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on March 12, 2021 A CALL TO ARMS Researchers are testing an arsenal of weapons against the pandemic coronavirus

n March 2020, as the scope of the By Robert F. Service setts biotech startup called 1910 Genetics, COVID-19 pandemic was coming asked a chemical company partner to syn- into view, Jen Nwankwo and col- hibitors to known targets. The two pro- thesize the compounds. A week or so later, leagues turned a pair of artificial grams searched for compounds able to her team received the orders, added each intelligence (AI) tools against SARS- block human enzymes that play essen- compound in turn to human cells, and CoV-2. One newly developed AI pro- tial roles in enabling the virus to infect learned that each blocked its target and gram, called SUEDE, digitally screens our cells. prevented viral entry into cells. 1910 Genet- all known druglike compounds for While SUEDE sifted through 14 billion ics is now looking to partner with antiviral likely activity against biomolecules compounds in just hours and spit out a hit, drug developers to pursue animal and hu- thought to be involved in disease. The BAGEL made equally fast work of design- man trials. “It shows that AI can massively

Iother, BAGEL, predicts how to build in- ing a lead. Nwankwo, CEO of a Massachu- accelerate drug design,” Nwankwo says. ART SCHMITZ/FOLIO STEPHAN ILLUSTRATION:

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immune protection wanes or viral variants bulky enough to prevent spike from binding emerge. “So, continuing development of anti- to the ACE2 receptor. virals is critical,” Denison says. David Baker, a computational biologist at To date, most of that search has centered the University of Washington, Seattle, and on “repurposed” compounds, antivirals origi- colleagues turned instead to miniproteins, nally developed to combat other diseases. each with about 60 amino acids, customized (Other repurposed drugs, such as the steroid to block protein-protein interactions. In late dexamethasone, target the body’s reaction to 2020, Baker’s team described miniproteins infection rather than the virus itself.) “Drug tailormade to bind tightly to the virus’ spike repurposing made sense as the first thing protein and block it from attaching to the to try,” Nwankwo says. Many repurposed ACE2 receptor (Science, 23 October 2020, antivirals have shown promise p. 426). The tiny proteins kept the against SARS-CoV-2 in cell and ani- virus from infecting human cells mal studies and are now in clinical Science’s in a test tube, and Baker says mini- trials. One, remdesivir, has already COVID-19 proteins could make ideal drugs proved to speed recovery by a few reporting is because they are far more stable days in very ill people. But several supported than conventional protein thera- by the other repurposed antivirals have Heising-Simons peutics, such as antibodies, which failed to prove effective. Foundation. must be refrigerated. Baker is in As a result, says Francis Collins, discussions with drug companies Downloaded from director of the National Institutes of Health to pursue his leads. (NIH), “We really, really need a bunch more Other researchers have a different strategy [antivirals].” Buoyed by almost daily ad- for interfering with viral binding. They are vances in understanding SARS-CoV-2, the designing ACE2 look-alikes to serve as de- rapidly growing list of new compounds that coys, drawing SARS-CoV-2 away from cells. might block it, and ongoing clinical trials— Researchers at Neoleukin Therapeutics and some of them late stage—Denison and others their partners, for example, reported creat- http://science.sciencemag.org/ hope to deliver effective drugs this year. Says ing a miniprotein, CTC-445.2d, that mimics Andrew Mesecar, a structural biologist from ACE2, binding tenaciously to spike (Science, Purdue University: “I am confident we will 4 December 2020, p. 1208). The compound have more treatments for coronavirus.” protected human cells from infection in vi- tro. When given to hamsters in a nasal spray, AS VIRUSES GO, SARS-CoV-2 is a behe- the decoy also prevented them from getting moth, with some 30,000 letters of RNA severe disease after they received a normally in its genetic code. Those letters encode lethal dose of the virus. Another “receptor 29 viral proteins that enable the virus to trap” molecule, described in November 2020

