A novel small-molecule enantiomeric analogue of traditional (-)-morphinans has specific TLR9 antagonist properties and reduces sterile inflammation-induced organ damage.

TLR9 is a key determinant of the innate immune responses in both infectious and sterile injury. Specific antagonism of TLR9 is of great clinical interest to reduce tissue damage in a wide range of pathologies, and has been approached by modification of nucleic acids, the recognized ligand for TLR9. Such oligonucleotide-derived pharmacotherapeutics have limitations in specificity for nucleic acid receptors, significant potential for immunologic recognition with generation of innate and adaptive immune responses, and limited bioavailability. We have identified enantiomeric analogues of traditional (-)-morphinans as having TLR9 antagonist properties on reporter cell lines. One of these analogues (COV08-0064) is demonstrated to be a novel small-molecule antagonist of TLR9 with greater specificity for TLR9 than oligo-based antagonists. COV08-0064 has wide bioavailability, including the s.c. and oral routes. It specifically inhibits the action of TLR9 antagonists on reporter cells lines and the production of cytokines by TLR9 agonists from primary cells. It also has efficacy in limiting TLR9-mediated sterile inflammation in in vivo models of acute liver injury and acute pancreatitis. The identification of a morphinan-based novel small-molecule structure with TLR9 antagonism is a significant step in expanding therapeutic strategies in the field of sterile inflammatory injury.

Author Correction: TLR9 activation via microglial glucocorticoid receptors contributes to degeneration of midbrain dopamine neurons.

The originally published version of this Article contained an error in the subheading "Microglial GR does not affect DN loss triggered by TLR4 and TLR7," which was incorrectly given as "Microglial GR does affect DN loss triggered by TLR2 and TLR4". This has now been corrected in both the PDF and HTML versions of the Article.

TLR9 activation via microglial glucocorticoid receptors contributes to degeneration of midbrain dopamine neurons.

Inflammation is a characteristic feature of Parkinson's disease (PD). We examined the role of TLR9 and its regulation by glucocorticoid receptors (GRs) in degeneration of substantia nigra dopamine neurons (DNs). TLR9 agonist, CpG-ODN, induced DN degeneration in mice lacking GR in microglia but not in controls. TLR9 deletion reduced DN loss in neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. GR regulates TLR9 activation during MPTP neurotoxicity as TLR9 antagonist suppressed increased DN loss in microglia/macrophage GR mutant mice. GR absence in microglia enhanced TLR9 translocation to endolysosomes and facilitated its cleavage leading to pro-inflammatory gene expression. GR-dependent TLR9 activation also triggered DN loss following intranigral injection of mitochondrial DNA. Finally, microglial GR sensitivity to A53T-alpha-synuclein induced DN degeneration as well as decreased microglial GR expression observed in SN of PD brain samples, all suggest that reduced microglial GR activity in SN can stimulate TLR9 activation and DN loss in PD pathology.

The novel TLR9 antagonist COV08-0064 protects from ischemia/reperfusion injury in non-steatotic and steatotic mice livers.

Ischemia/reperfusion (I/R) injury constitutes a major reason for failure of liver surgeries and transplantation. I/R injury is more severe in steatotic livers and limits their use in transplantation. Here, we present a novel and selective Toll-like receptor 9 (TLR9) antagonist COV08-0064 and test its potential to protect from I/R-induced injury in normal and steatotic livers. The in vivo effects of COV08-0064 pretreatment were investigated on normal chow diet (NCD) and high fat diet (HFD)-fed mice subjected to segmental (70%) warm hepatic I/R. Also, the in vitro effects of COV08-0064 were elucidated in murine macrophages and dendritic cells. Mice on a HFD had pronouncedly greater hepatic I/R injury than mice on a NCD. COV08-0064-pretreatment to both NCD and HFD-fed mice reduced hepatic I/R injury. COV08-0064-pretreatment was associated with less production of the liver inflammatory cytokines and mediators TNF-α, IL-1ß, IL-6, NLRP3, iNOS and MCP-1. These manifestations were preceded with inhibition of JNK and ERK phosphorylation and TLR9 cleavage in the liver. COV08-0064 enhanced the hepatic expression of the endogenous anti-inflammatory cytokines IL-10 and IL-1Ra at the early phase I/R injury. In vitro, COV08-0064 selectively blocked mRNA upregulation of TNF-α, IL-1ß, NLRP3 and MCP-1 in macrophages and IFN-ß mRNA in dendritic cells induced by the TLR9 agonist CpG-ODN. These effects were concordant with inhibition of JNK, ERK, IκBα and IKKα/ß phosphorylation. In conclusion, TLR9 signaling inhibition by COV08-0064 may be an effective approach in liver surgeries including transplantation to limit I/R-injury and overcome the shortages in the donor pool by incorporating steatotic livers.