Effective in 70 percent of cases, in 90 percent, even in 95 percent: The recent triumphant announcements from AstraZeneca, BioNTech and Moderna, rooted in preliminary results from ongoing clinical trials, make it look as though a vaccine against the coronavirus could soon be approved.
But there is another pharmaceutical footrace going on – one which will be just as crucial in the fight against SARS-CoV-2 and other, similar viruses. Largely under the public radar, researchers around the world are searching for a drug therapy to help those who have already contracted the virus. The hope is that patients will be able to take an inhibitor that binds to an important virus enzyme and paralyzes it. That, researchers hope, would then prevent the virus from replicating.
The German virologist Meike Dittmann of the New York University School of Medicine has tested a solution from Pfizer in her laboratory in Manhattan. In a petri dish, it was able to significantly reduce SARS-CoV-2 replication. “I believe the inhibitor is quite promising in the medium term,” Dittmann says.
Other laboratories have also reported encouraging findings. The University of Tübingen is conducting research similar to Dittmann’s, and the German biochemist Christoph Nitsche at Australian National University in Canberra has assembled a team to figure out how to block the important enzyme. The number of experiments with the same focus has exploded around the world.
But what should be the focus of such an agent? SARS-CoV-2 forces its way into host cells and takes control. The cells are reprogrammed and forced to produce viral genetic material and viral proteins – the building blocks of more viruses. A certain enzyme known as a protease, which has the ability to break down proteins, plays a key role here (see graphic).
It is possible to interrupt viral replication at a number of different stages in the process, but many researchers believe that protease present the best target. “It is my favorite target in the life-cycle of the virus,” says Meike Dittmann. “The virus needs the protease immediately after entering the cell, and if the protease is deactivated, the life-cycle is stopped before the virus can even begin to replicate.”
There are other advantages as well. The viral protease is significantly different from proteases found in humans, meaning an inhibitor would likely only bind the correct target. That would make undesirable side effects extremely unlikely. Furthermore, the coronaviruses that have thus far been identified all possess proteases that are more or less identical. That means that an inhibitor developed for SARS-CoV-2 could also be effective against other members of this family of viruses.
Plastic Balls and Sticks
Biochemist Rolf Hilgenfeld of the University of Lübeck saw all of this coming more than 20 years ago. He has always been particularly interested in proteases, he said during a March visit with him at his laboratory. “Back then, hardly any work was being done on coronavirus proteases. I thought: I’d really like to see one of these proteases in 3-D.”
It took a while, and it required the assistance of a Ph.D. student and a cooperation partner. Relying on biotechnology, Hilgenfeld and his team produced the protease of a harmless coronavirus. Then, the researchers were able to crystallize it, and they then exposed the crystals to X-rays. The rays were bent, creating a pattern from which a 3-D structure could be calculated. They were then able to examine the structure to determine which part could best be targeted with an inhibitor.
At his laboratory, Hilgenfeld proudly showed off a large model of the protease. Students of his had assembled it using plastic balls and sticks.
When the genetic sequence of SARS-CoV-2 was published on Jan. 11, 2020, Hilgenfeld immediately examined the sequence for the relevant protease. As expected, it was almost identical to the proteases of previously known varieties of coronavirus. There were, however, a couple of differences. Together with researchers Linlin Zhang and Xinyuanyuan Sun, Hilgenfeld got to work.
Just a few weeks later, the team from Lübeck submitted a manuscript to the journal Science, which was then published in March. In the article, they and other scientists from additional research institutes in Germany and China described in precise detail how the SARS-CoV-2 is constructed. Not only that, they were able to identify the site on the protease where it could best be inhibited. And they presented a possible compound for doing so.
Even as biochemist Hilgenfeld, who turned 66-years-old in April, was receiving his retirement certificate from the University of Lübeck, experts around the world pored over his paper. It provided a perfect manual for producing an inhibitor.
A physicist from the German Electron Synchrotron (DESY) in Hamburg came to Lübeck to pick up the protease gene. Together with scientists from other research institutions, the DESY physicist initially produced a large number of the protease. They then tested out 5,575 previously developed medical substances and allowed the preparations to crystallize. Using X-rays, they were then able to determine which of the substances had bonded with the protease. Ultimately, Alke Meents, one of the researchers who took part in the effort, says that six of the substances had formed a bond. She says that the project is currently in talks with a large pharmaceutical company about establishing a cooperation.
“The Most Promising Agent”
Meanwhile, the companies Novartis and Takeda are participating in the COVID R&D Alliance, a collaboration involving more than 20 of the most experienced medical drug researchers in the world. According to Takeda, there are seven different working groups, including one for “novel, small molecule antivirals.”
The researchers are hoping to “curb potential outbreaks in the future by developing a bespoke, antiviral therapy that can be used against any coronavirus that poses a threat in the future,” says Jay Bradner, president of the Novartis Institute for Biomedical Research. Some interesting inhibitors have already been found in cooperation with a university, he says.
Virologist Meike Dittmann has also joined a research alliance. She quickly realized that many companies don’t even have a virology department any longer. Dittmann, though, had access to a laboratory with a safety level of BSL-3 and offered to help. “We then chose what from our point of view was the most promising agent,” she says.
They opted for a substance called PF-00835231, which Pfizer had developed several years earlier for proteases, but then stopped pursuing because of a lack of demand. Following the successful experiments in Dittmann’s lab, Pfizer even apparently began testing the substance on people.
But PF-00835231 has a disadvantage: It has to be administered via injection. “Intravenous administration has to be done in a hospital, likely over the course of several days, which means it isn’t universally available,” says Dittmann. On top of that, the inhibitor has to be administered at the very beginning of an infection, when the virus is multiplying fastest.
The hurdles of availability and timing are most easily cleared with an inhibitor that can be administered orally. As such, most researchers are targeting such a drug.
“More and More Parameters”
The active agent must be able to find its way to the protease in the watery environment found in cells. But the protease is protected by a lipid membrane, meaning the substance cannot be too water soluble. “Otherwise, it wouldn’t be able to penetrate the membrane,” says the biochemist Hilgenfeld. “But it also can’t be too liposoluble, otherwise it will get stuck in the membrane.”
The next problem: Many cells seek to defend themselves against foreign substances. They are equipped with a kind of pump (P-glycoprotein), which pushes the undesirable substance back out of the cell. To counteract that reaction, it could be possible to combine the protease inhibitor with a P-glycoprotein inhibitor, Hilgenfeld says with a sigh. “But then, of course, you end up with more and more parameters.”
Researchers, of course, are no strangers to such complexities, and for much of his career, Hilgenfeld frequently faced an uphill battle in securing funding for his seemingly exotic research into coronaviruses. He led the Institute for Biochemistry in Lübeck for 17 years before he was sent into retirement – right at a time when he was suddenly in greater demand than ever before. The China Pharmaceutical University in Nanjing, for example, sought to recruit him and offered him an entire research group if he came to China. The Max Planck Institute of Molecular Physiology in Dortmund also wanted Hilgenfeld.
At the last moment, though, the Ministry of Education, Science and Cultural Affairs in his home state of Schleswig-Holstein found a way to continue paying him a salary. As a senior professor, he is now in the process of moving his protease laboratory into a new building on the university campus in Lübeck. He has also joined a consortium for the development of coronavirus therapies, which is financed to the tune of 77.7 million euros by the European Union and 11 different pharmaceutical companies.
The project will come to an end after five years, as will Hilgenfeld’s professorship. Does he believe that the first protease inhibitor will have been approved by then? “I certainly hope so,” says Hilgenfeld. “So much effort is being invested in the project that something has to come out of it.”