infect cells, reproduce, escape, and spread. in the Proceedings of the National Academy on March 12, 2021 “We’re fortunate this virus has provided us of Sciences, also diverted SARS-CoV-2, keep- with so many targets, so many opportuni- ing it from infecting cells in the test tube. ties for intervention,” says Sandra Weller, a Designing and developing a medicine is molecular biologist at UConn Health. AFTER ENTERING A CELL, a virus transforms its almost always painfully slow, regularly tak- The 29 proteins come in three main cat- host into a virus factory. That’s where SARS- ing at least a decade. Many steps—such as egories: structural proteins that make up the CoV-2’s NSPs come in. The viral proteins are animal studies, tweaking molecules to avoid outer coat; nonstructural proteins (NSPs), made by the host cell’s own protein facto- side effects, and clinical trials—can’t be ac- most of which help the virus replicate; and ries, the ribosomes, which translate the viral celerated. But the race toward new treat- accessory proteins, several of which appear RNA into two long “polyprotein” chains. The ments against COVID-19 is off to a blistering to subdue the host’s immune response. Thus chains spin off two smaller proteins, NSP3 start as researchers accelerate other parts of far, drug hunters have taken aim mainly at and NSP5, protein-cutting protease enzymes the search, deploying supercomputers, ro- the structural and replication proteins, con- that then chop up the rest of the polyproteins bots, synchrotrons, and every other tool they centrating on molecules similar to those that into independent, functioning proteins. have to find and lab test possible medicines have paid off in fighting other viruses. “These are absolutely critical functions at speed. According to a biotech industry SARS-CoV-2 has just four structural pro- that are highly conserved and should be very, drug tracker, some 239 antiviral molecules teins. The envelope and membrane proteins very vulnerable” to antivirals, Denison says. against COVID-19 are under development, make up the virus’ spherical shell, and the Drugs that block proteases have success- targeting multiple parts of the viral life cycle. nucleocapsid protein shields its genome. The fully fought HIV and hepatitis C and have Antivirals have proved critical in fighting fourth protein, spike, protrudes from the been among the most popular candidate other infections such as HIV and hepatitis shell, creating the crown of thorns that gives antivirals for SARS-CoV-2. Two repurposed C. Such drugs will be vital in the struggle the virus its name and enables it to bind to protease inhibitors for treating HIV, lopina- against the pandemic coronavirus, too, de- angiotensin-converting enzyme 2 (ACE2) re- vir and ritonavir, showed promise in vitro spite the ongoing rollout of COVID-19 vac- ceptors, its main entry point into cells. against SARS-CoV-2, but in October 2020, cines. “We know not everyone will be able to Spike is the primary target of many vac- the United Kingdom’s large Recovery clinical take the vaccine or respond to it,” says Mark cines and antivirals. However, small mol- trial reported they offered no benefit. Denison, a virologist at Vanderbilt Univer- ecules, the typical focus for drug discovery Researchers at the drug giant Pfizer are sity. Vaccines may also lose effectiveness as programs, won’t work because they aren’t pursuing an inhibitor that may work better

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Published by AAAS NEWS | FEATURES because it is designed to target NSP5, a pro- Antivirals take aim AFTER SARS-COV-2’S proteases have freed tease that is specific to SARS-CoV-2 and its As it infects cells, reproduces itself, and spreads, the coronavirus proteins from the original chains, coronavirus relatives. Pfizer scientists devel- pandemic coronavirus relies on dozens of viral and host 15 of them come together to form the repli- oped the drug in 2003 to block the molecule, proteins. They offer an array of targets for candidate cation transcription complex (RTC), which also known as main protease (Mpro), in se- drugs (blue boxes) that aim to block proteins key to copies the virus’ RNA genome to make new vere acute respiratory syndrome (SARS), the different stages in SARS-CoV-2’s life cycle. viruses (see graphic, left). Central to that ma- deadly coronavirus that emerged the previ- chinery are NSP9, which locks onto the virus’ ous year. That work was set aside when the 1 Attachment and entry RNA strand, and the RNA-dependent RNA SARS epidemic died out. Now, Pfizer has polymerase (RdRp) that copies the RNA. pulled the compound off the shelf and found The RTC’s critical role has made it, and that it stops SARS-CoV-2 from reproduc- RdRp in particular, the most popular treat- ing inside human cells. Pfizer researchers Miniproteins, ment bull’s-eye of all. It’s where remdesivir tweaked the structure to make a more sol- CTC-445.2d does its work. The drug is a nucleoside ana- uble version, known as PF-07304814. They log, a sort of imitation RNA building block showed that it sharply reduced viral load in that resembles adenosine (A), one of the mice; in other animals, high concentrations TMPRSS2 ACE2 four letters that make up RNA. The impos- of the drug could reach tissues. Viral ter tricks the RdRp into inserting remde- “It’s a promising lead,” says Celia Schiffer, RNA sivir molecules instead of A’s into growing a molecular biologist with the University of RNA strands, jamming RdRp and stopping Ribosomes Massachusetts Medical School. In September Translation of viral replication. 2 Downloaded from 2020, Pfizer launched a small clinical trial to viral proteins Polyprotein Researchers hope other repurposed nucle- test the safety of PF-07304814, delivered intra- chains oside and nucleotide analogs will be better at venously. But Annaliesa Anderson, who leads fooling the coronavirus’ RdRp. (Nucleotides Pfizer’s antiviral program, says recruiting vol- 3 Proteolysis are nucleosides with one or more phosphate unteers has been difficult. “Patients are either groups added.) The candidates include favipi- very sick, and it might be too late [to inhibit ravir and triazavirin, both originally designed viral replication], or they might not feel that PF-07304814, to combat flu viruses; ribavirin, a treatment http://science.sciencemag.org/ Main bad,” making an intravenous therapy less ap- protease boceprevir, for respiratory syncytial virus and hepatitis pealing. Given the slow recruitment, she ex- (NSP5) GC376 C; and galidesivir, which can block replica- pects results toward the end of this year. tion of Ebola, Zika, and yellow fever viruses. Nonstructural proteins Other researchers are also working on Researchers are guardedly optimistic Mpro inhibitors. In Nature Communications about molnupiravir, a nucleoside analog in September 2020, researchers in China re- that can be taken as a pill and was originally ported on two repurposed drugs, boceprevir developed to combat influenza. Last year, and GC376. Boceprevir is a hepatitis C drug, Cas13a, C5 concerns swirled around the drug after a whereas GC376 was designed to target a fe- 4 RNA replication whistleblower criticized what he saw as an

