Chan Michael, Ahmadi Alast, Yao Shiyin, Sato-Kaneko Fumi, Messer Karen, Pu Minya, Nguyen Brandon, Hayashi Tomoko, Corr Maripat, Carson Dennis A, Cottam Howard B, Shukla Nikunj M
Moores UCSD Cancer Center, University of California San Diego , La Jolla, California 92093, United States.
Division of Biostatistics, University of California San Diego , La Jolla, California 92093, United States.
ACS Comb Sci. 2017 Aug 14;19(8):533-543. doi: 10.1021/acscombsci.7b00080. Epub 2017 Jul 13.
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.
大多数疫苗佐剂直接刺激并激活抗原呈递细胞，但无法维持对这些细胞的免疫刺激。设计了一种高通量筛选（HTS）策略，以鉴定能够通过Toll样受体（TLR）4配体脂多糖（LPS）的刺激来维持NF-κB激活的化合物。多项初步研究优化了人单核细胞THP-1细胞中基于细胞的NF-κB报告基因检测的参数和条件。最终检测评估了LPS诱导的NF-κB激活在12小时时的延长情况。该检测的动态范围在对14631种化合物的初步筛选中得到确认，随后在对166304种化合物的主要广泛筛选中得到确认。使用基于无监督化学信息学聚类的富集策略以及简单的“前X名”方法鉴定出命中化合物。然后对总共2011种化合物进行重新筛选，以检测其在5小时和12小时时与LPS的共激活水平，从而获得动力学曲线。在407种经确认的命中化合物中，动力学曲线与结构相似性具有相关性的化合物导致鉴定出四种化学类型：嘧啶并[5,4-b]吲哚、4H-色烯-3-腈、苯并[d][1,3]二氧杂环戊烯-2-基脲和四氢噻吩并[2,3-c]吡啶，它们通过5小时和12小时的动力学特征进行区分。与先前研究中鉴定出的TLR4激动性嘧啶吲哚不同，本研究中揭示的嘧啶吲哚本身既不刺激TLR4也不诱导NF-κB，而是延长LPS诱导的NF-κB信号传导。合成了一个包含42个成员的组合文库，从而鉴定出有效的N3-烷基取代嘧啶吲哚，这些化合物不仅在体外具有活性，而且用作共佐剂时还能增强体内抗体反应。这种新颖的HTS策略导致鉴定出本身静止但在功能上延长TLR4配体刺激并从而增强疫苗效力的化合物。
