Researchers have designed a new class of TMPRSS2 inhibitors that have shown broad antiviral activity against coronaviruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Although several vaccines have now been approved for combating coronavirus disease 2019 (COVID-19), there are as yet no safe, targeted and effective drugs available for its treatment. Remdesivir and some other broad-spectrum antivirals have been repurposed (with emergency approval) to treat severe COVID-19, though has shown mixed success as a therapeutic avenue.

One potential target for drug development is viral proteases, like the papain-like protease (PLpro) or the 3C-like protease (3CL or Mpro), as well as other host proteases involved in viral entry and replication.

The transmembrane serine protease, TMPRSS2, is important for the entry of SARS-CoV-2 and influenza A viruses. The spike protein of the virus, which binds to the human angiotensin-converting enzyme 2 (ACE2), requires mediation by TMPRSS2 to enter host cells. Hence, small molecules that can target this protease may be useful in treating COVID-19.

In a new study, release as a preprint on the bioRxiv* server, researchers report a new class of ketobenzothiazole (kbt) inhibitors of TMPRSS2 that have strong antiviral activity.

Designing inhibitors

TMPRSS2 and other transmembrane serine proteases not only play a role in infectious diseases but also in cancers. This is believed to be because of its ability to activate hepatocyte growth factor (HGF). The researchers have reported the anticancer properties of ketothiazole and kbt inhibitors of HGF activator before. Since there is an overlap between the substrate specificity profile of TMPRSS2 with HGF-activating proteases, the team thought their synthetic HGF activation inhibitors (sHAIs) would also inhibit TMPRSS2.

The team tested two sHAI compounds and found them to inhibit viral entry into Calu-3 lung epithelial cells. These compounds block viral entry depending on the dose. They found no effect of the compounds in blocking viral entry in cells that did not express TMPRSS2. The team also found that the compounds inhibited viral entry in a human cell line.

The compounds developed have a serine trapping kbt that reacts reversibly with the protease but is a covalent reaction. This reversible reaction is in contrast to that of Camostat and Nafamostat, other known serine protease inhibitors, hence the authors continued their investigations using these compounds.

To design their kbt inhibitors, the authors used the crystal structure data of the previous inhibitors and modeled how they interact with TMPRSS2. In addition, they analyzed the peptide substrates preferred by TMPRSS2 and HGF activating proteases and found a notable overlap.

The team also performed substrate profiling using mass spectrometry to determine the substrate specificity of TMPRSS2. This helped them confirm that TMPRSS2 has high specificity toward the P1 position, which is occupied only by lysine or arginine. Peptide sequencing helped them identify the 25 most preferred substrates for TMPRSS2. Based on these analyses, the team synthesized four new analogs for TMPRSS2 inhibition.

Broad-spectrum antiviral activity

The authors tested viral inhibition using several analogs of the initial two compounds tested using a pseudotyped virus system, which helped them identify the compounds with the best inhibition.

When they tested the most potent compounds in inhibiting Middle Eastern respiratory syndrome (MERS) coronavirus entry into host cells, they found all inhibited viral entry similar to that of SARS-CoV-2. This suggests the compounds have broad activity against coronaviruses. They confirmed the antiviral activity of the compounds is related to TMPRSS2 using enzyme assays on recombinant TMPRSS2. All the compounds showed high potency, with the number of compounds needed to inhibit viral entry by half (IC₅₀) being less than nanomolar concentration.

The researchers also tested the activity of the most potent compounds against the wild-type SARS-CoV-2 in a cellular viability assay using human lung epithelial cells. The compounds were more than 20-fold effect as Remdesivir and showed no cellular toxicity up to 50 μM.

The team identified a relationship between the structure of the compounds and its potency. TMPRSS2 seems to prefer large groups that extend beyond the kbt C-terminal portion. Compounds with a smaller ketothiazole portion in the P1’ portion had lower potency.

All the compounds are also very stable in mice and human plasma. The most potent compounds also had good accumulation in mice lungs with a half-life of several hours. The lead compound was also tested for safety in mice, and the authors found no adverse effects in the tested mice. These compounds have potential not only for use against SARS-CoV-2 but also as broad-spectrum antivirals.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Lakshmi Supriya

Lakshmi Supriya got her BSc in Industrial Chemistry from IIT Kharagpur (India) and a Ph.D. in Polymer Science and Engineering from Virginia Tech (USA).