Replication Circulating RNA line coronavirus. Both compounds slowed improper effort to steer federal funding to it. on March 12, 2021 SARS-CoV-2 replication in cells. On 5 Febru- transcription complex (Science, 13 May 2020). But positive results ary, U.S. researchers reported in a bioRxiv zotatifn, plitidepsin have kept progress on track. preprint that most mice given GC376 after Early work showed molnupiravir inserts receiving a lethal dose of the pandemic vi- itself into RNA in place of the nucleoside NSP9 rus survived. And in August 2020 in Science cytidine, prompting errors in the copying Translational Medicine, U.S. researchers process and causing a lethal buildup of mu- described a GC376 analog that dramati- tations in the virus. That mechanism has cally boosted survival rates in mice infected RdRp raised worries that the drug might cause with Middle East respiratory syndrome similar mutations in host cells. But Richard and showed potent antiviral effects against remdesivir, favipiravir, triazavirin, Plemper, a cell biologist at Georgia State Uni- SARS-CoV-2 in cells. ribavirin, galidesivir, molnupiravir, AT-527 versity, says such problems have not been Other molecules designed specifically to in- seen in animal studies. hibit SARS-CoV-2’s Mpro remain at an earlier In April 2020 in Science Translational testing stage. In November 2020, for example, Medicine, Denison and colleagues reported Transcription and Charlotte Lanteri, a research microbiologist at 5 translation of structural that in mice, molnupiravir sharply reduced the Walter Reed Army Institute of Research, and accessory proteins replication of multiple coronaviruses, includ- reported discovering 807 Mpro inhibitors ing SARS-CoV-2; the drug also cut SARS- through an AI screen of 41 million com- CoV-2 replication in human airway epithelial pounds. Her team has identified seven as par- cells. A Nature paper added to the encourag- ticularly promising, but turning one or more 6 Assembly, packaging, ing data in February, showing the compound and release of those into drugs remains at least a couple decreased viral replication 100,000-fold in of years away, says Lanteri, who reported the mice engineered to have human lung tissue. SCIENCE findings at an antiviral drug summit at NIH. In December 2020, Plemper and colleagues She and colleagues are also looking for anti- reported in Nature Microbiology that mol- viral drugs effective against all coronaviruses. nupiravir might do more than just prevent “We want to be prepared as much as possible symptoms. The researchers gave the drug to

for the next emerging threat,” she says. ferrets, which readily spread the coronavirus, ALTOUNIAN/ V. GRAPHIC:

