Ten years ago, molecular biologist Pablo Gastaminza was looking for antivirals against the hepatitis C at the National Center for Biotechnology (CNB-CSIC). He did not imagine then that the virus, which became one of the most problematic in the world along with HIV, would end up being able to be effectively cured thanks to the advance in the development of these compounds. So Gastaminza was forced to retrain. The scientist went on to investigate drugs against viruses transmitted by mosquitoes, a growing problem in Spain due to largely due to climate change. But SARS-CoV-2 entered “like a gale”, in the words of the scientist, and with it the need for treatment. The CNB was one of the centers that got down to work. “From the CNB we made a call to institutions in the country to receive collections of compounds that could have antiviral activity. The response was fantastic and that allowed us to create an antiviral platform that has analyzed more than 8,000 different compounds”, explains the director of the center, Mario Mellado.
The screening platform or screening of antivirals is part of the Comprehensive Plan that the center proposed to the Ministry of Science and Innovation to fight the new coronavirus. Luis Enjuanes’ vaccine proposal or the covid-19 serological test ceded to WHO also came out of this draft. To start the project, the director of the CNB called Gastaminza and fellow molecular biologist Urtzi Garaigorta. “Because of the tradition of our laboratories”, explains the first. Garaigorta had also tried to give antiviral life to molecules, but for the hepatitis B virus. The necessary knowledge was obtained with the Nobel Prize in Chemistry Karl Barry Sharplessamong others, during his stays at the Scripps Research Institute, in La Jolla (United States).
When the screening platform began to roll, it did so by evaluating drugs already on the market used for other pathologies. In this way, if a drug was valid against SARS-CoV-2 infection, it could be used immediately as the relevant authorizations had been passed at the time. It is what is known as drug repositioning and it had about 2,400 molecules of the total. The scientists explain that they obtained similar results to those published by other research groups.
After the initial urgency, the project now works as a greased machine for testing and improving potential antivirals against the coronavirus. It uses collections of molecules designed and synthesized for other possible applications and that have not been released on the market.
The role of both biologists, together with the CNB research staff under their charge, consists of growing cells in the laboratory which they infect with SARS-CoV-2 and add the antiviral candidate. Given the danger of the virus, this work is carried out in the biosafety laboratory 3 (P3) of the CNB itself. The maximum security level in international standards is 4.
If you look through the portholes that separate the room from the corridor, you can see staff crossing dressed in a white jumpsuit and a respirator. Everything that comes out of the interior must be scrupulously disinfected, which includes everything from the person to plates with cultures, through the contact lenses that can be worn. That is why researchers choose to have some glasses inside and others outside.
The next step involves evaluating the infectivity of the virus in the presence of the compound. This work is done outside of P3 after virus inactivation. A device, called a multimodal reader, allows taking photos of the crops and counting the amount of infection. The less virus, the better antiviral activity. “A possible explanation for a lower virus signal is that the compound kills the cells,” says Garaigorta. For this reason, toxicity studies are carried out in parallel, as well as the mechanisms of action of the compounds.
The collection of substances has been obtained -and continues to be obtained- from 28 Spanish research centers and universities, two national hospitals and two foreign universities. Years of research condensed into thousands of molecules that can now have a second, or first, life. Marta Gutierrez of Peptidomimetics group of the Institute of Medicinal Chemistry (IQM-CSIC), is part of one of the teams in this network. “When we received the message from the management of our center, which was where the shipment was organized, we considered that we had to contribute our grain of sand”, he explains. This group of five researchers initially sent about 350 molecules in two different batches. From there, four families of promising molecules emerged, two of which have already obtained their European patent, and “six interesting compounds” in the words of Gutiérrez.
But the role of chemists is not limited to the mere delivery of molecules. The antiviral discovery process is an interactive and ongoing communication between chemists and biologists. “We medical chemists send the synthesized compounds, the platform evaluates their antiviral activity and they return the information to us,” explains Gutiérrez. With the information back, chemists improve the molecules with better antiviral activity and design other candidates from them.
The initial 8,000 compounds have resulted in four finalists that have already begun testing in mice for in vivo evaluation. Another four potential antivirals are waiting for it.
The team led by Miguel Ángel Martín Acebes, molecular biologist from the National Institute for Agricultural and Food Research and Technology (INIA-CSIC), is participating in this part of the chain. In a similar way to what happened to Marta Gutiérrez and her companions, Martín Acebes’ group felt “the responsibility to contribute” to the cause given their knowledge. So when the researcher received a call from Pablo Gastaminza to ask for his collaboration in this new project, the team signed up without hesitation.
The researcher has years of experience in the development and testing of antivirals in animals against flaviviruses, which are viruses transmitted by mosquitoes. Experimentation with these viruses, as in the case of SARS-CoV-2, also requires a biosafety laboratory 3, so the knowledge of the researcher and his team was even more valuable.
INIA has the Animal Health Research Center (CISA), located in Valdeolmos (Madrid). In Spain there are other biological safety facilities 3 that allow working with animals such as the Animal Health Research Center (IRTA-CReSA) or the Veterinary Sanitary Surveillance Center (VISAVET). As in the laboratory of the National Center for Biotechnology, the level of security at CISA requires more than half an hour of preparation and numerous levels of containment. These culminate in a suit that inflates with air to prevent any virus from sneaking into it if there is a cut. Even the mice are isolated with their own ventilation.
The antivirals are being tested in genetically modified mice so that they express the human ACE2 receptor in their cells. This is where the coronavirus sneaks into the body to infect it. Martín Acebes recalls that they had to genetically modify the animals, in collaboration with Alfonso Gutiérrez’s group from the INIA animal reproduction department, because it was not possible to get them on the market. It was the beginning of the pandemic and laboratories globally had mobilized in research.
“Our job is to infect the animal with SARS-CoV-2, treat it with the antiviral and then analyze its lungs,” says the biologist. Of the organs, they look at the viral load and their state of inflammation. An antiviral is expected to reduce the amount of virus and that the lungs are not as affected. The results of these first tests will still take time to arrive. “To say that something works or not, it is not enough to carry out an experiment”, says Garaigorta. Several tests with different concentration levels or different times of drug administration will be required to reach robust conclusions.
Here again the chemicals have a relevant role. As in the cases of cell cultures, the data obtained in mice returns to the chemistry laboratories to optimize the compounds. The screening platform is a project that does not intend to die with the end of the pandemic. All the investment in devices -such as the aforementioned multimodal reader-, personnel and knowledge wants to be used to test antivirals against other viruses. “The next potential challenge we see is mosquito-borne viruses,” says Gastaminza, who would pick up her previous line of work. Extending the platform to antibacterial compounds is also being considered. Superbugs already kill more than AIDS or malaria and in 30 years they will exceed the current mortality from cancer. Whether with viruses or bacteria, the working method will remain the same, with bidirectionality as the key.
This report has been carried out as part of the training activity of the CSIC-BBVA Foundation for Scientific Communication 2021 grants.
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