The overall goal of this procedure is to isolate broad spectrum antivirals from chemical libraries using a combination of in vitro cell based assays. This is accomplished by first selecting compounds that inhibit the in vitro replication of a recombinant strain of measles virus that encodes lucifers as a reporter. In parallel, the compound's toxicity is estimated using a luciferase based viability assay.
The combined phenotypic data from both assays is used to isolate HIIT molecules. The final steps are to retest the HIIT compounds for dose response replication, inhibition of measles and chicken guna virus, and to measure in vitro toxicity more accurately. Ultimately, broad spectrum antiviral compounds are identified and validated by the screen.
The main advantage of this technique over existing methods like standard virus replication assays based on cytopathic effects, is that it uses luminescence as a readout and combines to highly divergent viruses expressing luciferase as a reporter. Demonstrating the procedure will be Olivier Ellan, a technician from El Niemans team, and Marianne Luca Ori, an engineer working with me in f Fredrik JI'S research unit Begin by warming to room temperature. The 96 well mother plates containing 10 millimolar stock solutions of chemical compounds in DMSO.
Transfer five microliters of each compound to new 96 well plates already with 20 microliters of DMSO in each well, thus creating intermediate dilution plates. Now pipee one microliter of these dilution plates into dry wells of white barcoded tissue culture. 96 well plates.
These are the D one plates and are used to evaluate the toxicity of compounds at 20 micromolar. Store them at minus 20 degrees Celsius. Now make new plates from the intermediate dilution plates that are further diluted 10 times.
Take four microliters of each diluted compound and mix them into wells with 36 microliters of DMSO. From these plates, transfer one microliter per sample into dry wells of white flat bottom barcoded tissue culture. 96 well plates.
These are the D two plates and they are used to evaluate inhibition of MV replication. Store them at minus 20 degrees Celsius as well. Grow HEK 2 93 T cells in supplemented DMM with stabilized L-glutamine every four to five days, pass the cells by trypsin and make one 10th dilution into fresh flasks.
During their logarithmic growth phase, collect and count the cells for the experiment. Resuspend the cells at 300, 000 cells per milliliter. 12.5 milliliters of cells at this dilution are needed for each plate that will be screened.
Now, prepare the plates spike one set of control wells with one microliter of DMSO. Spike a second set of control wells with one microliter of DMSO and 2.5 microliters of a 0.5%ipol solution to kill the cells. Transfer the cell suspension to a trough and dispense 100 microliters to each well of the D one plates.
Regularly agitate the suspension to avoid sedimentation. Then incubate the prepared plates for 24 hours. Prepare the cells for the plates as done in the previous section.
The culture medium can be supplemented with uridine to filter out antiviral molecules that target the early steps of pyrimidine biosynthesis, as these are fairly common. Save one 10th of the cell suspension for the control wells with non-infected cells and infect the remaining volume. Thaw enough.
RMV two luxe stock solution to infect the culture with 0.1 infectious particles per target cell for a 0.1 multiplicity of infection. Add the calculated volume of virus to the cell suspension and mix it gently. Transfer the infected cells to a trough and dispense 100 microliters of infected cells to the D two plates.
Target columns two through 11, which contain the spiked chemical compounds and positive control wells in columns one and 12.Regularly. Gently mix the cells in the trough in alternate rows of columns one and 12, dispense 100 microliters of non-infected cells. Now incubate the plates for 24 hours.
After the incubation time, determine the viability of the D one plate cells. Mix 50 microliters of luciferase based viability reagent into each well and wait 10 minutes. Then read the plates with a luminometer.
Set the integration time to 100 milliseconds per well. Next, determine MV replication in the D two plates by mixing 50 microliters of firefly Lucifer substrate into each well and waiting six minutes at room temperature. Then consult a Z factor for each D one and D two plate of the toxicity assay.
Continue by calculating the inhibition of viral replication. Hi compounds are those that reduce viral replication to below the applied cutoff value and are not toxic for each HI compound. Make serial dilutions in DMSO starting at 500 micro molars and moving in half steps down to four micro molars along one column of a 96 well plate add one microliter from the compound dilution plate to the white barcoded tissue culture plate.
Repeat this process twice to create a triplicate set of test plates. Then fill tissue culture plates with 50 microliters of culture medium. Now prepare 37.5 milliliters of HEK 2 93 T cells, suspended in supplemented DM EM at 600, 000 cells per milliliter.
Load two 15 milliliter tubes with 12.5 milliliters of the cell suspension. Each infect one tube of cells with RMV two Luke at a multiplicity of infection of 0.1. Infect the second tube of cells with recombinant chivee expressing vanilla lucifers.
Use a multiplicity of infection of 0.2. Mix both tubes of virus infected cells using inversion. This should be performed in a biosafety level three laboratory because of the chivee pathogenicity now load the uninfected cells and the sets of infected cells into their own set of HIIT compound plates.
Dispense 50 microliters of cells per well and incubate the plates at 37 degrees Celsius for 24 hours. The next day, add 50 microliters of firefly luciferase to each well of RMV two Luke infected cells. Similarly, add 50 microliters of ran vanilla Lucifer substrate to the Chick V Ren infected cells to the uninfected control cells add 50 microliters of cruciferous base viability.
Reagent proceed by determining the hi compound concentrations that inhibit MV and Chick V replication by 50%In addition, disregard compounds showing cell toxicity. This screening pipeline relies on selecting compounds that do not show any significant cellular toxicity and inhibit both MV and Chick V replication as determined in the primary and secondary screens respectively, it takes advantage of recombinant viruses expressing firefly or ran vanilla lucifers. As a reporter.
Compound toxicity is assessed using a commercial lucifers based assay where luminescence correlates to living cells on this plate. 21 compounds were scored as toxic based on a threshold set by the positive control wells. Antiviral activity was first measured using RMV two.
Luke Luminescence was compared to the limit set by control wells. Here, four compounds were found able to inhibit viral replication by more than 75%in the other assay. Two of these were found to be toxic for all the HIIT compounds.
The half maximal inhibitory concentration was determined on both MV and Chick VRNA viruses expressing luciferase. These two viruses are unrelated and thus make the screen more robust. In parallel, the lack of cellular toxicity of selected compounds was confirmed in a dose response experiment.
In all 13 non-toxic compounds were identified from a chemical library of 10, 000 molecules. These compounds were novel in terms of both chemical structure and biological activity. Based on structural similarities, the compounds fell into three chemical families.
For reference, half maximal inhibitory concentration values were obtained with a substance with potent antiviral activity. When looking at these values, less than an order of magnitude, separated the reference molecule from most active hits identified through the screen. This demonstrates the relatively strong antiviral activity of the selected compounds Once mastered.
This technique can be used to screen large chemical libraries in a high throughput setting and select sets of small molecules and reach for broad spectrum antivirals.
