Most vaccine adjuvants directly stimulate and activate antigen presenting cells but do not sustain immunostimulation of these cells. A high throughput screening (HTS) strategy was designed to identify compounds that would sustain NF-κB activation by a stimulus from the Toll-like receptor (TLR)4 ligand, lipopolysaccharide (LPS). Several pilot studies optimized the parameters and conditions for a cell based NF-κB reporter assay in human monocytic THP-1 cells. The final assay evaluated prolongation of LPS induced NF-κB activation at 12 h. The dynamic range of the assay was confirmed in a pilot screen of 14 631 compounds and subsequently in a main extensive screen with 166 304 compounds. Hit compounds were identified using an enrichment strategy based on unsupervised chemoinformatic clustering, and also by a naı̈ve “Top X” approach. A total of 2011 compounds were then rescreened for levels of coactivation with LPS at 5 h and 12 h, which provided kinetic profiles. Of the 407 confirmed hits, compounds that showed correlation of the kinetic profiles with the structural similarities led to identification of four chemotypes: pyrimido[5,4-b]indoles, 4H-chromene-3-carbonitriles, benzo[d][1,3]dioxol-2-ylureas, and tetrahydrothieno[2,3-c]pyridines, which were segregated by 5 h and 12 h kinetic characteristics. Unlike the TLR4 agonistic pyrimidoindoles identified in previous studies, the revealed pyrimidoindoles in the present work did not intrinsically stimulate TLR4 nor induce NF-κB but rather prolonged NF-κB signaling induced by LPS. A 42-member combinatorial library was synthesized which led to identification of potent N3-alkyl substituted pyrimidoindoles that were not only active in vitro but also enhanced antibody responses in vivo when used as a coadjuvant. The novel HTS strategy led to identification of compounds that are intrinsically quiescent but functionally prolong stimulation by a TLR4 ligand and thereby potentiate vaccine efficacy.

中文翻译：

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鉴定可延长无内在活性的NF-κB活化的生物活性嘧啶并[5,4- b ]吲哚

大多数疫苗佐剂直接刺激并激活抗原呈递细胞，但不维持这些细胞的免疫刺激作用。设计了一种高通量筛选（HTS）策略，以鉴定通过Toll样受体（TLR）4配体脂多糖（LPS）的刺激而维持NF-κB活化的化合物。多项先导研究优化了人单核THP-1细胞中基于细胞的NF-κB报告基因检测的参数和条件。最终分析评估了LPS在12 h时诱导的NF-κB活化的延长。测定的动态范围在14 631种化合物的初步筛选中确认，随后在166 304种化合物的主要广泛筛选中确认。可以使用基于无监督化学信息聚类的富集策略和简单的“ Top X”方法来鉴定命中化合物。然后在5小时和12小时重新筛选了总共2011种化合物与LPS的共激活水平，从而提供了动力学特征。在407个已确认的命中物中，显示动力学特征与结构相似性相关的化合物可鉴定出四种化学型：嘧啶[5,4-b ]吲哚，4 H-亚甲基-3-腈，苯并[ d ] [1,3]二氧杂-2-基脲和四氢噻吩并[2,3- c ]吡啶，它们通过5 h和12 h动力学特征分离。与先前研究中确定的TLR4激动嘧啶并吲哚不同，本研究揭示的嘧啶并吲哚并没有内在地刺激TLR4或诱导NF-κB，而是延长了LPS诱导的NF-κB信号传导。合成了一个由42个成员组成的组合文库，该文库可鉴定出有效的N 3-烷基取代的嘧啶并吲哚类化合物，这些化合物不仅在体外具有活性，而且在体内具有增强的抗体应答能力当用作辅助剂时。新颖的HTS策略导致鉴定出本质上是静止的但功能上由TLR4配体刺激的化合物，从而增强了疫苗的效力。