Characterization of Toll-Like Receptor 8 and Its Modulation through Novel Small-Molecule Antagonists

Abstract

Toll-like receptor 8 (TLR8) represents a key endosomal pattern recognition receptor that senses RNA degradation products to initiate proinflammatory signaling in myeloid cells, linking innate immunity to adaptive responses. While essential for antimicrobial defense, dysregulated TLR8 activity drives chronic inflammation in autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and psoriasis, positioning selective antagonists as promising therapeutics to suppress pathologic cytokine release while preserving TLR7-mediated antiviral immunity. <br/> The first objective (chapter 2) of this thesis was to comprehensively review structure-guided modulation of endosomal TLR7/8/9, integrating cryo-EM insights, pharmacophores, and clinical candidates to highlight conserved activation mechanisms, selectivity challenges, and therapeutic opportunities in autoimmunity and cancer. This review integrated recent advances in agonist/antagonist design and identified regulatory nodes as novel intervention points. <br/> A second focus (chapter 3) addressed optimization of 6-(trifluoromethyl)pyrimidine scaffolds, yielding low-micromolar TLR8 antagonists with balanced potency and cellular viability, guided by structure–activity relationship analysis and molecular dynamics targeting the uridine-binding pocket. Biological profiling confirmed non-cytotoxic NF-κB inhibition, establishing a promising chemotype for further development despite modest potency relative to nanomolar benchmarks. <br/> The third part (chapter 4) reports virtual screening discovery of novel isoxazole-based antagonists, with the lead compound showing nanomolar potency, TLR8 selectivity over TLR7/9, and competitive blockade at the dimer interface confirmed by Schild analysis, targeted mutagenesis, and reduced MyD88 recruitment in co-IP assays. This scaffold suppressed TLR8-dependent cytokine production in primary immune cells and inflammasome priming without cytotoxicity, providing comprehensive mechanistic validation. <br/> Finally, rational scaffold hopping to pyrimidine and quinazoline chemotypes (chapter 5) identified low-nanomolar compound 35 with potent TLR8-dependent NF-κB inhibition across various assays and cell lines, high selectivity, and favorable drug-like properties (logD, microsomal stability, low plasma protein binding). Docking and molecular dynamics confirmed interactions with Gly351 and Glu427, supporting hit-to-lead advancement. <br/> In summary, this work delivers three novel TLR8 antagonist chemotypes (pyrimidine, isoxazole, quinazoline) with competitive uridine-site binding, myeloid cytokine suppression, and favorable early ADME profiles, addressing the unmet clinical need for selective TLR8 modulation in autoimmune therapy.