ACS Combinatorial Science 2017-06-28

Identification of Biologically Active Pyrimido[5,4-b] indoles that Prolong NF-κB Activation Without Intrinsic Activity

Michael Chan, Alast Ahmadi, Shiyin Yao, Fumi Sato-Kaneko, Karen Messer, Minya Pu, Brandon Nguyen, Tomoko Hayashi, Maripat Corr, Dennis Carson, Howard B. Cottam, Nikunj Shukla

文献索引：10.1021/acscombsci.7b00080

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摘要

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 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 12h (hours). 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 naïve ‘Top X’ approach. 2,011 compounds were then rescreened for levels of co-activation with LPS at 5h and 12h, 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 5h and 12h 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 co-adjuvant. 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.