| Tuberculosis(TB)is an infectious disease caused by Mycobacterium tuberculosis(MTB)and has shown an increasing incidence of new cases in recent years.The current treatment for TB relies primarily on first-line anti-tuberculosis drugs,but the cure rate for drug-resistant TB is lower.Developing novel anti-tuberculosis drugs with novel targets is a crucial strategy for addressing drug resistance.Cirsium japonicum DC.is a plant with abundant chemical components and diverse pharmacological activities.Previous investigations by the research group have revealed that quercetin isolated from Cirsium japonicum possesses anti-tuberculosis activity,with a minimum inhibitory concentration(MIC)of 160 μg/m L.The present study aims to isolate other anti-tuberculosis components from the ethyl acetate fraction of Cirsium japonicum and conduct structural modifications to preliminarily elucidate the antibacterial mechanism of the active compound,thus laying a foundation for the development of novel anti-tuberculosis drugs.By using an active tracking isolation method,the seventh component of the chromatographic column from the extraction of ethyl acetate from Cirsium japonicum was found to have antibacterial activity.The active monomer D-01 was then isolated from the seventh component.Based on spectroscopic data,the monomer was identified as caffeic acid,with MIC value of 125 μg/m L against Mtb.Structural modification of caffeic acid led to the synthesis of amide compounds 1 and 2,but these two compounds lack significant anti-tuberculosis activity(MIC=64 μg/m L).Quercetin represents an additional active constituent in Cirsium japonicum with potent anti-tuberculostatic effects,whose pharmacophore is 4H-chromen-4-one.However,the literature suggests that chroman-4-one,which has a similar structure to 4H-chromen-4-one,exhibits better antibacterial activity.Therefore,based on the principle of substitution,20 compounds were designed by utilizing chroman-4-one as a molecular framework combined with thiosemicarbazides compounds,featuring a pharmacophore analogous to that of quercetin.Subsequently,we used these compounds as ligands for molecular docking with M.tuberculosis Kat G and selected 13 of them(compounds 3-15)that exhibited higher scores for chemical synthesis,including 7 novel compounds.The results demonstrated that compounds3-15 displayed superior anti-tuberculostatic activity(MIC=1-32 μg/m L).Moreover,utilizing computer-assisted methods,we confirmed that all these compounds presented promising drug-like properties and satisfactory pharmacokinetic profiles.To elucidate the mechanism of action of the synthesized products,compound 9 was selected due to its superior activity(MIC=1 μg/m L)for preliminary mechanism exploration.Molecular docking results revealed that compound 9 can form critical hydrogen bonds with Trp107 at the active site of the Mycobacterium tuberculosis Kat G,potentially explaining its potent anti-tuberculostatic activity.Time-kill curves demonstrated that compound 9displayed time-and concentration-dependent killing effects on M.tuberculosis.SDS-PAGE analysis of Kat G indicated that in the presence of compound 9,M.tuberculosis employed negative feedback regulation and overexpressed the Kat G.Enzyme inhibition assays indicated that the M.tuberculosis Kat G activity was reduced with an increasing concentration of compound 9,displaying a clear dose-response relationship.As such,we speculated that compound 9 targets the M.tuberculosis Kat G.To substantiate this hypothesis,we measured the relative intracellular reactive oxygen species(ROS)content in bacterial cells.The accumulation of ROS in M.tuberculosis cells induced by compound 9 serves as evidence that Kat G inhibition by compound 9 inhibits the normal decomposition of hydrogen peroxide.Live-cell imaging revealed that post compound 9 treatment,M.tuberculosis exhibited morphological changes and even disintegration.In conclusion,we propose that compound 9 inhibits the M.tuberculosis Kat G,leading to an increase in intracellular ROS content,which disrupts the polymeric structure of the bacterial cell wall,ultimately resulting in bacterial death.This investigation has revealed that caffeic acid is the active anti-tuberculostatic compound present in Cirsium japonicum.The pharmacophore chroman-4-one has been identified as the optimal candidate for designing anti-tuberculostatic molecules.The synthesized compounds are expected to act on the Mycobacterium tuberculosis Kat G,thereby forming the basis for the development of novel drugs that exhibit innovative mechanisms of action against tuberculosis. |
