Identification of Substituted Pyrimido[5,4-b]indoles as Selective Toll-Like Receptor 4 Ligands

Abstract

A cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators. The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Specifically, a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could potentially be used as adjuvants or immune modulators.

Synthetic Toll-Like Receptor 4 (TLR4) and TLR7 Ligands as Influenza Virus Vaccine Adjuvants Induce Rapid, Sustained, and Broadly Protective Responses

“…By specifically targeting receptors in well-understood pathways, the mechanism of action for a small-molecule ligand is far easier to elucidate than for adjuvants without predefined receptor targets, such as oil-inwater emulsions, aluminum salts, or squalene-based emulsions. Accordingly, 1Z105 and 1V270 were optimized from lead compounds targeting TLR4 and TLR7, respectively, to potently activate their receptors while exhibiting little cytotoxicity (14,(17)(18)(19).…”

“…Accordingly, two low-molecular-weight synthetic Toll-like receptor (TLR) ligands, 1Z105 and 1V270, a TLR4 ligand and a TLR7 ligand, respectively, are being developed as novel vaccine adjuvants. 1Z105 is a substituted pyrimido [5,4-b]indole that was derived from a hit identified in a small-molecule screen for NF-B activators (14,15). At this time 1Z105, as well as its related compounds, is among the few small, synthetic, nonlipid-like TLR4 ligands described in the literature and perhaps the only one with demonstrated adjuvant properties (14).…”

“…1Z105 is a substituted pyrimido [5,4-b]indole that was derived from a hit identified in a small-molecule screen for NF-B activators (14,15). At this time 1Z105, as well as its related compounds, is among the few small, synthetic, nonlipid-like TLR4 ligands described in the literature and perhaps the only one with demonstrated adjuvant properties (14). The AS04 adjuvant licensed in GlaxoSmithKline's Cervarix vaccine provides a precedent for the safety and efficacy of a TLR4 ligand, namely, monophosphoryl lipid A (MPLA), as an adjuvant for a recombinant viral vaccine (16).…”

“…While various adjuvants under development exhibit efficacy in preclinical models, U.S. regulators have considerable safety concerns regarding adjuvants and vaccine reactogenicity that must be addressed (12,13,(25)(26)(27). 1Z105 and 1V270 demonstrate little in vitro toxicity (14,19). Moreover, intramuscular (i.m.)…”

CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance

“…However, the ability of UT12 to induce translocation of TLR4/MD2 into endosomes, as well as its potential for mediating TRIF-dependent signaling, has not been reported. Recently, a group of substituted pyrimido [5,4-b] indoles, synthetic ligands for TLR4 that activate NF-κB that act in a CD14-independent manner, were discovered by high-throughput screening (15). These synthetic ligands induced IL-6 and IP-10 in a TLR4/MD2-dependent, but CD14-independent manner (16).…”