Anti-metabolite Therapy Against Zika virus replication

Alondra D. Torres-Gerena Author

07/28/2021 Added

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Anti-metabolite Therapy Against Zika virus replication. Effects that 6-Aminonicotinamide has in infected cells & metabolic pathway(s).

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[00:00:00.660]Hi, my name is Alondra Torres.
[00:00:02.340]My project is "Anti-metabolite Therapy Against ZIKA virus replication."
[00:00:07.890]My mentor and PI is Dr. Rodrigo Franco
[00:00:12.940]Zika virus has had
[00:00:14.122]multiple outbreaks in every part of the world,
[00:00:18.640]particularly in Brazil, United States, Puerto Rico,
[00:00:22.600]and other countries.
[00:00:24.280]So you see microcephaly is one of the
[00:00:28.690]symptoms that could appear in babies. And you see,
[00:00:33.520]in 2014, the cases were lower, and 2015 and 16,
[00:00:38.440]they incremented dramatically.
[00:00:41.920]Also, this illness is associated with Guillain- Barre Syndrome.
[00:00:49.390]These results were the basis of my research.
[00:00:53.570]A grad student was already working with this drug called 6-aminonicotinamide
[00:00:59.387]also known as 6-AN.
[00:01:02.320]So this drug had an interesting effect. You see,
[00:01:06.010]this is non-treated with 6-AN
[00:01:09.160]nd infected with ZIKA virus, and this is infected but treated with 6-AN.
[00:01:12.400]You see the depletion in infection here.
[00:01:17.110]So what, so why does this happen? Well, you see,
[00:01:21.760]there is two enzymes called
[00:01:26.680]G6PD and 6PGD.
[00:01:31.270]These two enzymes. Drug is supposed to act on the pentose phosphate
[00:01:35.770]pathway.
[00:01:37.330]So what this drug was supposed to do,
[00:01:40.870]it was supposed to inhibit this, and this enzyme,
[00:01:45.820]and that's what we thought that
[00:01:50.260]occurred for this depletion.
[00:01:54.250]In this Western blot, you can see, we did a knockdown.
[00:01:58.630]So, in this Western blot, you can see the knockdown,
[00:02:03.100]but you can also see that it has no effect,
[00:02:06.880]cause this is the treated with 6-AN,
[00:02:11.260]and you can see the knockdowns,
[00:02:15.130]but you can also see that even if it's treated with 6-AN, there is
[00:02:20.260]no inhibition of the protein production of ZIKA virus.
[00:02:25.990]The purpose is to understand how 6-AN has an effect on ZIKA
[00:02:30.820]infection. Our hypothesis is that 6-AN
[00:02:34.060]interferes with NAD.
[00:02:36.820]This causes the depletion of multiple pathways since
[00:02:40.987]NAD is needed in most of them.
[00:02:46.060]Since we believe that 6-AN is affecting NAD,
[00:02:51.040]it affects multiple pathways as well.
[00:02:54.460]If it affects multiple pathway,
[00:02:57.610]it will change the metabolical processes.
[00:03:00.430]This is why we're doing the studies and observing which pathways and
[00:03:05.410]what it's doing to each pathway.
[00:03:09.060]The graph that we see here was provided by the 1D
[00:03:13.920]1H NMR machine that you can see here as well.
[00:03:18.374]This data shows that 6-AN
[00:03:23.190]and treated and non-treated cells have a drastic
[00:03:27.930]difference between the NAD production.
[00:03:31.620]As you can see here, there is significant statistical data.
[00:03:36.730]For us to compile the data that we needed for cell metabolism,
[00:03:40.480]we used a machine called Seahorse. This machine,
[00:03:45.160]what it does is it takes protein efflux rates through time.
[00:03:50.140]So it measures the flux. For first of all,
[00:03:53.320]you get the basal glycolysis, and then you treat it with
[00:03:58.090]Rotenone, and it blocks the carbons in going into the mitochondria,
[00:04:03.730]and that is why the lactate glycolysis
[00:04:09.550]is increased dramatically.
[00:04:13.780]If you can see how you can see what I am explaining,
[00:04:18.400]and also after this,
[00:04:20.770]you can have the compensatory glycolysis data.
[00:04:25.570]Then you treated with 2-DG, which what it does,
[00:04:30.520]it just inhibits this completely.
[00:04:34.600]So, it decreases immensely.
[00:04:37.860]So, in this graph,
[00:04:39.660]you can see the difference between treated and non-treated cells.
[00:04:43.980]You can see ZIKA plus 6-AN
[00:04:47.100]has a depletion significant in
[00:04:51.240]glycolosis,
[00:04:53.160]and here, in basal glycolysis,
[00:04:56.430]you can see the treated and non-treated,
[00:05:03.750]and here, in the compensatory glycolysis,
[00:05:06.690]you can see that treated and non-treated have a
[00:05:11.130]significant statistical data:
[00:05:15.420]is proof that 6-AN does have an
[00:05:21.180]effect on glycolysis,
[00:05:25.230]and it's demonstrated by the flux and Seahorse, because you
[00:05:30.150]can see that cells that have been treated have
[00:05:34.980]depleted.
[00:05:36.690]The data so far does demonstrate that 6-AN inhibits NAD,
[00:05:41.910]which is proven that our hypothesis is correct. However,
[00:05:46.740]we do need to keep experimenting and doing some measuring
[00:05:51.630]of any NAD with infected cells and adding precursors as
[00:05:56.220]well. I would like to thank my PI and mentor,
[00:06:00.680]Dr. Rodrigo Franco.
[00:06:02.720]I would also like to thank Dr. Bikash Sahoo,
[00:06:07.910]and I would also like to thank my grad student Alexandra Crook,
[00:06:13.490]and everyone else who had a part of this project and who helped me
[00:06:18.170]during the summer to complete and have the data that I needed for it,
[00:06:23.000]and I also like to thank UNL for the opportunity to work on this project.

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Anti-metabolite Therapy Against Zika virus replication

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