Discovery of Small Molecule Entry Inhibitors against Ebolavirus Infection

Colton Thompson Author

04/05/2021 Added

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Video Presentation: Discovery of Small Molecule Entry Inhibitors against Ebolavirus Infection

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[00:00:00.875]Hi, my name is Colton Thompson.
[00:00:02.761]Today, I'll be presenting my project
[00:00:04.659]on "Discovery of Small Molecule Entry Inhibitors
[00:00:07.770]against Ebolavirus Infection".
[00:00:10.580]For my background, Ebolavirus is a type of filovirus
[00:00:14.180]that can cause severe, hemorrhagic fever in humans
[00:00:17.180]and non-human primates with high mortality rates.
[00:00:19.950]Filoviruses are enveloped viruses
[00:00:22.526]with a single-stranded, negative-sense RNA genome.
[00:00:27.420]Ebolavirus has caused over 20 outbreaks
[00:00:29.990]since its discovery in 1975.
[00:00:32.766]The largest recent outbreak occurred from 2014 to 2016,
[00:00:37.967]infecting 28,600 people and causing 11,300 deaths.
[00:00:44.306]Ebolavirus is transmitted through contact with blood,
[00:00:48.170]secretions, or other bodily fluids of the infected people.
[00:00:52.026]A limited vaccine was approved for Ebolavirus
[00:00:55.180]in December of 2019 by the FDA.
[00:00:58.400]However, there are no FDA-approved drugs
[00:01:01.720]to treat filovirus infections at this time.
[00:01:04.840]Ebolavirus utilizes the cellular Niemann-Pick C1 receptor
[00:01:10.910]for viral entry.
[00:01:12.530]The viral envelope glycoprotein, GP, binds to NPC1
[00:01:17.685]to allow virus entry.
[00:01:19.570]Blocking the interaction between NPC1
[00:01:22.688]and the viral glycoprotein will prevent infection
[00:01:26.426]from spreading throughout the body.
[00:01:28.806]The overall goal of this project is
[00:01:31.390]to identify small organic molecules that will bind
[00:01:34.383]to the viral glycoprotein and prevent Ebolavirus
[00:01:38.110]from binding to the Niemann-Pick C1 receptor
[00:01:41.080]and entering the cells.
[00:01:43.980]In order to test the potential effectiveness
[00:01:46.590]of the candidate drugs developed for this project,
[00:01:49.984]cell viability assays
[00:01:51.724]and virus inhibition assays were performed.
[00:01:55.970]In order to do the cell viability assays, first,
[00:01:59.140]TZM-bl cells were seeded into 96 well plates
[00:02:03.250]at a rate of 3,000 cells per well
[00:02:06.066]and incubated for 24 hours.
[00:02:09.360]After this, the media was removed
[00:02:11.390]and replaced with 100 microliters of complete DMEM
[00:02:14.988]plus the candidate drug at the specified concentration
[00:02:18.889]and then incubated with this mixture for 48 hours.
[00:02:23.086]After this, the media was replaced with 100 microliters
[00:02:28.250]of complete DMEM only for an additional 24 hours.
[00:02:32.860]In order to read the results of this experiment,
[00:02:35.421]the media was replaced with 50 microliters of MTT solution
[00:02:40.878]at five milligrams per microliter
[00:02:43.780]which was diluted at a 1:10 ratio
[00:02:46.497]with expression media and incubated for three hours
[00:02:50.230]in order to allow time for the crystals to develop.
[00:02:53.231]After this, the crystals were dissolved in DMSO.
[00:02:57.978]The absorbance was read at 570 and 690 nanometers.
[00:03:02.942]In order to calculate the adjusted absorbance,
[00:03:05.618]the background absorbance, 690 nanometers,
[00:03:10.390]was subtracted from the 570 reading
[00:03:13.610]in order to determine the adjusted absorbance.
[00:03:16.879]This absorbance was proportional
[00:03:18.910]to the number of viable cells.
[00:03:22.500]After this, the virus inhibition assay was performed.
[00:03:25.987]To do this, TZM-bl cells were seeded in a 96 well plate
[00:03:30.900]at a rate of 6,000 cells per well
[00:03:33.610]and incubated for 24 hours.
[00:03:36.740]Infection media was prepared using complete DMEM
[00:03:40.040]plus the candidate drug at the specified concentration
