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The COVID pandemic illustrated how urgently we need antiviral medications capable of treating coronavirus infections. To aid this effort, researchers quickly homed in on part of SARS-Cov-2’s molecular structure known as the NiRAN domain—an enzyme region essential to viral replication that’s common to many coronaviruses. A drug targeting the NiRAN domain would likely work broadly to shut down a range of these pathogens, potentially treating known diseases like COVID as well as helping to head off future pandemics caused by related viruses,

In 2022, scientists in China (Yan et al.) published a structural model describing exactly how this domain works. It should have been a tremendous boon for drug developers.

But the model was wrong.

“Their work contains critical errors,” says Gabriel Small, a graduate fellow in the laboratories of Seth A. Darst and Elizabeth Campbell at Rockefeller. “The data does not support their conclusions.”

Now, in a new study published in Cell, Small and colleagues demonstrate exactly why scientists still don’t know how the NiRAN domain works. The findings could have sweeping implications for drug developers already working to design antivirals based on flawed assumptions, and underscore the importance of rigorous validation.

“It is absolutely important that structures be accurate for medicinal chemistry, especially when we’re talking about a critical target for antivirals that is the subject of such intense interest in industry,” says Campbell, head of the Laboratory of Molecular Pathogenesis. “We hope that our work will prevent developers from futilely trying to optimize a drug around an incorrect structure.”

A promising lead

By the time the original paper was published in Cell, the Campbell and Darst labs were already quite familiar with the NiRAN domain and its importance as a therapeutic target. Both laboratories study gene expression in pathogens, and their work on SARS-CoV-2 focuses in part on characterizing the molecular interactions that coordinate viral replication.

The NiRAN domain is essential for helping SARS-CoV-2 and other coronaviruses cap their RNA, a step that allows these viruses to replicate and survive. In one version of this process, the NiRAN domain uses a molecule called GDP to attach a protective cap to the beginning of the virus’s RNA. Small previously described that process in detail, and its structure is considered solved. But the NiRAN domain can also use a related molecule, GTP, to form a protective cap. Determined to develop antivirals that comprehensively shut down the NiRAN domain, scientists were keen to discover the particulars of the latter GTP-related mechanism.

In the 2022 paper, researchers described a chain of chemical steps, beginning with a water molecule breaking a bond to release the RNA’s 5′ phosphate end. That end then attaches to the beta-phosphate end of the GTP molecule, which removes another phosphate and, with the help of a magnesium ion, transfers the remaining portion of the GTP molecule to the RNA, forming a protective cap that allows the virus to replicate and thrive.

The team’s evidence? A cryo-electron microscopy image that showed the process caught in action. To freeze this catalytic intermediate, the team used a GTP mimic called GMPPNP.

Small read the paper with interest. “As soon as they published, I went to download their data,” he says. It wasn’t there. This raised a red flag—data is generally available upon release of a structural biology paper. Months later, however, when Small was finally able to access the data, he began to uncover significant flaws. “I tried to make a figure using their data, and realized that there were serious issues,” he says. Small brought his concerns to Campbell and Darst.

They agreed. “Something was clearly wrong,” Campbell says. “But we decided to give the other team the benefit of the doubt, and reprocess all of their data ourselves.”

An uphill battle

It was painstaking work, with Small leading the charge. Working frame by frame, he compared the published atomic model to the actual cryo-EM map and found something striking: the key molecules that Yan and colleagues claimed to have seen—specifically, the GTP mimic GMPPNP and a magnesium ion in the NiRAN domain’s active site—simply were not there.

Not only was there no supporting image data, but the placement of these molecules in the original model also violated basic rules of chemistry, causing severe atomic clashes and unrealistic charge interactions. Small ran additional tests, but even advanced methods designed to pick out rare particles turned up empty. He could find no evidence to support the model previously produced by Yan and colleagues.

Once the Rockefeller researchers validated their results, they submitted their findings to Cell. “It was very important that we publish our corrective manuscript in the same journal that published the original model,” Campbell says, noting that corrections to high-profile papers are often overlooked when published in lower tier journals.

Otherwise, this confusion in the field could cause problems that reach far beyond the lab bench, Campbell adds—a costly reminder that rigorous basic biomedical research is not just academic, but essential to real-world progress. “Companies keep their cards close to their chests, but we know that several industry groups are studying this,” she says. “Efforts based on a flawed structural model could result in years of wasted time and resources.”

Researchers have long focused on the STING (Stimulator of Interferon Genes) pathway as a way to harness the immune system’s natural defenses against cancer. This pathway, which plays a key role in helping the body defend against potential pathogens, can be leveraged to trigger an innate immune response that targets cancer cells. However, a study published July 3 in the journal Nature Chemical Biology, led by biochemist Lingyin Li (Bluesky: @lingyinli.bsky.social), is spearheading a new school of thought.

Historically, research on STING has overwhelmingly focused on activating the pathway to recruit immune cells that attack tumors. However, inhibiting the pathway is understudied and mounting evidence suggests that overactivation of STING may turn the immune system against healthy cells. This dual nature of activation and inhibition of STING make the pathway a powerful but complex target for drug development – one that has yet to be viable in humans.

“Our study evaluated the effectiveness of H-151, the most advanced inhibitor of human STING, which has shown promise in reversing cognitive decline in mice but has failed to block human STING signaling in purified human blood cells,” said senior author Li, an Arc Institute Core Investigator and professor in the Biochemistry Department and ChEM-H Institute at Stanford University. “Our results show that in humans the target site of H-151 lacks a pocket that is found in mouse STING – without it, drug tailoring is incredibly challenging.” 

H-151 is a powerful STING inhibitor because once it binds to its target, it does not let go. It also targets a section of the STING pathway that is necessary for mouse STING signalling, but not humans. This fundamental mechanistic difference explains the discrepancies in inhibitor effectiveness between the two species, highlighting the limitations of using mouse models to predict human outcomes in STING-targeted therapy development. 

To circumvent this mismatch, Li’s team rigorously dissected the essential steps required for human STING signalling. The team found that oligomerization,the process where STING molecules assemble to trigger immune signaling, is an essential checkpoint prior to activation. Drawing inspiration from STING’s natural autoinhibitory mechanism, Li’s team proposed targeting STING by directly preventing oligomerization and developed a proof-of-concept molecule that mimics this mechanism and prevents STING from forming the large complexes necessary for immune activation in humans.

“This work emphasizes the need to focus on developing STING inhibitors exclusively in humans,” says Xujun Cao, one of the first authors on the paper and a postdoctoral fellow in the Li Lab. “Our method for uncovering this distinct druggable pocket provides a blueprint for others seeking to identify context-independent targets that can prevent STING autoimmunity.” 

“For STING to function, it needs to oligomerize flawlessly,” says Rebecca Chan, the paper’s other first author, a former graduate student in the Li Lab. “This discovery reveals why STING activation has such a high threshold-if it were easy to activate, our immune system would be attacking our own cells all the time. It’s an exquisitely controlled process, which is actually a good thing.“

Looking ahead,the Li lab will explore whether this understanding of STING inhibition could expand treatment possibilities beyond cancer immunotherapy. Their research will focus on potential applications for neurodegeneration and autoimmune diseases, while simultaneously advancing the development of promising molecular candidates as human-ready STING inhibitors for future clinical trials.