Testing small molecule inhibitors of Zika virus RNA polymerase
Zika Virus (ZIKV) is a mosquito-transmitted virus capable of causing of severe neurological disease and neurocognitive disorders in infected adults and infants. From 2015 to 2016, a major outbreak in Brazil resulted in an estimated 1.5 million zika infections and at least 3,500 infants born with zika-related microcephaly or other symptoms of congenital zika syndrome. Presently, no vaccines or antivirals exist to treat ZIKV infection. The ZIKV RNA-dependent RNA polymerase (RdRp) enzyme is critical to replication of the single-stranded RNA virus and has previously been the target of in silico screening of a library of 100,000 small molecules for their potential to interfere with virus replication. We tested the seven lead compounds from this in silico screening for their ability to inhibit virus replication in cell-based in vitro assays. Compound 50 demonstrated high antiviral activity in infected cell cultures at a concentration 1.5 μM. Compound 50 has an (IC50) of 5.8 μM and a CC50 of no less than 60 μM, giving it a relatively high selectivity index of 10.4 and demonstrating its strong potential as an anti-ZIKV drug candidate.
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[00:00:01.060]Hello, and thank you
[00:00:02.460]for viewing my presentation.
[00:00:04.358]My name is Anika Jane Beamer,
[00:00:06.087]and my research in the Xiang lab
[00:00:07.812]this summer focused on the testing
[00:00:09.503]of small molecule inhibitors of
[00:00:11.258]the Zika virus RNA polymerase
[00:00:13.686]The Zika virus is a mosquito transmitted
[00:00:15.786]flavivirus with a positive sense
[00:00:17.549]single stranded RNA genome.
[00:00:20.345]As you may remember from the 2015-2016
[00:00:22.860]epidemic originating in Brazil,
[00:00:25.023]Zika virus infection can cause severe
[00:00:27.120]neurological disease and neurocognitive
[00:00:29.234]disorders, such as microcephaly
[00:00:31.204]and Guillan Barre syndrome.
[00:00:33.237]Climate change is actually expanding the
[00:00:35.294]range of the virus' mosquito vectors,
[00:00:37.338]yet there are no vaccines or antivirals
[00:00:39.619]to prevent or treat Zika infection
[00:00:42.678]Now, because Zika is an RNA virus
[00:00:45.247]and because human host cell machinery
[00:00:47.562]can only prepare RNA transcripts
[00:00:49.403]from a DNA genome template,
[00:00:51.135]each Zika virion must carry with it
[00:00:53.589]an enzyme that can synthesize
[00:00:55.545]RNA complementary to the virus’ RNA
[00:00:58.046]template and allow replication to occur.
[00:01:00.396]This enzyme is called
[00:01:02.696]RNA-dependent RNA polymerase, or RdRp.
[00:01:06.313]I have here two images of the Zika virus
[00:01:10.002]RDRP structure, and I’d like to draw your
[00:01:12.340]attention in particular to the encircled
[00:01:14.577]region on the right.
[00:01:16.041]This is the catalytic
[00:01:17.311]core of the RdRp enzyme, where single
[00:01:20.510]stranded RNA enters
[00:01:22.214]and where complementary
[00:01:23.484]strand synthesis is initiated.
[00:01:26.774]is critical to Zika virus replication and
[00:01:29.704]is therefore an ideal target for
[00:01:34.176]The Zika RdRp catalytic core has
[00:01:36.685]previously been the target of an in-silico
[00:01:39.149]screening, of a library of 100,000
[00:01:41.217]small molecules, in collaboration
[00:01:43.112]with the Xiang laboratory.
[00:01:45.252]In-silico screening is essentially
[00:01:47.256]computer modeling which allows
[00:01:48.975]researchers to predict the interactions
[00:01:50.835]between small molecules and a target,
[00:01:53.253]in this case the Zika RdRp, thereby
[00:01:56.438]identifying molecules which might inhibit
[00:01:58.502]the enzyme and be successful
[00:02:00.535]pharmaceuticals, before testing their
[00:02:02.476]efficacy in living cells.
