Structure–Activity Relationship Studies To Identify Affinity Probes in Bis-aryl Sulfonamides That Prolong Immune Stimuli

Abstract

Agents that safely induce, enhance, or sustain multiple innate immune signaling pathways could be developed as potent vaccine adjuvants or coadjuvants. Using high-throughput screens with cell-based nuclear factor κB (NF-κB) and interferon stimulating response element (ISRE) reporter assays, we identified a bis-aryl sulfonamide bearing compound 1 that demonstrated sustained NF-κB and ISRE activation after a primary stimulus with lipopolysaccharide or interferon-α, respectively. Here, we present systematic structure–activity relationship (SAR) studies on the two phenyl rings and amide nitrogen of the sulfonamide group of compound 1 focused toward identification of affinity probes. The murine vaccination studies showed that compounds 1 and 33 when used as coadjuvants with monophosphoryl lipid A (MPLA) showed significant enhancement in antigen ovalbumin-specific immunoglobulin responses compared to MPLA alone. SAR studies pointed to the sites on the scaffold that can tolerate the introduction of aryl azide, biotin, and fluorescent rhodamine substituents to obtain several affinity and photoaffinity probes which will be utilized in concert for future target identification and mechanism of action studies.

Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection

“…In the initial HTS, compound 81 emerged as a lead compound as it was able to enhance both ISRE and NF-κB activation (18,19). Based on these activities, 81 was suspected of being an innate immune activator.…”

“…Unlike most PRR ligands, these compounds can be easily synthesized in one to two synthetic steps from readily available commercial reagents. Additionally, compound 81, when used as a coadjuvant with MPLA, showed significant enhancement in antigen-specific immunoglobulin responses compared to MPLA alone (19).…”

“…[N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] that belonged to bis-aryl sulfonamide scaffold was identified as a low molecular weight compound that prolonged activation of ISRE induced by type I IFN (18) and was also found to enhance lipopolysaccharide (LPS)-induced NF-κB responses (19). Further structure-activity relationship (SAR) studies identified the sites critical for immune-stimulatory properties of derivatives of 81.…”

“…The above results showed that mitochondrial stress contributed to compound 81-induced immune responses. We further explored this activity using a chemical approach by examining eight derivatives of compound 81 from a prior SAR study for induction of mtROS levels (19), and ranked these compounds by mtROS levels (Table 1 and SI Appendix, Fig. S7).…”

Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release

“…Prior to treatment, CD63Tluc-CD9EmGFP reporter cells and parental THP-1 cells were washed with their base medium and resuspended in their complete culture medium substituted with exosome-depleted FBS (EXO-FBS-250A-1, System Biosciences, Mountain View, CA, United States, or Gibco ™ Exosome-Depleted FBS, Thermo Fisher Scientific). In the experiments performed for Figures 2A-G (Thermo Fisher), CD63Tluc-CD9EmGFP reporter cells were treated with graded concentrations of phorbol myristate acetate (PMA) (tlrl-pma, InvivoGen, San Diego, CA, United States) or immunostimulatory compounds (5 and 81) (Sato-Kaneko et al, 2018;Chan et al, 2019). Cell concentrations were 5 × 10 4 cells/200 µl/well for 96well format and 2.5 × 10 4 cells/80 µl/well for 384-well format unless otherwise indicated in the figure legends.…”