Session: Session 4

Study of the non canonical signalling induced by the binding of small molecules to CXCR4

The chemokine receptor CXCR4, known for its roles in cell migration and adhesion, is a promising target in immune regulation. Small molecules called minor pocket agonists (MiPAs) targeting CXCR4 exhibit potent anti-inflammatory effects by reversing TLR-mediated activation. This study investigates the involvement of a non-canonical signaling pathway in this process.

 

To confirm the broad-spectrum inhibitory effects of MiPAs, we monitored IRF7 phosphorylation and NF-kB activation via HTRF after TLR7/8 ligand activation. We focused on post-translational modifications such as phosphorylation and ubiquitination to capture comprehensive cell activation states. Using a PTM enrichment workflow with data-independent acquisition (DIA) mass spectrometry, we precisely quantified and identified diverse modified peptides. This facilitated the discovery of potential regulatory mechanisms within TLR signaling pathways and the identification of significant targets. Robust statistical analyses ensured the reliability and reproducibility of our findings. Key targets were validated through western blotting, flow cytometry, and RNA silencing, linking MiPA treatment to alterations in the PTM landscape.

 

MiPAs significantly decreased IRF7 and NF-kB phosphorylation, confirming their broad-spectrum anti-inflammatory efficacy. Phosphoproteomics identified >100 proteins with altered phosphorylation statuses between treated and untreated conditions. Validation through western blotting and flow cytometry provided insights into proteins involved in TLR pathway variations.

 

MiPAs diminish TLR7/8-mediated phosphorylation of transcription factors such as NF-kB and IRF7, possibly through a non-canonical pathway. DIA mass spectrometry was essential for profiling PTMs and revealing regulatory mechanisms, supported by robust statistical analyses. Future research should investigate CXCR4 and TLR pathway interactions, emphasizing PTMs' roles in immune regulation and utilizing DIA for enhanced understanding.