Dracorhodin targeting CMPK2 attenuates inflammation: A novel approach to sepsis therapy

BackgroundDespite all modern advances in medicine, an effective drug for treating sepsis has yet to be found. The discovery of CMPK2 spurred hopes for the treatment of sepsis. However, CMPK2-untapped target inhibitors are still an enormous obstacle that has hindered the CMPK2-centric treatment of sepsis.MethodsHere, we found that the CMPK2 gene is highly expressed in the whole blood of sepsis patients by RNA-Seq. First, recombinant CMPK2 was purified by a eukaryotic expression purification system, and the activity of recombinant CMPK2 was detected by the ADP-GLO assay. Second, we developed an affinity MS strategy combined with quantitative lysine reactivity profiling to discover CMPK2 ligands from the active ingredients of Chinese herbs. In addition, the dissociation constant Kd of the ligand and the target protein CMPK2 was further detected by microscale thermophoresis technology. Third, we used this strategy to identify a naturally sourced small molecule, dracorhodin (DP). Using mass spectrometry-based quantitative lysine reactivity profiling combined with a series of mutant tests, the results show that K265 acts as a bright hotspot of DP inhibition of CMPK2. Fourth, immune-histochemical staining, ELISAs, RT-qPCR, flow cytometry and immunoblotting were used to illustrate the potential function and related mechanism of DP in regulating sepsis injury.ResultsOur results suggest that DP exerts powerful anti-inflammatory effects by regulating the NLRP3 inflammasome via the lipopolysaccharide (LPS)-induced CMPK2 pathway. Strikingly, DP significantly attenuated LPS-induced sepsis in a mouse model, but its effect was weakened in mice with myeloid-specific Cmpk2 ablation.ConclusionWe provide a new framework that provides more valuable information for new therapeutic approaches to sepsis, including the establishment of screening strategies and the development of target drugs to provide a theoretical basis for ultimately improving clinical outcomes for sepsis patients. Collectively, these findings reveal that DP is a promising CMPK2 inhibitor for the treatment of sepsis. 1. The CMPK2 gene is highly expressed in the whole blood of sepsis patients.2. A two-step chemical tracking strategy was developed for the first time to identify and evaluate the inhibitor dracorhodin (DP) of CMPK2.3. DP, as the first CMPK2 inhibitor, regulates the NLRP3 inflammasome by lipopolysaccharide (LPS)-induced CMPK2 pathway in vivo and in vitro.image