Since the coronavirus pandemic began more than a year ago, only one antiviral drug, remdesivir, has been approved in the United States for treatment of COVID-19, but it barely works and is toxic to the liver.
After conducting a one-of-a-kind screen of drugs already approved or in the approval process in the United States, University of California, Berkeley, researchers have now found 20 compounds that, in combination with remdesivir, are much better than remdesivir alone in protecting human lung cells from SARS-CoV-2, the virus that causes COVID-19. The drugs could potentially be used in combination with lower doses of remdesivir to reduce the toxicity and dramatically improve the efficacy of the treatment.
“The data that we have is preliminary — it is in vitro data — but it is very promising, in the sense that, in tissue culture, the synergy is remarkable,” said Sarah Stanley, associate professor of infectious diseases and vaccinology at the UC Berkeley School of Public Health. “In terms of in vitro work, I haven’t seen anything more promising than what we’ve found, and that is really exciting. But the rubber meets the road in vivo, and we haven’t done that yet.”
Ironically, several of these drugs are currently used to cure infections by the hepatitis C virus, something remdesivir was designed for, but fell short of, in drug trials. The screen demonstrated that one three-drug combo — remdesivir with Epclusa, a trade-named, two-drug combination of sofosbuvir and velpatasvir used to treat hepatitis C — was more than 25 times better than remdesivir alone in preventing the virus from infecting human lung cells.
The results of the screen of more than 1,400 drugs were posted to BioRxiv in September, but since then, the UC Berkeley researchers have conducted further studies that confirm that this three-way treatment also prevents the virus from replicating within freshly isolated human lung cells, in addition to cultured lung-cell lines. The team is now planning studies in mice.
“I was thinking, first of all, that we would only find very few compounds, but we actually found a lot. That was definitely a surprise,” said Julia Schaletzky, executive director of UC Berkeley’s Henry Wheeler Center for Emerging and Neglected Diseases (CEND) and its Drug Discovery Center. “With this combinatorial screen, we could identify parallel pathways that the cell uses to circumvent the virus: Where if you knock out one, the other is still active, but if you knock out both at same time, you are dead. We were looking for molecules that, on their own, didn’t have much activity, but together, a whole lot of activity. That gave us a completely new avenue to find drugs that is different from the usual screens.”
The results complement the findings of Columbia University researcher Jingyue Ju, whose studies of the structure of the hepatitis C drugs sofosbuvir and velpatasvir a year ago suggested that they should interfere also with the new coronavirus. Subsequent studies in cell culture indicated that sofosbuvir in combination with daclatasvir, a drug similar to velpatasvir, demonstrated inhibitory activity for SARS-CoV-2 in lung cells, Ju said.
“There is a large-scale clinical trial underway of these hepatitis C drugs, like sofosbuvir and daclatasvir, but I think with an additional molecule added — remdesivir — it is probably going to improve the activity of the combination drugs, in particular, because the Berkeley work looked very systematically at this combination with remdesivir,” said Ju, professor of chemical engineering and pharmacology and director of Columbia’s Center for Genome Technology and Biomolecular Engineering.
Providing the foundations for clinical trials
After the UC Berkeley team’s results appeared on BioRxiv, Ju reached out to the UC Berkeley team and is currently collaborating with it to understand how these hepatitis C drugs and remdesivir interfere with the coronavirus’s lifecycle. Such molecular studies, combined with UC Berkeley’s cellular assays, are essential foundations for initiating clinical trials, he said.
Julia Schaletzky, CEND executive director, in UC Berkeley’s Drug Discovery Center with screening scientist Eddie Wehri. The researchers teamed up with colleagues in the campus’s BSL3 lab to find potential treatments for COVID-19. (Photo courtesy of CEND)
Schaletzky, Stanley and their colleagues urge the government office that coordinates the study of COVID-19 treatments and vaccines — the Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) public-private partnership — to fast-track these drugs for clinical evaluation.
Schaletzky suspects that the drugs work together, but not singly, because they target separate aspects of the virus’s machinery to copy its RNA genome. While remdesivir and sofosbuvir screw up the machinery that copies the RNA — like putting a slug in a vending machine, she said — velpatasvir could interfere with proofreading of the RNA. Together, that leads to defective RNA that is not fixed by the proofreading enzyme, stopping viral replication in its tracks. Recent data from Ju’s team indicated that velpatasvir and related molecules have inhibitory activity for both the SARS-CoV-2 polymerase and exonuclease, the enzymes that, respectively, carry out the RNA synthesis and proofreading.
Schaletzky noted that if this drug combination proves effective against COVID-19, delivery will still be a problem. Both the hepatitis C drugs and remdesivir work by interfering with viral replication, so they are most effective during the very early stages of infection, when viruses multiply quickly inside cells. Currently, however, remdesivir is approved for use only in hospitalized patients, most of whom are suffering from that early burst of virus production, but are no longer pumping out lots of new viruses, making the drug less effective.
