Toll-like receptor (TLR) 9 is an endolysosomal sensor of nucleic acids. It detects endosomal single strand deoxyribonucleic acid (ssDNA) leading to the production of proinflammatory cytokines and interferons through myeloid differentiation primary response gene 88 (MyD88) signalling. Excessive activation of TLR9-MyD88 mediated signalling has been implicated in several immune-mediated diseases including acute pancreatitis and systemic lupus erythematosus. Previous studies have shown that immunomodulators that block this pathway may be useful in the management of these conditions.
Our work focuses on a flavonoid derivative we discovered, referred to as ‘PHA’, which selectively inhibits TLR9 while largely sparing TLR7, when stimulated by their respective agonists, CpG DNA and R848. Inhibition of TLR9 signalling was observed across species in human HEK293 cells over-expressing human TLR9 and mouse bone marrow derived macrophages. PHA showed preferential inhibition of the signalling cascade downstream of TLR9 (IC50 ~100 nM) with only limited activity on TLR7 signalling, despite both receptors being thought to use similar machineries. The observations were validated with RT-qPCR and ELISA assays. This inhibitory effect of PHA was shared among various TLR9 agonists.
We are currently looking at whether PHA is impacting signalling at the levels of the ligands, the receptor, or their downstream effector molecules to unravel the mechanism underlying the selective activity on TLR9 over TLR7 signalling. Based on our observations thus far and a kinome-wide screen of PHA on 468 kinase enzymes, we hypothesize that PHA regulates a kinase activity uniquely contributing to the TLR9 signalling. This study has the potential for contributing to our understanding of TLR-MyD88 signalling pathway as well as showcasing targeted interventions for TLR9-mediated immune disorders.