Identification of a Human Toll-Like Receptor (TLR) 8-Specific Agonist and a Functional Pan-TLR Inhibitor in 2-Aminoimidazoles.

Activation of human toll-like receptor-8 (TLR8), expressed in myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells, evokes a distinct cytokine profile which favors the development of Type 1 helper T cells. Part-structures of the 2-aminobenzimidazole scaffold were examined with a view to identifying structural requisites corresponding to the smallest possible fragment of the benzimidazole core that would allow for retention of TLR8-agonistic activity. TLR8-specific agonistic activity was retained in 1-pentyl-4-phenyl-1H-imidazol-2-amine. The crystal structure of this compound bound to the TLR8 ectodomain displayed binding interactions that are common to other TLR8 agonists. This compound showed markedly attenuated proinflammatory properties in ex vivo human blood models. SAR studies revealed that 4-(2-(benzyloxy)phenyl)-1-pentyl-1H-imidazol-2-amine inhibited TLR signaling in a variety of TLR reporter cell lines, as well as in pharmacologically relevant human blood model systems. A kinase screen of this compound showed relative specificity for calmodulin kinases.

Identification of Adjuvantic Activity of Amphotericin B in a Novel, Multiplexed, Poly-TLR/NLR High-Throughput Screen.

Small-molecule agonists have been identified for TLR7, TLR8, TLR4 and TLR2 thus far, and chemotypes other than those of canonical ligands are yet to be explored for a number of innate immune receptors. The discovery of novel immunostimulatory molecules would enhance the repertoire of tools available for interrogating innate immune effector mechanisms, and provide additional venues for vaccine adjuvant development. A multiplexed, reporter gene-based high-throughput assay capable of detecting agonists of TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1 and NOD2 was utilized in screening 123,943 compounds, in which amphotericin B (AmpB) and nystatin were identified as prominent hits. The polyene antifungal agents act as TLR2- and TLR4-agonists. The TLR4-stimulatory activity of AmpB was similar to that of monophosphoryl lipid A, suggestive of TRIF-biased signaling. The adjuvantic activity of AmpB, at a dose of 100 micrograms, was comparable to several other candidate adjuvants in rabbit models of immunization. These results point to its potential applicability as a safe and effective adjuvant for human vaccines.

Human Toll-like receptor 8-selective agonistic activities in 1-alkyl-1H-benzimidazol-2-amines.

Toll-like receptor (TLR)-8 agonists strongly induce the production of T helper 1-polarizing cytokines and may therefore serve as promising candidate vaccine adjuvants, especially for the very young and the elderly. Earlier structure-based ligand design led to the identification of 3-pentyl-quinoline-2-amine as a novel, human TLR8-specific agonist. Comprehensive structure-activity relationships in ring-contracted 1-alkyl-1H-benzimidazol-2-amines were undertaken, and the best-in-class compound, 4-methyl-1-pentyl-1H-benzo[d]imidazol-2-amine, was found to be a pure TLR8 agonist, evoking strong proinflammatory cytokine and Type II interferon responses in human PBMCs, with no attendant CD69 upregulation in natural lymphocytic subsets. The 1-alkyl-1H-benzimidazol-2-amines represent a novel, alternate chemotype with pure TLR8-agonistic activities and will likely prove useful not only in understanding TLR8 signaling but also perhaps as a candidate vaccine adjuvant.

Structure-based design of novel human Toll-like receptor 8 agonists.

Toll-like receptor (TLR)-8 agonists activate adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds might be promising candidate vaccine adjuvants. Recently, a C2-butyl furo[2,3-c]quinoline was reported with purely TLR8 agonistic activity. This compound was successfully co-crystallized with the human TLR8 ectodomain, and the co-crystal structure revealed ligand-induced reorganization of the binding pocket of TLR8. The loss of a key hydrogen bond between the oxygen atom of the furanyl ring of the agonist and Thr 574 in TLR8 suggested that the furan ring is dispensable. Employing a disconnection strategy, 3- and 4-substituted aminoquinolines were investigated. Focused structure-based ligand design studies led to the identification of 3-pentyl-quinoline-2-amine as a novel, structurally simple, and highly potent human TLR8-specific agonist (EC50 =0.2 µM). Preliminary evaluation of this compound in ex vivo human blood assay systems revealed that it retains prominent cytokine-inducing activity. Together, these results indicate the suitability of this compound as a novel vaccine adjuvant, warranting further investigation.

Structure-activity relationships in Toll-like receptor 7 agonistic 1H-imidazo[4,5-c]pyridines.

