Discovery of substituted 4-aminoquinazolines as selective Toll-like receptor 4 ligands

Abstract

The Toll-like receptors (TLRs) are critical components of the innate immune system that regulate immune recognition in part through NF-κB activation. A human cell-based high throughput screen (HTS) revealed substituted 4-aminoquinazolines to be small molecular weight activators of NF-κB. The most potent hit compound predominantly stimulated through the human TLR4/MD2 complex, and had less activity with the mouse TLR4/MD2. There was no activity with other TLRs and the TLR4 activation was MD-2 dependent and CD14 independent. Synthetic modifications of the quinazoline scaffold at the 2 and 4 positions revealed trends in structure-activity relationships with respect to TLR dependent production of the NF-κB associated cytokine IL-8 in human peripheral blood mononuclear cells, as well as IL-6 in mouse antigen presenting cells. Furthermore, the hit compound in this series also activated the interferon signaling pathway resulting in type I interferon production. Substitution at the O-phenyl moiety with groups such as bromine, chlorine and methyl resulted in enhanced immunological activity. Computational studies indicated that the 4-aminoquinazoline compounds bind primarily to human MD-2 in the TLR4/MD-2 complex. These small molecules, which preferentially stimulate human rather than mouse innate immune cells, may be useful as adjuvants or immunotherapeutic agents.

Structure–Activity Relationship Studies of Pyrimido[5,4-b]indoles as Selective Toll-Like Receptor 4 Ligands

“…The extended C8-phenyl substituent of compound 36 is involved in additional interactions with hydrophobic residues Val82, Leu87, Ile124, Phe126, Ser127, and Tyr131 of MD-2 (cyan) and residues of the TLR4 (green) including Asn417, Phe440, Ser441, Leu444, and Phe463. Interestingly, several of these same hydrophobic residues of MD-2 were found to interact with the 4-aminoquinazolines 22 (such as 1Y136) in the human TLR4/MD-2 complex, suggesting that both chemotypes bind in the same approximate area of the complex. These interactions could explain the greater potency of 36 for TLR4 signaling compared to that of 1 .…”

“…Next, hTLR reporter cells and human peripheral blood mononuclear cells (hPBMC) were treated with compounds 1 , 21d , 36 , and 39 , and compared to 1Y136 , a 4-aminoquinazoline derivative was used as a positive control (Figure 3). 22 Previous studies from our laboratory 22 established 1Y136 as a TLR4 agonist with significant selectivity toward human versus mouse TLR4. After treatment, cell supernatants were assayed for IL-8 and IL-6 by ELISA, and results are displayed in Figure 3.…”

The Role of Self-Assembling Lipid Molecules in Vaccination

“…Likely the result of the complexity of interactions between LPS and the TLR4 signaling apparatus [15], relatively few families of small molecule TLR4 agonists have been reported to date. High-throughput screens have been successful in producing candidates for vaccine adjuvants including distinct, nonlipid chemical entities such as substituted pyramid [5,4-b]indole derivatives [48,49], and α-aminoacyl amides synthesized via the Ugi reaction [50], and small molecule TLR4 ligands have demonstrated preclinical potential as vaccine adjuvants with potent immunostimulatory potential with limited reactogenicity in mouse [7].…”

A Light‐Controlled TLR4 Agonist and Selectable Activation of Cell Subpopulations

“…TLR4 is activated by a diverse array of structures, including lipopolysaccharide and its variants [1–6] , peptides [7] , proteins [8–10] , and small molecules [11,12] . These agonists are used in vaccines against human papilloma virus [13] , and also contribute to the inflammation in opioid interactions [14] and many allergies [15] .…”

“…TLR4 is activated by a diversea rray of structures,i ncluding lipopolysaccharide and its variants, [1][2][3][4][5][6] peptides, [7] proteins, [8][9][10] and small molecules. [11,12] These agonists are used in vaccines against human papilloma virus, [13] and contribute to the inflammationi no pioid interactions [14] and many allergies. [15] Agonist stimulation of TLR4 leads to the activation of the MyD88 and/or TRIF signaling pathways, resulting in activation of transcriptionf actors, including nuclear factor kB( NF-kB) and interferon-regulatingf actors.…”