[00:02:04.475]In my research this summer,
[00:02:06.295]I tested seven promising
[00:02:07.556]compounds from this virtual screening for
[00:02:09.769]their ability to inhibit virus replication
[00:02:12.768]in vitro, that is, in living cells,
[00:02:14.937]and I further characterized the most
[00:02:17.054]promising molecule’s inhibitory
[00:02:18.965]and cytotoxic effects.
[00:02:21.511]To screen each of our compounds
[00:02:23.168]for their ability to inhibit viral
[00:02:25.048]replication, I used a traditional plaque
[00:02:28.895]I first infected monolayers of primate
[00:02:30.923]kidney cells (Vero 6) with equal amounts
[00:02:33.556]of Zika virus, and then incubated them
[00:02:36.002]with 1.5 um concentrations
[00:02:38.331]of our compounds of interest for 48 hours.
[00:02:41.077]After two days, I collected the
[00:02:43.226]supernatants from the cells,
[00:02:44.732]which now contain new viral
[00:02:46.421]particles that have been released
[00:02:48.100]by infected cells.
[00:02:50.127]If a compound strongly inhibits
[00:02:51.787]viral replication, the concentration
[00:02:53.881]of viral particles in the supernatant
[00:02:56.056]from that cell culture will be lower.
[00:02:59.292]I then performed 10-fold serial dilutions
[00:03:01.527]of the collected supernatants
[00:03:03.368]and used the dilutions to infect
[00:03:05.520]new Vero 6 cells.
[00:03:07.241]After being infected, these cells are
[00:03:09.625]covered with semi-solid media,
[00:03:11.449]which is a gummy solution that prevents
[00:03:13.712]new virions released from cells from
[00:03:15.586]traveling beyond the neighboring cells.
[00:03:18.019]This restricted transmission results in
[00:03:19.970]localized areas of infection, called,
[00:03:22.355]"plaques," which can be stained, seen,
[00:03:24.527]and counted with the naked eye
[00:03:26.227]after four days, as you can see in
[00:03:28.976]By counting these plaques, I could
[00:03:30.650]determine the number
[00:03:32.694]units per mL, or,
[00:03:33.937]infectious virions per mL,
[00:03:35.937]produced by infected cells treated
[00:03:38.064]with each of the compounds.
[00:03:39.809]After doing this for each of our
[00:03:41.737]compounds of interest, I found that
[00:03:43.972]Zika-infected cells treated with the
[00:03:45.909]compound we call, "Compound 50," produced
[00:03:48.625]far fewer plaque-forming units per mL
[00:03:50.592]than the untreated virus only control
[00:03:53.886]and also demonstrated
[00:03:55.550]similar but slightly
[00:03:56.550]weaker potency as our positive control,
[00:03:59.700]TPB is a compound with a very similar
[00:04:01.883]structure to Compound 50, and which
[00:04:04.166]has previously demonstrated
[00:04:05.888]anti-Zika RdRp inhibitory activity.
[00:04:08.992]Because Compound 50 appeared the
[00:04:10.858]most promising Zika inhibitor out of
[00:04:12.818]all of our compounds, we sought to
[00:04:14.517]further explore its potential as an
[00:04:16.440]antiviral by determining the IC50,
[00:04:18.903]the half maximal inhibitory concentration
[00:04:21.340]of the drug against Zika.
[00:04:23.998]The IC50 represents the concentration
[00:04:26.705]at which a drug inhibits Zika
[00:04:28.427]replication by 50%.
[00:04:30.435]I repeated the plaque assay protocol
[00:04:32.467]several times, however, this time
[00:04:34.516]I used different concentrations of
[00:04:36.267]compound 50, rather than distinct
[00:04:40.029]I found that the IC50
[00:04:41.459]of Compound 50 is about 5.8 micromolar.
[00:04:44.604]That is, a 5.8 micromolar concentration
[00:04:47.616]of this compound reduces viral replication
[00:04:52.351]Now, determining Ic50 requires
[00:04:54.616]extensive replication of this rather time
[00:04:57.042]consuming protocol, and so our
[00:04:58.598]laboratory will be continuing these
[00:05:00.324]experiments in the fall in order to
[00:05:02.038]further refine our calculation of
[00:05:05.876]When assessing the potential of a
[00:05:07.507]drug candidate, we are not only interested
[00:05:09.507]in its potency, how well it works, we are
[00:05:12.324]also interested in its cytotoxicity.
