Tuberculosis (TB) remains a major threat to human health. Due to the prevalence of drug-resistant Mycobacterium tuberculosis (Mtb), it is urgent to discover drugs with new mechanisms of action (MOA) to ensure effectiveness against strains that are resistant to existing TB drugs. Cynoglossum lanceolatum Forsk was used to treat TB in Traditional Chinese Medicine. In this article, shikonin, the anti-Mtb active component, was obtained from the whole herb extract of C. lanceolatum by bioassay-guided isolation. Using the microplate alamar blue assay (MABA), the minimum inhibitory concentration (MIC) of shikonin against Mtb was determined to be 128 μg/mL. In order to obtain a more efficient anti-Mtb molecule, (E)-1-(6-bromo-2,3-dihydrochromen-4-ylidene)thiosemicarbazide was synthesized based on the scaffold of shikonin, which exhibited potent activity against Mtb (MIC=4 μg/mL). These results highlight that both naphthalene-1,4-dione and chroman-4-one are pharmacophores with activities against Mtb. To investigate a plausible mechanism of action, the molecular docking was firstly performed against catalase-peroxidase enzyme (KatG) of Mtb using AutoDock 4 software. The results demonstrated that both shikonin and (E)-1-(6-bromo-2,3-dihydrochromen-4-ylidene)thiosemicarbazide could bind to the active site of Mtb KatG. KatG enzyme activity and intracellular reactive oxygen species (ROS) levels in Mtb cells were then measured by ultraviolet spectrophotometric method and fluorescence microplate reader assay, respectively. The experiments confirmed that above compounds could inhibit the catalytic activity of Mtb KatG, and cause the ROS accumulation in Mtb cells. Therefore, inhibition of KatG may be a novel mechanism of action for these two compounds to fight against Mtb.
Keywords: Cynoglossum lanceolatum; Mycobacterium tuberculosis; isolation; mechanism of action; synthesis of analog.
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