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Drug Discovery

Crystal structures of the novel coronavirus protease guide drug development

Medicinal chemists focus on the main protease of SARS-CoV-2 to develop antiviral treatments for the virus causing COVID-19

by Laura Howes
March 24, 2020

 

20200324lnp1-protease.jpg
Credit: © H. Tabermann/HZB
The novel coronavirus main protease dimer bound to an inhibitor (yellow).

With the novel coronavirus, SARS-CoV-2, spreading across the globe, labs are shutting down as people are told to stay at home. But some labs are still hard at work, looking for druggable targets to treat COVID-19, the illness caused by the virus. Two groups of researchers are using different approaches to find new inhibitors of a key part of the virus’ self-replication machinery.

There aren’t many targets for anticoronavirus drugs because the virus doesn’t produce many proteins, says Matthew Todd, an expert in drug discovery at University College London who isn’t involved with either of the two projects. But one focus for medicinal chemists has been the virus’s main protease known as Mpro or 3CLpro. This enzyme processes a polyprotein chain coded by the virus’s RNA, chopping up the chain into functional proteins that the virus then uses to assemble itself and multiply. Disrupting this key piece of the virus’s self-replication machinery could bring an infection screeching to a halt.

“The protease is essential, but has no human homologues,” Todd says. So inhibitors of the protease have less of a chance of hitting a human protease, he adds.

To try and develop inhibitors for Mpro, two different international collaborations have used synchrotron X-rays to get high-resolution structures of the protease with potential inhibitors bound. While the approaches the teams are taking are different, the researchers are keen to stress that their approaches are complementary, and that the lead compounds they are developing are a long way from being used on patients with COVID-19.

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At the University of Lübeck, Rolf Hilgenfeld has been studying coronaviruses for years. During the Middle East respiratory syndrome outbreak in 2013, which was caused by a coronavirus called MERS-CoV, Hilgenfeld’s team started working on protease inhibitors, giving the researchers a head start on SARS-CoV-2.

20200324lnp1-struct.jpg

Earlier this year, Hilgenfeld’s team used high-intensity synchrotron X-rays to get a crystal structure of the new virus’s Mpro at high resolution and used that information to optimize an existing α-ketoamide inhibitor developed for fighting other coronaviruses (Science 2020, DOI 10.1126/science.abb3405). The synthesis of the new inhibitors was delayed by shutdowns in China, a result of the outbreak, but recent lab tests have now shown that one of these optimized compounds (shown) can bind to and inhibit the protease. Tests on healthy mice also suggest that the molecule could be administered by inhalation.

Hilgenfeld hopes this lead compound will be developed by a consortium set up by the European Union to fight COVID-19, but he expects that the road from this molecule to a functional drug could take years, perhaps ready in time for the next coronavirus that pops up.

Meanwhile, another consortium of scientists is trying to speed up the process of developing a viral protease inhibitor with a huge crowdsourced initiative. Earlier in March, crystallographers at the Diamond Light Source also solved the structure of the SARS-CoV-2 main protease at high resolution. They then completed a large fragment-screen by soaking protein crystals with small molecules representing fragments of possible drugs to see which fragments bound to the enzyme.

Based on those data, the team started the crowdsourced initiative to combine the expertise of multiple labs and researchers around the world to process as many possible protease inhibitor structures as possible. Chemists are invited to design new compounds—or submit existing ones that might bind to the protease—on a website created by an artificial intelligence medicinal chemistry start-up called PostEra. Submitted structures get prioritized based on factors like ease of synthesis and possible toxicity before a custom-synthesis firm makes the molecules. Researchers then screen the molecules for binding activity.

“The goal of the project is to find an antiviral that can get to the clinical stage as soon as we can,” explains Alpha Lee, who is part of the team behind the initiative. The strategy, he says, is to open the funnel of candidate compounds as wide as possible. They hope to find several lead compounds and then use their AI algorithms to suggest changes to the molecules to help speed up drug development.

Todd describes the two approaches as “both interesting projects.” But he cautions that finding promising candidates is just one step of the process. The bottleneck is often optimizing those candidates into viable drugs and, he says, “that’s difficult to do under the right conditions.” With labs shutting down to reduce social contact, both teams hope they can keep their projects running.

“It’s always key to develop several things at once,” adds Nir London, whose lab is involved in screening crowdsourced fragments. “I think both approaches are required and necessary and we can learn from each other.”

“I think this is a wake up message for the world that infectious diseases have long been neglected and it’s time to not neglect them,” Lee says. Hilgenfeld agrees. The development of antiviral drugs should be decoupled from recurring outbreaks he says, so we can ensure the sustainable development of antiviral drugs.

