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Inhibiting SARS-CoV-2 protease with penicillin derivatives

Image by: Karl Harrison

Work from IOI researchers during lockdown has revealed that derivatives of the penicillins inhibit the main viral protease of SARS-CoV-2. Structural studies show that the mode of action of these drugs is related to that of the antibacterial activity of the penicillins.

The ongoing COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, remains a major public health problem worldwide. While multiple safe and effective vaccines for preventing infection are available, only a small number of antiviral drugs that can be used to treat COVID-19 have been approved – in the UK, these currently are limited to nirmatrelvir and ritonavir (Paxlovid), remdesivir (Veklury) and molnupiravir (Lagevrio). This small number of available drugs, coupled with the potential for emergence of resistance against these compounds, highlights the need to develop additional antivirals against SARS-CoV-2.

One SARS-CoV-2 protein that is a particularly attractive drug target is the main viral protease, Mpro. Once the virus infects cells, it produces long polypeptides that are then cut – mainly by Mpro – into different viral proteins that are responsible for viral replication. Therefore, Mpro is essential in the lifecycle of the virus and inhibiting this protease stops viral replication. Furthermore, previous studies have shown that drugs that inhibit Mpro, such as nirmatrelvir, can be useful in the clinic to control SARS-CoV-2 infection.

Most drugs targeting Mpro work by binding to its active site (i.e., the region of the protease that is involved in cutting the long polypeptides into individual proteins), thereby inhibiting its activity. During the lockdown period, researchers at the IOI developed a method to identify compounds that bind to the active site of Mpro, which led to the identification of a molecule similar to penicillins that can inhibit this viral protease. More specifically, they identified a compound related to penicillin V (called a derivative) that reacted with a key amino acid in the active site of Mpro (termed the nucleophilic cysteine) that is essential for the ability of this protease to cut long polypeptides. Since penicillins have a long history of being safe in humans – due to their widespread use to treat bacterial infections – the authors became interested in optimizing these drugs as inhibitors of the viral protease Mpro and understanding how they work.

In the new study, recently published in the Journal of Medicinal Chemistry, the researchers started by synthesizing a set of penicillin V derivates with diverse structures, based on the molecule they previously found to inhibit Mpro. These compounds were tested for their ability to block the cleaving activity of the viral protease, using a technique called mass spectrometry (SPE-MS) that enables researchers to study changes in proteins and polypeptides. This analysis between the structure of the different drugs and their inhibitory activity (called Structure Activity Relationship, or SAR analysis) revealed several important features of the compounds that are important for their ability to block Mpro activity, which the authors further explored to increase the potency of the inhibitors. As highlighted by Tika Malla, the DPhil student who co-led the study, the use of modern techniques was critical:

“The powers of modern methods, in our case mass spectrometry, to identify interesting mechanisms of inhibition was crucial to our work.”

To further clarify how the drugs inhibit the viral protease, the authors then used protein X-ray crystallography, a method that enabled them to directly visualize how one of the penicillin V derivates binds to Mpro. The resulting structure showed that the penicillin V derivative binds to and covalently modifies the nucleophilic cysteine in the active site of the viral protease, demonstrating the ability of these drugs to target a critical region of Mpro. Interestingly, the mode of Mpro binding is related to that by which penicillins act to kill bacteria.

As explained by Professor Christopher Schofield, who supervised the project:

“Their unique ring structure is central to the success of penicillins as antibacterials, of immense social importance. It seems that the same ring structure might be useful in fighting viral infections”

Altogether, the present study reveals not only a set of penicillin V derivatives that are potent Mpro inhibitors but also provides important insights into the mechanisms by which they interfere with this viral protease that is essential for SARS-CoV-2 replication. Therefore, while additional studies are needed to test the ability of these molecules to interfere with viral replication in vivo, the current findings, coupled with the proven efficacy and safety of penicillins in other settings (particularly as antibacterials), suggest that they should be further explored as antiviral drugs.

Dr. Lennart Brewitz, who co-led the study, summarized the importance of findings:

“Natural products, such as the penicillins, are a mainstay of modern medicine, and have been exploited most effectively as antibacterials. Our results are exciting because they suggest that modified penicillins have potential as antivirals”.

About the Ineos Oxford Institute for Antimicrobial Research at the University of Oxford

The Ineos Oxford Institute for Antimicrobial Research (IOI) was established at the University of Oxford in January 2021 to advance research, education and collaboration in the search for solutions to one of the biggest public health challenges of our time. Our mission is to lead science that will enable the development new of antibiotics and provide the evidence to support transformational change in how we address antimicrobial resistance. We are collaborating with world class academic talent across the University of Oxford, the UK and globally, with a primary research focus on antibiotic resistance in bacteria. You can learn more about the IOI here:

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