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

Abstract

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.

Keywords: aminoquinolines; innate immunity; structure-based drug design; toll-like receptor-8 (TLR8); vaccine adjuvants.

Figure 1

Representative heterocyclic small molecules with TLR8 agonistic activity.

Figure 2

Induced-fit docking^[8a] of 3 in human TLR8 (PDB ID: 3W3K) showing an salt bridge between the C4-amine and D543*, and an H-bond between the furanyl oxygen atom and T574. Interacting residues in protomers A and B (*) are highlighted in green and cyan, respectively.

Figure 3

A. Cα deviation in TLR8 bound to 3 versus unliganded TLR8. B. Regions undergoing ligand-induced Cα movements of more than 2.5 Ǻ are shown in red for the TLR8 protomers. C. TLR8 (protomers A and B represented in green and cyan, respectively) complexed with 3 showing the loss of H-bond of the furanyl oxygen atom due to reorganization of residues in the binding pocket (PDB code: 3WN4).

Figure 4

Disconnection strategy of 3 leading to substituted aminoquinolines.

Figure 5

Dose-response profiles of human TLR8 agonistic activities of 3-substituted 2-aminoquinolines. Error bars represent standard deviations obtained on quadruplicates. Compounds 1 and 3 were used as comparators.

Figure 6

Induction of cytokines (red) and chemokines (blue) in human PBMCs by the lead compound 14b. Means of triplicates are shown.

Scheme 1

Syntheses of 3-substituted quinolin-2-amine analogues. Reagents: (i) butyl iodide, K₂CO₃, DMSO; (ii) m-CPBA, CHCl₃; (iii) (a) benzoyl isocyanate, CH₂Cl₂; (b) NaOMe, MeOH; (iv) H₂, Pt/C, EtOH; (v) NH₃, MeOH; (vi) butylSH, NaH, DMSO; (vii) Pd(PPh₃)₄, RB(OH)₂, K₂CO₃, 1,4-dioxane, for 14f: Pd(PPh₃)₄, CuI, 1-pentyne, Et₃N:CH₃CN (1:3).

Scheme 2

4-alkyl-3-pentylquinolin-2-amines. Reagents: (i) POCl₃; (ii) Pd(PPh₃)₄, CuI, 1-pentyne, Et₃N:CH₃CN (1:3); (iii) Pd(PPh₃)₄, RB(OH)₂, K₂CO₃, 1,4-dioxane (iv) H₂, Pt/C, EtOH; (v) m-CPBA, CHCl₃; (vi) (a) benzoyl isocyanate, CH₂Cl₂; (b) NaOMe, MeOH.

Scheme 3

4-substituted quinolin-2-amines. Reagents: (i) butyl iodide, NaH, DMSO; (ii) m-CPBA, CHCl₃; (iii) (a) benzoyl isocyanate, CH₂Cl₂; (b) NaOMe, MeOH; (iv) POCl₃; (v) Pd(PPh₃)₄, RB(OH)₂, K₂CO₃, 1,4-dioxane.