A study revealed that Jun13296, a novel PLpro inhibitor, demonstrates potent antiviral and anti-inflammatory effects in vivo, offering hope for future COVID-19 therapies beyond Paxlovid.

Study: Design of quinoline SARS-CoV-2 papain-like protease inhibitors as oral antiviral drug candidates. Image Credit: Shutterstock AI Generator / Shutterstock.com

A study recently published in Nature Communications discusses the development of a quinoline-based drug candidate with inhibitory activity against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like protease (PL^pro). 

The development of novels antivirals against SARS-CoV-2

During the coronavirus disease 2019 (COVID-19) pandemic, researchers throughout the world explored the potential efficacy of existing antivirals and anti-inflammatory drugs against SARS-CoV-2 to reduce disease severity and mortality. Simultaneously, extensive efforts were made to develop novel broad-spectrum oral antivirals aimed at preventing and managing future pandemics caused by similar pathogens.

Broad-spectrum antivirals must target antigens like polymerases and proteases that are conserved over a wide range of viral pathogens. Remdesivir and molnupiravir, for example, exhibit conserved activity against the hepatitis C virus, filoviruses like Ebola virus, coronaviruses like SARS-CoV and SARS-CoV-2, Pneumoviruses like respiratory syncytial virus, and paramyxoviruses like mumps, measles, Nipah and Hendra viruses.

During the early drug development stages of the COVID-19 pandemic, the SARS-CoV-2 cysteine protease M^pro was identified as a potential target for novel therapeutics. Using high-throughput screening (HTS) methods coupled with drug design techniques, M^pro inhibitors like nirmatrelvir were developed.

Paxlovid, a combination of nirmatrelvir with the metabolic enhancer ritonavir, is currently approved by the United States Food and Drug Administration (FDA) for the treatment of mild to moderate COVID-19. Similarly, Ensitrelvir, a noncovalent M^pro inhibitor, received approval in Japan and Singapore.

Despite these advances, other therapeutic targets must be identified due to the emergence of SARS-CoV-2 strains with novel M^pro mutations. For example, P132H is a commonly observed M^pro mutation that has yet to develop resistance to nirmatrelvir .

Several studies have reported the presence of triple M^pro mutants like L50F/E166A/L167F that are reproductively fit but exhibit potent drug resistance. Likewise, a COVID-19 patient with weakened immunity was found to harbor the Nsp5-L50F/E166V mutant after prolonged Paxlovid treatment.

Papain-like protease inhibitors

The SARS-CoV-2 PL^pro is a cysteine protease required for viral replication and suppressing host immune responses. To date, experimental PL^pro inhibitors have demonstrated limited in vivo efficacy.

The authors of the current study previously designed a covalent PL^pro inhibitor Jun11313 that bound to the Val70^Ub binding site. To facilitate further aromatic substitutions in the ring while targeting the same site, the naphthalene-based structure was replaced with quinoline conjugated with a linker-amino acid moiety to improve its binding affinity.

Further optimization and testing led to the identification of a noncovalent biarylphenyl PL^pro inhibitor Jun12682. Thereafter, Jun12682 was designed to bind to both the blocking loop region 2 (BL2) and Val70^Ub.

A series of quinoline analogs were confirmed to bind to the Val70^Ub site through their 2-aryl substituents using X-ray crystallography. However, Jun12665 exhibited a flipped orientation, which causes its 2-pyrazolyl group to fit into the BL2 groove.

Jun13296 – promising drug candidate

Jun13296 was identified as the most potent compound, with ten-fold greater activity as compared to Jun12682 against both SARS-CoV-2 and PL^pro.  

Jun13296 has a biphasic pattern of plasma release, with two peaks observed at two and eight hours from oral administration, as well as two and four hours after intravenous administration, respectively. After oral dosing, Jun13296 levels remained higher than the effective antiviral concentration for more than eight hours.

In vivo studies confirmed the greater antiviral efficacy of Jun13296 as compared to Jun12682. Jun13296 also reduced the activity of inflammatory cytokines like interleukin 6 (IL-6) and interferon γ (IFN-γ), which may contribute to its superior survival benefit despite active viral replication at lower dosages.

The anti-inflammatory activity of Jun13296 may be the result of its inhibition of other enzymatic processes linked to PLpro like deubiquitinase and deISGylase. However, this hypothesis requires further investigation.

Due to its different mechanism of action, Jun13296 remains effective against SARS-CoV-2 variants that have developed resistance to nirmatrelvir, as well as recent SARS-CoV-2 Omicron variants. Jun13296 was also effective again a triple mutant SARS-CoV-2 variant isolated from an immunocompromised patient on prolonged Paxlovid treatment, as well as the quadruple mutant selected in viral passage under drug exposure conditions.

When administered to mice with SARS-CoV-2 infection, Jun13296 increased survival, reduced weight loss, decreased viral load in the lungs, as well as prevented lung inflammation and tissue damage.

These results underscore the potential of quinoline PL^pro inhibitors as promising oral SARS-CoV-2 antiviral candidates.”