Identification of Biologically Active Pyrimido[5,4-b]indoles That Prolong NF-?B Activation without Intrinsic Activity.

ABSTRACT: 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-?B 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-?B reporter assay in human monocytic THP-1 cells. The final assay evaluated prolongation of LPS induced NF-?B 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 na??ve "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-?B but rather prolonged NF-?B 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.

SUBMITTER: Chan M 

PROVIDER: S-EPMC5841913 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature