Design, synthesis and biological evaluation of 4-aminoquinoline derivatives as receptor-interacting protein kinase 2 (RIPK2) inhibitors.

Receptor-interacting protein kinase 2 (RIPK2) is an essential protein kinase mediating signal transduction by NOD1 and NOD2, which play an important role in regulating immune signalling. In this study, we designed and synthesised a novel series of 4-aminoquinoline-based derivatives as RIPK2 inhibitors. In vitro, compound 14 exhibited high affinity (IC50 = 5.1 ± 1.6 nM) and excellent selectivity to RIPK2 showing in a dendrogram view of the human kinome phylogenetic tree. Bearing favourable lipophilicity and eligible lipophilic ligand efficiency (LipE), compound 14 was selected to investigate cellular anti-inflammatory effect and was identified as a potent inhibitor to reduce the secretion of MDP-induced TNF-α with a dose-dependent manner. Moreover, compound 14 showed moderate stability in human liver microsome. Given these promising results, compound 14 could serve as a favourable inhibitor of RIPK2 for further physiological and biochemical research so as to be used in therapeutic treatment.

The protective effects of natural product tunicatachalcone against neuroinflammation via targeting RIPK2 in microglia BV-2 cells stimulated by LPS.

Microglia-induced neuroinflammation plays a critical role in neurological diseases. At present, RIPK2 is considered to participate in inflammatory and autoimmune cellular pathways and diseases. RIPK2 is found to be a pivotal therapeutic target in neurologic disorders related to inflammation. In our research, we discovered the protective function of tunicatachalcone (TC) against neuroinflammation. TC is a natural chalcone compound derived from Pongamia pinnata, a medicinal plant. The results revealed that TC (5-20 µM) ameliorated the activation of BV-2 microglia induced by lipopolysaccharide (LPS) in a dose-dependent way, which was proved by the reduced production of inflammation-related mediators. By using SPR-LC-MS/MS analysis, we revealed the potent inhibitory function of TC against neuroinflammation mediated by microglia via targeting RIPK2. A strong binding between TC and RIPK2 was further demonstrated based on the results of SPR, MST and molecular modeling. Through applying mRNA transcriptomics and bioinformatics analysis, it was demonstrated that TC could mediate RIPK2-dependent gene transcription to exert the neuroprotective effect. In summary, our research presented that RIPK2 was a possible therapeutic target of TC.

Prokaryotic expression, purification and functional characterization of recombinant human RIP2.

Receptor-interacting protein 2 (RIP2) is a member of the receptor interacting protein (RIP) family and plays an important role in the innate and adaptive immune responses. Overexpression of RIP2 mediates divergent signaling pathways including NF-κB activation and cell death. To further investigate the biological activity of RIP2 in vitro, a large amount of purified protein is required. For this purpose, the full length of RIP2 was cloned from human Ramos (human Burkitt lymphoma) tumor cells and inserted in a prokaryotic expression vector pET22b, and then the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. The expression of RIP2 was induced with IPTG. SDS-PAGE analysis showed that recombinant human RIP2 (rhRIP2) was mainly expressed as soluble fraction in the supernatant of the cell lysate. The recombinant protein was subsequently purified by His Trap FF crude to a purity of 90 %. MTT assay of the purified rhRIP2 showed its functional diversity in different cell lines, a specific inhibitory effect on MCF7 cells, but a promotion on the proliferation of Ramos cells. Furthermore, we identified that rhRIP2 could suppress activation of canonical NF-κB in MCF7 cells and activate non-canonical NF-κB signaling in Ramos cells, these data suggested that RIP2 participates in different signaling pathways contributing to its specific effects in vitro. Our results provided new clues to further explore the regulation mechanisms of RIP2 in tumorigenesis.