As the world awaits vaccines to bring the COVID-19 pandemic under control, scientists at UC San Francisco have devised a new approach to stopping the spread of SARS-CoV-2, the virus that causes the disease.
Led by UCSF graduate student Michael Schoof, a research team has created a fully synthetic, ready-to-manufacture molecule that narrows down important SARS-CoV-2 machinery that allows the virus to infect our cells. As reported in a new paper, now available on bioRxiv on the default server, experiments using live virus show that the molecule is among the most powerful SARS-CoV-2 antivirals yet detected.
In an aerosol formula they tested, called “AeroNabs” by the researchers, these molecules can be self-administered with a nasal spray or inhaler. Used once daily, AeroNabs can provide strong and reliable protection against SARS-CoV-2 until a vaccine becomes available. The research team is in active discussions with trading partners to increase the production and clinical testing of AeroNabs. If these tests are successful, scientists aim to make AeroNabs widely available as a cost-free, record-breaking drug to prevent and treat COVID-19.
“Much more effective than the wearable forms of personal protective equipment, we think of AeroNabs as a molecular form of PPE that can serve as an important stop until vaccines provide a more permanent solution to COVID-19,” he said. AeroNabs inventor Peter Walter, PhD, professor of biochemistry and biophysics at UCSF and a Researcher at the Howard Hughes Medical Institute. For those who cannot or do not respond to SARS-CoV-2 vaccines, Walter added, AeroNabs may be a more permanent line of defense against COVID-19.
“We have assembled an incredible group of talented biochemists, cell biologists, virologists and structural biologists to take the project from start to finish in just a few months,” said Schoof, a member of Walter Laboratory and co-inventor of AeroNabs.
Design inspired by Llama
Though fully designed in the lab, AeroNabs was inspired by nanobodes, antibody-like immune proteins that occur naturally in bulbs, camels, and related animals. Since their discovery in a Belgian laboratory in the late 1980s, the distinctive properties of nanobodes have intrigued scientists around the world.
“Although they function much like the antibodies found in the human immune system, nanobodes offer a number of unique advantages for effective therapy against SARS-CoV-2,” explained co-inventor Aashish Manglik, MD, PhD, assistant professor of pharmaceutical chemistry who often uses nanobodes as a tool in its research on the structure and function of proteins that send and receive signals across the cell membrane.
For example, nanobodes are an order of magnitude smaller than human antibodies, which makes them easier to manipulate and modify in the laboratory. Their small size and relatively simple structure also makes them significantly more stable than other mammalian antibodies. Plus, unlike human antibodies, nanobodes can be produced easily and cheaply: scientists insert genes that contain molecular schemes to build nanobodies into E. coli or yeast, and turn these microbes into high-yield nanobodic factories. The same method has been used safely for decades to mass produce insulin.
But as Manglik noted, “nanobodes were just the beginning for us. Although attractive in themselves, we thought we could improve on them through protein engineering. This eventually led to the development of AeroNabs.”
Spike is the key to infection
SARS-CoV-2 relies on its so-called spicy proteins to infect cells. These spikes study the surface of the virus and give a crown-like appearance when viewed through an electron microscope – hence the name “coronavirus” for the viral family that includes SARS-CoV-2. Spikes, however, are more than just a decoration – they are the essential key that allows the virus to enter our cells.
As an attractive tool, spikes can go from a closed, inactive state to an open and active state. When each of the virus particles is activated approximately 25 pins, those three “receptor binding domains,” or RBDs, become exposed and are prepared to attach to ACE2 (pronounced “ace two”), a receptor found in human cells of that line the lungs and airways.
Through a wrist-to-wrist interaction between an ACE2 receptor and an RBD lightning rod, the virus gains entry into the cell, where it then transforms its new host into a coronavirus producer. The researchers believed that if they could find nanobodes that inhibit spike-ACE2 interactions, they could prevent the virus from infecting cells.
Nanobodes Disable drops and prevent infection
To find effective candidates, scientists penetrated a recently developed library in Manglik’s laboratory with over 2 billion synthetic nanofibers. After successive rounds of testing, during which they set increasingly stringent criteria to eliminate weak or ineffective candidates, the scientists completed 21 nanobodes that prevented a modified form of the ACE2 interaction point.
Further experiments, including the use of cryo-electron microscopy to visualize the nanobody-spike interface, showed that more powerful nanobodes blocked interactions between point-ACE2 by strongly attaching themselves directly to peak RBDs. These nanobodes function a bit like a sheath that covers the RBD “key” and prevents its insertion into an ACE2 “blocker”.