Engagement of TLR7 in plasmacytoid dendritic cells leads to the induction of IFN-α/ß which plays essential functions in the control of adaptive immunity. We had previously examined structure-activity relationships (SAR) in TLR7/8-agonistic imidazoquinolines with a focus on substituents at the N(1), C(2), N(3) and N(4) positions, and we now report SAR on 1H-imidazo[4,5-c]pyridines. 1-Benzyl-2-butyl-1H-imidazo[4,5-c]pyridin-4-amine was found to be a pure TLR7-agonist with negligible activity on TLR8. Increase in potency was observed in N(6)-substituted analogues, especially in those compounds with electron-rich substituents. Direct aryl-aryl connections at C6 abrogated activity, but TLR7 agonism was reinstated in 6-benzyl and 6-phenethyl analogues. Consistent with the pure TLR7-agonistic behavior, prominent IFN-α induction in human PBMCs was observed with minimal proinflammatory cytokine induction. A benzologue of imidazoquinoline was also synthesized which showed substantial improvements in potency over the parent imidazopyridine. Distinct differences in N(6)-substituted analogues were observed with respect to IFN-α induction in human PBMCs on the one hand, and CD69 upregulation in lymphocytic subsets, on the other.

Exquisite selectivity for human toll-like receptor 8 in substituted furo[2,3-c]quinolines.

Toll-like receptor (TLR)-8 agonists activate adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds may be promising candidate adjuvants. We synthesized and evaluated hitherto unexplored furo[2,3-c]quinolines and regioisomeric furo[3,2-c]quinolines derived via a tandem, one-pot Sonogashira coupling and intramolecular 5-endo-dig cyclization strategy in a panel of primary screens. We observed a pure TLR8-agonistic activity profile in select furo[2,3-c]quinolines, with maximal potency conferred by a C2-butyl group (EC50 = 1.6 µM); shorter, longer, or substituted homologues as well as compounds bearing C1 substitutions were inactive, which was rationalized by docking studies using the recently described crystal structure of human TLR8. The best-in-class compound displayed prominent proinflammatory cytokine induction (including interleukin-12 and interleukin-18), but was bereft of interferon-α inducing properties, confirming its high selectivity for human TLR8.

Design and development of stable, water-soluble, human Toll-like receptor 2 specific monoacyl lipopeptides as candidate vaccine adjuvants.

Antigens in modern subunit vaccines are largely soluble and poorly immunogenic proteins inducing relatively short-lived immune responses. Appropriate adjuvants initiate early innate immune responses, amplifying subsequent adaptive immune responses. Agonists of Toll-like receptor 2 (TLR2) are devoid of significant proinflammatory activity in ex vivo human blood models and yet are potently adjuvantic, suggesting that this chemotype may be a safe and effective adjuvant. Our earlier work on the monoacyl lipopeptide class of TLR2 agonists led to the design of a highly potent lead but with negligible aqueous solubility, necessitating the reintroduction of aqueous solubility. We explored several strategies of introducing ionizable groups on the lipopeptide, as well as the systematic evaluation of chemically stable bioisosteres of the ester-linked palmitoyl group. These studies have led to a fully optimized, chemically stable, and highly water-soluble human TLR2-specific agonist, which was found to have an excellent safety profile and displayed prominent adjuvantic activities in rabbit models.

Toll-like receptor-8 agonistic activities in C2, C4, and C8 modified thiazolo[4,5-c]quinolines.

Toll-like receptor (TLR)-8 agonists typified by the 2-alkylthiazolo[4,5-c]quinolin-4-amine (CL075) chemotype are uniquely potent in activating adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds could be promising candidate vaccine adjuvants, especially for neonatal vaccines. Alkylthiazoloquinolines with methyl, ethyl, propyl and butyl groups at C2 displayed comparable TLR8-agonistic potencies; activity diminished precipitously in the C2-pentyl compound, and higher homologues were inactive. The C2-butyl compound was unique in possessing substantial TLR7-agonistic activity. Analogues with branched alkyl groups at C2 displayed poor tolerance of terminal steric bulk. Virtually all modifications at C8 led to abrogation of agonistic activity. Alkylation on the C4-amine was not tolerated, whereas N-acyl analogues with short acyl groups (other than acetyl) retained TLR8 agonistic activity, but were substantially less water-soluble. Immunization in rabbits with a model subunit antigen adjuvanted with the lead C2-butyl thiazoloquinoline showed enhancements of antigen-specific antibody titers.