Lab-made 'miniproteins' could block coronavirus from infecting cells

While the world waits for a Covid-19 vaccine, many researchers are focused on developing effective therapeutics that can be rolled out quickly and cheaply. Monoclonal antibodies — a potentially promising laboratory-manufactured therapy modelled on antibodies extracted from the blood of recovering patients — made headlines recently when President Trump received a not-yet-approved antibody cocktail made by the company Regeneron. And pharmaceutical giant Eli Lilly recently announced that its monoclonal antibody reduced the risk of hospitalisation in 300 people who had mild or moderate symptoms of Covid-19, in a small clinical trial.

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But David Baker, a biochemist at the University of Washington’s Institute for Protein Design, and his colleagues think they can produce an even better therapy. They have designed a synthetic peptide — a short string of amino acids, the building blocks of proteins — 20 times smaller than a monoclonal antibody that is designed to bind to the infamous “spike” protein on the surface of the SARS-CoV-2 virus particle. Doing so would directly block the virus from binding to the ACE-2 receptors on human cells, functioning much like an antibody produced by an infected person’s immune system. Baker and his colleagues described these “miniprotein inhibitors” in September in Science. Although the study only tested these synthetic proteins in the lab, mixing viral particles with monkey cells in vitro, he says that unpublished data show they can protect mice and hamsters from SARS-CoV-2 infection.

“We built these [tiny proteins] from scratch based on ‘first principles,’ using computers to model all the biochemical details of a theoretical protein that could stick to the virus,” explains Baker, who was awarded a $3 million Breakthrough Prize earlier in September for his decades of work pioneering the field of synthetic protein design. His team used computers to digitally design more than two million candidate “miniproteins,” crunched the data using algorithms, sifted out 118,000 candidate genes that encode these proteins, manufactured the proteins from scratch, and tested them directly against the virus in the lab—finding that seven designs could effectively bind to and thus disable the virus.

Over the course of 3.5 billion years, evolution has produced an incredible array of proteins and peptides. In recent years biochemists have tracked down and used some of these to create new drugs, such as Eptifibatide, an antiplatelet drug administered to prevent heart attacks whose active ingredient is extracted from the venom of the southern pygmy rattlesnake. The Protein Data Bank, an online repository of protein sequences and educational tools, contains the amino acid sequences and full 3-D structures for more than 160,000 peptides and proteins—but the natural world contains hundreds of millions of proteins.

“It’s very challenging to discover in nature a peptide that does exactly what you want it to do,” explains Gaurav Bhardwaj, also a biochemist at the Institute for Protein Design, but who was not involved in the Science study. He is trying to design a bespoke peptide that would prevent SARS-CoV-2 from replicating within human cells. “Now we can computationally explore the possible design configurations for a peptide in order to perform the exact functions that we want.”

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Every protein’s function depends on its structure. Interactions between the atoms of the protein’s amino acids cause these chains to self-assemble in less than a second into a complex array of spirals and pleats. As the chain of amino acids grows, these helices and rippled sheets stack on top of and around one another into a dizzyingly complex series of folds, and it is these folds that give proteins their shape and function. Yet figuring out how one amino acid sequence turns into a specific fold has been a torturously difficult task, and it was only in the 1990s—with ever expanding databases of protein information — that scientists could begin to link sequence to form.

“We can make up completely new proteins that have never been seen in nature because we now understand the nature of protein folding,” Baker says. “Our ability to use computers to design ‘de novo’ proteins has really only come into its own in the last few years – we might not have been able to apply ourselves to Covid-19 if the pandemic had happened five years ago.”

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Many organisations, including the Gates Foundation, the Open Philanthropy Foundation, and most recently, the committee of the Breakthrough Prize, have supported this work. Although monoclonal antibodies for SARS-CoV-2 are already in clinical trials, Baker says his miniprotein inhibitors have even greater potential to tackle the pandemic because they are 20 times smaller and thus would be cheaper to produce quickly and consistently.

Synthetic peptides show enormous potential to be scaled up at low cost to produce robust, bespoke treatments, says Sarel Fleishman of the Weizmann Institute of Science in Israel, who was not involved in the study. But they are still in uncharted territory, putting them at a disadvantage in the race for a cure, he says.

“The major advantage of monoclonal antibody treatments is that they are completely ‘human,’ meaning they are already compatible with our immune systems. So they carry a lot less risk than synthetic proteins,” he says.

Crossing regulatory hurdles will be a lot more straightforward with monoclonal antibodies, he says, because regulators will already understand what they are dealing with compared with a new and unproven technology.

Although synthetic peptides have enormous potential, we need to be cautious about being overly optimistic, adds biochemist Erik Procko of the University of Illinois, who worked as a postdoctoral researcher in Baker’s team, but was not part of this specific study. “The pharmacokinetics of miniproteins” — the ways the human body can metabolize, absorb and excrete them—“will be a barrier to their usefulness as drugs,” Procko says. “Eli Lily’s antibody drug persists in the body for a month; it will be challenging for a small designed miniprotein to match that stability in the blood.”

Baker acknowledges that both Fleishman and Procko are correct: “our miniproteins will have to go through the same scrutiny of clinical trials as monoclonal antibodies,” he says, “though it is worth noting that regulatory bodies like the FDA have vast experience with all sorts of drug and therapeutic modalities.”

Both Procko and Baker note that miniproteins will very likely need to be administered directly to the lungs by inhalation. Researchers at the University of California, San Francisco, have designed just such an aerosol formulation. The technology, called “AeroNabs,” would be administered by an inhaler or nasal spray. Roughly three times larger than Baker’s miniproteins, the U.C.S.F. ones are modelled on “nanobody” particles found in the immune systems of animals such as llamas, and function similarly: they bind to SARS-CoV-2’s “spike” protein and prevent it from fusing with the ACE-2 receptor on human cells.

“Monoclonal antibodies are unlikely to reach the airway spaces of the lungs when given as an injectable drug,” explains Aashish Manglik of U.C.S.F., part of the team that developed AeroNabs. He and his colleagues described their innovation in the preprint database bioRxiv in August. Only two per cent of monoclonal antibodies injected into the bloodstream tend to reach the pulmonary spaces, the regions of the lungs through which the virus gains entry in most people — but a drug delivered via aerosol would be able to reach these air sacs, and thus could serve both as a therapeutic and a prophylactic, Manglik says. “We see this as being useful with patients who are in the early stages of infection, or with people at high risk of becoming infected, such as frontline and healthcare workers,” he says. “However, from a technical perspective, what Baker has been able to pull off — designing everything prospectively and not based on an existing structure in nature — is just phenomenal. It’s an exciting time in protein science.”

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Beat Christen of the Institute of Molecular Systems Biology in Zurich, who was not involved in Baker’s or Manglik’s research, agrees it is an exciting time. “Synthetic biology is progressing very fast in developing vaccines and therapeutics — in a very short time frame we have seen many things pushed to the forefront, and the corporate world is reacting with many spinoffs and startups that have pivoted to this field,” he says.

With an increase in corporate interest, however, may come a decrease in public trust — as happened with genetically modified food two decades ago. The technology was largely seen as expensive and unnecessary, driven by corporate profit motives rather than public need. Synthetic peptides — many entirely “unnatural” and “never seen before on earth” — risk falling into the same trap.

“But with Covid-19, there is a clear, huge challenge facing humanity,” Christen says, “and if synthetic biology can contribute with new solutions and new therapies, people will easily see the need for it.”