Protocol for evaluation and validation of TLR8 antagonists in HEK-Blue cells via secreted embryonic alkaline phosphatase assay

Hu, Zhifeng; Zhang, Tuan; Jiang, Shuangshuang; Yin, Hang

doi:10.1016/j.xpro.2021.101061

Cited by 3 publications

(3 citation statements)

References 12 publications

(20 reference statements)

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“…The next day, the supernatant is collected and added to QUANTI-Blue detection solution and observed for change in color which is then quantitated by measuring the O.D. at 655 nm using a spectrophotometer . HEK-Blue hTLR9, HEK-Blue hTLR7, HEK-Blue hTLR8 reporter cells, QUANTI-Blue detection solution, Normocin, CpGB (ODN 2006), CL264, and CL075 were all purchased from Invivogen, USA.…”

Section: Methodsmentioning

confidence: 99%

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Development, Optimization, and In Vivo Validation of New Imidazopyridine Chemotypes as Dual TLR7/TLR9 Antagonists through Activity-Directed Sequential Incorporation of Relevant Structural Subunits

et al. 2022

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Undesirable activation of endosomal toll-like receptors TLR7 and TLR9 present in specific immune cells in response to host-derived ligands is implicated in several autoimmune diseases and other contexts of autoreactive inflammation, making them important therapeutic targets. We report a drug development strategy identifying a new chemotype for incorporating relevant structural subunits into the basic imidazopyridine core deemed necessary for potent TLR7 and TLR9 dual antagonism. We established minimal pharmacophoric features in the core followed by hit-to-lead optimization, guided by in vitro and in vivo biological assays and ADME. A ligand–receptor binding hypothesis was proposed, and selectivity studies against TLR8 were performed. Oral absorption and efficacy of lead candidate 42 were established through favorable in vitro pharmacokinetics and in vivo pharmacodynamic studies, with IC50 values of 0.04 and 0.47 μM against TLR9 and TLR7, respectively. The study establishes imidazopyridine as a viable chemotype to therapeutically target TLR9 and TLR7 in relevant clinical contexts.

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Section: Methodsmentioning

confidence: 99%

“…at 655 nm using a spectrophotometer. 76 HEK-Blue hTLR9, HEK-Blue hTLR7, HEK-Blue hTLR8 reporter cells, QUANTI-Blue detection solution, Normocin, CpGB (ODN 2006), CL264, and CL075 were all purchased from Invivogen, USA. DMEM was purchased from Gibco, Thermo Fisher Scientific.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Development, Optimization, and In Vivo Validation of New Imidazopyridine Chemotypes as Dual TLR7/TLR9 Antagonists through Activity-Directed Sequential Incorporation of Relevant Structural Subunits

et al. 2022

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“…Tests, such as secreted embryonic alkaline phosphatase (SEAP)-driven assay, cell proliferation assay, isothermal titration calorimetry (ITC), and immunoblotting, are also used in in vitro validations. A non-selective TLR7 agonist R848 (Resiquimod) can be commonly used as a positive control [ 17 ]. The inhibition of TLR7-induced pro-inflammatory cytokines can be measured through a real-time polymerase chain reaction (RT-PCR) [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

Recent Advances on Small-Molecule Antagonists Targeting TLR7

Zheng

Bonnet

2023

Molecules

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Toll-like receptor 7 (TLR7) is a class of pattern recognition receptors (PRRs) recognizing the pathogen-associated elements and damage and as such is a major player in the innate immune system. TLR7 triggers the release of pro-inflammatory cytokines or type-I interferons (IFN), which is essential for immunoregulation. Increasing reports also highlight that the abnormal activation of endosomal TLR7 is implicated in various immune-related diseases, carcinogenesis as well as the proliferation of human immunodeficiency virus (HIV). Hence, the design and development of potent and selective TLR7 antagonists based on small molecules or oligonucleotides may offer new tools for the prevention and management of such diseases. In this review, we offer an updated overview of the main structural features and therapeutic potential of small-molecule antagonists of TLR7. Various heterocyclic scaffolds targeting TLR7 binding sites are presented: pyrazoloquinoxaline, quinazoline, purine, imidazopyridine, pyridone, benzanilide, pyrazolopyrimidine/pyridine, benzoxazole, indazole, indole, and quinoline. Additionally, their structure-activity relationships (SAR) studies associated with biological activities and protein binding modes are introduced.

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