitors demonstrated high binding affinity to TNF-α in SPR assays, with dissociation constants (K _D ) ranging from nanomolar to sub-nanomolar levels. In functional assays, the inhibitors effectively blocked TNF-α-mediated signaling and cytotoxicity, with IC ₅₀ values ranging from 5 nM to 25 nM. These inhibitors significantly reduced IL-1β and IL-8 secretion in LPS- and TNF-α-stimulated human primary monocytes and suppressed IL-12 secretion during M1 macrophage differentiation. Moreover, these inhibitors induced a dose-dependent reduction of E-selectin expression in HUVECs. These inhibitors exhibited favorable PK profiles and demonstrated in vivo efficacy in autoimmune disease models, effectively mitigating disease progression.

Conclusion: A novel series of small-molecule TNF-α inhibitors has been discovered, featuring distinct molecular properties that exhibit potent biological activities in vitro and in vivo . These inhibitors show considerable promise as a new class of oral therapeutics for the treatment of RA, potentially overcoming the limitations associated with existing anti-TNF-α biologics and providing additional therapeutic options to meet the substantial unmet medical needs in oral AID treatment.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: Jinhua Chen IntelliGen Therapeutics Inc, Jingfeng Yu IntelliGen Therapeutics Inc., Jingjing Du IntelliGen Therapeutics Inc., Pu Shi IntelliGen Therapeutics Inc., Xin Wang IntelliGen Therapeutics Inc.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.