Design and Application of Conjugatable Small Molecule Toll-Like Receptor 4 Ligands

We identified structurally distinct pyrimido[5,4-<i>b</i>]indole derivatives as toll-like receptor 4 (TLR4) ligands. Previous structure-activity relationship studies revealed that C8-aryl derivatives in pyrimido[5,4-<i>b</i>]indole, especially phenyl and 2-naphthyl compounds, are more potent in the activation of TLR4 signaling. Molecular modeling of these compounds indicated that C8-aryl groups are important for the interaction of the TLR4/myeloid differentiation factor-2 (MD-2) complex. Additionally, the modeling suggested that the N5 position in pyrimido[5,4-<i>b</i>]indole could be used as a further modification site to develop various drug conjugates. In this study, we examined whether the N5 position in pyrimido[5,4-<i>b</i>]indole can be used for conjugation without losing potency. Since tetraethylene glycol (TEG) derivatives at the N5 position were predicted to bind to TLR4/MD-2 complex using in silico molecular docking analysis, the compounds with the TEG group at the N5 position were synthesized and evaluated for immunostimulatory activity by human TLR4 reporter cell assay. As a result of fine-tuning of the C8 substitution groups, we found that TLR4 ligand (TLR4L) <b>10</b> with a 3-thienylethynyl group at the C8 position maintained TLR4 potency and demonstrated agonistic activity in primary murine bone marrow dendritic cells (mBMDC) and human TLR4 reporter HEK-Blue cells. TLR4L <b>10</b> was conjugated to sugar-immobilized gold nanoparticles (SGNPs) by introducing thioctic acid as a spacer into the TEG moiety. The obtained TLR4L-SGNPs <b>17</b> were taken up and showed agonistic activity in mBMDC. Thus, our designed TLR4L <b>10</b> and TLR4L-SGNP <b>17</b> are new candidates as immunomodulators for novel class adjuvant systems.