I joined Dr. Evzen Boura’s research group when I was 15 years old. Ever since I have been exploring biochemical methods and working with viral enzymes.
On this page, I intend to briefly describe my research.
Also, I would be glad if read my first scientific article in which I share co-first authorship with Andrea Huskova.
Why viral enzymes interest me
Inhibitors are small molecules that bind to proteins and inhibit their function. Therefore, inhibitors of viral enzymes may be used to develop antiviral drugs.
How to obtain antiviral drugs?
We can obtain antiviral drugs by screening or design. In Dr. Boura’s group, we strive to do both. To achieve that, we study the functions and structures of viral proteins using biochemical and biophysical methods.
Screening
Screening requires the following steps:
- production of an active enzyme,
- optimization of the reaction conditions,
- employment of an assay which can be used in a high-throughput settings. Having both an active enzyme and a robust, rapid and economical assay, we can start screening thousands of existing molecules and monitor their effects.
Design
In drug design, we must subject an enzyme to biophysical methods, such as X-ray crystallography or cryo-genic electron microscopy, in order to solve its three-dimensional structure. Such techniques are often incredibly difficult or even impossible to conduct. Provided we obtain such structure, novel antiviral agents can be designed and synthesized by medicinal chemists such as Dr. Radim Nencka and his group, with whom Dr. Boura’s group cooperates.
There are currently no antiviral drugs against the Epstein-Barr and tick-borne encephalitis infections. Therefore, all steps mentioned above were part of my projects at the laboratory.
This structural analysis provides important information for drug development. From the recent paper by Krafcikova, P., Silhan, J., Nencka, R., and Boura, E. in Nature Communications 11, 3717 (2020). https://doi.org/10.1038/s41467-020-17495-9
My research on viral proteases
The tick-borne encephalitis virus (TBEV) is an RNA virus, belonging to the Flaviviridae family. Related viruses include virus Zika, dengue, or West Nile. Tick-borne encephalitis (TBE) is the most significant tick-borne disease that causes brain damage, paralysis and death.
Last year, I used the TBEV protease to develop a fluorescent high-throughput screening assay. Currently, I am striving to mutate and crystallize the protease. Hopefully, I will enable drug development and reveal the precise mechanism of its catalysis.
How I researched the tick-borne encephalitis protease
I carried out two projects within the research on the tick-borne encephalitis protease.
First Project: The development of an assay for drug discovery
To develop a brand new assay required the following steps:
- preparation of an active protease and its fluorescent substrate,
- assaying the proteolytic activity,
- establishment of the optimal conditions of the proteolysis,
- establishment of the optimal parameters of the measurement,
- testing several commercial inhibitors to verify its robustness.
It has been an exhilarating and challenging task, lasting moths of refining and repeating. It taught me to learn from failures and improve myself relentlessly. I would be glad if read my first scientific article.
Designing a fluorescent high-throughput assay
Our assay is based on the FRET (Förster resonance energy transfer) phenomenon: basically, if two specific (fluorescent) molecules are close enough together, one of them (the ‘donor’) can be excited by light of a particular wavelength and the energy gained by excitation can be transferred to the second molecule (the ‘acceptor’), which can be a quencher or a fluorophore emitting the light at its characteristic wavelength.
Therefore, we designed the protease substrates for this assay. The actual substrate, i.e. a peptide containing a site recognized and cleaved by the protease, was flanked by two fluorophores – the green fluorescent protein (GFP) and the red fluorescent protein (mCherry).
What is incredibly useful about using fluorescent proteins instead of other molecules is the low cost of production. Any biochemist can prepare them readily.
What I measured in the assay
In this assay, I measure the change of red light in the reaction time.
When I applied green light to the FRET substrate, the GFP molecule became excited and transferred the energy to mCherry, emitting red light. Therefore, the red light was roughly constant (actually, we must correct the data for diminishing fluorescence and bleaching).
Upon adding the protease to the substrate, the protease cleaves the link between GFP and mCherry molecules, terminating the FRET phenomenon. The energy is not being transferred to mCherry. Therefore, we observe the continuous decline of the red light in the reaction.
Using the measured data, I calculated reaction rates as the slope of the red fluorescence intensity versus time.
(A) Constructs of the fluorescent substrates. (B) Schematic representation of the assay. Left: excited donor (GFP) transferres its energy to the acceptor (mCherry), which emits light, due to the short distance between the molecules. FRET occurs. Right: Upon proteolysis, the distance between the two fluorophores increases. FRET ceases to occur. (C) Depiction of a concentration series in a microplate. Left: raw-data curve . Right: final graph showing the inhibition potency of the specific molecule tested (measured in IC50). (D) Raw data from the concentration series of the enzyme. (E) Verification of the measured data by SDS-PAGE (SDS-gel electrophoresis).
Optimizing reaction conditions
Before employing the assay for drug discovery and FRET measurements, I optimized the assay using gel electrophoresis (SDS-PAGE, in particular).
Optimization means finding the conditions that enable maximal enzymatic activity. Therefore, I tested different pH values (of the buffer, i.e. a solution providing a stable environment), and made a concentration series of various salts, ions and reduction agents.
After the termination of each reaction series, I applied the reaction mixtures to SDS polyacrylamide gels, which separate the proteins based on their molecular weight. I scanned the gels using a fluorescence scanner and determined which conditions produce the maximal amount of product.
Testing potential inhibitors
I used a microplate reader, which allows measuring 384 or even 1536 reaction mixtures at a time.
For the tests, I chose several commercially available inhibitors based on the type of the protease (serine) and past research of related enzymes (particularly, the Zika and dengue enzymes). Similarly to optimization, I made a concentration series of each inhibitor, observed decreasing red fluorescence and, based on the data, calculated the corrected reaction rates. The final result was a curve showing a relative percentage of inhibition, calculated with respect to control experiments.
(A), (C), (D) IC50 curve shows how much of the inhibitor is needed to inhibit the protease by 50%. (B) Verification of the reaction progress by SDS-PAGE (samples were taken after the measurements).
Second Project: Elucidating the TBEV protease mechanism for drug development
Currently, I strive to elucidate the mechanism by which the TBEV protease conducts cleavage. I am using mutagenesis and crystallography to achieve this. I believe that such a piece of information could serve to enable a specific drug design.
My research on a viral polymerase
The Epstein-Barr virus (EBV) is a DNA virus. It belongs to the Herpesviridae family which is known to cause sores and blisters. Also, the infection can be latent and lifelong as viral DNA integrates into the human genome. For the same reason, herpes viruses are thought to drive cancer.
How I researched the Epstein-Barr polymerase
In 2019, I worked with the EBV polymerase, which is now studied further by the group’s PhD candidate. My goal was to assay the activity of EBV polymerase and establish optimal conditions for its catalysis.
Designing a fluorescent assay
DNA polymerases catalyze the polymerization of dNTPs (deoxyribonucleotide triphosphates) in accordance with the complementary strand of DNA.
To monitor the progress of the polymerization, we designed a fluorescently-marked substrate. It was composed of a long strand of DNA hybridized to a smaller chain that served as a primer for the polymerase. A pink fluorescent mark (the HEX dye) tagged the 5’ terminus of the primer.
Afterwards, I set up different concentration series of the reaction components, for example, of the dNTPs and magnesium ions. After an SDS-PAGE analysis, I determined the optimal reaction conditions.
Further research
Currently, the polymerase is being subjected to further enzymatic assays and structural studies. Soon, these results will surely provide a novel basis for effective drugs.