In addition, remdesivir is delivered by an intravenous infusion, and the hepatitis C drugs are given in pill form. Both could be more effective if delivered directly to the first place the virus encounters: the airways and the lungs.
“My vision is to have an inhaler. People would just have it at home proactively, and once you feel something coming on, you just take a puff,” Schaletzky said. “Maybe you could hit it bad enough so that it it doesn’t break out into full disease.”
Inhalation strategies for delivering remdesivir are currently underway at a few medical centers in California, Schaletzky said, and she plans to pursue research on inhalation studies of hepatitis C drugs in mice.
Schaletzky and Stanley started working together in April, not long after the state instituted a shelter-in-place order that left both women at home with their school-aged kids. Schaletzky has a 10-year-old daughter and an 8-year-old son, while Stanley has a 10-year-old daughter.
“Women’s careers have really been taking a hit since COVID,” Schaletzky said, referring to studies showing that female scientists published a lower percentage of articles in 2020 than in a typical year, presumably because of childcare conflicts. “It has to do with responsibilities for kids — the teachers are out of the picture now, more or less, and you have to teach them yourself.”
Both women are married to UC Berkeley faculty members who also have full-time teaching and research responsibilities, requiring complex coordination of family and work schedules.
“My husband is a professor, too, but we split childcare responsibilities pretty equally, so I am one of the lucky ones,” said Stanley, who estimates that she spends two hours less per day on her research and teaching because of childcare duties, even with a very independent 10-year-old. “I think it is really hard if you have little kids and you don’t have child care, you don’t have help.”
Nevertheless, when the seriousness of the pandemic became obvious, Stanley and Schaletzky were quick to pivot from their normal research to a collaboration that could potentially discover new treatments for a disease that is 10 to 100 times more lethal than the seasonal flu.
It was good timing. Schaletzky had spent the previous two years setting up a drug discovery lab within CEND to allow faculty to access industry-quality infrastructure to screen small molecules in search of new treatments for diseases. Stanley, who studies tuberculosis, has one of the San Francisco Bay Area’s few Biosafety Level 3 (BSL3) labs, which are required to safely study pathogens as infectious and potentially deadly as SARS-CoV-2.
Within weeks, they had mobilized students and colleagues to assist — in particular, postdoctoral researchers Eddie Wehri, Scott Biering and Livia Yamashiro, graduate student Erik Van Dis and undergraduate Xammy Nguyenla. Stanley had presciently started to culture human lung epithelial cells (Calu 3 cells), which are easily infected by SARS-CoV-2, so she and Biering were quickly able to grow lung cells in well plates — 384 wells per plate — and deliver them to Schaletzky. She and Wehri then added different drugs to each well and dosed each with remdesivir — about 20% of the dose that would kill the virus.
The drugged plates were then sent to the BSL3 lab to be infected with SARS-CoV-2 by Stanley’s team. After three days, the cell cultures were tested to see how many cells had survived. The number of viable cells was assessed by measuring the amount each well glowed under UV light.
The team ran duplicate screens, along with controls, to confirm their findings and also reached out to the drug companies that make remdesivir — Gilead — and the hepatitis C drugs — both Gilead (Epclusa) and Merck (Zepatier, a combination of grazoprevir and elbasvir). The firms suggested additional experiments, which have been completed, and the work has been submitted to the journal Nature Communications, which has shown interest in publishing the research.
Despite vaccines, drug still needed for COVID-19
Schaletzky and Stanley also led a team that conducted screens of single drugs against SARS-CoV-2, finding seven not already identified, including a drug called B02 that is currently in clinical trials for other uses. A follow-up screen showed that B02 also works synergistically with remdesivir, boosting its effectiveness more than tenfold. The results have been submitted to the journal ACS Infectious Diseases.
The researchers noted that the rollout of vaccines to protect against COVID-19 does not mean that drugs to fight the diseases are no longer needed. The pandemic is expected to last through 2022, and the vaccinated population may not reach percentages essential for herd immunity for many months.
“I think a therapeutic option is important, in addition to the vaccine,” Schaletzky said. “We have to have a whole set of tools against this virus; it can’t just be one thing.”
Schaletzky is pleased that the Drug Discovery Center was available to use in the fight against COVID-19.
“It shows how investing in some kind of infrastructure for translational science on campus can be broadly fruitful and comes at the right time,” Schaletzky said. “I am happy about that, and that we pivoted really fast in the beginning and got started with this project.”
Stanley is excited by a new project initiated by a consortium of Northern California sequencing, epidemiology and BSL3 labs to test new variants of SARS-CoV-2 that are popping up around the world and could impact the spread of the pandemic and the effectiveness of vaccines and diagnostic tests for the virus.
“We are going to bring these new viral strains into BSL3 labs and test them to see whether they are more transmissible, using mouse and hamster models, to see whether they evade antibody responses, and to see if they are more virulent,” she said. ”I think this is super-important and very timely for public health. The idea that a new variant may arise that is able to evade the vaccine-induced antibody response is especially frightening.”