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Published by AAAS and transmission fell to zero within 24 hours. of SARS-CoV-2 RNA. The team’s Cas13a en- the same strategy against SARS-CoV-2. Rap- “This is the first demonstration of an orally zyme targets highly conserved regions of two idly reproducing viruses “have a great need available drug to rapidly block SARS-CoV-2 viral genes encoding the RdRp enzyme and for RNA,” says Marla Weetall, vice president transmission,” Plemper says. That’s crucial to the nucleocapsid protein. When hamsters of pharmacology with PTC Therapeutics. slowing the spread of disease. Because it is a infected with SARS-CoV-2 inhaled a vapor- The company’s compound, PTC299, was pill, molnupiravir can be given early in the ized formulation of the drug, it reduced viral originally designed as an oral drug to halt disease cycle, just when SARS-CoV-2 replica- replication and disease symptoms. cell proliferation in acute myeloid leuke- tion typically peaks, in contrast to injectable And in September 2020 in ACS Central mia. In an August 2020 preprint on bioRxiv, drugs such as remdesivir. “We want to start Science, Matthew Disney, a chemist at the Weetall and colleagues reported that treatment early and prevent people from ever Scripps Research Institute, and colleagues PTC299 sharply inhibited SARS-CoV-2 rep- showing up in a hospital,” Plemper says. reported discovering a compound called C5 lication in cells. The compound also blocked The same month, a medRxiv preprint re- that blocks a short, hairpin-shaped segment the production of immune molecules that ported that a small safety trial showed the of RNA involved in SARS-CoV-2 replication. cells build using RNA bases, hinting that drug was well tolerated, with no serious side “We have other segments of the viral genome PTC299 might help tame the immune over- effects in healthy volunteers. Molnupiravir is we think we can target as well,” he says. reaction seen in severe COVID-19. now in phase 2/3 clinical trials run by Merck Daniel Vitt, CEO of Immunic Therapeutics, and Ridgeback Biotherapeutics; in March, BECAUSE SARS-COV-2 relies on a host cell’s says his company has also seen promising re- scientists reported at a meeting that molnu- proteins to reproduce, disrupting those pro- sults in human trials of its oral compound, piravir reduced patients’ viral levels. teins could be another avenue to treatments— IMU-838, developed to treat inflammatory AT-527, another oral nucleoside analog with the advantage that not targeting the and autoimmune diseases. In February, the Downloaded from developed to treat hepatitis C by Atea Phar- virus directly could lower its odds of be- company reported preliminary results sug- gesting hospitalized patients on the drug had less need for ventilators. Trials continue for both companies.

ULTIMATELY, no one compound is likely to deliver a knockout punch to the pandemic http://science.sciencemag.org/ coronavirus, in part because drug-resistant viruses are likely to emerge. Collins and others argue that the best strategy takes a page from the treatment for HIV and hepatitis C: mixing and matching anti- virals aimed at several proteins, making it harder for the virus to evolve multiple workarounds at once. “We really need an arsenal,” says Lillian Chiang, CEO of Evrys

Bio, which is working on antivirals against on March 12, 2021 host-cell proteins. “This is going to take time,” says Michael Sofia, a chief scientific officer with Arbutus Biopharma, a Canadian antiviral com- pany. And money. According to recent esti- Pharmaceutical researchers use high-speed robotics and other tools to speed the search for antivirals. mates, bringing a new drug to market costs between $985 million and $2.8 billion. maceuticals and Roche, is also in a phase 2 coming resistant to the drugs. Their tar- Anderson says Pfizer, for one, is committing clinical trial against COVID-19. gets include host cell proteases TMPRSS2 company resources to defeating the pan- Scientists are trying to shut down other and furin, which the candidate drugs from demic without expectation of profit. Other RTC proteins, too. In recent results, two Nwankwo’s team block. Last month, NIH companies say the same. But during pre- compounds—zotatifin and plitidepsin— announced it was launching a phase 2/3 vious lulls in infectious disease outbreaks, appear to block viral replication by inter- trial for camostat mesilate, another TM- many drug companies abandoned work on fering with NSP9, the RNA-grabbing en- PRSS2 inhibitor. antivirals. “As soon as this stops being a hot zyme. Plitidepsin is in a phase 2/3 trial by Another target is a protein called di- area, people will move on,” Nwankwo says. the Spanish drug company PharmaMar. At hydroorotate dehydrogenase (DHODH). It’s In another disincentive, antiviral treat- least three other NSPs are considered good the linchpin in a pathway that cells use to ments for SARS-CoV-2 might be given for targets, says Tomáš Cihlárˇ, a virologist make two of RNA’s four bases when they need only a week or two, giving drugmakers a with Gilead Sciences, maker of remdesivir. extra RNA—for example, when proliferating. narrow window to reap returns. As a result, Eventually, drugs could target the corona- Viruses hijack that pathway to replicate. In Denison and others argue that more gov- virus’ RNA, not just its proteins. In Na- cell studies, blocking DHODH has halted ernment support is needed to keep stocking CH ture Biotechnology in February, Emmeline cancer and viral diseases, such as influenza the antiviral arsenal. Any lull in the battle

S E AR Blanchard, a biomedical engineer at the and cytomegalovirus. And DHODH blockers against SARS-CoV-2 and its kin is likely to

S R E Georgia Institute of Technology, and col- have thus far proved safe when tested in hun- be temporary, they say.

CR I P leagues reported creating a polymer-encased dreds of patients. “We are going to have another corona- O : S

T formulation of a gene-editing enzyme called Two biotech companies, PTC Therapeu- virus,” Mesecar says. “We just don’t know

P H O Cas13a that seeks out and chops up snippets tics and Immunic Therapeutics, are trying what it will look like.” j

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Published by AAAS A call to arms Robert F. Service

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