[00:03:43.650]plus pseudotyped Ebolavirus.
[00:03:46.810]50 microliters of this infection media was added
[00:03:50.370]to each well,
[00:03:51.400]and this was incubated for an additional 24 hours.
[00:03:55.377]After this, the infection media was removed
[00:03:58.690]and replaced with 100 microliters of complete DMEM
[00:04:02.265]and then incubated for another 48 hours.
[00:04:06.420]After this, the cells were lysed
[00:04:08.025]with passive lysis buffer diluted at a 1:5 ratio with PBS
[00:04:13.291]for 20 minutes in order to expose the luciferase enzyme.
[00:04:18.080]Luciferase activity was measured
[00:04:19.990]using the luciferin substrate and a Luminometer.
[00:04:24.070]To test the potential effectiveness of the 16 new analogs,
[00:04:27.664]a cytotoxicity screening was first performed
[00:04:30.509]at a rate of 10 micromolar concentration.
[00:04:34.180]The results of these experiments are shown in Figure 1.
[00:04:37.486]Following this, an inhibition screening
[00:04:40.209]of the 16 compounds were compared to the original compounds,
[00:04:43.709]again, at 10 micromolar concentration.
[00:04:46.989]Based on the results of these two screenings,
[00:04:49.503]we saw that compounds U 6-4
[00:04:52.701]and U 7-2 showed inhibition activity
[00:04:56.038]without high cytotoxicity.
[00:04:58.500]Therefore, these two compounds were selected
[00:05:02.010]for further testing.
[00:05:04.430]Cytotoxicity testing of U 7-2 and U 6-4 is shown
[00:05:10.100]in Figure 3.
[00:05:10.933]The CC50 of compound U 7-2 was determined
[00:05:15.890]to be 79.6 micromolar.
[00:05:19.068]The CC50 of compound U 6-4 was estimated
[00:05:23.613]at 96.51 micromolar.
[00:05:27.636]When it was discovered
[00:05:29.330]that U 6-4 showed little inhibition activity,
[00:05:34.184]testing of this compound was stopped.
[00:05:38.930]Next, virus innovation assays were performed
[00:05:42.510]on U 7-2 and U 6-4 in order to determine the IC50.
[00:05:48.093]The IC50 of compound U 7-2 was determined
[00:05:51.760]to be 32.37 micromolar.
[00:05:55.180]The IC50 of compound U 6-4 was not determined,
[00:06:00.460]and it did not show any inhibition activity
[00:06:03.560]even at test concentrations of up to 160 micromolar.
[00:06:09.310]In conclusion,
[00:06:10.340]16 analogs of previously tested compounds
[00:06:13.040]were selected based on in silico screening.
[00:06:16.750]Analogs, along with original compounds,
[00:06:19.090]were screened for cytotoxicity and inhibition activity
[00:06:22.180]at 10 micromolar.
[00:06:23.741]Based on the results of cytotoxicity
[00:06:26.379]and inhibition screening, U 6-4 and U 7-2 were selected
[00:06:31.450]for further screening.
[00:06:32.884]Both of these analogs displayed lower cytotoxicity
[00:06:37.140]and higher neutralization activity
[00:06:39.260]than the original compounds in these screenings.
[00:06:41.911]Further testing
[00:06:42.845]of U 6-4 showed insignificant inhibition activity,
[00:06:47.370]even at concentrations up to 160 micromolar.
[00:06:51.106]This information led to the decision to discontinue testing
[00:06:55.900]of U 6-4.
[00:06:57.576]Further testing of U 7-2 showed it had a CC50
[00:07:03.400]of 79.6 micromolar and an IC50 of 32.27 micromolar.
[00:07:10.454]The selectivity index of U 7-2 is 2.47.
[00:07:16.452]Further work will be conducted
[00:07:18.053]using structural activity relationship analysis
[00:07:21.174]to generate more analogs for testing.
[00:07:24.154]These analogs will be screened for cytotoxicity
[00:07:27.750]and inhibition activity.
[00:07:29.600]and top-performing compounds will be selected
[00:07:31.910]to determine the selectivity index.
[00:07:34.240]Thank you for funding provided by UCARE
[00:07:40.650]and by the National Institute of Health
[00:07:42.610]for the completion of this project.
[00:07:44.637]Thank you.

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Discovery of Small Molecule Entry Inhibitors against Ebolavirus Infection

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