Anti-inflammatory inhibitor screening model taking MyD88TIR (myeloid differentiation primary response protein 88 Toll/interleukin-1 receptor) dimerization as target point and application thereof

Abstract

The invention relates to an anti-inflammatory inhibitor screening model taking MyD88TIR (myeloid differentiation primary response protein 88 Toll / interleukin-1 receptor) dimerization as a target point and application thereof. The anti-inflammatory inhibitor screening model taking the MyD88TIR dimerization as the target point is characterized in that: an MyD88TIR builds fusion protein plasmids together with protein genes GFP / RFP (Green fluorescent protein / Red fluorescent protein) of a fluorescence donor and a fluorescent receptor, and the fusion protein plasmids are transfected into mammalian cells to build dual-positive expression cell strains; the FRET (fluorescence resonance energy transfer) phenomenon can be detected; when an MyD88TIR dimerization inhibitor exists in a culture medium, the cell strains which depend on dual-positive to express GFP-MyD88-TIR and RFP-MyD88-TIR are suggested, and whether the inhibitor directly blocks the interaction of the TIRs or not can be further determined according to in vitro binding analysis; and combined with the fluorescent FRET blocking results of eukaryotic cells and prokaryotic expression recombinant protein interaction analysis, the MyD88TIR dimerization inhibitor can be determined. The model can be used for widely screening commercialized small molecule libraries, self-prepared natural product components, or various chemical compounds, and modifiers, from which MyD88 dimerization inhibitory compounds are obtained to participate in the drug screening of MyD88 signal pathway-dependent chronic inflammation and autoimmune diseases.

Description

technical field [0001] The invention relates to a screening model and application of an anti-inflammatory inhibitor targeting MyD88 TIR dimerization, so as to realize high-throughput screening of MyD88 dimerization inhibitors in situ in living cells and based on changes in fluorescent signals. Background technique [0002] IL-1R receptors and Toll-like receptors (TLRs) belong to the same superfamily, and the IL-1R subfamily includes IL-1β and IL-18 receptors and other members, which mediate acute and chronic inflammatory responses. There are 10 members in the Toll-like receptor subfamily, which recognize pathogenic microorganisms or their metabolites (such as endotoxin LPS) and mediate innate immunity. When IL-1R receptors and Toll-like receptors bind to ligands, the intracellular TIR (Toll / IL-1 receptor homologous region) domain undergoes a conformational change, recruiting TIR structures that also exist in the cytoplasm Domain adapter protein molecules, this process is cr...

AI Technical Summary

Problems solved by technology

[0006] At present, many studies have designed and synthesized TIR structural analogs based on the amino acid sequence of the MyD88 TIR domain and introduced them into mammalian cells. Pro-inflammatory effects of 18

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