Abstract
Background: Toll-like receptor 4 (TLR4) initiates both innate and adaptive immune responses, which plays an important protective role in self-defense mechanisms. Excessive or inappropriate TLR4 activation causes the development of many autoimmune diseases. Dihydropyrimidinone derivatives are medicinally important molecules with diverse pharmacological activities, including anti-inflammatory activity. The present study focused on novel synthesized 3,4-dihydropyrimidinone derivatives and evaluated their inhibitory effects on TLR4. Methods: A series of 3,4-dihydropyrimidinone derivatives were recently synthesized and evaluated for their TLR4 inhibition activities and cytotoxic on HEK-BlueTM hTLR4 cells with the help of QUANTI-Blue assay and MTS assay. Selected compound 3 was analyzed for its molecular docking with TLR4 by using Autodock vina 1.1.2. Its effect on the TLR4 pathway related cytokines was also evaluated in THP-1 cells and human peripheral blood mononuclear cells by using real-time PCR, ELISA and western blot. Results: Five compounds were synthesized and characterized for effectiveness based on 3,4-dihydropyrimidinone. Compound 3 was found to be the potent hybrid among the synthesized compounds, with high TLR4 inhibition activities and low cytotoxic activities against HEK-BlueTM hTLR4 cells. Molecular docking analysis showed that two hydrogen bonds between compound 3 and residues Asp209(TLR4) and Asp99(MD-2) mainly contribute to the TLR4 inhibition. In addition, compound 3 suppressed LPS-induced of the mRNA expression of TLR4, IP-10, TNF-α, IL-6, IL-12A, and IL-12B, the protein expression of pIRF3 and pNFκB and the secretion of IP-10, TNF-α in THP-1 cell line. Compound 3 also inhibited LPS-induced expression of TNF-α, IL-6, and IL-1β but increased IP-10 at mRNA levels in human peripheral blood mononuclear cells. Conclusion: Our study reveals compound 3, a novel 3,4-dihydropyrimidinone derivative, is a potential TLR4 antagonist, which opens up new research avenues for the development of promising therapeutic agents for inflammatory and autoimmune diseases.
Highlights
The TLR family is a class of pattern recognition receptors (PRRs) of mammalian species, which can recognize many pathogen-associated molecular patterns (PAMPs) and is important in both innate and adaptive immune responses (Kawasaki and Kawai, 2014)
We identified and functionally characterized one of the compounds with low toxicity and relatively high Toll-like receptor 4 (TLR4) antagonistic activity, which suggests it can be used as a potent lead compound in the development of new TLR4 receptor antagonists
Two hydrogen bonds were observed between the compound 3 and residues Asp209(TLR4) and Asp99(MD-2), with the bond lengths of 2.99 and 2.93 Å, respectively, which was the main interaction between compound 3 and the TLR4-MD-2 complex
Summary
The TLR family is a class of pattern recognition receptors (PRRs) of mammalian species, which can recognize many pathogen-associated molecular patterns (PAMPs) and is important in both innate and adaptive immune responses (Kawasaki and Kawai, 2014). Activating of TLR4 by LPS after the infection of Gram-negative bacteria will result in the release of inflammatory cytokines and type I interferon, which plays a protective role in self-defense mechanism. Excessive activations of TLR4 by these endogenous ligands were related to many diseases such as arthritis, systemic sclerosis, and atherosclerotic (Erridge, 2010; Kiyeko et al, 2016; Bhattacharyya and Varga, 2018). Toll-like receptor 4 (TLR4) initiates both innate and adaptive immune responses, which plays an important protective role in self-defense mechanisms. Excessive or inappropriate TLR4 activation causes the development of many autoimmune diseases. The present study focused on novel synthesized 3,4dihydropyrimidinone derivatives and evaluated their inhibitory effects on TLR4
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