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Comments
Michael Vazquez (March 26, 2020 2:35 PM)
Unlike HIV protease inhibitors which may need to be taken for a lifetime, a Covid-19 protease inhibitor would probably need to be dosed for only 7-10 days. Just long enough to knock down the virus and allow ones immune system to get the virus under control. A Phase 1b clinical trial enrolling patients who are dosed for up to 10 days would only require 30 days toxicity testing in a rodent and non-rodent species. Under the current circumstances it would be a welcome effort to see big pharma aggressively evaluate the suitability of Covid-19 protease inhibitors for clinical use, scale-up GMP API, and petition the FDA to allow rapid testing. If successful in a Ph1b/Ph2 study, release the compound for compassionate use in the most severely affected patients.
Elizabeth Lacey (April 3, 2020 3:53 PM)
Has silicon dioxide (SiO2) been tested as a protease inhibitor in the search for developing a treatment for COVID-19.
Ildiko M. Kovach (April 30, 2020 11:38 PM)
I would like to call attention to a group of phosphonate esters developed for the reversible inhibition of serine hydrolase enzymes and studied during my academic career at The Catholic University of America. They would work with cysteine proteases as well. The compounds are 4-Nitrophenyl 4-substituted-phenacyl methylphosphonate esters. The leaving group could easily be changed to fluoride, for example, for better physiological compatibility. Their particular advantage is the ready modulation of the time scale (including of course long action times) for the return of enzyme activity, if desired. I do not have these compounds any more but their studies and synthesis are in the Literature. Publications and two patents from our work are listed below.

Publications

I.M. Kovach* and L. McKay, Reversible Modulation of Serine Protease Activity by Phosphonate Esters, Bioorg. Med. Chem. Lett. 2, 1735 (1992).

I.M. Kovach*, Q. Zhao, M. Keane and R. Reyes, Rate-Determining Carbonyl Hydration in the Intramolecular Hydrolysis of Phenacyl Phosphonate Esters: Isotopic Probes and Activation Parameters, J. Am. Chem. Soc.. 115, 10471 (1993).

Q. Zhao, I.M. Kovach*, A. Bencsura and A. Papathanassiu, Enantioselective and Reversible Inhibition of Trypsin and α-Chymotrypsin by Phosphonate Esters, Biochemistry 33, 8128 (1994).

A. Bencsura, I. Enyedy and I. M. Kovach*, Origins and Diversity of the Aging Reaction in Phosphonate Adducts of Serine Hydrolase Enzymes: What Characteristics of the Active Site Do They Probe? Biochemistry 34, 8989-8999 (1995).

Q. Zhao and I. M. Kovach*, Reversible Modification of Tissue-type Plasminogen Activator by Methylphosphonate Esters, Bioorg. Med. Chem 4, 523-529 (1996).

E. Enyedy and I. M. Kovach*, Modulation of the Activity of Human α-Thrombin with Phosphonate Ester Inhibitors, Bioorg. Med. Chem. 5, 1531-1541, (1997).

E. Enyedy and I. M. Kovach*, Reversible Modulation of Human Factor Xa Activity by Phosphonate Ester: Media Effects, Bioorg. Med. Chem. 8, 549-556 (2000).

Patents
I.M. Kovach, Temporary Inactivation of Serine Hydrolases Using Nitrophenyl Phenacyl Phosphonates. US patent 5,281,523 Ser.# 851,187, January 25, 1994.
US patent 5,324,815 Ser.# 850,112, May 24, 1994

I will be happy to respond to any inquiry about this.

Sincerely,

Ildiko M. Kovach, Ph.D.
Professor of Chemistry, Emerita
The Catholic University of America
Washington DC, 20064
Cell phone: 301-3850166
Dr.Paul C. Li (May 18, 2020 8:01 AM)
Dear Honorable C&EN Editors:
A zwitterion’s or amphoteric ion pairs’ positive and negative charges are separated by more than a few atoms but connected in the same main chain. It looks just like the emblem or totem of Tai Ji (太極).
The ion pairs’ role in biological kingdom and in the subnimal (neither an animal nor a plant) world of viruses might be the frontiers of conquest COVid19’s persistence.
Application of in-situ nano-magnetic excitation of this zwitterion or amphoteric ion pairs to cut through or unscrew the spikes (glycoproteins) on the COVid19’ spheres so as to allow your effective drugs (alpha keto-amide inhibitors) to get in holes through the lipid bilayers. The next drama is the migration of the membranes inside the sphere which might be exuded. Then, every one knows what’s going to happen next! Disintegration of the sphere is expected with simultaneous disabled RNA left like trash to be expelled. So Bingo. I believe humans are intelligent to outsmart the virus subnimal origins.
Do not look down the wisdom of I Ching, take it nonscientific side of mystic force that may be the fifth force of nature between you and me. The magnetized particles in the body fluid would rotate or scramble according to additional external field resulting in more advance disintegration with minimal hurt to the main body.
I used three known equations in mathematics applied simultaneously in treating diatomic molecules’ vibrational frequencies that made me courage enough to send you this letter in confidence with humble manners. Sincerely with highest level of honest by a life long chemist trained the US institution where honest is everywhere highly esteemed both faculty and the students.

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