Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

The HBX-DDB1 interaction is a crucial step for promoting HPV transcription from cccDNA, so this protocol may become a key asset to discover novel therapeutic agents for HPV functional cure. Our procedure is simple and requires only a short time to screen. The interaction that I mix can be detected in real-time without requiring cell lysis.

Moreover, the screening quality is satisfactory with a high Z prime score. Inhibition of the HBX-DDB1 interaction leads to restoration of Smc5/6, which results in suppression of HPV transcription, protein expression, and cccDNA production. This novel mechanism of antiviral action may overcome the inadequacies of current HPV therapies.

Protein-protein interactions are important class of drug targets. The split-luciferase-based acid described here, which targets the interactions between viral and hasS proteins, may provide a new strategy to develop cures for other infectious diseases. Although our protocol is simple and easy to understand, some steps may be hard to reproduce without the visual demonstration.

Thus, the visual demonstration will be a great help to understand our protocol. Begin by seeding five times 10 to the 5th HEK293T cells into a 100 millimeter dish with 10 milliliters of DMEM, and incubating them overnight at 37 degrees celsius. On the next day, dilute one microgram each of HBx-LgBit and SmBit-DDB1 expressing DNA plasmids and DNA condensation buffer for a total volume of 300 microliters.

Add 16 microliters of enhancer solution, and mix the tubes by vortexing for one second. Incubate the sample at room temperature for three minutes, then add 60 microliters of transfection reagent. Vortex the tube for 10 seconds, and incubate the sample for another eight minutes.

Meanwhile, aspirate the medium from the dish with the cells and wash them with five milliliters of PVS. Aspirate the PVS and add seven milliliters of DMEM. Add three milliliters of DMEM to the tube with the transfection complexes, pipette up and down to mix, and add the mixture to the cells.

Then, incubate the cells at 37 degrees celsius and five percent carbon dioxide for 10 hours. After the incubation, remove the spent culture medium, and wash the cells with five milliliters of PVS. Remove the PVS at one milliliter of 0.25%trypsin EDTA, and incubate the cells at 37 degrees celsius to detach them.

Next, add four milliliters of DMEM, and disperse the medium by pipetting it over the surface of the cell layer several times, and transfer the suspension to a tube. Centrifuge the cells at 500 times G for five minutes, then discard the supernatant, and re-suspend the cell pellet in one milliliter of PVS. Repeat the cetrifugation and discard the supernatant.

Then, re-suspend the cell pellet with buffered cell culture medium supplemented with 10%FBS to a seeding density of one million cells per milliliter. Pipette 50 microliters of the cell suspension into each well of a 96-well plate, and return the cells to the 37-degree-celsius incubator for 10 hours. While the cells are incubating, dilute the screening compounds and solvent to 13.5 X concentration.

Add 12.5 microliters of luminescent substrate to each well, and incubate the plate for five minutes at room temperature. Measure the baseline luminescence with a luminometer, and add five microliters of the compounds and controlled DMSO to each well immediately after. Measure luminescence every 30 minutes for two hours, then calculate the inhibitory effects of the compounds in comparison with DMSO treatment.

Baseline luminescence signals were measured, and the signal-to-background ratio was calculated to be greater than 80. The Z prime was greater than 0.5, indicating that this assay system is acceptable for high-throughput screening. By setting the threshold to greater than 40%inhibition compared to the DMSO-only control, nitazoxanide was identified as a candidate drug.

We previously identified nitazoxanide as an inhibitor of the HBX-DDB1 interaction by screening a reactivity small-scale compound library with this method. Using nitazoxanide, we confirm that inhibition of the HBX-DDB1 interaction, that reduction of viral transcription, and subsequent viral product levels. The optimal portion of the split luciferase fused to a target protein must be determined beforehand.

In this case, HPX fused to Large Bit at the C terminus of HPX and DDB1 fused to Small Bit at the end terminus of DDB1 provided the best results.