Discovery facilitates search for drug to sabotage replication of SARS-CoV-2

Scientists at a FAPESP-funded research center investigated the mechanism that produces the main enzyme involved in the virus’s replication in cells. The findings are reported in the Journal of Molecular Biology.

By Maria Fernanda Ziegler  |  Agência FAPESP – Scientists affiliated with the Center for Innovation in Biodiversity and Drug Discovery (CIBFar), in Brazil, have discovered details of the process of maturation of 3CL, the main protease involved in replication of the novel coronavirus. Their findings are reported in an article published in the Journal of Molecular Biology. The discovery facilitates the search for medications capable of sabotaging this process as soon as it begins.

“In the 18 months since the pandemic was declared, at least half a dozen vaccines have been approved for clinical use, but no drugs have proved effective and safe. Antivirals are indeed harder to develop, but it’s very important to obtain medications for COVID-19 because the virus can escape even the best vaccines,” said Glaucius Oliva, principal investigator for CIBFar, a Research, Innovation and Dissemination Center (RIDC) funded by FAPESP and hosted by the University of São Paulo’s São Carlos Institute of Physics (IFSC-USP).

The article describes the molecular mechanism whereby the main protease in SARS-CoV-2 matures, enabling the virus to self-assemble and replicate its genetic material (RNA) inside host cells.

“The more we understand about the metabolism of the virus and the stages of its replication, the better we’ll be able to identify targets in the process and develop molecules that block its inception,” said Gabriela Noske, a PhD candidate at CIBFar and first author of the article.

According to Oliva, the study was basic science but has immediate applications. “Unlike other viruses, such as zika, dengue or yellow fever, this coronavirus has a main protease that acts monomerically [as an isolated molecule]. To activate and multiply the virus’s RNA, the protease has to become dimeric. In other words, two copies of the protease are required so that it can cleave itself and the other proteins responsible for the viral metabolism inside the cell,” he explained.

Oliva leads a multidisciplinary project supported by FAPESP, with a team of researchers at the University of São Paulo’s Biomedical Sciences Institute (ICB-USP), São Carlos Institute of Chemistry (IQSC-USP) and Ribeirão Preto School of Pharmaceutical Sciences (FCFRP-USP), as well as colleagues at São Paulo State University (UNESP) and the University of Campinas (UNICAMP), in search of antivirals to treat COVID-19 (more at: agencia.fapesp.br/33509/).

Multiple stages

The RNA in SARS-CoV-2 is protected by an envelope made up of lipids and proteins, including the famous spikes that form the crown-like structure referred to in the name of the coronavirus family (Coronaviridae). The virus uses its spikes to penetrate cells. Once inside a cell, the capsid releases its RNA and begins to replicate. 

The structural proteins no longer play a fundamental role, especially RNA transport and immune system evasion, during this stage. Non-structural proteins are responsible for the metabolism of the virus once it has penetrated host cells.

“The virus must make copies of its RNA in order to replicate, but it doesn’t have all the requisite mechanisms and has to take over functions of the invaded cell. Other metabolic functions specific to the virus are performed by non-structural proteins, which include the main protease and 15 other molecules. Our study focused on the main protease,” said André Godoy, a researcher at IFSC-USP and a co-author of the article.

Structural proteins are typical targets for vaccines, he added, whereas non-structural proteins are a reference for antiviral drugs such as the cocktails used to treat HIV/AIDS, which attack the virus’s protease, among other targets.

The discovery that the main protease in SARS-CoV-2 undergoes different stages until it becomes mature and only then helps the virus replicate in infected cells was possible thanks to research conducted at Sirius, Brazil’s most complex science facility. An experiment performed last year by Godoy and researcher Aline Nakamura inaugurated the first workstation in the fourth-generation particle accelerator now being finalized at the Brazilian Center for Research in Energy and Materials (CNPEM) in Campinas, São Paulo state (more at: agencia.fapesp.br/34526). 

In three days, with the aid of a powerful beam of synchrotron light, they were able to determine the structure of more than 200 crystals belonging to two proteins from SARS-CoV-2, including the main protease in various forms and complexed with a range of ligands.

“Alongside the spike, the main protease is the most studied protein in SARS-CoV-2. Until now, no one knew how the virus processed two copies of the protease to create a region in its structure known as the ‘active site’, where it processes the other proteins synthesized from the information contained in its genome,” Godoy said. “Other viruses also have this trait. The novelty of this study is our understanding of how the process unfolds in its entirety.”

SARS-CoV-2’s RNA is transcribed by the ribosomes of each host cell so that it can begin producing non-structural proteins, the researchers explained. First, however, its structural and non-structural proteins must be decoded many times, because it has a single strand of RNA. This entails producing a long polyprotein, which is repeatedly cleaved to create the 16 molecules responsible for its metabolic mechanisms.

“The viral RNA is a very simple structure and encodes all the proteins in a chain like a long beaded necklace,” Oliva said. “They’re produced in the ribosomes of invaded cells as a single polyprotein, which has to be cut up into smaller pieces. The problem is that the cleaving is done by the main protease, which is also part of the ‘necklace’, so it first has to cleave itself and only then can cleave the others.”

The researchers analyzed the main protease using one of Sirius’s beams of synchrotron light, a type of very bright electromagnetic radiation widely used in structural biology. The analysis showed that the structure of the protease changes when it cleaves itself. “We found that to cleave one end of itself [called the C-terminal] it needs a dimeric partner, a similar protein capable of cutting the front end. It can cleave one end on its own but not the other, so it binds to the partner [the other mature protein], and can then do the requisite cutting,” Godoy said.

In sum, main protease maturation after a stage in which it is part of a long chain of proteins enables it to cleave itself at the N-terminal, join up with the other chain in the cell to form a dimer, and then process the C-terminal to form its mature active stage.

Light at the end of the tunnel

Pharmaceutical company Pfizer is conducting clinical trials of a drug that could block the main protease in its mature stage, Godoy added, and Merck is investing in a clinical trial of a molecule that could block the polymerases that synthesize copies of the viral RNA.

As Oliva noted, any antiviral must bind to a receptor. “The whole world is looking for candidate drugs that can stick to the protease, but people are looking at its structure when mature,” he said. “We showed that variations in previous stages of the protease could be more interesting targets for drug discovery. It’s best to remove weeds before they grow.”

The article “A crystallographic snapshot of SARS-CoV-2 main protease maturation process” is at: www.sciencedirect.com/science/article/pii/S0022283621003429.