High Throughput Screening Assay to Identify Modulators of IL-17 Expression.
Comb Chem High Throughput Screen
; 20(9): 804-819, 2017.
Article
in En
| MEDLINE
| ID: mdl-28571538
ABSTRACT
OBJECTIVE:
Herein we demonstrate the successful development of a new RORγt-enhanced IL-17F promoter-luciferase reporter assay and its use in a parallel high throughput screening approach, alongside a RORγt TR-FRET assay, to rapidly identify new small molecule RORγt/IL-17 inhibitors and evaluate their mode of action. MATERIAL &METHODS:
We sought to identify cell-permeable small-molecule inhibitors of RORγt for rapid progression into hit-to-lead chemistry. As such, we developed the IL-17F promoter luciferase reporter assay in a stable human T-cell (Jurkat) line expressing the RORγt receptor and miniaturised it to a final volume of 8 µL in 1536 well plates for HTS use in screening a library of > 350k compounds. In parallel, a RORγt TR-FRET binding assay was employed to cross-screen the same set of compounds. This enabled the rapid identification of a small number of cell permeable RORγt antagonists showing promising activity in both assays and also highlighted a larger group of potentially very interesting hits which inhibited IL-17 reporter activity, but did not appear to modulate RORγt directly.RESULT:
A rigorous triaging process of the novel non-RORγt IL-17 antagonists was followed, making use of in-silico filtering, historical screening data, selectivity screening using an IL-2 reporter assay with an identical cellular background, and final profiling in a phenotypic PBMC IL-17A production assay. This resulted in the identification of a set of promising small molecule compounds which show IL-17 inhibition via potentially novel pathways.CONCLUSION:
This technique for the fast identification of cell-permeable IL-17 modulators acting through different mechanisms, highlights the benefits of adopting a parallel approach combining high throughput profiling of hits in multiple assay formats, with robust in-silico triaging.Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Interleukin-17
/
Small Molecule Libraries
/
Nuclear Receptor Subfamily 1, Group F, Member 3
/
High-Throughput Screening Assays
Type of study:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Limits:
Humans
Language:
En
Journal:
Comb Chem High Throughput Screen
Journal subject:
BIOLOGIA MOLECULAR
/
QUIMICA
Year:
2017
Type:
Article
Affiliation country:
Saudi Arabia