[00:05:14.393]A good antiviral candidate must
[00:05:16.923]demonstrate low cellular toxicity,
[00:05:18.957]or it's not useful.
[00:05:20.595]This trait can be characterized by a
[00:05:22.708]compound's CC50, which is the
[00:05:24.897]concentration of the drug at which
[00:05:26.947]cell viability is reduced by 50%.
[00:05:29.503]So a higher CC50 value indicates
[00:05:35.030]Scientists in the Xiang laboratory
[00:05:36.786]have previously determined the CC50
[00:05:38.654]of Compound 50 via MTT cell viability
[00:05:41.704]assay, in which we measure the
[00:05:43.836]reduction of MTT to formazan by
[00:05:46.403]metabolically viable cells at
[00:05:48.063]varying compound concentrations.
[00:05:50.928]We have found the CC50 of Compound 50
[00:05:53.121]to be no less than 60 μM, indicating
[00:05:56.145]very low cytotoxicity.
[00:05:58.265]This low toxicity not only indicates that
[00:06:01.118]Compound 50 is safe for cells when
[00:06:03.087]used to treat Zika infection,
[00:06:04.613]it also suggests that the inhibitory
[00:06:07.087]effect of the compound against the virus
[00:06:09.341]cannot be attributed to any cytotoxicity
[00:06:13.927]We have therefore identified a
[00:06:15.942]small molecule with potent anti-Zika
[00:06:17.820]activity and very low cytotoxicity, which
[00:06:20.616]we believe targets the Zika virus
[00:06:22.959]RNA-dependent RNA polymerase,
[00:06:24.986]based upon prior in silico screening.
[00:06:27.776]Our next steps will include confirmation
[00:06:29.818]that Compound 50’s inhibitory activity
[00:06:31.974]is indeed due to its interaction and
[00:06:34.441]binding with the RdRp enzyme.
[00:06:36.526]If so, it's known that Zika RdRp structure
[00:06:40.050]is very very similar to the RdRp
[00:06:42.963]enzymes of other flaviviruses, such as
[00:06:45.657]Dengue virus, West Nile virus,
[00:06:47.425]Yellow Fever virus, et cetera,
[00:06:48.944]and so we are optimistic that
[00:06:50.640]a compound which successfully inhibits
[00:06:52.733]Zika replication by targeting the RdRp
[00:06:56.396]may also be an effective inhibitor of
[00:06:58.365]those other viruses as well.
[00:07:00.492]In my time in the Xiang laboratory,
[00:07:02.225]I have just begun
[00:07:03.081]to test the efficacy
[00:07:04.397]of Compound 50 against the Dengue
[00:07:06.022]virus, in hopes of demonstrating
[00:07:07.821]broad anti-flavivirus activity of the
[00:07:11.591]Should Compound 50 continue to display
[00:07:13.654]high potency and low cytotoxicity,
[00:07:15.966]mouse studies in the future will evaluate
[00:07:18.204]the compound’s efficacy in vivo.
[00:07:20.809]I would like to thank my mentors
[00:07:22.379]and collaborators on this project,
[00:07:24.103]including Leah Liu Wang, Colton Thompson,
[00:07:26.743]Leslie Estrada, Dr. Shi-Hua Xiang,
[00:07:29.472]and Dr. Corey Hopkins of UNMC for
[00:07:31.950]synthesizing our compound candidates,
[00:07:33.846]as well as Joshua Wiggins for his support
[00:07:35.939]in the laboratory.
[00:07:37.464]This project was funded by the Nebraska
[00:07:39.318]Center for Virology’s Undergraduate
[00:07:41.907]Experience in Virology and by the
[00:07:43.797]the University of Nebraska Collaboration
[00:07:47.753]Here are my references.
[00:07:49.127]Thank you so much for your time
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