The COVID-19 pandemic has painfully highlighted the global need for effective antiviral drugs that can target not only SARS-CoV-2, but also a broader spectrum of coronaviruses. In this race against time, the scientific community quickly turned its attention to a key part of the virus's molecular machinery known as the NiRAN domain. This is an enzymatic region that is crucial for viral replication and is common to many coronaviruses, making it an extremely attractive target. A drug that could successfully block the NiRAN domain could act as a universal key for treating existing diseases like COVID, but also as a first line of defense against future pandemics caused by related pathogens.

Three years ago, in 2022, news of a published study (Yan et al.) resonated throughout the scientific community, which, as it seemed at the time, detailed the structural model of this domain's function. It was expected that this work would represent a huge step forward and provide a solid basis for the development of new generations of drugs. However, it soon became apparent that this long-awaited breakthrough was built on a faulty foundation.

Scientific detective work reveals key errors

"Their work contains critical errors," stated Gabriel Small, a research associate in the laboratories of Seth A. Darst and Elizabeth Campbell at the prestigious Rockefeller University. "The data simply do not support the conclusions they have drawn." It was Small, along with his colleagues, who in a new study published in the same distinguished journal Cell, showed in detail why scientists, despite previous claims, still do not know the exact mechanism of action of the NiRAN domain. This discovery has far-reaching consequences, not only for the academic community but also for the pharmaceutical industry where teams may have already begun designing antiviral drugs based on false assumptions. It also serves as a powerful reminder of the importance of rigorous scientific review and validation.

"It is absolutely crucial that structural models be accurate for medicinal chemistry, especially when we are talking about a critical target for antiviral drugs that is the subject of such intense interest in the industry," emphasizes Elizabeth Campbell, head of the Laboratory of Molecular Pathogenesis. "We hope that our work will prevent development teams from futilely trying to optimize a drug around an incorrect structure."

A promising target and the delusion that followed

At the time of the original article's publication in 2022, the Campbell and Darst labs were already deeply involved in researching the NiRAN domain and its importance as a therapeutic target. Both labs study gene expression in pathogens, and their work on the SARS-CoV-2 virus is partly focused on characterizing the molecular interactions that coordinate viral replication.

The NiRAN domain plays a key role in a process known as "capping" on the virus's RNA. This process places a protective structure at the beginning of the virus's genetic material, allowing it to successfully replicate and survive within the host cell. One of the ways it does this is by using a molecule called guanosine diphosphate (GDP), and this process has already been described in detail and its structure is considered solved. However, the NiRAN domain can also use a related molecule, guanosine triphosphate (GTP), to form the protective cap. In their quest to develop comprehensive antiviral drugs that would completely "shut down" the NiRAN domain, scientists were extremely interested in discovering the details of this second, GTP-mediated mechanism.

In the controversial 2022 study, the researchers described a precise series of chemical steps. According to their model, a water molecule breaks a bond to release the 5'-phosphate end of the RNA. This end then binds to the beta-phosphate end of the GTP molecule, which, with the help of a magnesium ion, results in the transfer of the remaining part of the GTP to the RNA, thus forming a protective cap that allows the virus to continue multiplying. As crowning evidence, the team provided an image obtained by cryo-electron microscopy (cryo-EM), a cutting-edge technique that allows for the visualization of molecules at near-atomic resolution, which had supposedly caught the process "in the act." To "freeze" this key step, they used a synthetic GTP analog, the molecule GMPPNP.

Red flags and the painstaking path to the truth

Gabriel Small read the paper with great interest. "As soon as they published it, I tried to download their data," he recalls. But the data wasn't there. In the world of structural biology, where immediate data availability is standard, this was the first red flag. Months later, when he finally gained access to the data, he began to discover significant flaws. "I tried to create a map using their data and realized there were serious problems," says Small. He shared his concerns with Campbell and Darst, who agreed with his assessment.

"Something was obviously wrong," says Campbell. "But we decided to give the other team the benefit of the doubt and reprocess all their data from scratch ourselves."

A painstaking job ensued, with Small at the helm. Analyzing frame by frame, he compared the published atomic model with the actual cryo-EM density map and discovered something astonishing: the key molecules that Yan and colleagues claimed to have seen – specifically, the GTP analog GMPPNP and the magnesium ion in the active site of the NiRAN domain – simply weren't there. There was no imaging data to confirm their presence. Moreover, the positioning of these molecules in the original model violated basic rules of chemistry, causing severe atomic clashes and unrealistic charge interactions. Small also conducted additional, advanced tests designed to identify rare particles, but they too came up empty. He could not find a single piece of evidence to support the model presented by Yan and colleagues.

The importance of scientific correction and a look into the future

After the Rockefeller researchers confirmed their results, they sent their correction to the journal Cell. "It was very important that we publish our corrective manuscript in the same journal that published the original model," Campbell points out, noting that corrections to high-profile papers are often overlooked if published in lower-tier journals. Otherwise, the confusion in the scientific field could cause problems that extend far beyond the laboratory bench. This is an expensive reminder that rigorous basic biomedical research is not just an academic exercise, but is crucial for real progress in medicine.

"Companies keep their cards close to their chest, but we know that several industrial groups are studying this area," adds Campbell. "Efforts based on a faulty structural model could result in years of wasted time and resources." This situation highlights the critical role of the peer-review process and the self-correcting nature of science. Although mistakes can happen, transparency and a willingness to correct them are key to maintaining integrity and progress. The search for a complete understanding of the NiRAN domain continues, now with a clearer picture of what is not known, which takes scientists a step back but sets them on the right path toward the ultimate goal – a powerful antiviral drug of the future.

Source: Rockefeller University