With these findings in hand, researchers still had to demonstrate that these nanobodes could prevent the real virus from infecting cells. Dr. Veronica Rezelj, virologist at Marco Vignuzzi Laboratory, PhD, at the Pasteur Institute in Paris, tested three most promising nanobodies against live SARS-CoV-2, and found that nanobodes are extremely powerful, preventing infection even in extreme doses low.
The most powerful of these nanobisms, however, not only acts as a sheath over RBD, but also as a molecular mousetrap, choking on top in its closed, inactive state, which adds an extra layer of interaction protection. of point-ACE2 to lead to infection.
From nanobodet to AeroNabs
Scientists then engineered this dual-action nanobode in a number of ways to turn it into an even more powerful antiviral. In one set of experiments, they mutated each one of the amino-acid building blocks of the nanobode whose contacts dripped to detect two specific changes that yielded a 500-fold increase in strength.
In a separate set of experiments, they created a molecular chain that could bind three nanobodies together. As noted, each spike protein has three RBDs, each of which can be attached to ACE2 to allow the virus to enter the cell. The triple-bonded nanobode created by the researchers ensured that if one nanobode is attached to one RBD, the other two will be attached to the remaining RBDs. They found that this triple nanobode is 200,000 times more powerful than just a single nanobode.
And when they pulled in the results of both modifications, tying three of the strongly mutated nanobodys together, the results were “off the tables,” Walter said. “It was so effective that it surpassed our ability to measure its power.”
It would be easy to administer as an aerosol
This ultrapotent three-piece nanobode construction formed the basis for AeroNabs.
In a set of final experiments, the researchers placed the three-part nanobodies through a series of stress tests, subjecting them to high temperatures, turning them into a stable shelf powder, and making an aerosol. Each of these processes is very harmful to most proteins, but the scientists confirmed that, thanks to the inherent stability of nanobisms, there was no loss of antiviral power in aerosolized form, suggesting that AeroNabs are a potent antiviral SARS-CoV. -2 which may be practical to administer by means of a stable shelf inhaler or nasal spray.
“We are not alone in thinking that AeroNabs are an extraordinary technology,” Manglik said. “Our team is in ongoing discussions with potential trading partners who are interested in the production and distribution of AeroNabs, and we hope that human trials will begin soon. If AeroNabs proves to be as effective as we anticipate, they can help reshape the course of the pandemic throughout the world. “
Author: Additional authors include Bryan Faust, Reuben A. Saunders, Smriti Sangwan, Nick Hoppe, Morgane Boone, Christian Bache Billesbølle, Marcell Zimanyi, Ishan Deshpande, Jiahao Liang, Aditya A. Anand, Niv Dobzinski, Beth Shoshana Zha, Benjamin Vladisar UCSF Belyy, Silke Nock and Yuwei Liu; Camille R. Simoneau, Kristoffer Leon, Nevan J. Krogan, Danielle L. Swaney and Melanie Ott of the UCSF Institute for Quantitative Biology (QBI) and the J. David Gladstone Institute; Andrew W. Barile-Hill of Cytiva Life Sciences; Sayan Gupta and Corie Y. Ralston of Lawrence Berkeley National Laboratory; Kris M White and Adolfo García-Sastre from Icahn School of Medicine in Mount Sinai; and the QBI Coronavirus Structural Biology Consortium Consortium. Schoof, Faust, Saunders, Sangwan and Rezelj are the first authors of the manuscript.
funding: This work was supported by the UCSF COVID-19 Response Fund, a grant from Allen & Company, and sponsors of the UCSF Biomedical Research Program (PBBR), which was established with support from the Sandler Foundation. Other support included National Institutes of Health (NIH) Grants DP5OD023048, S10OD020054, S10OD021741, 1R01GM126218; Excellence Laboratory Grant ANR-10-LABX-62-IBEID; URGENCE COVID-19 Pasteur Institute fundraising campaign; Office of Science and Office of Biological and Environmental Research of the U.S. Department of Energy under contract DE-AC02-05CH11231; a postdoctoral fellowship of Helen Hay Whitney; Alfred Benzon Foundation; a gift from the Roddenberry Foundation; Howard Hughes Medical Institute; Pew Charitable Trusts; Fondi Esther A. & Joseph Klingenstein; and the Searle Scholars Program.
Disclosure: Schoof, Faust, Saunders, Hoppe, Walter, and Manglik are the inventors of a temporary patent outlining the anti-Spike nanobodes described in the manuscript.