Identification of novel and potent synthetic TLR agonists

Agonists and antagonists of Toll-like receptors (TLRs) may be useful as vaccine adjuvants or suppressors of inflammation, respectively. In an effort to identify compounds capable of activating macrophages via TLRs or other sensors, a synthetic compound library was screened using mouse peritoneal macrophages and human THP-1 cells. Through extensive SAR studies of initial hits, we developed two strong synthetic agonists: Neoseptin-3 and Diprovocim. Genetic studies established that neoseptin-3 is a mouse TLR4/MD-2 agonist with no structural similarity to LPS. It activates mTLR4/MD-2 independently of CD14 and triggers canonical MyD88- and TRIF-dependent signaling. Diprovocim was found by a combination of genetic and antibody blockade analyses to be a TLR1/2 agonist, active on both mouse and human receptors. Its EC50 in human THP-1 cells is 110 pM. Diprovocim showed TLR1/2 dependent adjuvant activity when co-administered with ovalbumin (OVA). It not only promoted antigen-specific humoral responses but also activated cytotoxic T lymphocyte responses in a TLR1/2 dependent manner. Since neither Neoseptin-3 nor Diprovocim resemble the natural ligands for TLR4/MD-2 nor TLR1/2, respectively, we surmise that other “unconventional” ligands for these TLRs may exist in nature. However, the exquisite SAR of both compounds makes it clear that TLRs are not highly promiscuous receptors; on the contrary, they are activated only by compounds that fulfill strict structural rules. In finding these agonists, we also identified antagonists that bind the TLR complexes but do not activate them. The parent compounds Neoseptin-3 and Diprovocim will be used to develop new agonists and antagonists optimized for clinical application.