A recently developed platform lets researchers watch any RNA virus — including the one that causes COVID-19 — in real time, quickly learn its behavior and fast-track screening of antiviral compounds for possible repurposing.
Described in a paper to be published in the July 1 edition of Biosensors and Bioelectronics, the novel method attaches fluorescence to an RNA virus injected in vitro into a human host cell. That fluorescence traces the pathogen's exact route and can indicate its replication abilities before and after an antiviral candidate is introduced.
"During the cells' life, we can image the location and the amount of the viral RNAs inside," lead author Dal-Hee Min told The Academic Times. "What that means is that we can measure the infectivity of the virus real time."
For instance, if fluorescence is reduced after an external molecule or compound is inserted, that means the material has inhibited the virus. Referred to as GOViRA, or Graphene Oxide-based Viral RNA Analysis system, the invention has already shown promise in tackling DENV, the virus that causes dengue fever, and is now having a go at SARS-CoV-2.
Min, a professor in the chemistry department at Seoul National University, is one among the cadre of scientists planning to prevent another pandemic, as her device could be applied to new RNA viruses that may surface, too.
"Infectious diseases, which have spread in tropical and sub-tropical regions, are becoming more serious," she said. "Global warming is one reason, and the other reason is frequent traveling."
She continued, "These kinds of diseases have risk to all of us — I think we should be prepared for that."
To discover a novel compound and then apply it commercially typically involves several important but time-consuming intermediate stages that can cost hundreds of millions of dollars. Before entering any phase of human clinical trial, the drug must be considered nontoxic, effective and have clear dosage restrictions. Then, even more toxicity tests must be done in early human trials, with the U.S. Food and Drug Administration standing between steps such as animal testing and first entry into humans as well as early phase trials and market availability.
Min says the technology is most useful for investigating compounds already approved by the FDA. If available medication can be repurposed, its secondary use would be cheaper than a novel drug option and simplify the process of moving toward public utility.
For proof of principle, the team used the tactic to screen FDA-approved drugs for one that inhibits DENV. According to the World Health Organization, the fatal mosquito-borne illness only affected nine countries prior to 1970, but it now puts about half the world's population at risk, with 100 million to 400 million cases reported per year, spread across more than 100 countries.
The team discovered that a compound commonly used for emergency contraception — Ulipristal — appeared to slow the replication of DENV. The next step was in vivo mouse models.
"In mice, the efficacy was so much more higher than I expected," Min highlighted, while warning that, "Mouse and human direct comparison is impossible."
However, if the researchers receive necessary grants, they will be able to take this compound to clinical trials without worrying about toxicity issues, because it is already FDA-approved.
"We can directly go to clinical phase two," she said. "We don't have to conduct phase one."
Min's sensor is what allowed the team to isolate and monitor Ulipristal and take it through the steps of drug discovery so quickly.
"Once the cells are infected by [the] virus," Min explained, "we add the platform to the surface — then we just wait about 30 minutes to one hour."
Presently, the most common method of assessing inhibitory properties of a drug, called the polymerase chain reaction test, or PCR, takes about six hours to complete and must be run many times.
Once the researchers infect an in vitro host cell with the RNA virus, it propagates accordingly and becomes rich in quantity. That's where the novel platform is brought into the picture. It's based on two main components. The first is an RNA probe, also called a peptide nucleic acid, which is an artificial molecule used in genetic engineering; it is tagged with the fluorescence. The second is a nanomaterial, graphene oxide.
The probe is given a sequence complementary to that of the RNA virus being studied, becoming something like a puzzle piece that fits the virus perfectly. However, the probe also requires transportation to the virus, and that's where graphene oxide comes in.
"This nucleic acid probe will bind to the graphene oxide — they make a complex," Min said, adding that after its introduction to the infected host cell, "The probe nucleic acid would bind to the target viral RNA rather than graphene oxide because the binding affinity of this probe toward the sequence-specific viral RNA would be higher."
From there, the virus's methods are exposed as the fluorescence travels along with it during its replication and movement throughout the host cell.
Min emphasized that she can also measure the fluorescence intensity and cellular localization of the fluorescence in individual places.
"If they are near mitochondria, for example, then that means something," she said, noting that this is a rather simplistic example.
Now, the team is using the same technology to search for antiviral drug candidates to fight against COVID-19, as this coronavirus is also RNA-based.
"By just changing the sequence of the probe, we can apply this platform towards newly emerging viruses," Min said. "So, that's a very important feature of this platform technology — it's a very simple system."
The study, "A graphene oxide-based fluorescent nanosensor to identify antiviral agents via a drug repurposing screen," published in the July 1 edition of Biosensors and Bioelectronics, was authored by Hojeong Shin, Se-Jin Park, Jungho Kim and Ji-Seon Lee, Seoul National University; and Dal-Hee Min, Seoul National University and Lemonex Inc.