Name: identifying inhibitors of the hbx-ddb1 interaction using a split luciferase assay system
Uploaded: 2019-12-21T14:00:00
Description: Watch this Scientific Journal Video about Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System at JoVE.com

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (#19H03430 and #17K09405 to M.O., and #19J11829 to K.S.), by a Grant-in-Aid for Scientific Research on Innovative Areas (#18H05024 to M.O.), by the Research Program on Hepatitis from Japan Agency for Medical Research and Development, AMED (to M.O., #JP19fk021005), by program on the Innovative Development and the Application of New Drugs for Hepatitis B (#JP19fk0310102 to K.K.) from AMED, by grants from the Japan Foundation for Applied Enzymology and from the Kobayashi Foundation for Cancer Research (to M.O.), by GSK Japan Research Grant 2018 (to K.S.), and by a grant from the Miyakawa Memorial Research Foundation (to K.S.).

NOTE: A schematic representation of the split luciferase assay is shown in Figure 1A, and the assay process is outlined in Figure 1B. The interaction dynamics can be measured in real time without cell lysis.
1. Cell Preparation
<ol>
	<li>Maintain cultured HEK293T cells in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS), 1x penicil...

<ol>
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We developed a convenient screening method using a split luciferase assay to find HBx-DDB1 binding inhibitors. The interaction dynamics can be detected in real time in living cells without the need for cell lysis. Inhibition of the HBx-DDB1 interaction leads to restoration of Smc5/6, which results in suppression of viral transcription, protein expression, and cccDNA production7. This novel mechanism of antiviral action may overcome the inadequacies of current HBV therapies.
<p class="jove_cont...

Hepatitis B virus (HBV) infection is a major public health concern worldwide, with annual estimates of 240 million people chronically infected with HBV and 90,000 deaths due to complications from the infection, including cirrhosis and hepatocellular carcinoma (HCC)1. Although the current anti-HBV therapeutic agents, nucleos(t)ide analogues, sufficiently inhibit viral reverse transcription, they rarely achieve elimination of viral proteins, which is the long-term clinical goal. Their poor effect on viral protein elimination is due to their lack of direct effect on viral transcription from episomal viral covalently closed circular DNA (cccDNA) minichromosomes in the hepatocyte nucleus2.
HBV transcription is activated by HBV regulatory X (HBx) protein3. Recent studies revealed that HBx degrades structural maintenance of chromosomes 5/6 (Smc5/6), a host restriction factor that blocks HBV transcription from cccDNA, via hijacking a DDB1-CUL4-ROC1 E3 ubiquitin ligase complex4,5,6. Therefore, a crucial step in promoting viral transcription from cccDNA is thought to be the HBx-DDB1 interaction. Compounds capable of inhibiting the binding between HBx and DDB1 may block viral transcription, and indeed nitazoxanide was identified as an inhibitor of the HBx-DDB1 interaction via a screening system developed in our laboratory7.
Here, we present our convenient screening system used to identify inhibitors of the HBx-DDB1 interaction, which utilizes a split luciferase complementary assay7,8. Split luciferase subunits are fused to HBx and DDB1, and the HBx-DDB1 interaction brings the subunits into close proximity to form a functional enzyme that generates a bright luminescent signal. As the interaction between the subunits is reversible, this system can detect rapidly dissociating HBx-DDB1 proteins (Figure 1). Using this system, a large compound library can be easily screened, which may result in the discovery of novel compounds capable of efficiently inhibiting the HBx-DDB1 interaction.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture microplate, 96 well, PS, F-BOTTOM</td>
			<td>Greiner-Bio-One GmbH</td>
			<td>655098</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D6046</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Tocris Bioscience</td>
			<td>3176</td>
			<td></td>
		</tr>
		<tr>
			<td>Effectene transfection reagent</td>
			<td>Qiagen</td>
			<td>301425</td>
			<td>Includes DNA-condensation buffer, enhancer solution and transfection reagent</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Nichirei</td>
			<td>175012</td>
			<td></td>
		</tr>
		<tr>
			<td>GloMax 96 microplate luminometer</td>
			<td>Promega</td>
			<td>E6521</td>
			<td></td>
		</tr>
		<tr>
			<td>HBx&#8211;LgBit expressing DNA plasmid</td>
			<td>Our laboratory</td>
			<td></td>
			<td>Available upon request</td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoBiT PPI starter systems</td>
			<td>Promega</td>
			<td>N2015</td>
			<td>Includes Nano-Glo Live Cell Reagent</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11058021</td>
			<td>Described as &#34;buffered cell culture medium&#34; in the manuscript</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Takara</td>
			<td>T900</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Screen-Well FDA-approved drug library V2 version 1.0</td>
			<td>Enzo Life Sciences</td>
			<td>BML-2841</td>
			<td>Compounds used here were as follows: mequinol, mercaptopurine hydrate, mesna, mestranol, metaproterenol hemisulfate, metaraminol bitartrate, metaxalone, methacholine chloride, methazolamide, methenamine hippurate, methocarbamol, methotrexate, methoxsalen, methscopolamine bromide, methsuximide, methyclothiazide, methyl aminolevulinate&#183;HCl, methylergonovine maleate, metolazone, metyrapone, mexiletine&#183;HCl, micafungin, miconazole, midodrine&#183;HCl, miglitol, milnacipran&#183;HCl, mirtazapine, mitotane, moexipril&#183;HCl, mometasone furoate, mupirocin, nadolol, nafcillin&#183;Na, naftifine&#183;HCl, naratriptan&#183;HCl, natamycin, nebivolol&#183;HCl, nelarabine, nepafenac, nevirapine, niacin, nicotine, nilotinib, nilutamide, nitazoxanide, nitisinone, nitrofurantoin, nizatidine, nortriptyline&#183;HCl, olsalazine&#183;Na, orlistat, oxaprozin, oxtriphylline, oxybutynin Chloride, oxytetracycline&#183;HCl, paliperidone, palonosetron&#183;HCl, paromomycin sulfate, pazopanib&#183;HCl, pemetrexed disodium, pemirolast potassium, penicillamine, penicillin G potassium, pentamidine isethionate, pentostatin, perindopril erbumine, permethrin, perphenazine, phenelzine sulfate, phenylephrine, phytonadione, pimecrolimus, pitavastatin calcium, and podofilox</td>
		</tr>
		<tr>
			<td>SmBit&#8211;DDB1 expressing DNA plasmid</td>
			<td>Our laboratory</td>
			<td></td>
			<td>Available upon request</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying inhibitors of the hbx-ddb1 interaction using a split luciferase assay system

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

Representative outcomes following the use of this protocol are shown in Figure 2A,B. The signal-to-background ratio was greater than 80 and the Z&#39; factor9 (the gold standard quality index for high-throughput screening) was greater than 0.5, indicating that this assay system was acceptable for high-throughput screening. With the threshold set to &#62;40% inhibition compared with the control (DMSO only), we identifie...

Watch this Scientific Journal Video about Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System at JoVE.com

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

Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently ...

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.

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