Acriflavine (Acriflavinium chloride 3,6-Acridinediamine mix) is a papain-like protease (PL(pro)) inhibitor, a fluorescent dye for labeling high molecular weight RNAs, has antimicrobial and anticancer activity, inhibits SARS-CoV-2 and other β-coronaviruses, and can be used for research on leukemia. be used to study leukemia.

A novel candidate drug, Acriflavine, exhibits significant dose-dependent inhibition of A549 cell viability at low concentrations (1, 2, 5, and 10 μM)[1].

Nude mice bearing xenografts of A549 cells were intraperitoneally injected with 2 mg/kg ACF daily for six weeks. The results indicate that treatment with ACF leads to a reduction in tumor size[1].

1. RNA marker
Experimental steps:
1. Prepare solution: Dissolve Acriflavine in an appropriate solvent, such as DMSO or water, and the concentration is adjusted according to the experimental needs.
2. Label RNA: Add solution (10-100uM) to the RNA sample and incubate for a certain period of time (usually 30 minutes to 1 hour).
3. Fluorescence measurement: Use a fluorescence spectrophotometer or fluorescence microscope to detect the fluorescence signal of RNA and evaluate labeling efficiency.
2. Antibacterial experiments
Experimental steps:
1. Dissolve Acriflavine: Dissolve Acriflavine in an appropriate solvent (usually in PBS or water).
2. Treat bacteria: Contact Acriflavine solution at different concentrations with the bacteria to be tested and incubate for a certain period of time.
3. Detection of inhibitory effect: Measure the antibacterial effect by bacterial growth curve or plate counting method.
III. Anti-cancer research
Experimental steps:
1. Dissolve Acriflavine: Dissolve Acriflavine in an appropriate solvent (such as DMSO).
2. Treat tumor cells: Add different concentrations of Acriflavine solution to cultured tumor cells and incubate for a certain period of time.
3. Cell proliferation detection: Use MTT method, cloning experiments or other cell proliferation detection methods to evaluate the anti-cancer activity of Acriflavine.
IV. SARS-CoV-2 inhibition experiment
Experimental steps:
1. Dissolve Acriflavine: Prepare Acriflavine solution in an appropriate solvent.
2. Contact with the virus: Add the Acriflavine solution to the virus culture system and contact with SARS-CoV-2 at different concentrations.
3. Viral inhibition detection: Viral replication level is detected by RT-PCR, virus titer experiments or immunofluorescence analysis.
5. Papain-like protease inhibition experiment:
Experimental steps:
1. Prepare Acriflavine solution: Dissolve Acriflavine in an appropriate solvent and adjust the concentration.
2. Reaction with enzymes: Add Acriflavine to the reaction system of papain-like protease and incubate.
3. Measuring enzyme activity: Evaluate the inhibitory effect of Acriflavine on enzyme activity by enzyme activity detection methods, such as substrate cleavage experiments.

CML mice are treated daily with acriflavine (8 mg/kg) or PBS via intraperitoneal injection, for 10 days starting from day 7 after bone marrow transplantation [2].